Publications

Scholarly Journals--Published

• Sheng MHC, Lau KHW, Rundle CH, Alsunna A, Wilson SM, and Baylink DJ (2022) Defective bone repletion in aged Balb/cBy mice was caused by impaired osteoblastic differentiation. J Bone Miner Metab 40 (6), 900-913. This study was undertaken to gain mechanistic information about bone repair using the bone repletion model in aged Balb/cBy mice. Briefly, 1-month-old (young) mice were fed a calcium-deficient diet for 2 weeks and 8-month-old (adult) and 21- to 25-month-old (aged) female mice for 4 weeks during depletion, which was followed by feeding a calcium-sufficient diet for 16 days during repletion. To determine if prolonged repletion would improve bone repair, an additional group of aged mice were repleted for 4 additional weeks. Control mice were fed calcium-sufficient diet throughout. In vivo bone repletion response was assessed by bone mineral density gain and histomorphometry. In vitro response was monitored by osteoblastic proliferation, differentiation, and senescence. There was no significant bone repletion in aged mice even with an extended repletion period, indicating an impaired bone repletion. This was not due to an increase in bone cell senescence or reduction in osteoblast proliferation, but to dysfunctional osteoblastic differentiation in aged bone cells. Osteoblasts of aged mice had elevated levels of cytosolic and ER calcium, which were associated with increased Cav1.2 and CaSR (extracellular calcium channels) expression but reduced expression of Orai1 and Stim1, key components of Stored Operated Ca2+ Entry (SOCE). Activation of Cav1.2 and CaSR leads to increased osteoblastic proliferation, but activation of SOCE is associated with osteoblastic differentiation. In summary, the bone repletion mechanism in aged Balb/cBy mice is defective that is caused by an impaired osteoblast differentiation through reduced expression of Orai1 and Stim1, key mediators of SOCE. (09/2022) (link)
• Ahmed ASI, Sheng MHC, Lau KHW, Wilson SM, Wongwarawat D, Tang X, Ghahramanpoui M, Nehme A, Xu Y, Abdipour A, Zhang XB, Wasnik S, and Baylink DJ (2022) Calcium released by osteoclastic resorption stimulates autocrine/paracrine activities in local osteogenic cells to promote coupled bone formation. Am J Physiol Cell Physiol 322 (5), C977-C990. A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, storeoperated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3–E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-.B ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.  (05/2022) (link)
• Stiffel V, Rundle CH, Sheng MHC, Das S, and Lau KHW (2022) A novel EphA4 signaling-based therapeutic strategy for osteoarthritis in mice. J Bone Miner Res 37 (4), 660-674. This study sought to develop a noninvasive, reliable, clinically relevant, and easy-to-implement mouse model that can be used for investigation of the pathophysiology of PTOA and for preclinical testing of new therapies of PTOA.  Accordingly, we have established a closed intraarticular tibial plateau compression loading-induced injury model of PTOA in C57BL/6J mice.  In this model, a single application of a defined loading force was applied with an indenter to the tibial plateau of the right knee to create injuries to the synovium, menisci, ligaments, and articular cartilage.  The limiting loading force was set at 55 N with the loading speed of 60 N/sec. This loading regimen limits the distance that the indenter would travel into the joint, but still yields substantial compression loading energy to cause significant injuries to the synovium, meniscus, and articular cartilage.  The joint injury induced by this loading protocol consistently yielded evidence for key histological hallmarks of PTOA at 5-11 weeks post-injury, including loss of articular cartilage, disorganization of chondrocytes, meniscal hyperplasia and mineralization, osteophyte formation, degenerative remodeling of subchondral bone.  These arthritic changes were highly reproducible and of a progressive nature.  Because 50% of patients with meniscal and/or ligament injuries without intraarticular fractures developed PTOA over time, this intraarticular tibial plateau compression loading-induced injury model is clinically relevant.  In summary, we have developed a noninvasive intraarticular tibial plateau compression loading-induced injury model in the mouse that can be used to investigate the pathophysiology of PTOA and for preclinical testing for new therapies. (04/2022) (link)
• Stiffel V, Thomas A, Rundle CH,  Sheng MHC, and Lau KHW (2020) The EphA4 signaling is anti-catabolic in synoviocytes but pro-anabolic in articular chondrocytes. Calcif Tissue Int 107 (6), 576-592. The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes.  In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases.  Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9).  EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies.  Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively.  However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage.  The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types. (08/2020) (link)
• Chen W, Wasnik S, Fu Y, Aranda L, Rundle CH, Lau KHW, Baylink DJ, and Zhang XB (2020) Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model. Bone Rep 12, 100236. In the present study we sought to improve the efficacy and safety of our Sca1+ PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1+PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1+ PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1+ PDGFB-DSS6 therapy but not by the positive control Sca1+ PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1+ PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeuticgene expression to avoid excess bone formation. (01/2020) (link)
• Li C-H, Tang X, Wasnik S, Wang X, Zhang J, Xu Y, Lau KHW, Nguyen HB, and Baylink DJ (2019) Mechanistic study of the cause of decreased serum 1,25-dihydroxyvitamin D in sepsis. BMC Infectious Diseases 19 (1), 1020. Background: Vitamin D deficiency, determined by blood levels of 25-hydroxyvitamin D [25(OH)D, i.e. the major vitamin D form in blood], has been shown to associate with all-cause mortalities. We recently demonstrated that blood levels of 1,25-dihydroxyvitamin D [1,25(OH)2D, i.e. the active vitamin D] were significantly lower in non-survivors compared to survivors among sepsis patients. Unexpectedly, despite the well documented roles of 1,25(OH)2D in multiple biological functions such as regulation of immune responses, stimulation of antimicrobials, and maintenance of barrier function, 1,25(OH)2D supplementation failed to improve disease outcomes. These previous findings suggest that, in addition to 1,25(OH)2D deficiency, disorders leading to the 1,25(OH)2D deficiency also contribute to mortality among sepsis patients. Therefore, this study investigated the mechanisms leading to sepsis-associated 1,25(OH)2D deficiency. Methods: We studied mechanisms known to regulate kidney 25-hydroxylvitamin D 1a-hydroxylase which physiologically catalyzes the conversion of 25(OH)D into 1,25(OH)2D. Such mechanisms included parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), fibroblast growth factor 23 (FGF-23), and kidney function. Results: We demonstrated in both human subjects and mice that sepsis-associated 1,25(OH)2D deficiency could not be overcome by increased production of PTH which stimulates 1a-hydroxylase. Further studies showed that this failure of PTH to maintain blood 1,25(OH)2D levels was associated with decreased blood levels of IGF-1, increased blood levels of FGF-23, and kidney failure. Since the increase in blood levels of FGF-23 is known to associate with kidney failure, we further investigated the mechanisms leading to sepsis-induced decrease in blood levels of IGF-1. Our data showed that blood levels of growth hormone, which stimulates IGF-1 production in liver, were increased but could not overcome the IGF-1 deficiency. Additionally, we found that the inability of GH to restore the IGF-1 deficiency was associated with suppressed expression and signaling of growth hormone receptor in liver. Conclusions: Because FGF-23 and IGF-1 have multiple biological functions besides their role in regulating kidney 1a-hydroxylase, our data suggest that FGF-23 and IGF-1 are warranted for further investigation as potential agents for the correction of 1,25(OH)2D deficiency and for the improvement of survival among sepsis patients. (12/2019) (link)
• Stiffel V, Rundle CH, Sheng MHC, Das S, and Lau KHW (2020) A mouse noninvasive intraarticular tibial plateau compression loading-induced injury model of posttraumatic osteoarthritis. Calcif Tissue Int 106(2), 158-171.  This study sought to develop a noninvasive, reliable, clinically relevant, and easy-to-implement mouse model that can be used for investigation of the pathophysiology of PTOA and for preclinical testing of new therapies of PTOA.  Accordingly, we have established a closed intraarticular tibial plateau compression loading-induced injury model of PTOA in C57BL/6J mice.  In this model, a single application of a defined loading force was applied with an indenter to the tibial plateau of the right knee to create injuries to the synovium, menisci, ligaments, and articular cartilage.  The limiting loading force was set at 55 N with the loading speed of 60 N/sec. This loading regimen limits the distance that the indenter would travel into the joint, but still yields substantial compression loading energy to cause significant injuries to the synovium, meniscus, and articular cartilage.  The joint injury induced by this loading protocol consistently yielded evidence for key histological hallmarks of PTOA at 5-11 weeks post-injury, including loss of articular cartilage, disorganization of chondrocytes, meniscal hyperplasia and mineralization, osteophyte formation, degenerative remodeling of subchondral bone.  These arthritic changes were highly reproducible and of a progressive nature.  Because 50% of patients with meniscal and/or ligament injuries without intraarticular fractures developed PTOA over time, this intraarticular tibial plateau compression loading-induced injury model is clinically relevant.  In summary, we have developed a noninvasive intraarticular tibial plateau compression loading-induced injury model in the mouse that can be used to investigate the pathophysiology of PTOA and for preclinical testing for new therapies. (09/2019) (link)
• Wasnik S, Lakhan R, Baylink DJ, Rundle CH, Xu Y, Zhang J, Qin X, Lau KHW, Carreon EE, and Tang X (2019) Cyclooxygenase 2 augments osteoblastic but suppresses chondrocytic differentiation of CD90+ skeletal stem cells in fracture sites. Science Advances 5 (7), eaaw2108.  Cyclooxygenase 2 (COX-2) is essential for normal tissue repair. Although COX-2 is known to enhance the differentiation of mesenchymal stem cells (MSCs), how COX-2 regulates MSC differentiation into different tissue-specific progenitors to promote tissue repair remains unknown. Because it has been shown that COX-2 is critical for normal bone repair and local COX-2 overexpression in fracture sites accelerates fracture repair, this study aimed to determine the MSC subsets that are targeted by COX-2. We showed that CD90+ mouse skeletal stem cells (mSSCs; i.e., CD45−Tie2−AlphaV+ MSCs) were selectively recruited by macrophage/monocyte chemoattractant protein 1 into fracture sites following local COX-2 overexpression. In addition, local COX-2 overexpression augmented osteoblast differentiation and suppressed chondrocyte differentiation in CD90+ mSSCs, which depended on canonical WNT signaling. CD90 depletion data demonstrated that local COX-2 overexpression targeted CD90+ mSSCs to accelerate fracture repair. In conclusion, CD90+ mSSCs are promising targets for the acceleration of bone repair. (07/2019) (link)
• Xu Y, Cheng Y, Baylink DJ, Wasnik S, Goel G, Huang M, Cao H, Cao H, Qin X, Lau KHW, Chan C, Koch A, Pham LHG, Zhang J,Li C-H, Wang X, Berumen EC, Smith J, and Tang X (2019) In vivo generation of gut-homing regulatory T cells for the suppression of colitis. J Immunology 202 (12), 3447-3457. Current therapies for the control of gut inflammation have not reached the desired specificity and are attended by unintended immune suppression. This study aimed to provide evidence for supporting a hypothesis that in vivo induction of gut-homing regulatory T (Treg) cells is a valid strategy for the treatment of chronic intestinal inflammation (e.g. inflammatory bowel disease). We showed that a dendritic cell (DC), engineered to de novo produce high concentrations of both 1,25(OH)2D (the active vitamin D metabolite) and retinoic acid (RA, an active vitamin A metabolite), augmented the induction of gut-homing Treg cells both in vitro and in vivo. In vivo, the newly generated gut-homing Treg cells homed to the intestine. Additionally, immunization with such engineered DC robustly suppressed ongoing experimental colitis. Furthermore, the colitis suppression was significantly reduced when foxp3(forkhead box P3)+ Treg cells were depleted. Collectively, we conclude that DC, de novo overproducing both 1,25(OH)2D and RA, is a promising novel therapeutic strategy for chronic intestinal inflammation. (06/2019) (link)
• Wasnik S, Rundle CH, Baylink DJ, Yazdi MS, Carreon EE, Xu Y, Qin X, Lau KHW, Tang X (2018) 1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites. JCI Insight 3(17):e98773. An indispensable role of macrophages in bone repair has been well recognized. Previous data have demonstrated the co-presence of M1 macrophages and mesenchymal stem cells (MSCs) during the pro-inflammatory stage of bone repair. However, the exact role of M1 macrophages in MSC function and bone repair is unknown. This study aimed to define the role of M1 macrophages at bone injury sites via the function of 1,25-Dihydroxyvitamin Din suppressing M1 but promoting M2 differentiation. We showed that 1,25(OH)2D suppressed M1 macrophage-mediated enhancement of MSC migration. Additionally, 1,25(OH)2D inhibited M1 macrophage secretion of osteogenic proteins (i.e. Oncostatin M, TNF-a, and IL-6). Importantly, the 1,25(OH)2D-mediated suppression of osteogenic function in M1 macrophages at pro-inflammatory stage was associated with 1,25(OH)2D-mediated reduction of MSC abundance, compromised osteogenic potential of MSCs, and impairment of fracture repair. More importantly, outside the pro-inflammatory stage, 1,25(OH)2D treatment did not suppress fracture repair. Accordingly, our data support two conclusions: one is that M1 macrophages are important for the recruitment and osteogenic priming of MSCs and hence are necessary for fracture repair; the other is that, under vitamin D sufficient condition, 1,25(OH)2D treatment is unnecessary and can be detrimental if provided during the pro-inflammatory stage of fracture healing. (09/2018) (link)
• Choudry M, Tang X, Santorian T, Wasnik S, Xiao J, Xing W, Lau KHW, Mohan S, Baylink DJ, and Qin X (2018) Deficient arginase II expression without alteration in arginase I expression attenuated experimental autoimmune encephalomyelitis in mice. Immunology 155 (1), 85-98. Studies support a role of Arginase II (Arg II) in vascular and endothelial disorders. However, the regulation and function of Arg II in autoimmune diseases are unclear. Here we report that a global Arg II null mutation in mice suppressed experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. During EAE both Arg I and Arg II were induced in spinal cords but only Arg II was induced in spleens and splenic DCs. DC activation by LPS, CD40L or TLR8 agonist significantly enhanced Arg II expression without affecting Arg I expression. Conversely, DC differentiating cytokines (IL4 and GM-CSF) yielded opposite effects. In addition, Arg I and Arg II were regulated differentially during Th1 and Th17 cell polarization. Arg II deficiency in mice delayed EAE onset, ameliorated clinical symptoms and reduced myelin loss accompanied by a remarkable reduction in the EAE-induced spinal cord expression of Th17 cell markers (IL17 and RORγt). The abundance of Th17 cells and IL23+ cells in relevant draining lymph nodes were significantly reduced in Arg II KO mice. In activated DCs, Arg II deficiency significantly suppressed the expression of Th17-differentiating cytokines IL23 and IL6. Interestingly, Arg II deficiency did not lead to any compensatory increase in Arg I expression in vivo and in vitro. In conclusion, Arg II was identified as a factor promoting EAE likely via an Arg I-independent mechanism. Arg II may promote EAE by enhancing DC production of Th17-differentiating cytokines. Specific inhibition of Arg II could be a new approach to treat MS. (09/2018) (link)
• Lau KHW, and Sheng MHC (2018) A novel miR17/protein tyrosine phosphatase-oc/EphA4 regulatory axis of osteoclast activity. Archives of Biochemistry and Biophysics 650, 30-38. Information about the molecular mechanisms leading to the activation of the osteoclast is relatively limited. While there is compelling evidence that the signaling mechanisms of Src and integrin β3 are essential for osteoclast activation, the regulation of these two signaling mechanisms is not fully understood. In this review, evidence supporting a novel regulatory axis of osteoclast activation that plays an upstream regulatory role in both the Src and integrin β3 signaling during osteoclast activation is discussed. This regulatory axis contains three unique components: a structurally unique transmembrane protein-tyrosine phosphatase, PTP-oc, EphA4,and miR17. In the first component, PTP-oc activates the Src signaling through dephosphorylation of the inhibitory tyr-527 of Src. This in turn activates the integrin β3 signaling, enhances the JNK2/NFκB signaling, promotes the ITAM/Syk signaling, and suppresses the ITIM/Shp1 signaling; the consequence of which is activation of the osteoclast. In the second component, EphA4 inhibits osteoclast activity by suppressing the integrin β3 signaling. PTP-oc relieves the suppressive actions of EphA4 by directly dephosphorylating EphA4. In the third component, PTP-oc expression is negatively regulated by miR17. Accordingly, suppression of miR17 during osteoclast activation upregulates the PTP-oc signaling and suppresses the EphA4 signaling, resulting in the activation of the osteoclast. This regulatory axis is unique, in that each of the three components acts to exert suppressive action on their respective immediate downstream inhibitory step. Because the final downstream event is the EphA4-mediated inhibition of osteoclast activation, the overall effect of this mechanism is the stimulation of osteoclast activity. (06/2018) (link)
• Lau KHW, Stiffel VM, Rundle CH, Amoui M, Tapia J, White TD, and Sheng MHC (2017) Conditional disruption of miR17~92 in osteoclasts led to osteoclast activation and loss of trabecular bone in part through suppression of the miR17-mediated downregulation of protein-tyrosine phosphatase-oc in mice. Journal of Bone and Minereral Research Plus 1 (2), 73-85. This study sought to understand the regulation of an osteoclastic protein-tyrosine phosphatase (PTP-oc), a positive regulator of osteoclast activaty. Our past studies suggested that PTP-oc is regulated post-transcriptionally. The 3’-UTR of PTP-oc mRNA contains a target site for miR17. During osteoclastic differentiation, there was an inverse relationship between the cellular levels of miR17 (expressed as one of the six cluster genes of miR17~92) and PTP-oc mRNA. Overexpression of pre-miR17~92 in mouse osteoclast precursors reduced PTP-oc mRNA level and the size of the derived osteoclasts; whereas deletion of miR17~92 or inhibition of miR17 resulted in the formation of larger osteoclasts containing more nuclei that expressed higher PTP-oc mRNA levels and created larger resorption pits. Thus, PTP-oc-mediated osteoclast activation is modulated in part by miR17~92, particularly miR17. The miR17~92 osteoclast conditional knockout (cKO) mutants, generated by breeding miR17~92loxp/loxp mice with Ctsk-Cre mice, had lower Tb.BV/TV, Tb.BMD, Tb.Conn-Dens, Tb.N, and Tb.Th, but larger Tb.Sp, and greater bone resorption without a change in bone formation compared to littermate controls. The cKO marrow-derived osteoclasts were twice as large, contained twice as many nuclei, and produced twice as large resorption pits as osteoclasts of littermate controls. The expression of genes associated with osteoclast activation was increased in cKO osteoclasts, suggesting that deletion of miR17~92 in osteoclasts promotes osteoclast activation. The cKO osteoblasts did not show differences in cellular miR17 level, alkaline phosphatase activity, and bone nodule formation ability. In conclusion, miR17-92 negatively regulates the osteoclast activity, in part via the miR17-mediated suppression of PTP-oc in osteoclasts. (10/2017) (link)
• Li C-H, Zhang J, Baylink DJ, Wang X, Goparaju NB, Xu Y, Wasnik, S, Cheng Y, Berumen EC, Qin X, Lau KHW, Tang X (2017) Dendritic cells, engineered to overexpress 25-hydroxyvitamin D 1alpha-hydroxylase and pulsed with a myelin antigen, provide myelin-specific suppression of ongoing experimental allergic encephalomyelitis. FASEB Journal 31 (7), 2996-3006.  Multiple sclerosis (MS) is caused by immune-mediated damage of myelin sheath. Current therapies aim to block such immune responses. However, this blocking is not sufficiently specific and hence compromises immunity, leading to severe side effects. Additionally, blocking medications usually provide transient effects and require frequent administration, which further increases the chance to compromise immunity. In this regard, myelin-specific therapy may provide the desired specificity and a long-lasting therapeutic effect by inducing myelin-specific T regulatory (Treg) cells. Tolerogenic dendritic cell (TolDC) is one such therapy. However, ex vivo generated TolDCs may be converted into immunogenic DCs in a proinflammatory environment. Here we describe a potential novel myelin-specific therapy that works with immunogenic DCs, hence without the in vivo conversion concern. We showed that immunization with DCs, engineered to overexpress 25-hydroxyvitamin D 1alpha-hydroxylase for de novo synthesis of a focally high 1,25-dihydroxyvitamin D concentration in the peripheral lymphoid tissues, induced Treg cells. Additionally, such engineered DCs, when pulsed with a myelin antigen, led to myelin-specific suppression of ongoing experimental allergic encephalomyelitis (an MS animal model) and the disease suppression depended on forkhead-box-protein-P3(foxp3)+ Treg cells. Our data support a novel concept that immunogenic DCs can be engineered for myelin-specific therapy for MS. (07/2017) (link)
• Ahmed ASI, Sheng MHC, Wasnik S, Baylink DJ, and Lau KHW (2017) The effect of aging on stem cells. World Journal of Experimental Medicine 7(1):1-10. Pluripotent stem cells have the remarkable self-renewal ability and are capable of differentiating into multiple diverse cells. There is increasing evidence that the aging process can have adverse effects on stem cells. As stem cells age, their renewal ability deteriorates and their ability to differentiate into the various cell types is altered. Accordingly, it is suggested aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Understanding the role of the aging process in deterioration of stem cell function is crucial, not only in understanding the pathophysiology of aging-associated disorders, but also in future development of novel effective stem cell-based therapies to treat aging-associated diseases. This review article first focuses on the basis of the various aging disease-related stem cell dysfunction. It then addresses the several concepts on the potential mechanism that causes aging-related stem cell dysfunction. It also briefly discusses the current potential therapies under development for aging-associated stem cell defects. (02/2017) (link)
• Sheng MHC, Lau KHW, Lakhan R, Ahmed ASI, Rundle CH, Biswanath P, and Baylink DJ (2017) Unique regenerative mechanism to replace bone lost during dietary bone depletion in weanling mice. Endocrinology 158 (4), 714-729. This study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes involved in bone repletion, a regenerative bone process that restores the bone lost to calcium deficiency. To initiate depletion, weanling mice with a mixed C57BL/6 (75%) and CD1 (25%) genetic background were fed a calcium-deficient diet (0.01%) for 14 days. For repletion, the mice were re-fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma PTH, 1,25(OH)2D, and CTX. These plasma parameters quickly returned toward normal upon repletion. Trabecular bone volume and connectivity decreased drastically during depletion, but were completely restored by the end of repletion.  This bone repletion process was largely due to new bone formation.  When BrdU was administered in the middle of depletion for 3 days and examined by FACS at 7 days into repletion, significant increases in BrdU incorporation were seen in several CD105 subsets of cells of osteoblastic lineage. When BrdU was administered on days 1-3 of repletion and examined 11 days later, no increases in BrdU in these subsets were seen. Additionally, osteocytes stained positively for BrdU were increased during depletion. In summary, this study establishes a unique regenerative mechanism to initiate bone repair during the bone insult.  Calcium homeostatic mechanisms and the bone repletion mechanism are opposing functions but are simultaneously orchestrated in such a way as to optimize both endpoints. These results have potential clinical relevance to disease entities such as type II osteoporosis. (01/2017) (link)
• Lau KHW, Rundle CH, Zhou XD, Baylink DJ, and Sheng MHC (2016) Conditional deletion of IGF-I in osteocytes unexpectedly accelerates bony union of the fracture gap in mice. Bone 92, 18-28. This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28 days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and >2 fold increase in the number of osteoclasts in cKO calluses at 14 days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) were greater in cKO calluses than in WT calluses. µ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14 days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair. (08/2016) (link)
• Satoorian T, Li B, Tang X, Xiao J, Xing W, Shi W, Lau KHW, Baylink DJ, and Qin X (2016) MicroRNA233 promotes pathogenic T-cell development and autoimmune inflammation in central nervous system in mice. Immunology 148 (4), 326-338. Multiple sclerosis (MS) is an incurable central nerve system autoimmune disease. Understanding the MS pathogenesis is essential for development of new MS therapy. In the present study, we identified a novel microRNA (miR) that regulates the experimental autoimmune encephalomyelitis (EAE), an animal model of MS. miR223 expression was up-regulated specifically in spinal cords and lymphoid organs but not in other examined tissues. A global miR223 knockout (miR233-/-) in mice led to a significant delay in EAE onset reduction in spinal cord lesion, and lessening in neurological symptoms. These protective effects could be reproduced in bone marrow chimeras reconstituted with miR223-/- hematopoietic stem cells. We also found that miR223 deficiency reduced Th1 and Th17 infiltration into spinal cords. To address underlying mechanisms, we investigated the role of miR223 in regulating the function, development and interaction of the major immune cells. Expression of the genes associated with dendritic cell (DC) activation (CD86 and MHC II) and Th1 and Th17 differentiation (IL12 and IL23, respectively) was significantly decreased in the spleens of miR223-/- mice bearing EAE. miR233-/- DCs expressed significantly lower levels of basal and LPS-induced IL12 and IL23 as compared to the wild-type DC2. This data is consistent with the observed lower efficiency of miR233-/- DCs to support Th1 and Th17 differentiation from naive T cells overexpressing an EAE antigen-specific T cell receptor. Our data suggest that miR223 promotes EAE likely through enhancing DC activation and subsequently the differentiation of naive T cells toward Th1 and Th17 effector cells. (08/2016) (link)
• Rundle CH, Xing W, Lau KHW, and Mohan S (2016) Eph-ephrin bidirectional signaling in bone.  Osteoporos Sarcopenia 2(2), 65-76. The interaction between ephrin ligands and their receptors (Eph) is capable of inducing forward signaling, from ligand to receptor, as well as reverse signaling, from receptor to ligand. They are widely expressed in many tissues, where they mediate cell migration and adherence, properties that make the ephrin-Eph signaling critically important in establishing and maintaining tissue boundaries. The ephrin-Eph system has also received considerable attention in skeletal tissues, as ligand and receptor combinations are predicted to mediate interactions between the different types of cells that regulate bone development and homeostasis. This review summarizes our current understanding of Eph-ephrin signaling with a particular focus on expression and functions of ephrins and their receptors in bone. (06/2016) (link)
• Lau K-HW, Chen ST, Wang X, Mohan S, Wergedal JE, Kesavan C, Srivastava AK, Gridley DS, Hall SL (2016) Opposing effects of Sca-1+ cell-based systemic FGF-2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse. Gene Therapy 23(6), 500-509. Our previous work showed that a Sca-1+ cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1+ cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age, are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W41/W41 mice. The Sca-1+ cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1+ cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1+ cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow. (06/2016) (link)
• Chen W, Baylink DJ, Lau KHW, Zhang XB (2016) Generation of mesenchymal stem cells by blood cell reprogramming. Current Stem Cell Research & Therapy 11 (2), 114-121. Mesenchymal stem cells (MSCs) have been successfully used to treat multiple diseases in animal studies and clinical trials. Currently, the commonly used MSCs are derived from bone marrow and adipose tissue. Alternative approaches include differentiation of induced pluripotent stem cells (iPSCs) into MSCs, or direct reprogramming of blood cells into MSCs. This review summarizes recent progresses concerning how to generate MSCs by blood cell reprogramming and how studies in cellular reprogramming may help identify new factors to expand or even rejuvenate adult MSCs. (01/2016) (link)
• Lakhan R, Baylink DJ, Lau KHW, Tang X, Sheng MHC, Rundle CH, Qin X (2015) Local administration of AAV DJ pseudo serotype expressing COX2 provided early onset of transgene expression and promoted bone fracture healing in mice. Gene Therapy  22 (9), 721-728. We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein we reported that AAV-DJ, an artificial AAV pseudo-serotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared to AAV-2, the use of AAV-DJ led to approximately 5-fold increase in infectivity in MSCs and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5x1011 genomic copies led to high COX2 level at the fracture site on day 3 post-injections and significantly promoted fracture union at 21 days, as analyzed by radiography and μ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans. (09/2015) (link)
• Lau K-HW, Amoui M, Stiffel V, Chen S-T, and Sheng MH-C (2015) An osteoclastic transmembrane protein-tyrosine phosphatase enhances osteoclast activity in part through the dephosphorylation of the EphA4 in osteoclasts. Journal of Cellular Biochemistry (2015) 116(8), 1785-1196. We have previously shown that PTP-oc is an enhancer of the functional activity of osteoclasts and that EphA4 is a suppressor. Here, we provide evidence that PTP-oc enhances osteoclast activity in part through inactivation of EphA4 by dephosphorylating key phosphotyrosine (pY) residues of EphA4. We show that EphA4 was pulled down by the PTP-oc trapping mutant but not by the wild-type (WT) PTP-oc and that transgenic overexpression of PTP-oc in osteoclasts drastically decreased pY602 and pY779 residues of EphA4. Consistent with the previous findings that EphA4 deficiency increased pY173-Vav3 level [Rac-GTP exchange factor (GEF)] and enhanced bone resorption activity of osteoclasts, reintroduction of WT-Epha4 in Epha4 null osteoclasts led to ~50% reduction in the pY173-Vav3 level and ~2-fold increase in bone resorption activity. Overexpression of Y779F-Epha4 mutant in WT osteoclasts markedly increased in pY173-Vav3 and reduced bone resorption activity, but overexpression of Y602F-Epha4 mutant had no effect, suggesting that pY779 residue plays an important role in the EphA4-mediated suppression of osteoclast activity. Deficient EphA4 in osteoclasts has been shown to up-regulate Rac-GTPase and down-regulate Rho-GTPase. PTP-oc overexpression in osteoclasts also increased the GTP-Rac level to 300% of controls, but decreased the GTP-Rho level to ~50% of controls. PTP-oc overexpression or deficient Epha4 each also reduced pY87-Ephexin level, which is a Rho GEF. Thus, PTP-oc may differentially regulate Rac signaling vs. Rho signaling through dephosphorylation of EphA4, which has shown to have opposing effects on Rac-GTPase vs. Rho-GTPase through differential regulation of Vav3 vs. Ephexin. (08/2015) (link)
• Chen W, Baylink DJ, Brier-JonesJ, Neises A, Kiroyan JB, Rundle CH, Lau KHW, and Zhang XB (2015) Stem cell gene therapy increases bone strength in the mouse. Proceedings of National Academy of Sciences U. S. A. 112 (29), E3893-E3900. Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1+ cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity, leading to a 45% increase in the bone strength. Transplantation of PGK-PDGFB-transduced Sca1+ cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning. (07/2015) (link)
• Chen W, Baylink DJ, Lau KHW, Zhang XB (2015) Generation of mesenchymal stem cells by blood cell reprogramming. Curr Stem Cell Res Ther 11(2), 114-121. Mesenchymal stem cells (MSCs) have been successfully used to treat multiple diseases in animal studies and clinical trials. Currently, the commonly used MSCs are derived from bone marrow and adipose tissue. Alternative approaches include differentiation of induced pluripotent stem cells (iPSCs) into MSCs, or direct reprogramming of blood cells into MSCs. This review summarizes recent progresses concerning how to generate MSCs by blood cell reprogramming and how studies in cellular reprogramming may help identify new factors to expand or even rejuvenate adult MSCs. (05/2015) (link)
• Lau KHW, Baylink DJ, and Sheng MHC (2015) Osteocyte-derived insulin-like growth factor I is not essential for the bone repletion response in mice. PLoS One10 (1): e0115897. The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both Igf1 osteocyte conditional knockout (cKO) mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by the reduction in osteoclast number, incdicating the deficient Igf1 expression in osteocytes also did not hace deleterious effects on the bone repletion response. The lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected since previous studies show that these Igf1 osteocyte cKO mice exhibited impaired developmental growth and displayed complete resistance to bone anabolic effects of loading. These studies suggest that there is a dichotomy between the mechanisms necessary for anabolic skeletal repletion following low dietary calcium challenge. In conclusion, to our knowledge this study has demonstrated for the first time that osteocyte-derived IGF-I, which is essential for anabolic bone response to mechanical loading, is not a key regulatory factor for bone repletion after a low calcium challenge. (01/2015) (link)
• Li B, Baylink DJ, Walter MH, Lau KHW, Meng X, Wang J, Cherkas A, Tang X, and Qin X (2015) Targeted 25-hydroxyvitamin D3 1α hydroxylase adoptive gene therapy ameliorates DSS-induced colitis without causing hypercalcemia in mice. Molecular Therapy 23 (2), 339-351. Systemic 1,25(OH)2D3 treatment ameliorating murine inflammatory bowel diseases (IBD) could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b+/Gr1+ monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b+/Gr1+ monocytes migrated initially to inflamed colon and some healthy tissues in dextran sulfate sodium (DSS) colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1+ monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (interleukin (IL)-17a and interferon (IFN)-γ) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases. (10/2014) (link)
• Lau KHW, Popa NL, and Rundle CH (2014) Microarray analysis of gene expression reveals that Cyclo-oxygenase-2 gene therapy up-regulates hematopoiesis and down-regulates inflammation during endochondral bone fracture healing. Journal of Bone Metabolism 21 (3), 169-188. Background: Cyclo-oxygenase-2 (Cox-2) is an inflammatory mediator that is necessary for the tissue repair, including bone fracture healing. Although the application of Cox-2 gene therapy to a murine closed femoral fracture has accelerated bony union, but the beneficial effect was not observed until the endochondral stage of bone rpair that is well after the inflammatory stage normally subsides. Methods: To identify the molecular pathways through which Cox-2 regulates fracture healing, we examined gene expression profile in fracture tissues in response to Cox-2 gene therapy during the endochondral bone repair phase. Cox2 gene therapy was applied to the closed murine femur fracture model. Microarray analsis was performed at 10 days post-fracture to examine global gene expression profile in the fracture tissues during the endochondral bone repair phase. The entire repertoire of significantly expressed genes was examined by gene set enrichment analysis, and the most up-regulated individual genes were evaluated further. Results: The genes that normally promote inflammation were under-represented in the microarray analysis, and the expression of several inflammatory chemokines was significantly down-regulated. There was an up-regulation of two key transcription factor genes that regulate hematopoiesis and erythropoiesis. More surprisingly, there was no significant up-regulation in the genes that are normally involved in angiogenesis or bone formation. However, the expression of two tissue remodeling genes was up-regulated. Conclusions: The down-regulation of the inflammatory genes in response to Cox-2 gene therapy was unexpected, given the pro-inflammatory role of prostaglandins. Cox-2 gene theapy could promote bony union through hematopoietic precursor proliferation during endochondral bone repair and thereby enhances subsequently fracture callus remodeling that leads to bony union of the fracture gap. (08/2014) (link)
• Rundle CH, Chen S-T, Coen MJ, Wergedal JE, Stiffel V, and Lau KHW (2014) Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis. PLoS One 9 (5), e98004. This study sought to determine if direct application of the lentiviral (LV)-cyclooxygenase 2 (COX2) vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis.  The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an effective therapy to promote tendon-to-bone healing after tenodesis or related surgeries. (05/2014) (link)
• Stiffel V, Amoui M, Sheng MH-C, Mohan S, and Lau KHW (2014) EphA4 receptor is a novel negative regulator of osteoclast activity. Journal of Bone and Mineral Research 29 (4), 804-819. Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast-like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4-fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild-type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast-lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild-type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF-kB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein-tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the β3-integrin signaling through reduced phosphorylation of the tyr-747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to β3-integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to β3-integrin via an increase in EphA4-dependent tyr-747 phosphorylation. (03/2014) (link)
• Popa NL, Wergedal JE, Lau KHW, Mohan S, and Rundle CH (2014)  Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling . Journal of Bone and Mineral Metabolism 32 (2), 124-135. Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair. (03/2014) (link)
• Sheng MH-C, Lau KHW, and Baylink DJ (2014) Role of osteocyte-derived insulin-like growth factor I in developmental growth, modeling, remodeling, and regeneration of the bone. Journal of Bone Metabolism 21 (1), 41-54. The osteocyte has long been considered to be the primary mechanosensory cell in the bone. Recent evidence has emerged that the osteocyte is also a key regulator of various bone and mineral metabolism and that its regulatory effects are in part mediated through locally produced osteocyte-derived factors, such as sclerostin, receptor activator of nuclear factor k-B ligand (RANKL), and fibroblast growth factor-23 (FGF-23). Osteocytes secrete large amounts of insulin-like growth factor-I (IGF-I) in bone. Although IGF-I produced locally by other bone cells, such as osteoblasts and chondrocytes, has been shown to play important regulatory roles in bone turnover and developmental bone growth, the functional role of osteocyte-derived IGF-I in the bone and mineral metabolism has not been investigated and remains unclear. However, results of recent studies in osteocyte Igf1 conditional knockout transgenic mice have suggested potential regulatory roles of osteocyte-derived IGF-I in various aspects of bone and mineral metabolism. In this review, evidence supporting a regulatory role for osteocyte-derived IGF-I in the osteogenic response to mechanical loading, the developmental bone growth, the bone response to dietary calcium depletion and repletion, and in fracture repair is discussed. A potential coordinated regulatory relationship between the effect of osteocyte-derived IGF-I on bone size and the internal organ size is also proposed. (02/2014) (link)
• Su R-J, Baylink DJ, Neises A, Meng X, Kiroyan JB, Payne KJ, Tschudy-Seney B, Duan Y, Appleby N, Kearns-Jonker M, Gridley DS, Wang J, Lau K-HW, and Zhang XB (2013) Efficient generation of integration-free iPS cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. PLoS One 8 (5), e64496. The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source - peripheral blood - has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3-/CD19- cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine. (05/2013) (link)
• Lau K-HW, Baylink DJ, Zhou XD, Rodriguez D, Bonewald LF, Li Z, Ruffoni D, Müller R, Kesavan C, and Sheng M-HC (2013) Osteocyte-derived insulin-like growth factor-1 is essential for determining bone mechanosensitivity. American Journal of Physiology: Endocrinology and Metabolism 305 (2), E271-E281. This study sought to determine whether deficient Igf1 expression in osteocytes would affect loading-induced osteogenic response. Tibias of osteocyte Igf1 conditional knockout (KO) mice (generated by cross-breeding Igf1 floxed mice with Dmp1-Cre transgenic mice) and wild-type littermates were subjected to four-point bending for 12 days. Micro-computer tomography analyses confirmed that the size of tibias of conditional mutants was smaller. Loading with an equivalent mechanical strain increased periosteal woven bone and endosteal lamellar bone formation in wild-type mice but not in conditional KO mice. Consistent with the lack of an osteogenic response, the loading failed to up-regulate expression of early mechanoresponsive genes (Igf1, Cox-2, c-fos) and osteogenic genes (Cbfa-1, and osteocalcin) in conditional KO bones. The lack of osteogenic response was not due to reduced osteocyte density or insufficient loading strain. Deficient osteocyte Igf1 expression also reduced the loading-induced upregulation of expression of canonical Wnt signaling genes (Wnt10b, Lrp5, Dkk1, sFrp2). Intriguingly, the loading upregulated (by ~1.5-fold) in conditional KO mice, but reduced (by 40%) in wild-type mice, the expression of sclerostin (Sost) [a key inhibitor of the Wnt signaling]. Conditional disruption of Igf1 in osteocytes also completely abolished the loading-induced increase in the bone level of β-catenin protein, suggesting an impaired response in the loading-induced activation of the canonical Wnt signaling in conditional KO mice. In summary, conditional disruption of Igf1 in osteocytes completely abolished the loading-induced upregulation of the canonical Wnt signaling and the corresponding osteogenic response. In conclusion, osteocyte-derived IGF-1 plays a key determining role in bone mechanosensitivity. (05/2013 - 07/2013) (link)
• Nguyen HB, Eshete B, Lau K-HW, Sai A, Villarin M, and Baylink DJ (2013) Serum 1,25-dihydroxyvitamin D: an outcome prognosticator in human sepsis. PLoS One 8 (5), e64348. In sepsis, the vitamin D active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) may play a crucial role by its action to produce cathelicidin and improve endothelial barrier function, such that a deficiency in 1,25(OH)2D is associated with poor outcome. To test our hypothesis, we performed analysis of stored plasma samples from a prospective observational study in 91 patients with sepsis, age of 59.1+/- 2.0 years, 52.7% females, and 11.0% deaths at 30 days. Vitamin D status, including 25-hydroxyvitamin D (25(OH)D), 1,25(OH)2D, 24,25-dihydroxyvitamin D (24,25(OH)2D), and parathyroid hormone (PTH), were measured daily over 3 days after hospital admission. At baseline, 1,25(OH)2D was significantly different between survivors vs. non-survivors. But there was no significant difference in 25(OH)D, 24,25(OH)2D, and PTH. In a multivariable binomial logistic regression model, age, total calcium and 1,25(OH)2D were significant predictors of 30-day mortality. Kaplan Meier analysis showed that patients with 1,25(OH)2D < 15.4 pg/mL measured at hour 48 had 57.1% 30-day survival compared to 91.5% in patients with 1,25 (OH)2D level >= 15.4 pg/mL (p < 0.01). From repeated measures regression analysis, there was significant increase in 1,25(OH)2D for increases in 25(OH)D in both survivors and non-survivors. However, compared to survivors, the low 25(OH)D in non-survivors was insufficient to account for the larger decrease in 1,25(OH)2D, indicating a dysfunctional 1α-hydroxylase. Additionally, there was a significant negative correlation between PTH and 1,25(OH)2D in both survivors and non-survivors, suggesting a severe impairment in the effect of PTH to increase renal 1α-hydroxylase activity. In conclusion, low 1,25(OH)2D levels are associated with increased 30-day mortality in sepsis patients, likely due to impaired 25(OH)D hydroxylation and PTH insensitivity. Our data also suggest that the active metabolite 1,25(OH)47 2D may be an important therapeutic target in the design of sepsis clinical trials. (05/2013) (link)
• Meng X, Su R-J, Baylink DJ, Neises A, Kiroyan JB, Lee WY-W, Payne KJ, Gridley DS, Wang J, Lau K-HW, Li G, and Zhang X-B (2013) Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor. Cell Research 23 23 (5), 658-672. The direct conversion of skin or blood cells into somatic stem cells opens new therapeutic possibilities. Here we show that human induced mesenchymal stem cells (iMSCs) can be efficiently generated from cord blood or adult peripheral blood CD34 + cells by direct reprogramming with a single factor, OCT4. In the presence of a GSK3 inhibitor, 16% of OCT4-transduced CD34+ cells are converted into iMSCs in 2 weeks. Episomal vector-mediated transient OCT4 expression is sufficient for direct reprogramming. iMSCs express MSC markers, resemble bone marrow (BM) MSCs in morphology and in vitro multilineage differentiation capacity, yet have a greater proliferative capacity. Similar to BM MSCs, implanted iMSCs form bone and connective tissues and are nontumorigenic. However, BM MSCs do not while iMSCs do form muscle fibers, indicating a potential functional advantage of iMSCs. In addition, an OCT4 dosage effect was observed: high levels favor reprogramming and self-renewal, while low levels allow for multilineage differentiation. Our method for highly efficient generation of iMSCs from blood CD34+ cells could have important implications for future applications in regenerative medicine that require patient-specific MSCs. This discovery would also help the development of strategies for direct conversion of blood cells into other cells of clinical importance. (03/2013 - 05/2013) (link)
• Li B, Baylink DJ, Deb C, Zannetti C, Rajaallah F, Xing W, Walter MH, Lau K-HW, and Qin X (2013) 1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo. PLoS One 8 (3), e58808. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis. (03/2013) (link)
• Lau K-HW, Kothari V, Das A, Zhang X-B, and Baylink DJ (2013) Cellular and molecular mechanisms of accelerated fracture healing by COX2 gene therapy. Studies in a mouse model of multiple fractures. Bone 53 (2), 369-381. This study sought to determine cellular and molecular mechanisms of cyclooxygenase-2 (COX2) gene therapy to accelerate fracture repair in a multiple fractures model. The lenti-COX2 or control vector was injected into fractures on day 1 of the fracture. At days 3-7, the COX2 treatment increased Sdf1-, Cxcr4-, Nes-, and Podxl-expressing mesenchymal stem cells (MSCs) within the fracture calluses, suggesting an enhancement in recruitment or expansion of MSCs. The COX2-treated mice formed smaller cartilaginous calluses and had less cartilage content than control mice. The expression of Sox9 mRNA was 7-fold less in COX2-treated calluses than in control calluses at day 14, implying that COX2 reduces chondrogenic differentiation of MSCs. The therapy also enhanced angiogenesis as reflected by increased immunostaining of CD31, vWF, and α-SMA over controls in the cartilaginous callus at day 14-21. At which time, the COX2 gene therapy promoted bony remodeling of the cartilaginous callus to bridge the fracture gap that was accompanied by 2-fold increase in osteoclasts along the surface of the woven bone and an onset of osteogenesis. Blocking angiogenesis with daily injection of endostatin from day 4 to day 10 into fracture sites blocked the COX2-mediated reduction of callus size that was associated with a marked increase in hypertrophic chondrocytes and concomitant reduction in osteoclasts. In conclusion, COX2 promotes fracture healing involving three actions: 1) recruitment/expansion of MSCs; 2) decreased cartilaginous callus formation; and 3) promoted angiogenesis-dependent cartilage remodeling that was associated with an earlier onset of bony bridging of the fracture gap. (01/2013) (link)
• Sheng MH-C, Zhou X-D, Bonewald LF, Baylink DJ, and Lau K-HW (2013) Disruption of the insulin-like growth factor-1 gene in osteocytes impairs developmental bone growth in mice. Bone 52 (1), 133-144. This study evaluated the role of osteocyte-derived insulin-like growth factor 1 (IGF-1) in developmental bone growth by assessing the bone phenotype of osteocyte Igf1 conditional knockout (KO) mice, generated by crossing the Dmp1-driven Cre-expressing transgenic mice with Igf1 floxed mice containing loxP sites that flank exon 4 of the Igf1 gene. The periosteal diameter of femurs of homozygous conditional KO mutants was 8-12% smaller than wild-type (WT) littermates. The conditional mutants had 14-20%, 10-21%, and 15-31% reduction in total, trabecular, and cortical bone mineral contents, respectively. However, there were no differences in the total, trabecular, or cortical bone mineral densities, or in trabecular bone volume, thickness, number, and separation at secondary spongiosa between the mutants and WT littermates. The conditional KO mutants showed reduction in dynamic bone formation parameters at both periosteal and endosteal surfaces at the mid-diaphysis and in trabecular bone formation rate and resorption parameters at secondary spongiosa. The lower plasma levels of PINP and CTx in conditional KO mice support a regulatory role of osteocyte-derived IGF-1 in the bone turnover. The femur length of conditional KO mutants was 4-7% shorter due to significant reduction in the length of growth plate and hypertropic zone. The effect on periosteal expansion appeared to be bigger than that on longitudinal bone growth. The conditional KO mice had 14% thinner calvaria than WT littermates, suggesting that deficient osteocyte IGF-1 production also impairs developmental growth of intramembraneous bone. Conditional disruption of Igf1 in osteocytes did not alter plasma levels of IGF-1, calcium, or phosphorus. In summary, this study shows for the first time that osteocyte-derived IGF-1 plays an essential role in regulating bone turnover during developmental bone growth. (01/2013) (link)
• Meng X, Baylink DJ, Sheng M, Wang H, Gridley DS, Lau K-HW, and Zhang X-B (2012) Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation. PLoS One 7 (5), e37569. Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice received Sca1+ cells that were transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved FGF2-based stem cell gene therapy strategy should have important implications in clinical therapy for systemic skeletal augmentation. (05/2012) (link)
• Lau K-HW, Stiffel V, and Amoui M (2012) An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, Syk, and Shp1 signaling through respective Src-dependent phosphorylation in osteoclasts. American Journal of Physiology - Cell Physiology 302 (11), C1676-C1686. This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β3-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β3 integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β3-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β3-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCg levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, the PTP-oc plays a central regulatory role in the concerted regulation of the β3-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through the activation of Src PTK. (03/2012 - 06/2012) (link)
• Sheng MH-C, Wergedal JE, Mohan S, Amoui M, Baylink DJ, and Lau K-HW (2012) Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption and bone boss in mice. PLoS One 7 (4), e35280. This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ~2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by µ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (cathepsin K, MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW246.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Serum c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and serum levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption. (03/2012 - 04/2012) (link)
• Linares GR, Brommage R, Powell D, Xing W, Chen S-T, Alshbool F, Lau K-HW, Wergedal JE, and Mohan S (2012) Claudin 18 is a novel negative regulator of bone resorption and osteoclast differentiation. Journal of Bone and Mineral Research 27 (7), 1553-1565. Claudin 18 (Cldn-18) belongs to a large family of transmembrane proteins that are important components of tight junction strands. Although several claudin members are expressed in bone, the functional role for any claudin member in bone is unknown. Here we demonstrate that disruption of Cldn-18 in mice markedly decreased total body bone mineral density, trabecular bone volume, and cortical thickness in Cldn-18 (-/-) mice. Histomorphometric studies revealed that bone resorption parameters were increased significantly in Cldn-18 (-/-) mice without changes in bone formation. Serum levels of TRAP5b and mRNA expression levels of osteoclast specific markers and signaling molecules were also increased. Loss of Cldn-18 further exacerbated calcium deficiency induced bone loss by influencing bone resorption, thereby resulting in mechanically weaker bone. In vitro studies with bone marrow macrophages revealed Cldn-18 disruption markedly enhanced RANKL-induced osteoclast differentiation but not MCSF-induced BMM proliferation. Consistent with a direct role for Cldn-18 in regulating osteoclast differentiation, overexpression of wild type but not PDZ binding motif deleted Cldn-18 inhibited RANKL-induced osteoclast differentiation. Furthermore, our findings indicate that Cldn-18 interacts with Zonula occludens 2 (ZO-2) to modulate RANKL signaling in osteoclasts. In conclusion, we demonstrate that Cldn-18 is a novel negative regulator of bone resorption and osteoclast differentiation. (03/2012 - 07/2012) (link)
• Meng X, Neises A, Su R, Payne K, Ritter L, Gridley D, Wang J, Sheng M, Lau K-HW, Baylink DJ, Zhang X-B  (2012) Efficient reprogramming of human cord blood CD34+ cells into induced pluripotent stem cells with OCT4 and SOX2 alone. Molecular Therapy 20 (2), 408-416. The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck posttranscriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs. (11/2011 - 02/2012) (link)
• Coen MJ, Chen S-T, Rundle CH, Wergedal JE, and Lau K-HW (2011) Lentiviral-based BMP4 in vivo gene transfer strategy increases pull-out tensile strength without an improvement in the osteointegration of the tendon graft in a rat model of biceps tenodesis. Journal of Gene Medicine 13 (10), 511-521. Background: Methods: A rat model of biceps tenodesis was developed with an interference fit open surgical technique. A LV vector expressing a BMP4 gene or b-galactosidase (b-gal) control gene was applied to the bone tunnel and the tendon graft before its insertion into the bone tunnel. Osteointegration was assessed by histology and pull-out tensile strength was measured by a biomechanical test suitable for small rat biceps tendon grafts. Results: Neo-chondrogenesis was seen at the tendon–bone interface of LVBMP4-treated but not control rats. The LV-BMP4-treated rats showed 32% (p<0.05) more newly-formed trabecular bone at the tendon–bone junction than the LV-b-gal-treated controls after 3weeks. However, the sites of neochondrogenesis and new bone formation in the LV-BMP4-treated tenodesis were highly spotty. Although the LV-BMP4 strategy did not promote bony integration of the tendon graft, it yielded a 29.511.8% (p=0.066) increase in improvement the pull-out strength of rat biceps tendons compared to the LVb-gal treatment after 5 weeks. Conclusions: Although the LV-BMP4 in vivo gene transfer strategy did not enhance osteointegration of the tendon graft, it yielded a marked improvement in the return of the pull-out strength of the tendon graft. This presumably was largely a result of the bone formation effect of BMP4 that traps or anchors the tendon graft onto the bony tunnel. The present study aimed to develop a rat model of biceps tenodesis and to assess the feasibility of a lentiviral (LV)-based bone morphogenetic protein (BMP) 4 in vivo gene transfer strategy for healing of biceps tenodesis. (09/2011 - 10/2011) (link)
• Kesavan C, Wergedal JE, Lau K-HW, and Mohan S (2011) Conditional disruption of IGF-I gene in type 1α2-collagen expressing cells shows an essential role of IGF-I in skeletal anabolic response to loading. American Journal of Physiology - Endocrinology and Metabolism 301 (6), E1191-E1197. To provide a causal role for locally produced IGF-I in the mechanical strain response in the bone, we have generated mice with conditional disruption of the IGF-I gene in type 1α2 collagen expressing cells using the Cre-loxp approach. At 10 weeks of age, loads adjusted to account for bone size difference were applied via four-point bending (4.pt) or axial loading (AL). Two weeks of 4.pt bending and AL produced significant increases in BMD and bone size at the mid-diaphysis of wild type (WT) but not knockout (KO) mice. In addition, AL produced an 8 to 25% increase in trabecular parameters (BV/TV, Tb.Th, Tb.BMD) at the secondary spongiosa of WT but not KO mice. Histomorphometric analysis at the trabecular site revealed that AL increased osteoid width by 60% and decreased TRAP labeled surface by 50% in the WT mice but not KO mice. Consistent with the in vivo data, blockade of IGF-I action with inhibitory IGFBP4 in vitro completely abolished the fluid flow stress induced MC3T3-E1 cell proliferation. One way ANOVA analysis revealed that expression levels of EFNB1, EFNB2, EFNA2, EphB2 and NR4a3 were different in the loaded bones of WT versus KO mice and may in part be responsible for the increase in bone response to loading in the WT mice. In conclusion, IGF-I expressed in type 1 collagen producing bone cells is critical for converting mechanical signal into anabolic signal in bone, and other growth factors cannot compensate for the loss of local IGF-I. (08/2011 - 12/2011) (link)
• Hall SL, Chen S-T, Wergedal JE, Gridley DS, Mohan S, and Lau K-HW (2011) Stem cell antigen-1 positive cell–based systemic human growth hormone gene transfer strategy increased endosteal bone resorption and bone loss in mice. Journal of Gene Medicine 13 (2), 77-88. Background. This study assesses the effect of the stem cell antigen-1 positive (Sca-1+) cell-based human growth hormone (hGH) ex vivo gene transfer strategy on endosteal bone mass in the mouse. Methods. Sublethally irradiated recipient mice were transplanted with Sca-1+ cells transduced with lentiviral vectors expressing hGH or β-galactosidase (β-gal) control gene. Bone parameters were assessed by μCT and histomorphometry. Results. This hGH strategy drastically increased hGH mRNA levels in bone marrow cells and serum IGF-I (by ~50%, p<0.002) in hGH recipient mice. Femoral trabecular bone volume of the hGH mice was significantly reduced by 35% (p<0.002). The hGH mice also had decreased trabecular number (by 26%, p<0.0001), increased trabecular separation (by 38%, p < 0.0002), and reduced trabecular connectivity density (by 64%, p<0.001), as well as significantly more osteoclast number (2.5-fold, p<0.05) and greater osteoclastic surface per bone surface (2.6-fold, p<0.01). Conclusion. Targeted expression of hGH in cells of marrow cavity through the Sca-1+ cell-based gene transfer strategy increased circulating IGF-I and decreased endosteal bone mass through an increase in resorption in recipient mice. These results indicate that high local levels of hGH or IGF-I in the bone marrow microenvironment enhanced resorption, which are consistent with previous findings in transgenic mice with targeted bone IGF-I expression that high local IGF-I expression increased bone remodeling that favored a net bone loss. Thus, the GH and/or IGF-I would not be an appropriate transgene for use in this Sca-1+ cell-based gene transfer strategy to promote endosteal bone formation. (02/2011) (link)
• Kapur S, Amoui M, Kesavan C, Wang X, Mohan S, Baylink DJ, and Lau K-HW (2010) Leptin receptor (Lepr) is a negative modulator of bone mechanosensitivity and genetic variations in Lepr may contribute to the differential osteogenic response to mechanical stimulation in the C57BL/6J and C3H/HeJ pair of mouse strains. Journal of Biological Chemistry 285 (48), 37607-37618. This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ~2,500 me, which was insufficient to produce a bone formation response in B6 mice, increased significantly bone formation parameters in leptin-deficient ob-/ob- mice, and that a loading strain of ~3,000 µε also yielded greater osteogenic responses in Lepr-deficient db-/db- mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [3H]thymidine incorporation and Erk1/2 phosphorylation in ob-/ob- osteoblasts and db-/db- osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced, but in osteoblasts of C3H [the mouse strain with poor bone mechanosensitivity] restored, their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts.  In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which results in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice. (11/2010) (link)
• Lau K-HW, Gysin R, Chen S-T, Wergedal JE, Baylink DJ, and Mohan S (2009) Marrow stromal cell-based cyclooxygenase 2 ex vivo gene transfer strategy surprisingly lacks bone regeneration effects and suppresses the bone regeneration action of bone morphogenetic protein 4 in a mouse critical-sized calvarial defect model. Calcified Tissue International 85 (4), 356-367. This study sought to evaluate whether the murine leukemia virus(MLV)-based cyclooxygenase-2 (Cox-2) ex vivo gene transfer strategy promotes healing of calvarial defects and/or synergistically enhances the bone morphogenetic protein (BMP) 4-mediated bone regeneration. Gelatin scaffolds impregnated with mouse marrow stromal cells (MSCs) transduced with MLV-expressing BMP4, Cox-2, or a control gene were implanted into mouse calvarial defects. Bone regeneration was assessed by X-ray, dual-energy X-ray absorptiometry, and histology. In vitro, Cox-2 or prostanglandin (PG) E2 enhanced synergistically the osteoblastic differentiation action of BMP4 in mouse MSCs. In vivo, implantation of BMP4-expressing MSCs yielded massive bone regeneration in calvarial defects after 2 weeks, but the Cox-2 strategy surprisingly did not promote bone regeneration even after 4 weeks. Staining for alkaline phosphatase-expressing osteoblasts showed strong staining throughout the defect of animals receiving BMP2/4-expressing cells, but defects receiving Cox-2-expressing cells displayed weak ALP staining along the edge of original intact bone, indicating that the Cox-2 strategy lacked bone regeneration effects. The Cox-2 strategy not only lacked bone regeneration effects but also suppressed the BMP4-induced bone regeneration. In vitro co-culture of Cox-2-expressing MSCs with BMP4-expressing MSCs in gelatin scaffolds reduced of BMP4 mRNA transcript levels, suggesting that Cox-2 may promote BMP4 gene silencing in BMP4-expressing cells, which may play a role in the suppressive action of Cox-2 on BMP4-mediated bone formation. In summary, the Cox-2 ex vivo gene transfer strategy not only lacks bone regeneration effects, but also suppresses the bone regeneration action of BMP4, in healing of calvarial defects. (10/2009) (link)
• Hall SL, Chen S-T, Gysin R, Gridley DS, Mohan S, Lau K-HW (2009) Stem cell antigen-1+ cell-based bone morphogenetic protein-4 gene transfer strategy in mice failed to promote endosteal bone formation. Journal of Gene Medicine 11 (10), 877-888. Background: This study assessed whether a Sca-1+ cell-based ex vivo gene transfer strategy, which has been shown to promote robust endosteal bone formation with a modified fibroblast growth factor-2 (FGF2) gene, can be extended to use with bone morphogenetic protein (BMP)2/4 hybrid gene.  Methods: Sublethally-irradiated recipient mice were transplanted with lentiviral (LV)-BMP2/4-transduced Sca-1+ cells. Bone parameters were monitored by pQCT and µCT. Gene expression was assessed by the real-time reverse transcriptase polymerase chain reaction.  Results: Recipient mice of LV-BMP2/4-transduced Sca-1+ cells yielded high engraftment and increased BMP4 mRNA levels in marrow cells; but exhibited only insignificant increases in serum and bone alkaline phosphatase activity compared to control mice. pQCT and µCT analyses of femurs showed that, with the exception of small changes in trabecular bone mineral density and cortical bone mineral content in LV-BMP2/4 mice, there were no differences in measured bone parameters between mice of the LV-BMP2/4 group and controls. The lack of large endosteal bone formation effects with the BMP4 strategy could not be attributed to ineffective engraftment or expansion of BMP4-expressing Sca-1+ cells, an inability of the transduced cells to secrete active BMP4 proteins, or to use of the LV-based vector.   Conclusions: Sca-1+ cell-based BMP4 ex vivo strategy did not promote robust endosteal bone formation, raising the possibility of intrinsic differences between FGF2- and BMP4-based strategies in their ability to promote endosteal bone formation. It emphasizes the importance of choosing an appropriate bone growth factor gene for delivery by this Sca-1+ cell-based ex vivo systemic gene transfer strategy to promote bone formation. (09/2009) (link)
• Sheng MH-C, and Lau K-HW (2009) Role of protein-tyrosine phosphatases in regulation of osteoclastic activity. Cellular and Molecular Life Sciences 66 (11-12), 1946-1961. Osteoclasts, the primary cell type mediating bone resorption, are multinucleated, giant cells derived from hematopoietic cells of monocyte-macrophage lineage. Osteoclast activity is, in a large part, regulated by protein-tyrosine phosphorylation. While information about functional roles of several protein-tyrosine kinases (PTK), including c-Src, in osteoclastic resorption has been accumulated, little is known about the roles of protein-tyrosine phosphatases (PTPs) in regulation of osteoclast activity. Recent evidence implicates important regulatory roles for four PTPs (SHP-1, cyt-PTP-e, PTP-PEST, and PTP-oc) in osteoclasts. Cyt-PTP-e, PTP-PEST, and PTP-oc are positive regulators of osteoclast activity, while SHP-1 is a negative regulator. Of these PTPs in osteoclasts, only PTP-oc is a positive regulator of c-Src PTK through dephosphorylation of the inhibitory phosphotyrosine-527 residue. Although some information about mechanisms of action of these PTPs to regulate osteoclast activity is reviewed in this article, much additional work is required to provide more comprehensive details about their functions in osteoclasts. (05/2009) (link)
• Sheng MH-C, Amoui M, Stiffel V, Srivastava AK, Wergedal JE, and Lau K-HW (2009) Targeted transgenic expression of an osteoclastic transmembrane protein-tyrosine phosphatase in cells of osteoclastic lineage increased bone resorption and bone loss in young adult male mice. Journal of Biological Chemistry 284 (17), 11531-11545. This study evaluated whether transgenic expression of PTP-oc (osteoclastic transmembrane protein-tyrosine phosphatase) in cells of the osteoclast lineage would affect bone resorption and bone density in young adult mice. Transgenic mice were generated with a transgenic construct using a tartrate-resistant acid phosphatase exon 1C promoter to drive expression of rabbit of femurs of young adult male progeny of three lines showed that transgenic mice had reduced bone volume and area, cortical and trabecular bone mineral content, and density. Histomorphometric analyses at secondary spongiosa of the femur and at metaphysis of the L4 vertebra confirmed that male transgenic mice had decreased trabecular surface, reduced percentage of trabecular area, decreased trabecular number, increased trabecular separation, and increased osteoclast number per bone surface length. Consistent with an increase in bone resorption, the serum C-telopeptide level was 25% higher in transgenic mice than in wild-type littermates. However, the bone phenotype was not readily observed in female young adult transgenic mice. This could in part be due to potential interactions between estrogen and PTP-oc signaling, since the bone loss phenotype was seen in young adult ovariectomized transgenic mice by microcomputed tomography analysis. In vitro, the average pit area per resorption pit created by marrow-derived transgenic osteoclasts was ~50% greater than that created by wild-type osteoclasts. Transgenic osteoclasts showed a lower c-Src phosphotyrosine 527 level, greater c-Src kinase activity, and increased tyrosine phosphorylation of paxillin. In summary, this study provides compelling in vivo evidence that PTP-oc is a positive regulator of osteoclasts. (04/2009) (link)
• Rundle CH, Chen S-T, Porte R, Wergedal JE, and Lau K-HW (2008) Retroviral-based BMP-4 In vivo gene transfer strategy with intramedullary viral delivery optimizes transgene expression in rat femur fractures. The Open Tissue Enginerring and Regenerative Medicine Journal 1, 14-22. We have developed an intramedullary delivery strategy to administer retroviral vectors expressing a therapeutic gene to promote healing in a closed rat femur fracture. This strategy involves implantation of an indwelling catheter with the stabilizing Kirschner (K)-wire during the surgery prior to fracture of the femur by the three-point bending technique.  It uses the openings in the bone that were already created for the stabilizing K-wire and the catheter insertion.  In this study, transgene expression and callus bone formation induced by intramedullary delivery of MLV-based vectors expressing the bone morphogenetic protein-2/4 (BMP-2/4) hybrid gene or b-galactosidase (b-gal) gene were compared with those produced by percutaneous injections of the same vectors at the periosteum of the fracture site.  The percutaneous injections of MLV-BMP-4 vector led to massive but asymmetric transgene expression in surrounding tissues within the fracture callus and large amounts of supraperiosteal as well as asymmetric callus bone formation. In contrast, the intramedullary administration produced a robust and symmetric pattern of transgene expression at the fracture site with very minimal transduction at cells of surrounding tissues, resulting in normal subperiosteal bone development around the entire fracture callus without supraperiosteal bone formation.  In summary, we have developed an intramedullary retroviral vector delivery strategy with a rat femur fracture model that led to uniformed transgene expression around the entire fracture site, which optimizes the gene therapy-enhanced fracture repair. This strategy should readily be adapted to administer large dosages of any therapeutic vehicle (therapeutic molecules, peptides, or proteins, as well as viral or non-viral vectors) throughout much of early fracture repair, and thus it would be an ideal rat model for in vivo testing of various therapeutic agents to promote fracture repair. (11/2008) (link)
• Rundle CH, Wang X, Sheng MH-C, Wergedal JE, Lau K-HW, and Mohan S (2008) Bax deficiency in mice increases cartilage production during fracture repair through a mechanism involving increased chondrocyte proliferation without changes in apoptosis. Bone 43 (5), 880-888.  This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair. (11/2008) (link)
• Rundle CH, Wang X, Wergedal JE, Mohan S, and Lau K-HW (2008) Fracture healing in mice deficient in plasminogen activator inhibitor-I. Calcified Tissue International 83 (4), 276-284. To evaluate the role of plasminogen activator inhibitor (PAI)-1, a key negative regulator of the plasmin system of extracellular matrix proteases in developmental bone growth and fracture repair, the bone phenotype of male adult PAI-1-deficient mice was determined and their femoral fracture healing was compared with that of age- and sex-matched wild-type C57BL/6J control mice. Regarding bone phenotype, the length and size (but not cortical thickness) of the femur of male PAI-1-deficient mice were smaller than those of wild-type controls. Although the total bone mineral content of PAI-1-deficient mice was not significantly different from wild-type mice, the total bone area in PAI-1-deficient mice was smaller, leading to an increase in the total bone mineral density. With respect to fracture healing, PAI-1-deficient mice developed fracture calluses that were larger and more mineralized than wild-type mice, but only at 14 days post-fracture. These changes were even greater given the smaller size of the normal femur in PAI-1-deficient mice. Surprisingly, the larger fracture callus remodeled rapidly to normal size and mineral content by 21 days post-fracture. Examination of the fracture histology revealed that these changes were associated with a dramatic increase followed by a rapid remodeling of the fracture callus cartilage. The remodeling of fracture callus cartilage in PAI-1-deficient mice also displayed an abnormal pattern. These findings demonstrate for the first time that PAI-1 (and potentially the plasminogen extracellular matrix proteases system) is an important regulator of bone size during developmental growth and plays a regulatory role in determination of fracture callus size, cartilage formation and resorption during bone fracture repair. (10/2008) (link)
• Strohbach CA, Rundle CH, Wergedal JE, Chen S-T, Linkhart TA, Lau K-HW, and Strong DD (2008) LMP-1 retroviral gene therapy stimulates osteoblast differentiation and fracture healing: a preliminary study. Calcified Tissue International 83 (3), 202-211. LIM mineralization protein-1 (LMP-1) is a novel intracellular osteogenic factor associated with bone development that has been implicated in the bone morphogenetic protein (BMP) pathway. This preliminary study evaluated the possibility of LMP-1-based retroviral gene therapy to stimulate osteoblast differentiation in vitro and fracture repair in vivo. A Moloney leukemia virus (MLV)-based retroviral vector to express LMP-1 with a hemagglutinin (HA) tag was developed, and its effects were evaluated on MC3T3-E1 cell differentiation and in the rat femur fracture model. MC3T3-E1 osteoblasts transduced with the MLV-HA-LMP-1 vector demonstrated significantly increased osteoblast marker gene expression (P < 0.05) and mineral deposition compared to control transduced cells. Femoral midshaft fractures were produced in Fischer 344 rats by the three-point bending technique. The MLV-HA-LMP-1 or control vector was applied at the fracture site through percutaneous injections 1 day postfracture. Analysis of fracture healing of 10 MLV-HA-LMP-1-treated and 10 control MLV-beta-galactosidase (beta-gal)-treated animals was completed at 3 weeks by X-ray, peripheral quantitative computed tomography, and histology. MLV-HA-LMP-1-treated animals had 63% more bone mineral content at the fracture site (P < 0.01), 34% greater total hard callus area (P < 0.05), and 45% less cartilage in the fracture callus (P < 0.05) compared to MLV-beta-gal-treated animals. There was no effect of LMP-1 treatment on the density of the hard callus. Immunohistochemistry revealed expression of the LMP-1 transgene in the fracture callus at 21 days postfracture. Immunohistochemistry also revealed that LMP-1 transgene expression did not result in an increase in BMP-4 expression in the fracture callus. Compared to MLV-BMP-4 gene therapy studies, MLV-HA-LMP-1 gene therapy improved bony union of the fracture gap to a greater extent and did not cause heterotopic bone formation. This suggests that LMP-1 may be a better potential candidate for gene therapy for fracture repair than BMP-4. These exciting, albeit preliminary, findings indicate that LMP-1-based gene therapy may potentially be a simple and effective means to enhance fracture repair that warrants further investigation. (09/2008) (link)
• Sheng MH-C, Wergedal JE, Mohan S, and Lau K-HW (2008) Osteoactivin is a novel osteoclastic protein and plays a key role in osteoclast differentiation and activity." FEBS Letters 582 (10), 1451-1458. This study presents gene expression, protein expression, and in situ immunohistochemical evidence that osteoclasts express high levels of osteoactivin (OA), which had previously been reported to be an osteoblast-specific protein in bone. OA expression in osteoclasts was up-regulated upon receptor activator of NFkappaB ligand-induced differentiation. Suppression of functional activity of OA with neutralizing antibody reduced cell size, number of nuclei, fusion, and bone resorption activity of osteoclasts. OA was co-immunoprecipitated with integrin beta(3) and beta(1), indicating that OA co-localizes with integrin beta(3) and/or beta(1) in a hetero-polymeric complex in osteoclasts. These findings indicate that OA is a novel osteoclastic protein and plays a role in osteoclast differentiation and/or activity. (04/2008) (link)
• Rundle CH, Strong DD, Chen S-T, Linkhart TA, Sheng M-HC, Wergedal JE, Lau K-HW, and Baylink DJ  (2008) Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat. Journal of Gene Medicine 10 (3), 229-241. BACKGROUND: An in vivo gene therapy strategy was developed to accelerate bone fracture repair. METHODS: Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3''''''''''''''''''''''''''''''''-untranslated region and to improve protein translation. RESULTS: In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. CONCLUSIONS: This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair. (03/2008) (link)
• Baylink DJ, Lau K-HW, Mohan S (2007) The role of IGF system in the rise and fall in bone density with age. Journal of Musculoskeletal and Neuronal Interactions 7 (4), 304-305. (10/2007 - 12/2007)
• Hall SL, Lau K-HW, Chen S-T, Wergedal JE, Srivastava A, Klamut H, Sheng MH-C, Gridley DS, Mohan S, Baylink DJ (2007) Sca-1+ hematopoietic cell-based gene therapy with a modified FGF-2 increased endosteal/trabecular bone formation in mice. Molecular Therapy 15 (10), 1881-1889. This study assessed the feasibility of using an ex vivo stem cell antigen-1-positive (Sca-1(+)) cell-based systemic fibroblast growth factor-2 (FGF-2) gene therapy to promote endosteal bone formation. Sca-1(+) cells were used because of their ability to home to, and engraft into, the bone marrow cavity. The human FGF-2 gene was modified to increase protein secretion and stability by adding the bone morphogeneic protein (BMP)-2/4 hybrid signal sequence and by mutating two key cysteines. Retro-orbital injection of Sca-1(+) cells transduced with a Moloney leukemia virus (MLV)-based vector expressing the modified FGF-2 gene into sub-lethally irradiated W(41)/W(41) recipient mice resulted in long-term engraftment, more than 100-fold elevation in serum FGF-2 level, increased serum bone-formation markers, and massive endosteal bone formation. In recipient mice showing very high serum FGF-2 levels (>2,000 pg/ml), this enhanced endosteal bone formation was so robust that the marrow space was filled with bony tissues and insufficient calcium was available for the mineralization of all the newly formed bone, which led to secondary hyperparathyroidism and osteomalacia. These adverse effects appeared to be dose related. In conclusion, this study provided compelling test-of-principle evidence for the feasibility of using an Sca-1(+) cell-based ex vivo systemic FGF-2 gene therapy strategy to promote endosteal bone formation. (10/2007) (link)
• Yang JH, Amoui M, Strong DD, and Lau K-HW (2007) Characterization and comparison of the intronic promoter of murine osteoclastic protein-tyrosine phosphatase, PTP-oc, with the human PTP-oc promoter. Archives of Biochemistry and Biophysics 465 (1), 72-81. This study characterized the murine osteoclastic protein-tyrosine phosphatase (PTP-oc) promoter located within intron 12 of the Glepp1 gene. A 2-kb DNA fragment containing the putative intronic promoter showed strong promoter activity in pre-osteoclastic RAW264.7 and U937 cells, but not in non-osteoclastic cells. Deletion analyses identified a proximal region with elements required for basal activity, and upstream repressor and enhancer elements. The cell-type-specificity of the promoter was conferred by upstream domains. At least nine conserved response elements, with potential transcription factor binding sites, were identified in both human and murine promoters. EMSA and ChIP indicate the presence of occupied binding sites for Pit-1a, Ikaros-1/2, and D1DR transcription factors in the murine promoter. Site-directed mutagenesis of response elements resulted in down- or up-regulation of promoter activity: some of the effects were different between the murine and human promoter, suggesting that there may be inter-species differences in the regulation of the PTP-oc promoter. (09/2007) (link)
• Amoui M, Sheng MH-C, Chen S-T, Baylink DJ, and Lau K-HW  (2007) A transmembrane osteoclastic protein-tyrosine phosphatase regulates osteoclast activity in part by promoting osteoclast survival through c-Src-dependent activation of NF-kappaB and JNK2. Archives of Biochemistry and Biophysics 463 (1), 47-59. This study evaluated the effects of overexpression of wild-type (WT) or phosphatase-deficient (PD) mutant of an osteoclastic protein-tyrosine phosphatase (PTP-oc) in RAW/C4 cells. Osteoclast-like cells derived from WT-PTP-oc overexpressing clones increased, while those derived from PD-PTP-oc expressing clones decreased, their resorption activity. WT-PTP-oc clones had lower apoptosis, lower caspase 3/7 activity, reduced c-Src tyr-527 phosphorylation (PY527) and IkappaBalpha cellular levels, and increased NFkappaB activation and JNK phosphorylation. Overexpression of PD-PTP-oc or PTP-oc siRNA treatment increased apoptosis, caspase 3/7 activity, PY527 and IkappaBalpha levels, and decreased NFkappaB and JNK2 activation. Inhibition of the c-Src kinase blocked the PTP-oc-mediated NFkappaB and JNK2 activation. Blocking the NFkappaB activation had no effect on the JNK2 activation. Inhibiting the NFkappaB and/or JNK2 pathway prevented the PTP-oc-mediated reduction in apoptosis. In conclusion, PTP-oc activates osteoclast activity in part by promoting osteoclast survival through the PTP-oc-mediated c-Src-dependent activation of NFkappaB and JNK2. (07/2007) (link)
• Wang X, Rundle CH, Wergedal JE, Srivastava AK, Mohan S, and Lau K-HW (2007) Loss of sex-specific difference in femoral bone parameters in male leptin knockout mice. Calcified Tissue International 80 (6), 374-382. Sex-dependent differences were identified in the femoral bone parameters of male and female ob/ob (leptin knockout) mice as compared with their C57BL/6 wild-type control background strain. Total fat, lean and body weight were not different between adult male and female leptin knockout mice but significantly lower than adult male and female C57BL/6 mice. pQCT measurements at the femoral midshaft revealed that the normal differences in the periosteal circumference, endosteal circumference, total bone mineral content and polar moment of inertia normally observed between adult male and female wild-type mice were lost between adult male and female ob/ob mice. Significant reductions in these bone parameters were seen in male ob/ob mice as compared to male wild-type mice, but not in female ob/ob mice as compared to female wild-type mice. In pre-pubertal mice, there were no differences in phenotype and femoral bone parameters between male and female mice within any strain, suggesting sex hormone functions. Serum free testosterone levels were 5.6-fold higher in adult male ob/ob mice than in adult male C57BL/6 wild-type mice, and serum estradiol levels were 1.8 and 1.3-fold greater in adult male and female ob/ob mice, respectively, than in their wild-type counterparts. Androgen receptor gene expression was not different in the periosteal cells of male ob/ob mice as compared with wild-type mice. The loss of sex-related differences in these bone parameters in adult male ob/ob mice might result from deficient signaling in the androgen signaling pathway, and that leptin functions are permissive for androgen effects on bone development. (06/2007) (link)
• Yang JH, Amoui M, Lau K-HW (2007) Targeted deletion of the osteoclast protein-tyrosine phosphatase (PTP-oc) promoter prevents RANKL-mediated osteoclastic differentiation of RAW264.7 cells. FEBS Letters 581 (13), 2503-2508. An osteoclastic protein-tyrosine phosphatase, PTP-oc, shares the same gene with a renal PTP, Glepp1. This study demonstrated that targeted deletion of PTP-oc promoter by homologous recombination in RAW264.7 cells completely abolished PTP-oc expression without affecting Glepp1 expression. This strategy to inhibit PTP-oc function has three advantages over commonly used gene knock down strategies (e.g., small interference RNA). This strategy: (1) yielded cells completely devoid of PTP-oc, (2) had no off-target gene silencing effects, and (3) did not affect Glepp1 expression. The inability of PTP-oc-deficient RAW264.7 cells to undergo RANKL-mediated osteoclastic differentiation confirmed a regulatory role for PTP-oc in RANKL-mediated osteoclast differentiation. (05/2007) (link)
• Chen S-T, Gysin R, Sonia Kapur S, Baylink DJ, and Lau K-HW (2007) Modifications of the fibroblast growth factor-2 gene led to a marked enhancement in secretion and stability of the recombinant fibroblast growth factor-2 protein. Journal of Cellular Biochemistry 100 (6), 1493-1508. Progress in FGF-2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF-2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF-2 gene and mutation of cys-70 and cys-88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF-2 protein in mammalian cells using MLV-based vectors. Single or double mutations of cys-70 and cys-88 to ser-70 and asp-88, respectively, markedly increased the amounts of FGF-2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp-88 residue. Addition of BMP2/4 secretion signal increased FGF-2 secretion, but also suppressed FGF-2 biosynthesis. The combination of BMP2/4 secretion signal and double cys-70 and cys-88 mutation increased the total amount of secreted FGF-2 protein > 60-fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF-2 protein was as functionally as effective as the unmodified FGF-2. An ex vivo application of rat skin fibroblasts transduced with the modified FGF-2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF-2 vector increased serum FGF-2 level >15-fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing ?-galactosidase- or wild-type FGF-2-transduced control cells. This modified vector may be useful in FGF-2 gene therapy investigations. (04/2007) (link)
• Hall SL, Lau K-HW, Chen S-T, Felt JC, Gridley DS, Yee J-K, and Baylink DJ (2007) An improved mouse Sca-1+ cell-based bone marrow transplantation model for use in gene- and cell-based therapeutic studies. Acta Haematologica 117 (1), 24-33. This study sought to develop a murine bone marrow transplantation strategy that would yield consistently high levels of long-term engraftment without significant morbidity and mortality. Hematopoietic stem cell (HSC)-enriched Sca-1+ cells were used for transplantation because of their propensity of homing to bone marrow. Green fluorescent protein (GFP)-expressing transgenic mice were used as donors. Murine Sca-1+ cells were enriched 13-fold from whole bone marrow with immuno-magnetic column chromatography. Retroorbital injections yielded highly reproducible and higher levels of engraftment compared to tail vein injections. The combination use of W41/W41 recipient mice and sublethal irradiation preconditioning produced long-term engraftment with minimal morbidity and mortality. A 24-hour delay between the sublethal irradiation and transplantation did not affect the efficiency and level of engraftment, but provided flexibility with respect to the timing of transplantation. Based on these findings, a mouse Sca-1+ cell-based strategy, involving the retroorbital injection of Sca-1+ cells into sublethally irradiated, myelosuppressed W41/W41 recipient mice within 24 hours post-irradiation was developed. Transplantation of lentiviral vector-transduced wild-type Sca-1+ cells expressing GFP by this strategy led to consistently high levels of long-term engraftment. In summary, this murine Sca-1+ cell-based strategy could be used in studies of HSC-based gene or cell therapies. (01/2007) (link)
• Sheng MH-C, Lau K-HW, Mohan S, Baylink DJ, and Wergedal JE (2006) High osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is associated with a low apoptosis in C3H/HeJ osteoblasts. Calcified Tissue International 78 (5), 293-301. This study sought to confirm that osteoblasts of C3H/HeJ (C3H) mice, which have higher differentiation status and bone-forming ability compared to C57BL/6J (B6) osteoblasts, also have a lower apoptosis level and to test whether the higher differentiation status and bone-forming ability of C3H osteoblasts were related to the lower apoptosis. C3H mice had 50% fewer (P < 0.01) apoptotic osteoblasts on the endocortical bone surface than B6 mice as determined by the TUNEL assay. Primary C3H osteoblasts in cultures also showed a 50% (P < 0.05) lower apoptosis level than B6 osteoblasts assayed by acridine orange/ethidium bromide staining of apoptotic osteoblasts. The lower apoptosis in C3H osteoblasts was accompanied by 22% (P < 0.05) and 56% (P < 0.001) reduction in the activity of total caspases and caspases 3/7, respectively. C3H osteoblasts also displayed greater alkaline phosphatase (ALP) activity (P < 0.001) and higher expression of Cbfa1, type-1 collagen, osteopontin, and osteocalcin genes (P < 0.05 for each). To assess if an association existed between population apoptosis and the differentiation status (ALP-specific activity) and/or bone-forming activity (insoluble collagen synthesis), C3H and B6 osteoblasts were treated with several apoptosis enhancers (tumor necrosis factor-alpha, dexamethasone, lipopolysaccharide, etoposide) and inhibitors (parathyroid hormone, insulin-like growth factor I, transforming growth factor beta1, estradiol). Both ALP (r = -0.61, P < 0.001) and insoluble collagen synthesis (r = -0.61, P < 0.001) were inversely correlated with apoptosis, suggesting that differentiation (maturation) and/or bone-forming activity of these mouse osteoblasts were inversely associated with apoptosis. In conclusion, these studies support the premise that higher bone density and bone formation rate in C3H mice could be due in part to lower apoptosis in C3H osteoblasts. (05/2006) (link)
• Lau K-HW, Kapur S, Kesavan C, and Baylink DJ (2006) Upregulation of the wnt, estrogen receptor, insulin-like growth factor-I and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the differential anabolic response to fluid shear. Journal of Biological Chemistry 281 (14), 9567-9588. This study tested the hypothesis that shear stress interacts with the IGF-I pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm2 for 30 min followed by 24-hr incubation with IGF-I (0 to 50 ng/ml). IGF-I increased proliferation dose-dependently (1.5- to 2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2- to 5.3-fold and that of IGF-I receptor (IGF-IR) by 2- to 4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12-fold and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test if the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress ± IGF-I was measured. Echistatin reduced basal proliferation by ~60%, and the shear stress-induced mitogenic response by ~20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and SHP-1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and SHP-1 away from IGF-IR (04/2006) (link)
• Rundle CH, Wang H, Yu H, Chadwick RB, Davis EI, Wergedal JE, Lau K-HW, Mohan S, Ryaby JT, and Baylink DJ (2006) Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair. Bone 38 (4), 521-529. Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6,555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes were restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms. (04/2006) (link)
• Lau K-HW, Wu L-W, Sheng, MH-C, Amoui M, Suhr SM, and Baylink DJ (2006) An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity. Journal of Cellular Biochemistry 97 (5), 940-955. This study sought to determine the mechanism whereby an osteoclastic protein-tyrosine phosphatase, PTP-oc, regulates osteoclast activity by testing the hypothesis that PTP-oc enhances osteoclast activity through c-Src activation. The effects of several resorption activators and inhibitors on PTP-oc expression, resorption activity, and c-Src activation were determined in rabbit osteoclasts. PTP-oc expression was assayed with immunoblots and semi-quantitative RT-PCR. Osteoclastic resorption was determined by resorption pit formation; and c-Src activation was monitored by dephosphorylation of P-tyr527 (PY527) and in vitro kinase assay. Treatment of osteoclasts with some test resorption activators (PTH, PGE2, 1,25(OH)2D3, IL-1), but not RANKL or IL-6, significantly stimulated resorption activity, increased PTP-oc mRNA and protein levels, and reduced c-Src PY527 level with corresponding activation of c-Src PTK activity. The PTP-oc antisense phosphorothioated oligo treatment blocked the basal and IL-1?-mediated but not RANKL-mediated resorption activity of isolated osteoclasts. Calcitonin and alendondrate, resorption inhibitors, significantly reduced resorption activity and PTP-oc expression, and increased c-Src PY527 with corresponding reduction in its PTK activity. The cellular PTP-oc protein level correlated with the resorption activity. Among the various signaling proteins co-immunoprecipitated with PTP-oc, the resorption effectors caused corresponding changes in the tyrosyl phosphorylation level of only c-Src. The GST-PTP-oc fusion protein dephosphorylated PY-527-containing c-Src peptide in time- and dose-dependent manner in vitro. In summary, 1) PTP-oc is regulated in part at transcriptional level, 2) upregulation of PTP-oc in osteoclasts led to c-Src activation, and 3) PY527 of c-Src may be a cellular substrate of PTP-oc. These findings are consistent with the hypothesis that PTP-oc is a positive regulator of c-Src in osteoclasts. (04/2006) (link)
• Kapur S, Mohan S, Baylink DJ, and Lau K-HW (2005) Fluid shear stress synergizes with IGF-I on osteoblast proliferation through integrin-dependent activation of IGF-I mitogenic signaling pathway. Journal of Biological Chemistry 280 (20), 20163-20170. This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR. (05/2005) (link)
• Amoui M, Suhr SM, Baylink DJ, and Lau K-HW (2004) An osteoclastic protein-tyrosine phosphatase may play a role in the differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells. American Journal of Physiology. Cell Physiology 287 (4), C874-C884. This study investigated if an osteoclastic protein-tyrosine phosphatase (PTP), PTP-oc, plays a role in the functional activity and differentiation of osteoclastic cells by determining the effects of overexpression of wild-type (WT)- or phosphatase-deficient (PD)-PTP-oc on bone resorption activity and differentiation of human promyelomonocytic U-937 cells, which could be induced to differentiate into "osteoclast-like" cells by phorbol ester/1,25(OH)(2)D(3) treatment. U-937 cells overexpressing WT- or PD-PTP-oc were produced with a transposon-based vector. The size and depth of resorption pits created by WT-PTP-oc-overexpressing osteoclast-like cells were greater, while those by PD-PTP-oc-overexpressing osteoclast-like cells were less, than those created by control osteoclast-like cells. Overexpression of WT-PTP-oc also enhanced, while overexpression of PD-PTP-oc suppressed, their differentiation into osteoclast-like cells. Overexpression of WT-PTP-oc increased apoptosis and proliferation of U-937 cells, and overexpression of PD-PTP-oc reduced cell proliferation. Cells overexpressing WT-PTP-oc has also led to greater c-Src and NF-kappabeta activation, whereas cells overexpressing PD-PTP-oc resulted in less c-Src and NF-kappabeta activation. c-Src activation and NF-kappabeta activation each correlated with resorption activity and differentiation into osteoclast-like cells. In summary, these results show that 1) PTP-oc regulates both the activity and the differentiation of osteoclast-like cells derived from U-937 cells; 2) PTP-oc enzymatic activity is important to these processes; 3) high PTP-oc enzymatic activity caused an increase in U-937 cell apoptosis and proliferation, leading to no significant changes in the number of viable cells; and 4) some of the PTP-oc actions are mediated in part by the c-Src and/or NF-kappabeta pathways. (10/2004) (link)
• Sheng MH-C, Lau K-HW, Beamer WG, Baylink DJ, and Wergedal JE (2004) In vivo and in vitro evidence that the high osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is intrinsic to bone cells. Bone 35 (3), 711-719. Two inbred mouse strains, C3H/HeJ (C3H) and C57BL/6J (B6), displayed a profound difference in femoral peak bone density. We have previously shown that the difference could be attributed to a greater bone formation rate (BFR) that was due to a higher osteoblastic activity [measured by a mineral apposition rate (MAR)] in the C3H (high density) than B6 (low density) mice. The present study sought to determine (1) whether the BFR/MAR differences between the two mouse strains present in weight-loaded endochondral bones are also seen in less weight-loaded membranous bones and (2) whether the difference in osteoblastic activity was seen in vitro in the absence of systemic factors. To address the first objective, we performed histomorphometric measurements on the weakly loaded membranous bones (i.e., parietal bones of the calvaria) to determine if there were similar differences in MAR and BFR of membranous bones as those of highly loaded, endochondral bones. The parietal bones of adult C3H mice showed similar increases in MAR and BFR as the endochondral bones, compared to B6 mice of same age, suggesting that the differences in the MAR and BFR in the two mouse strains are probably not related to differences in mechanical strain. These findings also suggest that the gene(s) responsible for the difference in MAR between strains may not be a mechanical response gene. With respect to the second objective, we isolated osteoblasts from the parietal bones and determined their differentiation status (i.e., ALP-specific activity) and bone-forming ability (i.e., mineralized nodule formation) in vitro. Consistent with the premise that C3H osteoblasts have an intrinsic, higher differentiation status and bone-forming ability than B6 osteoblasts, osteoblasts isolated from C3H mice as compared with those from B6 mice had a significantly greater ALP-specific activity and a greater ability to form mineralized nodules in vitro in the absence of systemic factors. Because differences in ALP activity, bone-forming ability, cortical bone width, and osteoblastic activity were detected at birth, the different MAR/BFR phenotypes develop at very early life and even perhaps during embryogenesis. In conclusion, we have for the first time provided evidence that the genetic differences responsible for the observed MAR/BFR phenotype in the C3H-B6 strains are intrinsic to osteoblasts and might not depend on responses to mechanical loading and/or alterations in systemic factors. (09/2004) (link)
• Klamut HJ, Chen S-T, Lau K-HW, and Baylink DJ (2004) Progress toward skeletal gene therapy. Critical Reviews in Eukaryotic Gene Expression 14 (1&2), 89-136. Skeletal gene therapy is an attractive new approach to the treatment of bone disorders. Impressive advances in our knowledge of the molecular genetic basis of skeletal disorders and fracture healing have led to the development of novel therapeutics based on ectopic expression of one or more genes in patient cells that can influence repair or regenerative processes in bone. Although still a relatively immature field, proof-of-principle for enhanced bone formation through skeletal gene therapy has already been established. The challenge now is to more precisely define optimal cellular targets and therapeutic genes, and to develop safe and efficient ways to deliver therapeutic genes to target cells. In this review, we will highlight some of the exciting advances that have been made in skeletal gene therapy in recent years, with a focus on treatment of localized skeletal lesions. Strengths and weaknesses of current approaches will be discussed, as will strategies for improved safety and therapeutic outcome in the future. Skeletal gene therapy can have an enormous impact on patient care. The next 5 years will present us with unparalleled opportunities to develop more effective therapeutic strategies and overcome obstacles presented by current gene transfer technologies. (08/2004) (link)
• Kapur S, Chen S-T, Baylink DJ, and Lau K-HW (2004) Extracellular signal-regulated kinase-1 and -2 are both essential for the shear stress-induced human osteoblast proliferation. Bone 35 (2), 525-534. Extracellular signal-regulated kinases (Erk)-1 and -2 are key mediators of various mitogenic signaling pathways, including mechanical stress-induced osteoblast proliferation. Mechanical stimuli, such as flow shear stress, simultaneously activate both Erk-1 and -2 in osteoblasts, resulting in stimulation of osteoblast proliferation. This study sought to test whether Erk-1, -2, or both are essential for the fluid flow shear stress-induced osteoblast proliferation. Moloney leukemia virus (MLV)-based vectors expressing wild-type (wt)- or kinase-deficient (kd) Erk-1 and Erk-2, respectively, were constructed and used to transduce human TE85 osteosarcoma cells with an MOI of 30. An MLV-red fluorescent protein (RFP) vector was included as a control. Effects of Erk-1 and -2 overexpression on cell proliferation in response to a 30-min constant fluid flow shear stress at 20 dynes/cm2 were determined with [3H]thymidine incorporation 24 h after the shear stress. The MLV-Erk vector-transduced TE85 cells showed a >10- and approximately 2-fold overexpression of Erk-1 and -2 protein, respectively. The RFP expressing control cells and the parental TE85 cells each showed an approximately twofold increase (P < 0.01) in [3H]thymidine incorporation in response to the shear stress. Cells overexpressing wt-Erk-1 or -2 showed small enhancing effects on the response to the shear stress in the increases in [3H]thymidine incorporation and cell number. Conversely, overexpression of kd-Erk-1 or -2 each alone completely abolished the shear stress-induced osteoblast proliferation. Overexpression of either kd-Erk-1 or kd-Erk-2 alone did not have a significant effect on basal osteoblast proliferation, suggesting that the Erk signaling pathway may not be essential for basal cell proliferation. In summary, this study demonstrates for the first time that Erk-1 and -2 are both required for the mitogenic response to fluid flow shear stress in human osteoblasts and that blocking Erk-1 or -2 each alone is sufficient to completely block the mitogenic response to shear stress-induced proliferation. (08/2004) (link)
• Sivanandam AS, Mohan S, Kapur S, Kita H, Lau K-HW, Bagi G, Baylink DJ, and Qin X (2004) Covalent interaction between proform of eosinophil major basic protein (proMBP) and pregnancy-associated plasma protein-A (PAPP-A) is a cell-mediated event and required for proMBP inhibition of the catalytic activity of PAPP-A. Archives of Biochemistry and Biophysics 423 (2), 343-350. This study was undertaken to determine the mechanism by which proform of eosinophil major basic protein (proMBP) inhibits the IGFBP-4 proteolytic activity of pregnancy-associated plasma protein (PAPP)-A. Co-overexpression of PAPP-A with proMBP in 293T cells, or co-incubation of 293T cells, respectively, overexpressing proMBP and PAPP-A resulted in the formation of a covalent proMBP-PAPP-A complex and inhibition of IGFBP-4 proteolysis. Similar results were obtained when recombinant proMBP and PAPP-A were incubated in the presence of U2 osteosarcoma cells or when recombinant proMBP was added to the U2 cells overexpressing PAPP-A. In contrast, no formation of covalent proMBP-PAPP-A complex or inhibition of IGFBP-4 proteolysis was observed when recombinant proMBP and PAPP-A were incubated under cell-free conditions, although proMBP was able to interact with PAPP-A in a non-covalent manner. These new findings suggest that formation of covalent proMBP-PAPP-A complex is a cell-mediated event and is required for proMBP to inhibit the catalytic activity of PAPP-A. (03/2004)
• Zhang XS, Linkhart TA, Chen S-T, Peng H, Wergedal JE, Guttierez GG, Sheng MH-C, Lau K-HW, and Baylink DJ (2004) Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice. Journal of Gene Medicine 6 (1), 4-15. BACKGROUND: Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV-based retroviral vector to express human bone morphogenetic protein 4 (BMP4). METHODS: We assessed two approaches for administering transduced MSCs. beta-Galactosidase (beta-Gal) transduced C57BL/6J mouse MSCs were injected intravenously via tail vein or directly injected into the femoral bone marrow cavity of non-marrow-ablated syngenic recipient mice and bone marrow cavity engraftment was assessed. BMP4- or beta-Gal-transduced cells were injected into the femoral bone marrow cavity and effects on bone were evaluated by X-ray, peripheral quantitative computed tomography (pQCT), and histology. RESULTS: After tail-vein injection less than 20% of recipient mice contained beta-Gal-positive donor cells in femur, humerus or vertebra marrow cavities combined, and in these mice only 0.02-0.29% of injected cells were present in the bone marrow. In contrast, direct intramedullary injection was always successful and an average of 2% of injected cells were present in the injected femur marrow cavity 24 hours after injection. Numbers of donor cells decreased over the next 14 days. Intramedullary injection of BMP4-transduced MSCs induced bone formation. Trabecular bone mineral density (BMD) determined by pQCT increased 20.5% at 14 days and total BMD increased 6.5% at 14 days and 10.4% at 56 days. CONCLUSIONS: The present findings support the feasibility of using ex vivo MSC-based retroviral gene therapy to induce relatively sustained new bone formation, with normal histological appearance, at endosteal bone sites. (01/2004) (link)
• Amoui M, Baylink DJ, Tillman JB, and Lau K-HW (2003) Expression of a structurally unique osteoclastic protein-tyrosine phosphatase is driven by an alternative intronic, cell-type-specific promoter. Journal of Biological Chemistry 278 (45), 44273-44280. An osteoclastic protein-tyrosine phosphatase (PTP-oc), essential for osteoclast activity, shows sequence identity with the intracellular domain of GLEPP1, a renal receptor-like transmembrane PTP. PTP-oc has been assumed to be a truncated variant of GLEPP1, resulting from alternative splicing. However, the 5''-untranslated region sequence of PTP-oc mRNA contains 217 bp from an intron of GLEPP1. There are no splicing acceptor sites at the PTP-oc transcription site. The intronic sequence flanking the 5'' end of the PTP-oc transcription start site contains potential promoter elements essential for transcriptional initiation. To test the hypothesis that the PTP-oc gene has an alternative, tissue-specific, intronic promoter, the promoter activity of a 1.3-kb PCR fragment covering the 5''-flanking region of the PTP-oc gene was measured. The putative PTP-oc promoter fragment showed strong promoter activity in U937 cells. Mutation of the putative TATA box within the PTP-oc promoter abolished 60-90% of its promoter activity. The PTP-oc promoter fragment showed strong promoter activity in cells that express PTP-oc (U937 cells and RAW264.7 cells) but not in cells that do not express the enzyme (skin fibroblasts, TE85 cells, and HEK293 cells). These findings strongly support the conclusion that the 1.3-kb intronic fragment contains the tissue-specific, PTP-oc proximal promoter. Deletion and functional analyses indicate that the proximal 5'' sequence flanking the TATA box of the PTP-oc contains potential repressor elements. The removal of the putative repressor elements led to the apparent loss of tissue specificity. In summary, we conclude that an intronic promoter within the GLEPP1 gene drives the expression of the PTP-oc in a cell type-specific manner. This GLEPP1/PTP-oc gene system is one of the very few systems in which two important tissue-specific enzymes are derived from the same gene by the use of alternative intronic promoters. (11/2003) (link)
• Lau K-HW and Baylink DJ (2003) Osteoblastic tartrate-resistant acid phosphatase: its potential role in the molecular mechanism of osteogenic action of fluoride. Journal of Bone and Mineral Research 18 (10), 1897-1900. Although type 5 TRACP is recognized as a histochemical and biochemical marker of osteoclasts, there is evidence that bone forming cells, osteoblasts, and osteocytes also express a type 5 TRACP. Accordingly, an osteoblastic type 5 TRACP has been purified from human osteoblasts and from bovine cortical bone matrices. Comparison of biochemical properties of osteoblastic type 5 TRACP with those of osteoclastic type 5 TRACP suggests that osteoblastic type 5 TRACP is a different isoenzyme from osteoclastic type 5 TRACP. Two properties of osteoblastic type 5 TRACP may be relevant to its physiological functions: (1) it acts as a protein-tyrosine phosphatase (protein tyrosine phosphorylation) under physiologically relevant conditions, and (2) it is sensitive to inhibition by clinically relevant concentrations of fluoride. Because fluoride is a stimulator of osteoblastic proliferation and differentiation and a potent osteogenic agent and because protein tyrosine phosphorylation plays an important regulatory role in cell proliferation and differentiation, these unique properties and other evidence summarized in this review led to the proposal that the osteogenic action of fluoride is mediated, at least in part, by the fluoride-mediated inhibition of osteoblastic type 5 TRACP/protein tyrosine phosphorylation, which leads to a stimulation of osteoblast proliferation and differentiation, and subsequently, an increase in bone formation. (10/2003)
• Rundle CH, Miyakoshi N, Kasukawa Y, Chen S-T, Sheng MH-C, Wergedal JE, Lau KHW, and Baylink DJ (2003) In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with BMP-4. Bone 32 (6), 591-601. This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation. (06/2003) (link)
• Tai M-F, Chi K-M, Lau K-HW, Baylink DJ, and Chen S-T (2003) Generation of magnetic retroviral vectors with magnetic nanoparticles. Reviews in Advanced Material Sciences 5 (1), 319-323. We have successfully synthesized iron and gamma ferric oxide magnetic nanoparticles with particle sizes of 4±0.8 nm through a high-yield chemical thermal reflux method. Polyethyleneimine (PEI) was used to enhance biologically compatibility and monodispersion of nanoparticles. The PEI coated magnetic nanoparticles have sizes between 100 to 200 nm. More importantly, we formed magnetic retroviral vectors through combining Moloney leukemia virus based retroviral vectors with the particles. An external magnetic field can bring the magnetic retroviral vectors to the targeted cells and significantly raised the gene transduction efficiency. We present physical properties and biological activities of magnetic nanoparticles associated with retroviral vectors. (06/2003) (link)
• Kapur S, Baylink DJ, and Lau K-HW (2003) Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone 32 (3), 241-251. This study sought to assess the role of several signaling pathways in the fluid flow shear stress-induced proliferation and differentiation of normal human osteoblasts. We evaluated the effects of an effective dose of selective inhibitors of the extracellular signal-regulated kinases (ERK) pathway (PD98059 and U0126), the nitric oxide synthase pathway (N(omega)-nitro-L-arginine methyl ester), the cyclo-oxygenase pathway (indomethacin), or the Gi/o pathway (pertussis toxin [PTX]) on the flow-mediated effects. A 30-min steady flow shear stress at 20 dynes/cm(2) increased significantly [(3)H]thymidine incorporation (an indicator of proliferation), alkaline phosphatase activity (an index of osteoblast differentiation), phosphorylation of ERK, and expression of integrin beta1. PD98059, U0126, and N(omega)-nitro-L-arginine methyl ester completely blocked the shear stress-induced increases in ERK phosphorylation, [(3)H]thymidine incorporation, and alkaline phosphatase, but without an effect on integrin beta1 expression, indicating that the ERK and nitric oxide synthase pathways are essential for the shear stress-induced proliferation and differentiation of normal human osteoblasts and that each involves ERK activation but not integrin beta1 upregulation. Indomethacin blocked the shear stress-induced osteoblast proliferation and differentiation and integrin beta1 upregulation but not ERK activation, suggesting that the cyclo-oxygenase pathway (i.e., prostacyclin and/or prostaglandin E(2)) mediates the shear stress-induced osteoblast proliferation in an ERK-independent manner. In contrast, PTX completely blocked the flow-induced increase in integrin beta1 expression but had no effect on the increase in the ERK phosphorylation or [(3)H]thymidine incorporation. PTX not only did not inhibit but also significantly enhanced the stimulatory effect of shear stress on alkaline phosphatase activity, suggesting that a PTX-sensitive signaling pathway may have an inhibitory role in osteoblast differentiation. In summary, this study shows, for the first time, that the signal transduction mechanism of shear stress in osteoblasts is complex and involves multiple ERK-dependent and independent pathways, and provides circumstantial evidence that there may be a PTX-sensitive pathway that has completing effects with an unknown pathway on the differentiation of normal human osteoblasts. (03/2003)
• Lau KHW (2003) Osteoblastic tartrate-resistant acid phosphatase: characterization and potential functions. Recent Research and Development in Biochemistry 4 (1), 503-517. Although the type-5 tartrate-resistant acid phosphatase (TRACP) is recognized as a histochemical and biochemical marker of osteoclasts, there is evidence that bone forming cells, osteoblasts and osteocytes, also express a type-5 TRACP. Accordingly, an osteoblastic type-5 TRACP has been purified from human osteoblasts and also from bovine cortical bone matrices. Comparison of biochemical properties of osteoblastic type-5 TRACP suggests that osteoblastic typr-5 TRACP may be relevant to its physiological functions: 1) it acts as a protein-tyrosine phosphatase (PTP) under physiologically relevant conditions, and 2) it is sensitive to inhibition by clinically relevant concentrations of fluoride. Because fluoride is a stimulator of osteoblastic proliferation and differentiation and a potent osteogenic agent, and because PTP plays an important regulatory role in cell proliferation and differentiation, these unique properties and other evidence summarized in this review led to the proposal that the osteogenic action of fluoride is mediated, at least in part, by the fluoride-mediated inhibition of osteoblastic type-5 TRACP, which leads to a stimulation of osteoblast proliferation and differentiation and, subsequently, an increase in bone formation. (01/2003)
• Gu W-K, Li X-M, Roe BA, Lau K-HW, Edderkaoui B, Mohan S, and Baylink DJ (2003) Application of genomic resources and gene expression profiles to identify genes that regulate bone density. Current Genomics 4 (1), 75-102. Inadequate bone density is the strongest determinant of subsequent osteoporotic fracture. More than 70% of the variability in human bone density has been attributed to genetic factors. Therefore, the identification of genes regulating peak bone density represents a major advance in both the understanding of pathways that regulate bone density and the pathogenesis of diseases such as osteoporosis. Although association studies have revealed many candidate genes, the exact roles of these genes in the regulation of bone density are not clearly defined. Recently, a large number of bone density quantitative trait loci (QTLs) have been identified using mouse models and human populations. However, none of the genes responsible for these QTLs have been identified. Thus, the regulation of bone density is likely far more complicated than previously anticipated. Over the next decade, DNA microarrays, combined with sophisticated informatics and genomic databases, will provide a new generation of molecular tools for the identification and functional studies of genes responsible for bone density. This review intends to provide an update on the application of genomic resources and gene expression profiles to identify genes that regulate bone density. First, the progress and problems with association studies for QTL identification of bone density will be summarized. Then current resources of genomic sequences and ESTs that can be used for the identification of QTL genes will be discussed. Finally information on the 207 ESTs that are expressed in the bone and 39 ESTs that we have identified within the QTL regions will be presented. It is anticipated that this review will stimulate further studies on candidate genes that regulate bone density by taking advantage of the rapidly emerging genomic data. (01/2003)
• Harris JW, Strong DD, Amoui M, Baylink DJ, and Lau K-HW (2002) Construction of a Tc1-like transposon Sleeping Beauty-based gene transfer plasmid vector for generation of stable transgenic mammalian cell clones. Analytical Biochemistry 310 (1), 15-26. We have constructed a single plasmid-, Tc1-like transposon-based gene transfer vector, termed the Prince Charming vector (pPC). The pPC vector was constructed by ligating the CMV-driven "Sleeping Beauty" transposase gene downstream to the Tc1-like transposon inverted repeat (IR) elements and by inserting the RSV promoter (to drive expression of the gene-of-interest) along with a multiple cloning site (MCS), a polyadenylation signal, and the SV40 promoter-driven neomycin gene, at a site flanked by the transposon IR elements. To assess the utility of the pPC vector, we cloned a red fluorescent protein (RFP) gene into the pPC vector at the MCS and transfected human TE85 osteosarcoma cells with the pPC-RFP expression vector using Effectene. Stable transgenic cell clones expressing RFP were selected with G418 sulfate and individual clones were isolated. After 4 weeks of clonal isolation and expansion, 99% of cells in each randomly selected clone expressed RFP strongly. Aliquots of each clone were then maintained in either the presence or the absence of G418 sulfate and were passaged weekly. Even after 6 months in culture in the absence of G418 sulfate, approximately 90% of the cells in each clone still maintained a strong expression level of RFP, indicating that these transgenic cell clones were stable and that the clonal stability of these clones did not require a constant selection pressure. In conclusion, we have developed a single plasmid-, Tc1-like transposon-based gene transfer vector that can be used to generate stable transgenic mammalian cell clones. (11/2002)
• Gu WK, Li XM, Lau K-HW, Edderkaoui B, Donahue LR, Rosen CJ, Beamer WG, Mohan S, and Baylink DJ  (2002) Gene expression between a congenic strain that contains a QTL locus of high bone density from CAST/EiJ and its wild type strain C57BL/6J. Functional Integrative Genomics 1 (6), 375-386. Peak bone density is an important determining factor of future osteoporosis risk. We previously identified a quantitative trait locus (QTL) that contributes significantly to high bone density on mouse chromosome 1 from a cross between C57BL/6J (B6) and CAST/EiJ (CAST) mouse strains. We then generated a congenic strain, B6.CAST-1T, in which the chromosomal fragment containing this QTL had been transferred from CAST to the B6 background. The congenic mice have a significantly higher bone density than the B6 mice. In this study we performed cDNA microarray analysis to evaluate the gene expression profile that might yield insights into the mechanisms controlling the high bone density by this QTL. This study led to several interesting observations. First, approximately 60% of 8,734 gene accessions on GEM I chips were expressed in the femur of B6 mice. The expression and function of two-thirds of these expressed genes and ESTs have not been documented previously. Second, expression levels of genes related to bone formation were lower in congenic than in B6 mice. These data are consistent with a low bone formation in the congenic mice, a possibility that is confirmed by reduced skeletal alkaline phosphatase activity in serum compared with B6 mice. Third, expression levels of genes that might have negative regulatory action on bone resorption were higher in congenic than in B6 mice. Together these findings suggest that the congenic mice might have a lower bone turnover rate than B6 mice and raise the possibility that the high bone density in the congenic mice could be due to reduced bone resorption rather than increased bone formation. (06/2002) (link)
• Lau K-HW, Goodwin C, Arias M, Mohan S, and Baylink DJ (2002) Bone cell mitogenic action of fluoroaluminate and aluminum fluoride but not that of sodium fluoride involves the upregulation of the insulin-like growth factor system. Bone 30 (5), 705-711. (05/2002) (link)
• Sheng MH-C, Baylink DJ, Beamer WG, Donahue LR, Lau K-HW, and Wergedal JE (2002) Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice. Bone 30 (3), 486-491. The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice. (03/2002) (link)
• Gu WK, Li XM, Edderkaoui B, Strong DD, Lau K-HW, Beamer WG, Donahue LR, Mohan S, and Baylink DJ (2002) Construction of a BAC contig for a 3 cM biologically significant region of mouse chromosome 1. Genetica 114 (1), 1-9. One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature. (01/2002) (link)
• Koyama H, Nakade O, Takada Y, Kaku T, and Lau K-HW (2002) Melatonin, at pharmacologic doses, increases bone mass in mice by suppressing resorption through down-regulation of the RANKL-mediated osteoclast formation and activation. Journal of Bone and Mineral Research 17 (7), 1219-1229. This study evaluated if melatonin would increase bone mass in mice. Four groups of 4-week-old male ddy mice received daily injections of vehicle or 1, 5, or 50 mg/kg of melatonin, respectively, for 4 weeks. Treatment with 5 mg/kg per day or 50 mg/kg per day of melatonin significantly increased bone mineral density (BMD; by 36%, p < 0.005) and bone mass (bone volume per tissue volume [BV/TV] by 49%, p < 0.01, and trabecular thickness [Tb.Th] by 19%, p < 0.05). This treatment significantly reduced bone resorption parameters (i.e., osteoclast surface [Oc.S/bone surface [BS]] by 74%,p < 0.05, and osteoclast number [N.Oc/BS] by 76%,p < 0.005) but did not increase histomorphometric bone formation parameters (i.e., bone formation rate [BFR/ BS], mineral apposition rate [MAR], and osteoid volume [OV/TV]), indicating that melatonin increases bone mass predominantly through suppression of bone resorption. Melatonin (1-500 microM) in vitro caused dose-dependent reduction (p < 0.001 for each) in the number and area of resorption pits formed by osteoclasts derived from bone marrow cells but not those formed by isolated rabbit osteoclasts. Because RANKL increases, while osteoprotegerin (OPG) serves as a soluble decoy receptor for RANKL to inhibit osteoclast formation and activity, the effect of melatonin on the expression of RANKL and OPG in mouse MC3T3-E1 osteoblastic cells was investigated. Melatonin (5-500 microM) increased in a dose-dependent manner and reduced the mRNA level of RANKL and both mRNA and protein levels of OPG in MC3T3-E1 cells (p < 0.001 for each). In summary, these findings indicated for the first time that melatonin at pharmacologic doses in mice causes an inhibition of bone resorption and an increase in bone mass. These skeletal effects probably were caused by the melatonin-mediated down-regulation of the RANKL-mediated osteoclast formation and activation. (01/2002) (link)
• Gysin R, Wergedal JE, Sheng MH-C, Kasukawa Y, Miyakoshi N, Chen S-T, Peng H, Lau K-HW, Mohan S, and Baylink DJ (2002) Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats. Gene Therapy 9 (15), 991-999. In order to develop a successful gene therapy system for the healing of bone defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human bone morphogenetic protein (BMP) 4 transgene with high transduction efficiency. The bone formation potential of BMP4 transduced cells was tested by embedding 2.5 x 10(6) transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new bone in experimental rats after 4 weeks, while limited bone formation occurred in either control group. Bone mineral density (BMD) of the defect in the gene therapy group was 67.8 +/- 5.7 mg/cm(2) (mean +/- s.d., n = 4), which was 119 +/- 10% of the control BMD of bone surrounding the defect (57.2 +/- 1.5 mg/cm(2)). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 +/- 7.4 mg/cm(2), P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2-4 weeks after inoculation of the critical size defect with 2.5 x 10(6) implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects. (01/2002) (link)
• Rundle CH, Miyakoshi N, Ramirez E, Wergedal JE, Lau K-HW, and Baylink DJ (2002) Expression of fibroblast growth factor receptor genes in fracture repair. Clinical Orthopaedics and Related Research (403), 253-263. The spatial and temporal expression domains of the fibroblast growth factor receptor genes were examined in the healing rat femur fracture by in situ hybridization. Fibroblast growth factor receptor gene expression was detected in diverse fracture tissues throughout healing. Fibroblast growth factor receptor 1 and 2 expression was present throughout fracture repair, in the early proliferating periosteal mesenchyme, in the osteoblasts during intramembranous bone formation, and in the chondrocytes and osteoblasts during endochondral bone formation. Fibroblast growth factor receptor 3 expression colocalized with fibroblast growth factor receptor 1 and 2 expression in the chondrocytes and osteoblasts beginning at 10 days of healing, and persisted throughout endochondral bone formation. Fibroblast growth factor receptor 3 recapitulated its expression in fetal skeletal development, suggesting that it has a similar function in the control of endochondral bone growth during fracture repair. Fibroblast growth factor receptor 4 expression was not observed at any time. The extensive colocalized expression of the fibroblast growth factor receptors in healing indicates that fibroblast growth factor regulation of fracture callus maturation is extensive, and accurate identification of the receptor isoforms is necessary to establish the functions of fibroblast growth factor family members in fracture repair. (01/2002)
• Sheng MH-C, Taper LJ, Veit H, Qian H, Ritchey SJ, and Lau K-HW (2001) Dietary boron supplementation enhanced the action of estrogen, but not that of parathyroid hormone, to improve bone quality in ovariectomized rats. Biological Trace Element Research 82 (1-3), 109-123. This study investigated whether boron would enhance the ability of 17beta-estradiol (E2) or parathyroid hormone (PTH) to improve bone quality in ovariectomized OVX rats. Adult OVX rats were treated for 5 wk with vehicle, boron (5 ppm as boric acid), E2 (30 microg/kg/d, sc), PTH (60 microg/kg/d, sc), or a combination of boron and E2 or PTH, respectively. The E2 treatment corrected many adverse effects of OVX on bone quality, increased bone Ca, P, and Mg contents, and decreased trabecular plate separation. Dietary boron supplementation had no effects on these bone parameters in OVX rats. When OVX rats were treated with boron and E2 together, trabecular bone volume (Tb.BS/TV) and plate density were increased significantly more than that caused by E2 alone. The boron and E2 combination also increased trabecular bone surface (Tb.BV/TV) and decreased trabecular plate separation in OVX rats. In contrast, whereas daily PTH injection also increased bone Ca, Mg, and P contents, Tb.BV/TV, Tb.BS/TV, trabecular plate density and thickness, and decreased trabecular plate separation in OVX rats, the combination of boron and PTH had no additional improvement in bone quality over that achieved by PTH alone. In summary, this study shows for the first time that boron enhanced the action of E2, but not that of PTH, to improve trabecular bone quality in OVX rats. (12/2001) (link)
• Dimai H-P, Domej W, Leb G, and Lau K-HW (2001) Bone loss in patients with untreated chronic obstructive pulmonary disease is mediated by an increase in bone resorption associated with hypercapnia. Journal of Bone and Mineral Research 16 (11), 2132-2141. This study sought to determine whether the bone loss in untreated chronic obstructive pulmonary disease (COPD) is associated with hypercapnia and/or respiratory acidosis. Bone mineral density (BMD) measured at the distal forearm of the nondominant arm (with peripheral quantitative computed tomography [pQCT]) and serum markers of bone turnover were determined in 71 male patients with untreated COPD and 40 healthy male subjects who matched the patients in age, weight, and body mass index (BMI). The COPD patients, compared with controls, had reduced pulmonary functions, lower arterial pH, and elevated arterial partial pressure of CO2 (PCO2) The BMD (in T score) was significantly lower in COPD patients than that in control subjects (-1.628 +/- 0.168 vs. -0.058 +/- 0.157; p < 0.001). The BMD of COPD patients correlated positively with arterial pH (r = 0.582; p < 0.001), negatively with PCO2 (r = -0.442; p < 0.001), and negatively with serum cross-linked telopeptide of type I collagen (ICTP), a bone resorption marker (r = -0.444; p < 0.001) but not with serum osteocalcin, a bone formation marker. Serum ICTP, but not osteocalcin, correlated with PCO2 (r = 0.593; p < 0.001) and arterial pH (r = -0.415; p < 0.001). To assess the role of hypercapnia, COPD patients were divided into the hypercapnic (PCO2 > 45 mm Hg; n = 35) and eucapnic (PCO2 = 35-45 mm Hg) group (n = 36). Patients with hypercapnia had lower BMD, lower arterial pH, and higher serum ICTP than did patients with eucapnia. Arterial pH and serum ICTP of eucapnic patients were not different from those of controls. To evaluate the role of uncompensated respiratory acidosis, COPD patients with hypercapnia were subdivided into those with compensatory respiratory acidosis (pH > or = 7.35; n = 20) and those with uncompensated respiratory acidosis (pH < 7.35; n = 15). The BMD and serum ICTP were not different among the two subgroups. In conclusion, this study presents the first associative evidence that the bone loss in COPD is at least in part attributed to an increased bone resorption that is associated primarily with hypercapnia rather than uncompensated respiratory acidosis. (11/2001) (link)
• Hashimoto H and Lau K-HW (2001) Differential effects of bacterial toxins on mitogenic actions of sodium fluoride and those of aluminum fluoride in human TE85 osteosarcoma cells. Molecular and Cellular Biochemistry 228 (1-2), 91-98. This study compared the effects of cholera toxin (CTX) and pertussis toxin (PTX) on the actions of sodium fluoride (NaF) and those of aluminum fluoride (AlF3) on cell proliferation and differentiation, as well as tyrosine phosphorylation level of mitogen activated protein kinase (MAPK) in human bone cells. NaF and AlF3 each significantly stimulated the proliferation of human TE85 osteosarcoma cells, increased cellular alkaline phosphatase (ALP) activity, and increased MAPK tyrosine phosphorylation level. CTX completely blocked the bone cell anabolic activities of both NaF and AlF3. While PTX (2 ng/ml) inhibited the bone cell actions of NaF, it had no significant effect on those of AlF3. Both CTX and PTX completely blocked the stimulatory action of AlF3 on MAPK tyrosine phosphorylation, but neither toxin had an effect on the action of NaF on MAPK tyrosine phosphorylation. In conclusion, PTX and CTX had contrasting effects on the anabolic bone cell actions of NaF and AlF3 actions. These findings argue against the hypothesis that the osteogenic activity of NaF is mediated via the formation of AlF3 in human TE85 osteosarcoma cells. (11/2001) (link)
• Suhr SM, Pamula S, Baylink DJ, and Lau K-HW  (2001) Antisense oligodeoxynucleotide evidence that a unique osteoclastic protein-tyrosine phosphatase is essential for osteoclastic resorption.  Journal of Bone and Mineral Research 16 (10), 1795-1803. This study tested the hypothesis that a unique osteoclastic transmembrane protein tyrosine phosphatase (PTP-oc) is involved in osteoclastic resorption by determining whether suppression of PTP-oc expression with a specific phosphorothioated 20-mer PTP-oc antisense oligodeoxynucleotide (oligo) would inhibit basal, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated, and PTH-stimulated osteoclastic resorption. Treatment of rabbit osteoclasts with 1 microM of the antisense oligo for up to 4 days showed a time-dependent reduction in PTP-oc protein level, indicating that this PTP-oc antisense oligo was effective. To assess the effect of PTP-oc antisense oligo on osteoclastic resorption, rabbit osteoclasts were pretreated for 3 days with 1 microM of the antisense, a scramble oligo, or vehicle, respectively, followed by a 3-day treatment with vehicle, 10 nM of 1,25(OH)2D3, or 10 nM of parathyroid hormone (PTH). 1,25(OH)2D3 and PTH each alone increased PTP-oc cellular level and stimulated resorptive activity of rabbit osteoclasts. The antisense oligo treatment, but not the scramble oligo, decreased the basal and the stimulated resorption activity and reduced the PTP-oc protein level. Treatment with the PTP-oc antisense oligo, but not the scramble oligo, also markedly increased the Y527 phosphorylation level of c-src in rabbit osteoclasts. In conclusion, these results provide the first antisense oligo evidence that PTP-oc plays an essential role in osteoclastic resorption. (10/2001) (link)
• Peng H, Chen S-T, Wergedal JE, Polo JM, Yee J-K, Lau K-HW, and Baylink DJ (2001) Development of an MFG-based retroviral vector system for high secretion of functionally active human BMP-4.  Molecular Therapy 4 (2), 95-104. We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/10(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein. (08/2001) (link)
• Sheng MH-C, Taper LJ, Veit H, Thomas EA, Ritchey SJ, and Lau K-HW (2001) Dietary boron supplementation enhances the effects of estrogen on bone mineral balance in ovariectomized rats. Biological Trace element Research 81 (1), 29-45. The present study investigated whether boron would enhance the action of 17beta-estradiol (E2) or parathyroid hormone (PTH) on bone mineral balance in ovariectomized (OVX) rats. Forty-three days after OVX, the rats were treated for 5 wk with vehicle, boron (5 ppm as boric acid), E2 (30 microg/kg/d, sc), PTH (60 microg/kg/d, sc), or a combination of boron and E2 or PTH. Bone mineral balance was assessed by measuring apparent absorption, excretion, and retention of calcium (Ca), phosphorus (P), and magnesium (Mg). Serum Ca, P, Mg, and osteocalcin were also measured in this experiment. Boron alone had no effects on food consumption, weight gain, bone mineral balance, and serum levels of Ca, P, Mg, and osteocalcin. E2 alone increased serum P and Mg and decreased serum osteocalcin, but it had no effect on bone mineral balance. The combination of boron and E2 markedly improved apparent absorption of Ca, P, and Mg. In addition, the combination treatment increased the apparent retention of Ca and Mg (but not P) and also increased serum Ca and Mg but not serum P. On the other hand, boron cotreatment did not prevent the E2-induced reduction in serum osteocalcin in OVX rats. PTH alone significantly increased serum Ca, P, Mg, and osteocalcin concentrations, although it had no effect on bone mineral balance. Contrary to the boron-E2 combination treatment, the combination of boron and PTH did not enhance bone mineral balance. However, inasmuch as boron-PTH cotreatment did not enhance the stimulatory action of PTH on serum Ca, P, and osteocalcin, boron completely abolished the stimulatory effect of PTH on serum Mg. In conclusion, we have demonstrated for the first time that although boron by itself has no effect on bone mineral homeostasis, it appears to have synergistic enhancing effects on the action of E2 on Ca and Mg homeostasis in OVX rats. (08/2001) (link)
• Lau K-HW (2001) Role of 1,25(OH)2D3 action deficiency in the pathophysiology of senile osteoporosis. Italian Journal of Mineral and Electrolyte Metabolism 15 (1-4), 49-56. The aging-associated intestinal calcium (Ca) malabsorption, which has been implicated as a major cause of senile osteoporosis, is usually caused by some forms of vitamin D deficiency: 1) primary vitamin D deficiency due to insufficient supply of precursor (vitamin D or 25(OH)D3) of the active form of vitamin D, 12,25(OH)2D3, and 2) 1,25(OH)2D3 deficiency and/or resistance due to inadequate renal production of 1,25(OH)2D3 and/or decreased responsiveness of target organs to 1,25(OH)2D3. Both types of vitamin D deficiency could occur with aging, and each has been implicated as a potential cause of intestinal Ca malabsorption. Regardless of the etiology, the overall effect of vitamin D deficiency is an insufficient biological action of 1,25(OH)2D3. Insufficient 1,25(OH)2D3 action would lead to intestinal Ca malabsorption and a decrease in serum Ca, which would result in secondary hyperparathyroidism, which would in turn increase bone resorption. Insufficient 1,25(OH)2D3 action also causes an inhibition of bone formation, presumably through the reduction in the bone content and action of bone growth factors. The combination of increased bone resorption and reduced bone formation leads to significant loss of bone and, consequently, osteoporosis. Propensity to falls is also an important risk factor contributing to osteoporotic fractures. In this regard, 1,25(OH)2D3 has an effect on muscle strength. Thus, inadequate 1,25(OH)2D3 could reduce muscle strength and could lead to increased incidence of falls. As a result, the aging-related deficiency in 1,25(OH)2D3 action, not only could reduce bone mass, but also could increase fall incidence, which in turn results in an increase in fracture risks. Consequently, primary vitamin D deficiency and/or 1,25(OH)2D3 deficiency/resistance could play an important role in the pathogenesis of senile osteoporsis. Regarding therapies for vitamin D deficiency, primary vitamin D deficiency can be corrected effectively by daily oral supplements of 1,000 IU of plain vitamin D. In contrast, 1,25(OH)2D3 deficiency/resistance would require calcitriol or active analog (e.g., alfacalcidol) therapy to correct the secondary hyperparathroidism and the Ca malabsorption. In summary, it is important from a clinical standpoint to recognize the type of vitamin D deficiency in patients so that primary D deficiency can be treated with plain vitamin D, whereas 1,25(OH)2D3 deficiency/resistance would be appropriately treated with calcitriol or alfacalcidol. (04/2001)
• Lau K-HW and Baylink DJ (2001) Treatment of 1,25(OH)2D3 (D-hormone) deficiency/resistance with D-hormone and analogs. Osteologie 10 (1), 28-39. The aging-associated reduction in intestinal Ca absorption has been implicated in causing bone loss as well as senile osteoporosis. The pathogenesis of the aging-related intestinal Ca malabsorption is related to some forms of vitamin D deficiency. There are two types of vitamin D deficiency: 1. primary vitamin D deficiency due to insufficient supply of precursors of 1,25(OH)2D3 (D-hormone), and 2. D-hormone action deficiency due to deficiency of and/or resistance to D-hormone. Either an inadequate renal production of D-hormone or a decreased responsiveness of target organs to D-hormone causes D-hormone action deficiency. Both types of vitamin D deficiency can occur with aging. Each has been implicated as a potential cause of intestinal Ca malabsorption, secondary hyperparathyroidism, and senile osteoporosis. The overall effect of vitamin D deficiency, regardless of the etiology, is a reduced biological action of D-hormone. The vitamin D deficiency-associated calcium malabosrption would lead to secondary hyperparathyroidism, which would in turn increase bone resorption. Insufficient D-hormone action would also cause an inhibition of bone formation, presumably through the reduction in the bone content and action of bone growth factors, such as TGF-beta and IGFs. The combination of increased bone resorption and reduced bone formation leads to bone loss and, consequently, could reduce muscle strength and, as such, may lead to increased incidence of falls, which significantly increases fracture risks. Therefore, the aging-related D-hormone action deficiency could increase (in addition to bone loss) fall incidence. Consequently, D-hormone action deficiency may play an important role in the pathogenesis of senile osteoporosis. Regarding therapies for vitamin D deficiency, primary vitamin D deficiency can be effectively corrected by daily oral supplements of 1,000 I.U. of plain vitamin D. In contrast, D-hormone deficiency/resistance requires D-hormone or analog therapy to correct the high serum PTH and the Ca malabsorption. In this regard, elderly patients with D-hormone deficiency/resistance exhibit a decrease in serum PTH, and increase in Ca absorption, and/or a reduction in falls in response to therapy with D-hormone or D-hormone analog (but not with plain vitamin D). In summary, it is important from a clinical standpoint to recognize the type of vitamin D deficiency in patients so that primary vitamin D deficiency can be treated with plain vitamin D therapy, whereas D-hormone deficiency/resistance would be appropriately treated with D-hormone or D-hormone analog therapy. (01/2001)
• Qin X, Byun D, Lau K-HW, Baylink DJ, and Mohan S (2000) Evidence that the interaction between insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the action of the IGF-II dependent IGFBP-4 protease. Archives of Biochemistry and Biophysics 379 (2), 209-216. A variety of human cell types, including human osteoblasts (hOBs), produce an IGFBP-4 protease, which cleaves IGFBP-4 in the presence of IGF-II. Recently, the pregnancy-associated plasma protein (PAPP)-A has been determined to be the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. This study sought to define the mechanism by which IGF-II enhances IGFBP-4 proteolysis. Addition of PAPP-A antibody blocked the IGFBP-4 proteolytic activity in hOB conditioned medium (CM), suggesting that PAPP-A is the major IGFBP-4 protease in hOB CM. Pre-incubation of IGFBP-4 with IGF-II, followed by removal of unbound IGF-II, led to IGFBP-4 proteolysis without further requirement of the presence of IGF-II in the reaction. In contrast, prior incubation of the partially purified IGFBP-4 protease from either hOB CM or human pregnancy serum with IGF-II did not lead to IGFBP-4 proteolysis unless IGF-II was re-added to the assays. To further confirm that the interaction between IGF-II and IGFBP-4 is required for IGFBP-4 protease activity, we prepared IGFBP-4 mutants, which contained the intact cleavage site (Met135-Lys136) but lacked the IGF binding activity, by deleting the residues Leu72-His74 in the IGF binding domain or Cys183-Glu237 that contained an IGF binding enhancing motif. The IGFBP-4 protease was unable to cleave these IGFBP-4 mutants, regardless of whether or not IGF-II was present in the assay. Conversely, an IGFBP-4 mutant with His74 replaced by an Ala, which exhibited normal IGF binding activity, was effectively cleaved in the presence of IGF-II. Taken together, these findings provided strong evidence that the interaction between IGF-II and IGFBP-4, rather than the direct interaction between IGF-II and IGFBP-4 protease, is required for optimal IGFBP-4 proteolysis. (07/2000) (link)
• Dimai H-P, Wirnsberger G, and Lau K-HW (2000) Measurements of circulating ionized magnesium level: fresh whole blood samples versus serum samples. Clinical Biochemistry 33 (4), 329-332. (06/2000) (link)
• Yoon HK, Baylink DJ, and Lau K-HW (2000) Protein tyrosine kinase inhibitors block the stimulatory actions of phosphotyrosine phosphatase inhibitors to increase cell proliferation, alkaline phosphatase activity, and collagen synthesis in normal human bone cells. American Journal of Nephrology 20 (2), 153-162. The present study sought to test whether inhibition of phosphotyrosine phosphatases (PTPs) would stimulate proliferation and differentiation of normal bone cells, and whether the PTP inhibitor-mediated effects would be blocked by protein tyrosine kinase (PTK) inhibitors. Three inhibitors [phenylarsine oxide (PAO), orthovanadate (VO(4)), and molybdate (MoO(4))] and two normal human bone cells with different basal differentiation status (i.e., mandible- and vertebra-derived bone cells) were used. Cell proliferation was determined with [(3)H]thymidine incorporation, and confirmed by cell counting. Bone cell differentiation was assessed by increases in alkaline phosphatase (ALP) specific activity and collagen synthesis. The three test PTP inhibitors each stimulated [(3)H]thymidine incorporation in both human bone cell types in a biphasic, dose-dependent manner with optimal doses of 20 nM PAO, 1 microM VO(4) and 2 microM MoO(4), respectively. These PTP inhibitors at mitogenic doses each significantly and reproducibly increased ALP specific activity and collagen synthesis. To determine whether the stimulatory effects of PTP inhibitors could be blocked by PTK inhibitors, the effects of tyrphostin A51 and erbstatin, two potent PTK inhibitors, on the actions of PTK inhibitors on [(3)H]thymidine incorporation and ALP specific activity were evaluated. Both tyrphostin A51 and erbstatin, which by themselves alone significantly inhibited human bone cell proliferation and increased ALP specific activity, completely abolished the stimulatory effects of each of the three test PTP inhibitors on bone cell proliferation and ALP specific activity. In conclusion, these findings confirm the premise that inhibition of PTP activities in normal human bone cells could lead to increases in cell proliferation and differentiation, effects that are independent of basal differentiation status of the cells. More importantly, this study demonstrates for the first time that the stimulatory actions of the PTP inhibitor on bone cell proliferation and ALP could be blocked by a PTK inhibitor, suggesting that the osteogenic effects of PTP inhibitors may depend on PTK activities, presumably to increase basal tyrosyl phosphorylation level. Accordingly, one should interpret results of studies using PTK inhibitors with caution in that an inhibition by a PTK inhibitor does not necessarily indicate the requirement of PTK activities, as it could also suggest involvement of an inhibition of PTPs. (02/2000) (link)
• Chen W, Wasnik S, Fu Y, Aranda L, Rundle CH, Lau KHW, Baylink DJ, and Zhang XB (2020) Unique anabolic action of stem cell gene therapy overexpressing PDGFB-DSS6 fusion protein in OVX osteoporosis mouse model. Bone Rep 12, 100236. In the present study we sought to improve the efficacy and safety of our Sca1+ PDGFB stem cell gene therapy for osteoporosis in ovariectomized (OVX) mouse model. This therapy is administered by marrow transplantation. We established the promise of this approach by previously showing that this therapy in normal mice increase bone density, increased endosteal cortical and trabecular bone formation, caused de novo trabecular bone formation, increased cortical thickness and improve bone strength. In the current study we produced a fusion gene, PDGFB-DSS6. We reasoned that the DSS6, calcium binding protein would trap the PDGFB at the bone surface and thereby limit the amount of PDGFB required to produce an optimal bone formation response, i.e. efficacy with a lower engraftment. The result shows that indeed with a very low level of engraftment we achieved a large increase in bone formation in the OVX model of bone loss. Serum analysis for biochemical marker of new bone formation showed an approximate 75% increase in alkaline phosphatase levels in Sca1+PDGFB-DSS6 group as compared to other groups. Quantitative analysis of bone by microCT showed a massive increase in trabecular bone density and trabecular connectivity of the femur in the metaphysis in Sca1+ PDGFB-DSS6 group. The increased cortical porosity produced by OVX was replaced by the Sca1+ PDGFB-DSS6 therapy but not by the positive control Sca1+ PDGFB. Additionally, an increase in the femur bone strength was also observed specifically in Sca1+ PDGFB-DSS6 as compared to other treatment groups, emphasizing the functional significance of the observed anabolic action is on bone formation. In future work we will focus on nontoxic preconditioning of our marrow transplantation procedure and also on transcriptional control of therapeutic gene expression to avoid excess bone formation. (01/2000) (link)
• Baylink DJ and Lau K-HW (2000) The diagnosis and management of osteoporosis. Zeitschrift fur Rheumatologie 59 (Suppl 1), 42-44 Osteoporosis is a prevalent disease, which can cause considerable morbidity due to osteoporotic fractures. Kowledge of the cause of osteoporotic bone loss would help implementation of the appropriate treatment. There are two pathogenic mechanisms involved in the development of osteoporosis: 1) achievement of a relatively low peak bone density in young adulthood which increases the risk of osteoporosis because only a modest bone loss later in life results in bone densities in the osteoporotic range, and 2) a rapid bone loss rate at menopause and/or later in life. Most patients exhibiting osteoporosis have some combination of these two pathogenic mechanisms. There are basically two causes for osteoporosis: 1) a normal peak bone mass with rapid bone loss, and 2) low peak bone mass with a normal bone loss rate. Proper management of osteoporosis involves early diagnosis of the disease and appropriate treatments. With respect to diagnosis of osteoporosis, we now have two important diagnostic tools: a) instruments that measure bone density with high precision, and b) serum and urine biochemical markers of bone formation and resorption assays that allows rapid and frequent monitoring of bone turnover rates. Bone density measurements allow the diagnosis of osteoporosis before fractures occur. Biomarkers can identify patients with a high bone turnover, a situation that leads to rapid bone loss. These two diagnostic tools together can help identify osteoporosis in its earliest form so that preventative measures can be instituted. With respect to treatment of osteoporosis, we now have several forms of effective therapies, such as estrogens, bisphosphonates, calcitonins, and vitamin D (and/or active vitamin D metabolites), that are approved for the use in the prevention and/or treatment of osteoporosis. consequently, most patients with osteoporosis can now be treated with effective therapies that will have a positive impact on the disease. (01/2000)
• Sheng MH-C, Baylink DJ, Beamer WG, Donahue LR, Rosen CJ, Lau K-HW, and Wergedal JE (1999) Histomorphometric studies show that bone formation and bone mineral apposition rate are greater in C3H/HeJ (high density) than C57BL/6J (low density) mice during growth. Bone 25 (4), 421-429. High-density C3H/HeJ (C3H) and low-density C57BL/6J (B6) mice, with femoral bone density differing by 50%, were chosen as a model to investigate the mechanisms controlling peak bone density and to map peak bone density genes. The present longitudinal study was undertaken to further establish the bone biologic phenotypes of these two inbred strains of mice. To evaluate phenotypic differences in bone formation parameters in C3H and B6 mice between the ages of 6 and 26 weeks, undecalcified ground sections from the diaphyses of the tibia and femur were prepared from mice receiving two injections of tetracycline. Histomorphometric analyses revealed that the cortical bone area was significantly greater (16%-56%, p < 0.001) in both the femur and tibia of the C3H mice than in the B6 mice at all timepoints. This difference in cortical bone area was due to significantly smaller medullary areas in the C3H mice than in the B6 mice. The bone formation rates (BFR) at the endosteum in both the femur and tibia were significantly greater (28%-117%,p < 0.001) in the young C3H mice (6-12 weeks old) than in B6 mice. The higher bone formation in C3H mice was associated with higher values of the bone mineral apposition rate (25%-94%, p < 0.001), and was not associated with higher values of the forming surface length as measured by tetracycline label length. Similar interstrain differences in mineral apposition and bone formation rates were observed in the periosteum of the femur and tibia. In conclusion, the greater bone area in the high-density C3H mice vs. the low-density B6 mice was, in part, due to the greater periosteal and endosteal bone formation rates during growth in the C3H mice. Because the C3H and B6 mice were maintained under identical environmental conditions (diet, lighting, etc.), the observed interstrain differences in bone parameters were the result of the action of genetic factors. Consequently, these two inbred strains of mice are suitable as a model to identify genetic factors responsible for high bone formation rates. (10/1999) (link)
• Lau K-HW and Baylink DJ (1999) Vitamin D therapy of osteoporosis: plain vitamin D therapy versus active vitamin D analog (D-hormone) therapy. Calcified Tissue International 65 (4), 295-306. Normal intestinal calcium (Ca) absorption is an essential feature of bone homeostasis. As with many other organ systems, intestinal Ca absorption declines with aging, and this is one pathological factor that has been identified as a cause of senile osteoporosis in the elderly. This abnormality leads to secondary hyperparathyroidism, which is characterized by high serum parathyroid hormone (PTH) and an increase in bone resorption. Secondary hyperparathyroidism due to poor intestinal Ca absorption has been implicated not only in senile osteoporosis but also in age-related bone loss. Accordingly, in population-based studies, there is a gradual increase in serum PTH from about 20 years of age onward, which constitutes a maximum increase at 80 years of age of 50% of the basal value seen at 30 years of age. The cause of the increase in PTH is thought to be partly due to impaired intestinal Ca absorption that is associated with aging, a cause that is not entirely clear but at least in some instances is related to some form of vitamin D deficiency. There are three types of vitamin D deficiency: (1) primary vitamin D deficiency, which is due to a deficiency of vitamin D, the parent compound; (2) a deficiency of 1,25(OH)(2)D(3) resulting from decreased renal production of 1,25(OH)(2)D(3); and (3) resistance to 1,25(OH)(2)D(3) action owing to decreased responsiveness to 1, 25(OH)(2)D(3) of target tissues. The cause for the resistance to 1, 25(OH)(2)D(3) could be related to the finding that the vitamin D receptor level in the intestine tends to decrease with age. All three types of deficiencies can occur with aging, and each has been implicated as a potential cause of intestinal Ca malabsorption, secondary hyperparathyroidism, and senile osteoporosis. There are two forms of vitamin D replacement therapies: plain vitamin D therapy and active vitamin D analog (or D-hormone) therapy. Primary vitamin D deficiency can be corrected by vitamin supplements of 1000 U a day of plain vitamin D whereas 1,25(OH)(2)D(3) deficiency/resistance requires active vitamin D analog therapy [1, 25(OH)(2)D(3) or 1alpha(OH)D(3)] to correct the high serum PTH and the Ca malabsorption. In addition, in the elderly, there are patients with decreased intestinal Ca absorption but with apparently normal vitamin D metabolism. Although the cause of poor intestinal Ca absorption in these patients is unclear, these patients, as well as all other patients with secondary hyperparathyroidism (not due to decreased renal function), show a decrease in serum PTH and an increase in Ca absorption in response to therapy with 1, 25(OH)(2)D(3) or 1alpha(OH)D(3). In short, it is clear that some form of vitamin D therapy, either plain vitamin D or 1,25(OH)(2)D(3) or 1alpha(OH)D(3), can be used to correct all types of age-dependent impairments in intestinal Ca absorption and secondary hyperparathyroidism during aging. However, from a clinical standpoint, it is important to recognize the type of vitamin D deficiency in patients with senile osteoporosis so that primary vitamin D deficiency can be appropriately treated with plain vitamin D therapy, whereas 1,25(OH)(2)D(3) deficiency/resistance will be properly treated with 1,25(OH)(2)D(3) or 1alpha(OH)D(3) therapy. With respect to postmenopausal osteoporosis, there is strong evidence that active vitamin D analogs (but not plain vitamin D) may have bone-sparing actions. However, these effects appear to be results of their pharmacologic actions on bone formation and resorption rather than through replenishing a deficiency. (04/1999) (link)
• Lau K-HW and Baylink DJ (1998) Molecular mechanism of action of fluoride on bone cells. Journal of Bone and Mineral Research 13 (11), 1660-1667 Fluoride is an effective anabolic agent to increase spinal bone density by increasing bone formation, and at therapeutically relevant (i.e., micromolar) concentrations, it stimulates bone cell proliferation and activities in vitro and in vivo. However, the fluoride therapy of osteoporosis has been controversial, in large part because of a lack of consistent antifracture efficacy. However, information regarding the molecular mechanism of action of fluoride may improve its optimum and correct usage and may disclose potential targets for the development of new second generation drugs that might have a better efficacy and safety profile. Accordingly, this review will address the molecular mechanisms of the osteogenic action of fluoride. In this regard, we and other workers have proposed two competing models, both of which involve the mitogen activated protein kinase (MAPK) mitogenic signal transduction pathway. Our model involves a fluoride inhibition of a unique fluoride-sensitive phosphotyrosine phosphatase (PTP) in osteoblasts, which results in a sustained increase in the tyrosine phosphorylation level of the key signaling proteins of the MAPK mitogenic transduction pathway, leading to the potentiation of the bone cell proliferation initiated by growth factors. The competing model proposes that fluoride acts in coordination with aluminum to form fluoroaluminate, which activates a pertussis toxin-sensitive Gi/o protein on bone cell membrane, leading to an activation of cellular protein tyrosine kinases (PTKs), which in turn leads to increases in the tyrosine phosphorylation of signaling proteins of the MAPK mitogenic signal transduction pathway, ultimately leading to a stimulation of cell proliferation. A benefit of our model, but not the other model, is that it accounts for all the unique properties of the osteogenic action of fluoride. These include the low effective fluoride dose, the skeletal tissue specificity, the requirement of PTK-activating growth factors, the sensitivity to changes in medium phosphate concentration, the preference for undifferentiated osteoblasts, and the involvement of the MAPK. Unlike fluoride, the mitogenic action of fluoroaluminate is not specific for skeletal cells. Moreover, the mitogenic action of fluoroaluminate shows several important, different characteristics than that of fluoride. Thus, it is likely that our model of a fluoride-sensitive PTP represents the actual molecular mechanism of the osteogenic action of fluoride. (11/1998) (link)
• Yoon H-K, Chen K, Baylink DJ, and Lau K-HW (1998) Differential effects of two protein tyrosine kinase inhibitors, tyrphostin and genistein, on human bone cell proliferation as compared with differentiation. Calcified Tissue International 63 (3), 243-249 Protein tyrosyl phosphorylation is a key determinant of cell proliferation and differentiation. The aim of this study was to test the hypothesis that the signal transduction pathway(s) responsible for human bone cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve this, we investigated the effects of two structurally different PTK inhibitors, viz, tyrphostin A51 and genistein, on the proliferation ([3H]thymidine incorporation) and differentiation [alkaline phosphatase (ALP) specific activity and collagen synthesis] of two normal human bone cell types: mandible-derived and vertebra-derived bone cells. Tyrphostin A51 and genistein each markedly reduced cellular tyrosyl phosphorylation level (assessed by Western analysis using a commercial anti-phosphotyrosine antibody and the enhanced chemiluminescence detection assay), confirming that these two effectors are potent PTK inhibitors in human bone cells. Regarding bone cell proliferation, tyrphostin A51 (5-30 microM) caused, a dose-dependent inhibition of basal [3H]thymidine incorporation of both human bone cell types. In contrast, genistein (5-20 microM), not only did not inhibit, but significantly stimulated [3H]thymidine incorporation of these same cell types in a dose-dependent, biphasic manner, with the optimal stimulatory dose between 10 and 20 microM. These effects on cell proliferation were confirmed by cell number counting. In addition, whereas the mitogenic activity of 10 ng/ml epidermal growth factor (EGF) on human mandible-derived bone cells was completely abolished by 5-30 microM tyrphostin A51, genistein at 5-30 microM enhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and genistein each significantly increased basal ALP specific activity and collagen synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphostin-sensitive PTKs are involved in basal and EGF-induced human bone cell proliferation; (3) genistein stimulated basal proliferation and enhanced EGF-mediated cell proliferation, suggesting that genistein-sensitive PTKs may play an inhibitory role in human bone cell proliferation; and (4) these differential effects of PTK inhibitors on human bone cell proliferation and differentiation are independent of basal differentiation status of the cells. (09/1998) (link)
• Dimai H-P, Porta S, Wirnsberger G, Lindschinger M, Pamperl I, Dobnig H, Wilders-Truschnig M, and Lau K-HW (1998) Daily oral magnesium supplementation suppresses bone turnover in young adult males. Journal of Clinical Endocrinology and Metabolism 83 (8), 2742-2748 This study examined the effects of daily oral magnesium (Mg) supplementation on bone turnover in 12 young (27-36 yr old) healthy men. Twelve healthy men of matching age, height, and weight were recruited as the control group. The study group received orally 15 mmol Mg (Magnosolv powder, Asta Medica) daily in the early afternoon with 2-h fasting before and after Mg intake. Fasting blood and second void urine samples were collected in the early morning on days 0, 1, 5, 10, 20, and 30, respectively. Total and ionized Mg2+ and calcium (Ca2+), and intact PTH (iPTH) levels were determined in blood samples. Serum biochemical markers of bone formation (i.e. C-terminus of type I procollagen peptide and osteocalcin) and resorption (i.e. type I collagen telopeptide) and urinary Mg level adjusted for creatinine were measured. In these young males, 30 consecutive days of oral Mg supplementation had no significant effect on total circulating Mg level, but caused a significant reduction in the serum ionized Mg+ level after 5 days of intake. The Mg supplementation also significantly reduced the serum iPTH level, which did not appear to be related to changes in serum Ca2+ because the Mg intake had no significant effect on serum levels of either total or ionized Ca2+. There was a strong positive correlation between serum iPTH and ionized Mg2+ (r = 0.699; P < 0.001), supporting the contention that decreased serum iPTH may be associated with the reduction in serum ionized Mg2+. Mg supplementation also reduced levels of both serum bone formation and resorption biochemical markers after 1-5 days, consistent with the premise that Mg supplementation may have a suppressive effect on bone turnover rate. Covariance analyses revealed that serum bone formation markers correlated negatively with ionized Mg2+ (r = -0.274 for type I procollagen peptide and -0.315 for osteocalcin), but not with iPTH or ionized Ca2+. Thus, the suppressive effect on bone formation may be mediated by the reduction in serum ionized Mg2+ level (and not iPTH or ionized Ca2+). In summary, this study has demonstrated for the first time that oral Mg supplementation in normal young adults caused reductions in serum levels of iPTH, ionized Mg2+, and biochemical markers of bone turnover. In conclusion, oral Mg supplementation may suppress bone turnover in young adults. Because increased bone turnover has been implicated as a significant etiological factor for bone loss, these findings raise the interesting possibility that oral Mg supplementation may have beneficial effects in reducing bone loss associated with high bone turnover, such as age-related osteoporosis. (08/1998) (link)
• Åkesson K, Lau K-HW, Johnston P, Imperio E, and Baylink DJ (1998) Effects of a short term calcium depletion and repletion on biochemical markers of bone turnover in young adult women. Journal of Clinical Endocrinology and Metabolism 83 (6), 1921-1927 The skeletal responses to calcium depletion and repletion in rodents have been well characterized, but those in humans are poorly understood. The present study sought to evaluate the effects of short term dietary calcium depletion and repletion on biochemical markers of bone turnover in 15 young Caucasian women (age, 21-30 yr). The study contained 3 phases: 1) 5 days of a regular diet containing more than 800 mg/day calcium to establish baseline values (baseline phase), 2) 22 days of a restricted diet containing less than 300 mg/day calcium (depletion phase), and 3) 7 days of a normal diet containing more than 800 mg/day calcium (repletion phase). Serum and urine samples were obtained from each subject during the baseline phase; on the first, second, and last days of the depletion phase; and on the third and last days of the repletion phase. Serum levels of calcium, PTH, 1,25-dihydroxyvitamin D3, osteocalcin, and C-terminal type I procollagen peptide (PICP) and urinary levels of calcium and deoxypyridinoline were determined. Serum and urinary calcium levels were significantly reduced, and serum PTH and 1,25-dihydroxyvitamin D3 levels were markedly increased during depletion. These changes were completely reversed after 1 week of repletion. Depletion also rapidly and significantly increased the urinary deoxypyridinoline level, indicating increased bone resorption. The increase also returned rapidly to baseline upon repletion. Calcium depletion had contrasting effects on bone formation markers; whereas depletion significantly reduced the serum PICP level, it significantly increased serum osteocalcin level. Past histomorphometric studies in rodents indicated that the number of mature but inactive osteoblasts was increased during depletion despite an inhibition of bone formation. Thus, it is speculated that although the reduction in serum PICP reflected the depletion-associated inhibition of bone formation, the increase in serum osteocalcin could represent this depletion-related increase in osteoblast number. During repletion, serum osteocalcin remained elevated above baseline. PICP recovered from its depressed level and increased above baseline, a finding consistent with past histomorphometric findings of increased bone formation during repletion. In summary, this study confirms that 1) a short calcium depletion period produces calcium stress in young women, which leads to rapid stimulation of bone resorption and inhibition of bone formation; and 2) a subsequent calcium repletion period could lead to a compensatory increase in bone formation. In conclusion, the skeletal responses to calcium depletion/ repletion in young women may be similar to those in rodents. (06/1998) (link)
• Yoo A, Tanimoto H, Åkesson K, Baylink DJ, and Lau K-HW (1998) Effects of calcium depletion and repletion on serum insulin-like growth factor I and binding protein levels in weanling rats. Bone 22 (3), 225-232 Previous studies in a weanling rat model indicated that dietary calcium depletion not only stimulated osteoclastic resorption but also inhibited bone formation. The present study sought to test whether the depletion-associated inhibition of bone formation is related to a reduction in serum insulin-like growth factor-I (IGF-I) and/or an increase in its binding proteins (IGFBPs). Twenty male weanling rats were divided into two weight-matched groups. The study group was subjected to a semisynthetic diet deficient in calcium (0.02% calcium) for 28 days, while the control group was pair-weighed on the same diet but containing 0.62% calcium. After the depletion phase, all rats were fed the same calcium-containing diet for an additional 14 days. Serum samples were obtained from each animal on a weekly basis and assayed for IGF-I and IGFBPs. During depletion, there was no statistically significant difference in serum IGF-I level between the study group and the control group. In contrast, the study group showed a statistically significant increase in several serum IGFBPs with apparent molecular size of 30-38 kD (IGFBP-3), 26-28 kD (IGFBP-1, -2, -5, and/or -6), and 24-25 kD (IGFBP-4), respectively, compared to the control group. There was no difference in nutritional intakes between the two groups of rats during depletion. During repletion, there was also no significant difference in serum IGF-I level between the control and study group. However, during the first 7 days of repletion, serum IGFBP-3 and the 26-28 kD IGFBP of the study group was significantly less than those of the control group, which then returned to the control level after 2 weeks of repletion. In summary: (1) calcium depletion in weanling rats increased several serum IGFBPs without an effect on IGF-I; and (2) calcium repletion induced an acute reduction in serum IGFBP-3. In conclusion, these findings represent the first evidence that the depletion-related inhibition of bone formation in the rat may be associated with an increase in several serum IGFBPs, which may act to inhibit the osteogenic actions of IGFs. (03/1998) (link)
• Lau K-HW and Baylink DJ (1998) Pros and cons of fluoride therapy. Osteologie 7 (3), 157-163. We have developed a set of strategies to improve the advantage-todisadvantage of fluoride therapy. The first principle of the strategy we propose is to avoid a high fluoride deposition in bone. We think this Congress has demonstrated this, and it is very important for us to acknowldege this and find ways to reduce the amount of fluoride deposited in bone. we can achieve this by keeping the blood level as low as possible while still achieving a mitogenic action. We should use a slow release preparation because, if we do that, we can eliminate the blood fluoride peak value that is unnecessary for the mitogenic action of fluoride. In addition, we should use intermittent (cyclic) therapy to reduce further the total amount of fluoride administered without impairing the mitogenic effect. Secondary, we need to avoid calcium deficiency which can occur, particularly in the elderly who already have impaired calcium absorption. Therefore, calcium deficiency should be carefully monitored, and this is even more important in Europe than in Southern California, because vitamin D deficiency in the elderly is very frequent in Europe. However, even if the patient had a normal vitamin D level like the patients in Southern California, they can still have calcium malabsorption that is correctable only with active vitamin D metabolites. Finally, we propose to add an antiresorptive therapy to reduce the high bone resorption rate down to the premenopausal level.  If we apply this strategy, we believe it will increase bone density without increasing the fluoride concentration in the bone up to a level that will impair bone quality. If successful, this approach will lead to an increase in bone density and, at the same time, preserve the biomechanical competence of the bone, whic together should decrease the fracture rate. That this is a realistic option is demonstrated by the data which are summarized in this report. Consequently, it looks as though, if we follow the aforementioned suggestions, that we may be able to accentuate the mitogenic action of fluoride and reduce the adverse effect of fluoride on bone biomechanical quality. (03/1998)
• Wu L-W, Yoon HK, Baylink DJ, Graves LM, and Lau K-HW (1997) Fluoride at mitogenic doses induces a sustained activation of p44mapk, but not p42mapk, in human TE85 osteosarcoma cells. Journal of Clinical Endocrinology and Metabolism 82 (4), 1126-1135 Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 mumol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, with the maximal increase (4- to 13-fold) after 1-3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44mapk was dose dependent, with the optimal dose being 100 mumol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44mapk, but not that of p42mapk, in a time-dependent manner similar to the effect on the p44mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity. (04/1997) (link)
• Åkesson K, Lau K-HW, and Baylink DJ (1997) Rationale for active vitamin D analogue therapy in senile osteoporosis. Calcified Tissue International 60 (1), 100-105 (01/1997) (link)
• Nakade O, Baylink DJ, and Lau K-HW (1996) Osteogenic actions of phenytoin in human bone cells are mediated in part by TGF-beta 1. Journal of Bone and Mineral Research 11 (12), 1880-1888 We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes. Treatment with 5-50 microM phenytoin reproducibly and markedly increased (up to 6-fold, p < 0.001) the mRNA of transforming growth factor (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II, platelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and IGF binding proteins (i.e., IGFBP-3, -4, and -5). The stimulation was dose dependent, with an optimal dose of 10-50 microM. Maximal increase was seen after 1 h of phenytoin treatment. The release of biologically active TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenytoin treatment significantly increased the production of biologically active TGF-beta (2-fold, p < 0.05) with the optimal dose between 5-50 microM. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respective optimal doses had similar maximal stimulatory effects on [3H]thymidine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells. The stimulatory effects of phenytoin on [3H]thymidine incorporation and ALP-specific activity were completely blocked by a neutralizing anti-TGF-beta antibody. In conclusion, these findings demonstrate for the first time that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1. (12/1996) (link)
• Lau K-HW, Song XD, Ochi M, and Wergedal JE (1996) Mitogenic action of hydrochlorothiazide on human osteoblasts in vitro: requirement for platelet-derived growth factor. Calcified Tissue International 59 (6), 505-510 Long-term use of hydrochlorothiazide (HCTZ), a common diuretic agent for hypertension, has been associated with increased bone density and reduced hip fracture rates in patients. In this study, we sought to examine whether HCTZ has an anabolic effect on the proliferation of human osteoblasts (derived from either vertebrate or rib bone samples) in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell number counting. In medium supplemented with 1% bovine calf serum, HCTZ significantly and reproducibly increased [3H]thymidine incorporation and cell number. The stimulatory effect was dose dependent in a biphasic manner, with the maximal stimulation (approximately 60% above control, P < 0.001) seen at 1 microM HCTZ. In fresh serum-free medium, HCTZ was ineffective as a bone cell mitogen, indicating that the bone cell mitogenic activity of HCTZ required a serum growth factor (GF). HCTZ at doses greater than 10 microM was inhibitory in the presence or the absence of serum, presumably because of the cytotoxic effects. The serum requirement for the bone cell mitogenic activity of HCTZ could be replaced with a conditioned medium (conditioned with normal human osteoblasts for 24 hours), or with a mitogenic dose (1 ng/ml) of PDGF. The GF requirement was specific for PDGF, because other bone cell-derived growth factors (i.e., TGFbeta, IGF-I, IGF-II, and bFGF) were unable to replace serum for the bone cell mitogenic activity of HCTZ. In summary, this study shows that (1) HCTZ stimulated the proliferation of normal, untransformed, human osteoblasts in vitro; (2) the bone cell mitogenic effect of HCTZ required the presence of a serum GF; (3) the serum requirement could be replaced with a bone cell GF in conditioned medium; (4) the GF requirement was specific for PDGF. In conclusion, we have demonstrated for the first time that HCTZ has a direct anabolic effect on human osteoblasts in vitro, and that the mitogenic activity is dependent on the presence of PDGF. Because increased bone cell proliferation is a key determinant of bone formation, these observations raise the interesting possibility that HCTZ could act directly on bone cells to stimulate bone formation in patients. (12/1996) (link)
• Thomas AB, Hashimoto H, Baylink DJ, and Lau K-HW (1996) Fluoride at mitogenic concentrations increases the steady state tyrosyl phosphorylation level of cellular proteins in human bone cells. Journal of Clinical Endocrinolory and Metabolism 81 (7), 2570-2578 This study was designed to test the hypothesis that treatment of human bone cells with mitogenic concentrations of fluoride would lead to an increase in the steady state level of tyrosyl phosphorylation of specific cellular proteins. With an immunoblot assay method, it was found that mitogenic concentrations of fluoride (i.e. 50-200 mumol/L) induced a dose- and time-dependent increase in the level of tyrosyl phosphorylation of at least 13 cellular proteins in both normal human bone cells and human TE85 osteosarcoma cells. Time-course studies revealed that a statistically significant increase in tyrosyl phosphorylation of these 13 cellular proteins in human bone cells was observed after 3-6 h of fluoride treatment and was sustained for up to 24 h. This time course was not compatible with a direct activation of tyrosyl kinases, as epidermal growth factor, which activates tyrosyl kinase activity, induced an immediate and acute response that was rapidly reversible within 1 h. Although fluoride increased the steady state tyrosyl phosphorylation of the cellular proteins in human bone cells, the same micromolar doses of fluoride had no effect on human skin fibroblasts, which are fluoride-nonresponsive cells. The effects of fluoride were rapidly reversible in the absence of fluoride and could be acutely potentiated by pretreatment with epidermal growth factor. In summary, we have shown for the first time that mitogenic concentrations (i.e. 50-200 mumol/L) of fluoride increased the steady state level of tyrosyl phosphorylation of at least 13 cellular proteins in human bone cells, and that the increases were relatively show in onset and sustained. In conclusion, these findings are consistent with the hypothesis that the osteogenic actions of fluoride are mediated at least in part by an inhibition of the activity of one or more fluoride-sensitive phosphotyrosyl protein phosphatases in human bone cells. (07/1996) (link)
• Wu L-W, Baylink DJ, and Lau K-HW (1996) Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase. Biochemical Journal 316 (Pt 2), 515-523 Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single PTP catalytic domain. The PTP catalytic domain shows 45-50% sequence identity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in osteoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclusion, PTP-oc represents a structurally unique subfamily of transmembrane PTPs. (06/1996) (link)
• Takada J, Chevalley T, Baylink DJ, and Lau K-HW (1996) Dexamethasone enhances the osteogen­ic effects of fluoride in human TE85 osteosarcoma cells in vitro. Calcified Tissue International 58 (5), 355-361 The in vitro osteogenic effects of fluoride have not always been consistently observed in human bone cells. The present study sought to test if dexamethasone (Dex) could potentiate the action of fluoride to increase the detectability of the stimulatory effects of fluoride on [3H]thymidine incorporation, alkaline phosphatase (ALP) specific activity, collagen synthesis, and osteocalcin secretion in human TE85 osteosarcoma cells. Neither Dex at 10(-10)-10(-6) M or fluoride at a mitogenic dose (100 microM) had any consistent stimulatory effects on thymidine incorporation. When the cells were treated with both agents simultaneously, significant and highly reproducible stimulations were observed. The mitogenic effects of the two agents were confirmed with cell number counting. Analysis of variance (ANOVA) revealed a significant interaction (P < 0.001) between fluoride and Dex on cell proliferation. The enhancing effect of Dex on [3H]thymidine incorporation was not due to a shift of the optimal dose response of fluoride. Though fluoride alone or Dex alone also had no consistent effect on ALP specific activity, the co-treatment with fluoride and Dex for 24 hours produced significant (P < 0.001, ANOVA) stimulation in ALP specific activity. Fluoride alone had no consistent effect on collagen synthesis and on 1, 25(OH)2D3-dependent osteocalcin secretion, whereas Dex treatment consistently inhibited these two osteoblastic parameters in a dose-dependent manner. However, both the collagen synthesis and osteocalcin secretion rates were significantly higher (P < 0.001 ANOVA for each) when the cells were co-treated with Dex and fluoride (100 microM) than when they were treated with Dex alone. Thus, these data indicate that the response in collagen synthesis and osteocalcin secretion to fluoride stimulation was more readily observed in the presence of Dex than in its absence. ANOVA analysis revealed that the interaction between fluoride and Dex on collagen synthesis, but not the 1,25(OH)2D3-dependent osteocalcin secretion, was significant (P < 0.02). In summary, we have demonstrated for the first time that in TE85 cells (1) Dex potentiated the effects of fluoride on cell proliferation, ALP specific activity, and collagen synthesis; (2) while Dex at 10(-7)-10(-6) M alone inhibited the collagen synthesis and at 10(-9)-10(-6) M reduced osteocalcin secretion, Dex at 10(-8)-10(-6) M significantly stimulated the proliferation of TE85 cells; and (3) Dex interacted with fluoride to increase the percentage of experiments showing an osteogenic action of fluoride. In conclusion, the in vitro osteogenic actions of fluoride in human TE85 cells are more consistently observed in the presence than in the absence of Dex. (05/1996) (link)
• Takada J, Baylink DJ, and Lau K-HW (1995) Pretreatment with low doses of norethindrone potentiates the osteogenic effects of fluoride on human osteosarcoma cells. Journal of Bone and Mineral Research 10 (10), 1512-1522 We recently reported that picomolar doses of norethindrone (NET), a synthetic analog of 19-nortestosterone, significantly stimulated human TE85 osteosarcoma cell proliferation, differentiation, and activity in vitro. In the present study, we investigated the possibility that NET interacts with another osteogenic agent, i.e., fluoride, to stimulate human TE85 osteosarcoma cell proliferation, differentiation, and activities. Bone cell proliferation was measured by the stimulation in [3H]thymidine incorporation. Differentiation was monitored by the increase in alkaline phosphatase-specific activity. Osteoblastic activity was assessed by the stimulations in collagen synthesis and in osteocalcin secretion (in the presence of 1 nM 1,25-dihydroxyvitamin D3). When the human TE85 cells were incubated with mitogenic doses of NET and fluoride concurrently, the stimulatory effects of the two agents on these parameters exhibited no significant interaction. The enhancing effect of NET on the osteogenic effect of fluoride was not due to a shift of the fluoride dose response curve. Pretreatment with NET for 24 h followed by a treatment with a mitogenic dose (i.e., 100 microM) of fluoride for an additional 24 h significantly and synergistically potentiated the effects of fluoride on the [3H]thymidine incorporation, alkaline phosphatase-specific activity, collagen synthesis, and osteocalcin secretion, compared with those with the subsequent vehicle (0.05% ethanol) treatments. In contrast, pretreatment with fluoride for 24 h before the addition of NET for 24 h did not produce significant synergistic stimulations in the test parameters. Pretreatment of TE85 cells with the same doses of dihydrotestosterone or progesterone prior to treatment with fluoride under the same conditions did not induce synergistic potentiation of fluoride in [3H]thymidine incorporation, suggesting that the synergistic interaction with fluoride is probably not a common property of anabolic sex steroids. In summary, we found that: (1) the osteogenic effects of fluoride and NET were additive when cells were treated with both agents concurrently; (2) a 24-h pretreatment with picomolar doses of NET potentiated the osteogenic actions of fluoride in human TE85 osteosarcoma cells; and (3) pretreatment with NET produced a subsequent fluoride response that was synergistic. In conclusion, these findings led us to speculate that the osteogenic actions of NET and fluoride act through different mechanisms, and that NET at low doses has a permissive effect on the osteogenic effects of fluoride, and as such NET may be used in concert with fluoride to increase osteoblast proliferation, differentiation, and activity. (10/1995) (link)
• Lau K-HW, Nakade O, Barr B, Taylor AK, Houchin K, and Baylink DJ (1995) Phenytoin increases markers of osteogenesis for the human species in vitro and in vivo. Journal of Clinical Endocrinology and Metabolism 80 (8), 2347-2353 Phenytoin therapy is a well recognized cause of gingival hyperplasia, a condition characterized by increased gingival collagen synthesis, and may also cause acromegalic-like facial features. Based on these clinical findings suggestive of anabolic actions, we sought to test the hypothesis that phenytoin acts on normal bone cells to induce osteogenic effects. To test the direct actions of phenytoin on human bone cells, we measured the dose responses to phenytoin for [3H]thymidine incorporation, cell number, alkaline phosphatase specific activity, and collagen synthesis in human hip bone-derived cells. Phenytoin significantly and reproducibly increased [3H]thymidine incorporation, cell number, alkaline phosphatase specific activity, and collagen synthesis in a biphasic manner with optimal stimulatory doses between 5-10 mumol/L. Thus, micromolar concentrations of phenytoin can act directly on human bone cells to stimulate osteoblast proliferation and differentiation. We next sought to test the hypothesis that phenytoin stimulates bone formation in humans in vivo. Accordingly, three serum biochemical markers of bone formation, i.e. osteocalcin, skeletal alkaline phosphatase, and procollagen C-terminal extension peptide, were measured in 39 male epileptic patients, 20-60 yr of age, with an average duration of phenytoin therapy of 10.5 +/- 1.62 yr (mean +/- SEM). In this group of patients, the mean serum phenytoin level was 9.56 +/- 0.90 mg/L (mean +/- SEM; equivalent to 34.9 +/- 3.3 mumol/L). Thirty apparently healthy male subjects of similar age and taking no medication were included as controls. Serum calcium, 25-hydroxyvitamin D3, and PTH levels in the phenytoin-treated patients were not significantly different from those in the age-matched controls and were within the clinical laboratory normal range of our hospitals, indicating that the patients did not develop hypocalcemia, vitamin D deficiency, or secondary hyperparathyroidism. Serum levels of osteocalcin, skeletal alkaline phosphatase, and procollagen peptide in the phenytoin-treated patients were significantly increased compared to those in the age-matched subjects; in each case these biochemical markers were significantly correlated with the serum phenytoin level, but not with the dose or duration of phenytoin treatment. These findings are consistent with the interpretation that phenytoin increases the bone formation rate in humans in vivo.(ABSTRACT TRUNCATED AT 400 WORDS) (08/1995) (link)
• Ohta T, Wergedal JE, Matsuyama T, Baylink DJ, and Lau K-HW (1995) Phenytoin and fluoride act in concert to stimulate bone formation and to increase bone volume in adult rats. Calcified Tissue International 56 (5), 390-397 We have recently demonstrated that phenytoin is an osteogenic agent at low doses. The present paper describes observations that a mitogenic dose (i.e., 20 microM in BGJb medium) of fluoride significantly augments the phenytoin-dependent stimulation of normal human bone cell proliferation and alkaline phosphatase (ALP) activity in cell culture. Additionally, the present study was designed to investigate whether fluoride and phenytoin would interact to increase bone formation in rats in vivo. Four groups of weight-matched adult male rats received daily I.P. injection of (1) vehicle (10% DMSO), (2) 5 mg/kg/day phenytoin, (3) 5 mg/kg/day phenytoin and 50 ppm NaF, and (4) 50 ppm NaF and vehicle, respectively, for 36 days. Sodium fluoride (NaF) was delivered in drinking water. Blood samples were drawn weekly and analyzed for serum osteocalcin, ALP, calcium, phosphorus, and 25(OH)D3. Rats were labeled with tetracycline at day 21 and 30 and histomorphometric analysis was carried out on the tibia at the end of the experiment. Neither agent by itself or together affected the serum calcium, phosphorus, or 25(OH)D3 levels. All measures of bone formation, i.e., serum osteocalcin level and ALP activity, bone ALP specific activity, mineral apposition rate, bone formation rate, and % bone formation surface, were increased by each agent. Fluoride and phenytoin together produced bigger increases in each parameter than did each agent alone. Trabecular bone volume was increased in the tibial metaphysis by fluoride or phenytoin alone; and when administered together, the two agents produced a greater increase.(ABSTRACT TRUNCATED AT 250 WORDS) (05/1995) (link)
• Schwender CF, Beers SA, Malloy E, Demarest K, Minor L, and Lau K-HW (1995) 1-Naphthylmethylphosphonic acid derivatives as osteoclastic acid phosphatase inhibitors. Bioorganic and Medicinal Chemistry Letters 5 (16), 1801-1806 Inhibition of the enzyme, osteoclastic acid phosphatase (OAP) may be a viable approach to the treatment of osteoporosis. A series of arylmethylphosphonic acids were synthesized and shown to be inhibitors of OAP. The most potent inhibitor, bis-benzoyl-1-naphthylmethylphosponic acid (7a) had an IC50 = 1.4 µM. (05/1995) (link)
• Thomas AB, Hall SL, Nicolas V, Lau K-HW, and Farley JR (1995) Calcitonin has acute effects to increase tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells. Calcified Tissue International 56 (4), 268-273 In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10(-13) to 10(-9) mol/liter, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with 125I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and 125I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the level of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses--time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.05 for each).(ABSTRACT TRUNCATED AT 250 WORDS) (04/1995) (link)
• Lau KHW, Baylink DJ, and Sheng MHC (2015) Osteocyte-derived Insulin-like growth factor I is not essential for the bone repletion response in mice. PLoS One 10 (1): e0115897. The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both Igf1 osteocyte conditional knockout (cKO) mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by the reduction in osteoclast number, indicating that deficient Igf1 expression in osteocytes also did not have deleterious effects on the bone repletion response. The lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected since previous studies show that these Igf1 osteocyte cKO mice exhibited impaired developmental growth and displayed complete resistance to bone anabolic effects of loading. These studies suggest that there is a dichotomy between the mechanisms necessary for anabolic responses to mechanical loading and the regulatory hormonal and anabolic skeletal repletion following low dietary calcium challenge. In conclusion, to our knowledge this study has demonstrated for the first time that osteocyte-derived IGF-I, which is essential for anabolic bone response to mechanical loading, is not a key regulatory factor for bone repletion after a low calcium challenge.Bone has two major skeletal anabolic functions apart from fracture healing: 1) bone formation response to mechanical loading and 2) repletion of bone mass after a period of mineral deficiency. We recently reported that mice with conditional knockout (cKO) of Igf1 in osteocytes displayed complete resistance to bone anabolic effects of loading. The present study sought to determine whether deficient expression of Igf1 in osteocytes would also impair the bone repletion response to two-week of dietary calcium restriction followed by one-week of dietary calcium repletion. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both the cKO mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by a sharp reduction in osteoclast number, indicating that deficient Igf1 expression in osteocytes also did not have deleterious effects on the bone repletion response. Because deficient osteocyte-derived IGF-I expression completely abolished the bone formation response to mechanical loading, the lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected. It is possible that while osteocyte-derived IGF-I is a key growth factor involving in local bone regeneration, this osteocytic growth factor may only have limited functional endocrine regulation of bone turnover. In conclusion, this study has demonstrated for the first time that 1) there is a dichotomy between the mechanisms necessary for anabolic responses to mechanical loading and the regulatory hormonal and anabolic skeletal repletion following low dietary calcium challenge; and 2) osteocyte-derived IGF-I is essential for anabolic bone response to mechanical loading but not for bone repletion after a low calcium challenge. (01/1995) (link)
• Nakade O, Baylink DJ, and Lau K-HW (1995) Phenytoin at micromolar concentrations is an osteogenic agent for human mandible-derived bone cells in vitro. Journal of Dental Research 74 (1), 331-337 The present study sought to test the hypothesis that phenytoin acts on normal human-mandible-derived bone cells to induce osteogenic effects. To test the effects of phenytoin on bone cell proliferation, we measured [3H]-thymidine incorporation into cell DNA during the final four hr of a 24-hour incubation with phenytoin. Phenytoin at micromolar concentrations significantly stimulated the [3H]-thymidine incorporation in a dose-dependent, biphasic, manner with a maximal effect at from 10 to 50 microM. We confirmed the proliferative effect of phenytoin by counting cell number. To evaluate the effects of phenytoin on osteoblastic differentiation, we determined alkaline-phosphatase specific activity and found that phenytoin at micromolar concentrations significantly increased that activity in a dose-dependent manner, with maximal stimulation at approximately 1 microM. To investigate the effects of phenytoin on mature osteoblastic activities, we measured de novo collagen synthesis and osteocalcin secretion. Mitogenic doses of phenytoin significantly increased collagen synthesis and osteocalcin secretion in a dose-dependent, biphasic, manner, with the maximal stimulatory dose at from 5 to 10 microM. In summary, phenytoin at micromolar ranges increased (a) [3H]-thymidine incorporation and cell number, (b) alkaline-phosphatase specific activity, (c) collagen synthesis, and (d) osteocalcin secretion in monolayer cultures of normal human-mandible-derived bone cells. These observations are consistent with the premise that low doses of phenytoin act on human craniofacial bone cells to stimulate cell proliferation, differentiation, and mature osteoblastic activities to stimulate bone formation. (01/1995) (link)
• Ohta T, Wergedal JE, Gruber HE, Baylink DJ, and Lau K-HW (1995) Low dose phenytoin is an osteogenic agent in the rat. Calcified Tissue International 56 (1), 42-48 Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)2D3 levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D3 levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin bone ALP, periosteal MAR, and trabecular bone volume.(ABSTRACT TRUNCATED AT 250 WORDS) (01/1995) (link)
• Wang SP, Demarest KT, Gunnet JW, Baylink DJ, and Lau K-HW (1994) Develop­ment of a heterologous radioimmunoassay for canine osteocalcin. Calcified Tissue International 55 (2), 134-140. The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (> 1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin cross-reacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The inter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA. Application of the heterologous assay to dogs of different age revealed that yound dogs (3 months old) had 15-fold higher serum osteocalcin level than adult (>2 years old) dogs. In summary, we have (1) purified dog osteocalcin; (2) produced an antiserum against it: and (3) developed a heterologous RIA that could accurately measure dog osteocalcin cin, and could be used routinely to measure dog osteocalcin. (08/1994) (link)
• Lau K-HW, Wang SP, Linkhart TA, Demarest KT, and Baylink DJ (1994) Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line, and collagen synthesis without an effect on bone resorption. Journal of Bone and Mineral Research 9 (5), 695-703. To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10(-6)-10(-12) M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions. In conclusion, these in vitro osteogenic activities of NET suggest that it may be a useful therapeutic agent to increase bone formation in patients with low bone mass. (05/1994) (link)
• Lau K-HW, Utrapiromsuk S, Yoo A, Mohan S, Strong DD, and Baylink DJ (1993) Mechanism of mitogenic action of aluminum ion on human bone cells: potential involvement of the insulin-like growth factor regulatory system. Archives of Biochemistry and Biophysics 303 (2), 267-273. Aluminum ion at micromolar concentrations significantly stimulated the [3H]thymidine incorporation into human TE85 osteosarcoma cell DNA. Cells treated with mitogenic concentrations of aluminum ion for 48 h showed biphasic stimulation in secretion of IGFs (insulin-like growth factors) into the conditioned medium. Treatment of the human osteosarcoma TE85 cells with mitogenic doses of aluminum ion for 24 h also markedly and reproducibly increased the steady-state level of IGF-II mRNA in a dose-dependent, biphasic manner. The effect of aluminum ion on the steady-state level of IGF-I mRNA could not be determined since the IGF-I mRNA in these cells was not detectable with our oligodeoxynucleotide probes. To test whether the mitogenic effects of aluminum ion could be mediated through IGFs, the stimulation of [3H]thymidine incorporation of TE85 cells was evaluated in the presence and the absence of an inhibitory IGF binding protein (i.e., IGFBP-4). The presence of IGFBP-4 significantly reduced the stimulation in thymidine incorporation by a mitogenic concentration of aluminum ion. Western ligand blot analysis revealed that mitogenic concentrations of aluminum ion also inhibited the secretion of IGF-binding proteins, particularly the inhibitory IGFBP-4, which could lead to the potentiation of the overall activity of IGFs. In conclusion, these findings are consistent with the premise that the mitogenic action of aluminum ion on human bone cells is, in part, mediated by an increased local bone cell production and activity of IGFs. (06/1993) (link)
• Lau K-HW and Baylink DJ (1993) Phosphotyrosyl protein phosphatases: potential regulators of cell proliferation and differentiation. Critical Reviews in Oncogenesis 4 (4), 451-471. Tyrosyl phosphorylated proteins have been associated with the regulation of cellular activities, including proliferation, differentiation, and transformation. The overall cellular levels of tyrosyl phosphorylated proteins are regulated by the balance of the activities of both tyrosyl protein kinases and phosphotyrosyl protein phosphatases. Until recently, it was generally assumed that the kinase reaction was the key regulatory process for determining the level of tyrosyl phosphorylated proteins and that the dephosphorylation reaction was an unregulated event. However, recent evidence strongly suggest a regulatory role for phosphotyrosyl protein phosphatases for determination of the overall level of tyrosyl phosphorylated proteins. This review focuses on the discussion of the properties, the regulation, and the primary structure of phosphotyrosyl protein phosphatases. Evidence that supports an important role for phosphotyrosyl protein phosphatases on cell proliferation and differentiation also is presented. (04/1993)
• Lau K-HW, Thomas AB, Yoo A, Nguyen TSA, and Wergedal JE (1992) Conversion of skeletal tartrate-sensitive acid phosphatases into tartrate-resistant isoenzymes in vitro. International Journal of Biochemistry 24 (11), 1815-1824. 1. Chicken skeletal tartrate-sensitive (TsACP) and -resistant (TrACP) acid phosphatase isoenzymes could be separated from each other by carboxylmethyl-sepharose ion exchange chromatography. 2. Chicken skeletal TsACP showed a gradual time-dependent loss of sensitivity to tartrate inhibition when incubated at room temperature, but not at 4 degrees C. 3. The loss of sensitivity to tartrate inhibition was associated with an activation of the enzyme activity. 4. These changes were accompanied with a shift in the electrophoretic mobility of the enzyme activity from a large molecular sized form to a smaller molecular sized form that resembled the freshly prepared TrACP on the native acidic polyacrylamide electrophoresis gels, and on molecular sieve Superose-12 Fast Protein Liquid Chromatography. 5. Kinetic evaluations of the biochemical properties of the "converted" TsACP activity resembled the TrACP. 6. The apparent "conversion" was not unique to chicken TsACP, since similar "conversion" was observed with partially purified preparations of bovine bone matrix TsACP and of human osteoblastic TsACP. 7. Addition of several serine protease inhibitors did not prevent the "conversion". 8. These findings are consistent with the possibility that skeletal TsACPs are precursors of skeletal TrACPs. (11/1992) (link)
• Finkelman RD, Lau K-HW, Abraham SM, and Baylink DJ (1992) Evidence for lack of receptors for nerve growth factor in bone cells. Molecular and Cellular Biochemistry 115 (2), 129-136. Nerve growth factor (NGF) is essential for the development and differentiation of sympathetic and sensory neurons. Recently, NGF receptors were demonstrated in non-neural cells, and several mesenchymal cell types including lymphocytes and skeletal myotubes were shown to be stimulated to proliferate by NGF. Our purpose was to examine for the presence of functional NGF receptors in osteoblasts. Bone cells from chick calvaria were used as a model; PC-12 cells derived from rat adrenal pheochromocytoma were used as positive controls. NGF was examined for functions in chick bone cells by studying effects on (1) [3H]-thymidine incorporation; (2) alkaline phosphatase (ALP) activity; and (3) protein tyrosine phosphorylation. Effects of NGF on thymidine incorporation and protein tyrosine phosphorylation by PC-12 cells were also measured. A radioreceptor assay was used to test for the presence of receptors. In chick calvarial cells, NGF had no effect on thymidine incorporation, ALP activity or protein tyrosine phosphorylation. Radioreceptor assay with bone cells showed no evidence of NGF receptors. In contrast, in PC-12 cells, NGF (1) decreased thymidine incorporation; (2) increased protein tyrosine phosphorylation; and (3) showed receptor activity by radioreceptor assay. In conclusion, unlike several other mesenchymal cell types, chick bone cells show no evidence of NGF receptors or functional responses to NGF in vitro. (10/1992) (link)
• Wergedal JE and Lau K-HW (1992) Human bone cells contain a fluoride sensitive acid phosphatase. Evidence that this enzyme functions at neutral pH as a phosphotyro­syl protein phosphatase. Clinical Biochemistry 25 (1), 47-53. Fluoride is a potent therapeutic agent that increases spinal bone density in osteoporotic subjects. Based on work with animal cells previously, we proposed fluoride acts by inhibiting phosphotyrosyl protein phosphatase (EC 3.1.3.48) activity in bone cells. The presence of fluoride sensitive acid phosphatase (EC 3.1.3.2) activity was characterized in extracts of cultured human bone cells. Crude extracts contained acid phosphatase activity that was inhibited by fluoride with an apparent Ki of 12 mumol/L. The activity was investigated further by separating the acid phosphatase isoenzymes using CM Sepharose chromatography and a gradient of acetate pH 4.8-6.5. The major peak of activity recovered from CM Sepharose chromatography was characterized for stability, Km and inhibition by fluoride. The enzyme was sensitive to inhibition by tartrate, had a high affinity for paranitrophenylphosphate (apparent Km = 0.158 mupmol/L) and an apparent pH optimum of 4.8. Fluoride was a strong competitive inhibitor with an apparent Ki of 12.4 mumol/L. The column fractions containing the acid phosphatase were tested further for phosphotyrosyl protein phosphatase activity using [32P]labeled phosphotyrosyl histone as the substrate. Release of [32P]phosphate from this substrate at pH 7.0 was proportional to enzyme concentration and incubation time, demonstrating the presence of phosphotyrosyl protein phosphatase activity. The phosphotyrosyl protein phosphatase activity was inhibited by fluoride and had a pH optimum of approximately 7. These observations indicate that human osteoblasts contain a fluoride-sensitive phosphotyrosyl protein phosphatase. Thus, these results are consistent with the hypothesis that fluoride stimulates human bone cell proliferation by inhibiting the action of phosphotyrosyl protein phosphatase, thereby increasing the level of phosphorylated tyrosine residues which are known to play a role in increasing cell proliferation. (02/1992) (link)
• Kyeyune-Nyombi E, Lau K-HW, Baylink DJ, and Strong DD (1991) 1,25-dihydroxyvitamin D3 stimulates both alkaline phosphatase gene transcription and mRNA stability in human bone cells. Archives of Biochemistry and Biophysics 291 (2), 316-325. We have previously reported that 1,25(OH)2D3 stimulated the cellular alkaline phosphatase (ALP) activity and increased the steady-state level of ALP mRNA in a human osteosarcoma cell line (TE-85), under serum-free conditions. To define the molecular mechanism by which 1,25(OH)2D3 acts to stimulate ALP activity, the time courses of the increases in ALP activity and in the steady-state ALP mRNA level in response to 1,25(OH)2D3 were evaluated. 1,25(OH)2D3 progressively increased the steady-state level of ALP mRNA from 5 to 24 h of treatment, at which time a plateau was observed. In contrast, no significant increase in ALP-specific activity was detected until after 10 h of treatment, at which time the activity increased linearly with time up to 72 h. These time courses are consistent with the premise that the increased ALP activity was the result of increased gene expression. Nuclear runoff analysis indicated that the transcription rate of the ALP gene was more than five-fold higher in the 1,25(OH)2D3-treated cells than in the control cells. In addition, it was found that 1,25(OH)2D3 treatment increased ALP mRNA stability. The 1,25(OH)2D3-induced increase in ALP mRNA stability was not due to an interaction of the 1,25(OH)2D3-receptor complex with the ALP mRNA, since the removal of 1,25(OH)2D3 did not abolish its stabilizing effect. In the presence of cycloheximide, the stabilizing effect of 1,25(OH)2D3 was abolished, suggesting that a 1,25(OH)2D3-inducible protein factor was involved. Based on these findings, we have proposed a model in which 1,25(OH)2D3 stimulated ALP activity in human bone cells through mechanisms involving both (1) increased transcription of the ALP gene and (2) increased stability of ALP mRNA, an effect which requires the de novo synthesis of a protein, a putative ALP mRNA "stabilizing factor." (12/1991) (link)
• Lau K-HW, Stepan JJ, Yoo A, Mohan S, and Baylink DJ (1991) Evidence that tar­trate-resistant acid phosphatases from osteoclastomas and hairy cell leukemia spleen are members of a multigene family. International Journal of Biochemistry 23 (11), 1237-1244. 1. Osteoclasts and hairy cell leukemia spleen both contain large amounts of a band 5-tartrate-resistant acid phosphatase (TrACP). 2. We have recently purified to homogeneity a band 5 TrACP from human osteoclastomas and two isoforms of band 5 TrACP (5a and 5b) from the spleen of a patient with hairy cell leukemia. 3. Although the N-terminal amino acid sequences and the apparent molecular weights of the osteoclastoma, hairy cell leukemia spleen TrACPs were identical, there were several differences in the physical and biochemical properties between the three isoenzymes. 4. Based on these findings, it is concluded that these isoenzymes are different enzymes, but that they could have originated from a similar ancestral gene. 5. It is proposed that the osteoclastoma and hairy cell leukemia band 5 TrACPs are members of a multigene family. (11/1991)
• Lau K-HW, Yoo A, and Wang SP (1991) Aluminum stimulates the proliferation and differentiation of osteoblasts in vitro by a mechanism that is different from fluoride. Molecular and Cellular Biochemistry 105 (2), 93-105. Micromolar concentrations of aluminum sulfate consistently stimulated [3H]thymidine incorporation into DNA and increased cellular alkaline phosphatase activity (an osteoblastic differentiation marker) in osteoblast-line cells of chicken and human. The stimulations were highly reproducible, and were biphasic and dose-dependent with the maximal stimulatory dose varied from experiment to experiment. The mitogenic doses of aluminum ion also stimulated collagen synthesis in cultured human osteosarcoma TE-85 cells, suggesting that aluminum ion might stimulate bone formation in vitro. The effects of mitogenic doses of aluminum ion on basal osteocalcin secretion by normal human osteoblasts could not be determined since there was little, if any, basal secretion of osteocalcin by these cells. 1,25 Dihydroxyvitamin D3 significantly stimulated the secretion of osteocalcin and the specific activity of cellular alkaline phosphatase in the human osteoblasts. Although mitogenic concentrations of aluminum ion potentiated the 1,25 dihydroxyvitamin D3-dependent stimulation of osteocalcin secretion, they significantly inhibited the hormone-mediated activation of cellular alkaline phosphatase activity. Mitogenic concentrations of aluminum ion did not stimulate cAMP production in human osteosarcoma TE 85 cells, indicating that the mechanism of aluminum ion does not involve cAMP. The mitogenic activity of aluminum ion is different from that of fluoride because (a) unlike fluoride, its mitogenic activity was unaffected by culture medium changes; (b) unlike fluoride, its mitogenic activity was nonspecific for bone cells; and (c) aluminum ion interacted with fluoride on the stimulation of the proliferation of osteoblastic-line cells, and did not share the same rate-limiting step(s) as that of fluoride. PTH interacted with and potentiated the bone cell mitogenic activity of aluminum ion, and thereby is consistent with the possibility that the in vivo osteogenic actions of aluminum ion might depend on PTH. In summary, low concentrations of aluminum ion could act directly on osteoblasts to stimulate their proliferation and differentiation by a mechanism that is different from fluoride. (07/1991) (link)
• Finkelman RD, Linkhart TA, Mohan S, Lau K-HW, Baylink DJ, and BellNH (1991) Vitamin D deficiency causes a selective reduction in the deposition of transforming growth factor b in rat bone: possible mechanism for impaired osteoinduction. Proceedings of National Academcy of Sciences USA 88 (9), 3657-3660. We demonstrated previously that implants of bone matrix prepared from vitamin D-deficient (-D) rats were less osteoinductive and contained less extractable mitogenic activity compared with control implants prepared from vitamin D-replete (+D) rats and proposed that bone from -D rats is deficient in one or more specific growth factors. To test this hypothesis, bones from rats that were fed either +D or -D diets and kept in the dark for 8 wk were extracted and assayed for insulin-like growth factors I and II (IGF-I and IGF-II) and transforming growth factor beta (TGF-beta), the three most abundant growth factors in rat bone, and osteocalcin. Serum calcium, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were determined at sacrifice. In -D rats, there were significant reductions in serum calcium, 25-hydroxyvitamin D3, and 1,25(OH)2D3 and skeletal TGF-beta but no differences in extractable skeletal protein, IGF-I, IGF-II, or osteocalcin compared with +D rats. To determine whether 1,25(OH)2D3 increased TGF-beta production by bone cells, we treated mouse calvaria for 6 days and mouse osteoblasts for 2 days with 10 nM 1,25(OH)2D3. Production of TGF-beta was increased almost 100% by 1,25(OH)2D3. We conclude that vitamin D deficiency reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could contribute to the previously observed decrease in osteoinduction in implants from -D rat bone. The findings support the possibility that vitamin D and bone-derived TGF-beta are required for normal repair of the skeleton. (05/1991)
• Tanimoto H, Lau K-HW, NishimotoSK, Wergedal JE, and Baylink DJ (1991) Evaluation of the usefulness of serum phosphatases and osteocalcin as serum bone markers using a calcium-depletion and -repletion rat model. Calcified Tissue International 48 (2), 101-110. The present study examined the usefulness of the serum alkaline phosphatase (ALP) activity, osteocalcin, and tartrate-resistant acid phosphatase (TrACP) activity as bone turnover markers in a calcium depletion/repletion rat model. Weanling rats were fed calcium-deficient diet for 4 weeks, followed by 2 weeks of dietary calcium repletion. Serum phosphatases and osteocalcin were determined and compared with those of corresponding age-matched, pair-weighted controls. Rats were sacrificed at the end of each phase of the study, and bone phosphatase activities in tibiae and vertebrae were measured. During calcium depletion, rats developed hypocalcemia and lost significant bone calcium, which were reversed with dietary calcium repletion. During depletion when previously published histologic studies indicated a suppressed bone formation and stimulated bone resorption, serum ALP activity and osteocalcin levels were significantly elevated and serum TrACP activity reduced; at the same time, the bone ALP and TrACP activities were increased. Because the serum level of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) which has been shown to stimulate the synthesis of skeletal ALP and of osteocalcin, was also significantly increased during depletion, the increased serum ALP and osteocalcin level could be indirect consequences of the hypocalcemia-mediated elevation in 1,25(OH)2D3 level. These effects were reversed upon calcium repletion, during which previously published histologic studies demonstrated a stimulated bone formation and a suppressed resorption in these rats. In conclusion, although there is increasing evidence for the usefulness of these serum proteins as markers of bone metabolism in humans, a great deal more work is required before we can understand the significance of these assays. Until such is accomplished, these assays should not be assumed to be validated. (02/1991) (link)
• Matsuyama T, Lau K-HW, and Wergedal JE (1990) Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells. Calcified Tissue International 47 (5), 276-283. Cytochemical staining of normal human bone cells in monolayer cultures for alkaline phosphatase (ALP) indicated that the cultures contained mixed-cell populations. Time course evaluations of the cytochemical staining revealed, in addition to the ALP-negative cell population, at least two subpopulations of ALP-positive human bone cells with different levels of ALP. A cytochemical method has been developed which separates the ALP-positive cells into high and intermediate ALP subpopulations. In this method, human bone cells were stained for ALP using an azo-dye method and incubating at 4 degrees C for 10 and 30 minutes, respectively. We defined the cell population that stained positively for ALP at 10 minutes as strong ALP-positive cells, and both strong and intermediate cells were stained at 30 minutes. The intermediate cells were determined from the difference between the values at the two time points. The intra- and interassay variations of the assay, with the same investigator in blinded investigations, were both less than 10% and the interobserver variation was approximately 25%. Analysis of the distribution of ALP levels in cells with a laser densitometer confirmed the presence of at least three cell subpopulations. 1,25(OH)2D3 treatment increased the proportions of both ALP-positive cell populations, whereas TGF-beta treatment increased only the intermediate ALP-positive cell population. On the contrary, fluoride increased the proportion of the strong ALP cells, and IGF-1 had no effect on the proportions of either ALP-positive subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS) (11/1990) (link)
• Kraenzlin ME, Lau K-HW, Liang L, Freeman TK, Singer FR, Stepan JJ, and Baylink DJ (1990) Development of an immunoassay for human serum osteoclastic tartrate-re­sistant acid phosphatase. Journal of Clinical Endocrinology and Metabolism 71 (2), 442-451. A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget''''s disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption. (08/1990)
• Pacifici R, Rothstein M, Rifas L, Lau K-HW, Baylink DJ, Avioli LV, and Hruska K (1990) Increased monocyte interleukin-1 secretion and decreased vertebral bone density in fasting idiopathic hypercalciuria. Journal of Clinical Endocrinology and Metabolism 71 (1), 138-145. Idiopathic hypercalciuria (IH) is a heterogeneous disorder frequently observed in patients with nephrolithiasis. At one extreme of its clinical spectrum is fasting hypercalciuria (FH), a condition characterized by increased bone resorption and turnover. In previous studies we have shown that monocytes from patients with high turnover osteoporosis and from women in early postmenopause elaborate increased amounts of interleukin-1 (IL-1), a cytokine that stimulates bone resorption in vitro and in vivo. Since IL-1 could also mediate the resorptive mechanism of FH and cause a clinically significant bone loss, we have studied the relationship of IH, vertebral mineral density, bone turnover, and monocyte IL-1 activity in 47 patients with absorptive hypercalciuria (AH), 23 with FH, and 38 nonhypercalciuric subjects with recurrent nephrolithiasis (controls). Vertebral mineral density, as measured by quantitative computer tomography, was decreased in each of the three patient groups, but was significantly lower in FH patients than in AH patients or control subjects. Twenty-four-hour total urinary hydroxyproline excretion was increased in FH patients compared to that in AH patients or controls, but blood levels of osteocalcin were not. Monocytes from FH subjects yielded significantly more IL-1 (alpha + beta) activity than those from AH patients or controls; levels of IL-1 activity in monocytes of AH and control patients were similar. In IH subjects, significant correlations were found between IL-1 and hydroxyproline (r = 0.70; P less than 0.0001), IL-1 and quantitative computer tomography values (r = -0.49; P less than 0.005), and IL-1 and urinary calcium (r = -0.36; P less than 0.05). Serum PTH levels were within normal limits in all subjects and were similar in the three study groups, 1,25-Dihydroxyvitamin D3 levels, although higher in IH patients than in controls, were not significantly different in FH and AH subjects. Increased IL-1 activity and decreased vertebral mineral density are features of a subset of patients with IH. Although a cause-effect relationship remains to be established, increased monocytic IL-1 activity, rather than elevated PTH or 1,25-dihydroxyvitamin D3 levels, could underlie the resorptive component of FH. (07/1990) (link)
• Stepan JJ, Lau K-HW, Mohan S, Singer FR, and Baylink DJ (1990) Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma. Evidence for a single structure. Biochemical and Biophysical Research Communication 168 (2), 792-800. Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase. (04/1990) (link)
• Kasperk CH, Wergedal JE, Mohan S, Long DL, Lau K-HW, and Baylink DJ (1990) Interactions of growth factors present in bone matrix with bone cells: effects on DNA synthesis and alkaline phosphatase. Growth Factors 3 (2), 147-158. It has been shown that bone cells produce and secrete several growth factors (GFs) which are also found in the bone matrix. To investigate the role of these growth factors in bone cell metabolism, we compared the effects of different factors separately and in combination with respect to osteoblastic cell proliferation and differentiation. While basic fibroblast GF (FGF), transforming GF beta-1 (TGF beta), and platelet-derived GF (PDGF) enhance DNA synthesis, they had the opposite effect on alkaline phosphatase (ALP) activity in cell extracts: FGF, TGF beta, and PDGF inhibited cell ALP but strongly stimulated DNA synthesis. The IGFs had little effect on cell ALP but increased the release of ALP into the conditioned medium. In mitogenic tests of combinations of GFs, most had at least additive effects at low concentrations, and FGF, TGF beta, and IGF2 produced synergistic effects. Evidence is presented for (1) the modulation of the effects of one GF by the action of other GF, (2) synergistic interactions between FGF, TGF beta, and IGF2, and (3) a possible role for the observed interactions among GF for the mitogenic effect of human bone extract. (02/1990)
• Kyeyune-Nyombi E, Lau K-HW, Baylink DJ, and Strong DD (1989) Stimulation of cellular alkaline phosphatase activity and its messenger RNA level in a human osteosarcoma cell-line by 1,25(OH)2D3. Archives of Biochemistry and Biophysics 275 (2), 363-370. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) significantly stimulated cellular alkaline phosphatase activity in a human osteoblastic osteosarcoma cell line (TE-85 cells) in serum-free medium with 0.1% bovine serum albumin as the hormone carrier in a dose- and a time-dependent manner. The extent of the maximal stimulation was greater and the minimal dose that was required for stimulation was lower than those previously reported for TE-85 cells in the presence of serum. The magnitude of the stimulation of alkaline phosphatase activity by 1,25(OH)2D3 varied with the cell density. Daily changes of conditioned medium, as compared with no medium changes, significantly reduced the magnitude of the stimulation, suggesting that endogenous factors secreted into culture medium could play an enhancing role. Finally, application of Northern blot analysis using an oligodeoxynucleotide probe corresponding to a unique sequence of the human bone/liver/kidney alkaline phosphatase cDNA coding region revealed that 1,25(OH)2D3 increased the alkaline phosphatase mRNA level, suggesting that the increase in alkaline phosphatase activity was a result of either an increase in the rate of transcription or an increase in message stability. (12/1989) (link)
• Stepan JJ, Lau K-HW, Mohan S, Kraenzlin M, and Baylink DJ (1989) Purification and N-terminal sequence of two tartrate-resistant acid phosphatases type-5 from the hairy cell leukemia spleen. Biochemical and Biophysical Research Communication 165 (3), 1027-1034. Tartrate-resistant acid phosphatases types 5a and 5b were purified from human hairy cell leukemia spleen by sequential chromatography on Phenyl-Sepharose, CM-Sepharose, concanavalin A-Sepharose, FPLC Superose-12 and FPLC Mono-S. The purification over the original tissue extract was 1150- and 3300-fold, with a yield of 2.1% and 2.5%, respectively. Gel filtration indicated an Mr of about 30000 for both forms. There was a N-terminal sequence identity between the two enzymes. However, they appeared to be different as assessed by cation exchange chromatography and amino acid composition. (12/1989) (link)
• Lau K-HW, Farley JR, Freeman TK, and Baylink DJ (1989) A proposed mechanism of the mitogenic action of fluoride on bone cells: inhibition of the activity of an osteo­blastic acid phosphatase. Metabolism 38 (9), 858-868. Fluoride (F) is a potent inhibitor of osteoblastic acid phosphatase activity with an apparent Ki value (10 to 100 mumol/L) that corresponds to F concentrations that increase bone cell proliferation and bone formation in vivo and in vitro. This high sensitivity of acid phosphatase to F inhibition appeared to be specific for skeletal tissues. Mitogenic concentrations of F did not increase cellular cAMP levels but significantly stimulated net protein phosphorylation in intact calvarial cells and in isolated calvarial membranes. These concentrations of F also stimulated net membrane-mediated phosphorylation of angiotensin II (which contains tyrosyl but no seryl or threonyl residues), suggesting that some of the F-stimulated protein phosphorylations could occur on tyrosyl residues. F had no apparent effect on thiophosphorylation of membrane proteins, suggesting that the F-stimulated net protein phosphorylation in bone cells was probably not mediated via activation of protein kinases. Orthovanadate or molybdate at concentrations that inhibit bone acid phosphatase activity also stimulated bone cell proliferation, supporting the idea that inhibition of bone acid phosphatase would lead to stimulation of bone cell proliferation. Mitogenic concentrations of F potentiated the mitogenic activities of insulin, EGF, and IGF-1 (ie, growth factors the receptors of which are tyrosyl kinases) to a greater extent than they potentiated the action of basic FGF (a growth factor that does not appear to stimulate tyrosyl protein phosphorylation). Based on these findings, a model is proposed for the biochemical mechanism of the osteogenic action of F in which F stimulates bone cell proliferation by a direct inhibition of an osteoblastic acid phosphatase/phosphotyrosyl protein phosphatase activity, which in turn increases overall cellular tyrosyl phosphorylation, resulting in a subsequent stimulation of bone cell proliferation. (09/1989) (link)
• Lau K-HW, Farley JR, and Baylink DJ (1989) Phosphotyrosyl protein phosphatases. Biochemical Journal 257 (1), 23-36. (01/1989) (link)
• Lau K-HW, Tanimoto H, and Baylink DJ (1988) Vanadate stimulates bone cell proliferation and bone collagen synthesis in vitro. Endocrinology 123 (6), 2858-2867. We recently proposed a hypothesis for the molecular mechanism of the osteogenic action of fluoride in which it stimulates osteoblast proliferation via the inhibition of an osteoblastic acid phosphatase-like phosphotyrosyl protein phosphatase activity. To test this hypothesis, we investigated whether orthovanadate, a known phosphotyrosyl protein phosphatase inhibitor, would mimic fluoride in the stimulation of bone cell proliferation and bone collagen synthesis in vitro. Orthovanadate inhibited the osteoblastic acid phosphatase activity and stimulated bone cell proliferation at the same low concentrations (i.e. 5-15 microM). At the mitogenic doses, orthovanadate also showed a dose-dependent increase in alkaline phosphatase (a marker of mature osteoblasts) in cultured calvarial cells and stimulated bone collagen synthesis, as measured by the incorporation of [3H]proline and the conversion into [3H] hydroxyproline in organ calvaria cultures. Therefore, orthovanadate stimulated bone formation by increasing the number of mature osteoblasts mediated via stimulation of cell proliferation and differentiation. Orthovanadate was dependent on the presence of a mitogen in cell medium for its mitogenic action in vitro and synergistically potentiated the mitogenic actions on osteoblasts of those growth factors, i.e. insulin, epidermal growth factor, insulin-like growth factor I, and skeletal growth factor, whose mitogenic action involved tyrosyl protein phosphorylation. However, the interaction between orthovanadate and basic fibroblast growth factor, a growth factor that does not appear to involve tyrosyl protein phosphorylation, on bone cell proliferation was additive. In summary, these data are consistent with the hypothesis that inhibition of the osteoblastic phosphotyrosyl protein phosphatases can prolong and/or potentiate the mitogenic actions of growth factors, and thereby stimulates cell proliferation. (12/1988)
• Lau K-HW, Jennings JC, and Baylink DJ (1988) Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro. International Journal of Biochemistry 20 (12), 1443-1450. 1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues. (12/1988)
• Lundy MW, Lau K-HW, Blair HC, and Baylink DJ (1988) Chicken osteoblasts contain a fluoride-sensitive acid phosphatase activity. Journal of Histochemistry and Cytochemistry 36 (9), 1175-1180. We used histological and biochemical methods to determine the cellular origin of bone matrix fluoride-sensitive acid phosphatase in chicken bone. Embryonic chicken calvariae were embedded in plastic and sections stained for acid phosphatase at various concentrations of substrate and fluoride. Acid phosphatase activity was observed in osteoblasts and osteoclasts but not in fibroblasts. Striking inhibition of osteoblastic acid phosphatase occurred at 100 µM fluoride, a concentration that had no apparent effect on osteoclastic acid phosphatase. Inhibition of osteoblastic and osteoclastic acid phosphatase by fluoride was also examined using extracts of embryonic chicken calvarial cells, mouse osteoblasts (MC3T3-El cell line), and purified chick osteoclasts, respectively. Fluoride is a partial competitive inhibitor of both chicken and mouse osteoblastic acid phosphatases, with apparent inhibition constants of 10-100 µM. These concentrations of fluoride correspond to those that increase bone formation in vitro and in vivo. In contrast, the apparent inhibition constant for fluoride of osteoclastic acid phosphatase was much higher (i.e., 0.5 mM). In summary, this study demonstrates that chicken osteoblasts contain an acid phosphatase that is sensitive to inhibition by low concentrations (i.e., µM) of fluoride. (09/1988) (link)
• Wergedal JE, Lau K-HW, and Baylink DJ (1988) Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. Clinical Orthopaedics and Related Research 233, 274-282. The effects of fluoride (20 µmol/L) and bovine bone extract (17 µgrams/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of factors. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 µmol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract. (08/1988) (link)
• Carmel R, Lau K-HW, Baylink DJ, Saxena S, and Singer FR (1988) Cobalamin and osteoblast specific proteins. New England Journal of Medicine 319 (2), 70-75. Cobalamin deficiency has well-known hematologic and neurologic effects, but little is known about its other effects. We therefore studied the effect of cobalamin on osteoblast-related proteins. We found that mean (+/- 1 SD) levels of skeletal alkaline phosphatase in the blood were lower in 12 cobalamin-deficient patients (3.89 +/- 2.19 units per liter) than in 5 nondeficient and 5 iron-deficient control subjects (7.55 +/- 3.99 units per liter). The degree of the megaloblastic anemia correlated with the reduction in skeletal alkaline phosphatase levels (r = 0.67, P less than 0.01). With cobalamin therapy, levels of skeletal alkaline phosphatase rose in 11 of the 12 cobalamin-deficient subjects but not in the controls. The cobalamin-deficient patients also had significantly lower osteocalcin levels than the control subjects (1.11 +/- 0.77 vs. 1.84 +/- 0.49 nmol per liter). During cobalamin therapy, these levels rose in the cobalamin-deficient patients but not in the controls. In contrast to the levels of osteoblast-related proteins, hepatic alkaline phosphatase levels were similar in the patients and controls and were usually unaffected by cobalamin therapy. In vitro studies of calvarial cells from chicken embryos showed that their alkaline phosphatase content was cobalamin-dependent, thus supporting our in vivo observations in humans. Our findings suggest that osteoblast activity depends on cobalamin and that bone metabolism is affected by cobalamin deficiency, but we do not yet know whether cobalamin deficiency produces clinically important bone disease. (07/1988)
• Lau K-HW, Jennings JC, and Baylink DJ (1988) Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro. International Journal of Biochemistry 20 (12), 1443-1450. 1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues. (06/1988)
• Lau K-HW, Farley JR, and Baylink DJ (1987) Bone acid phosphatase is a neutral pH phosphotyrosyl protein phosphatase. Adv Prot Phosphatases 4 (1), 165-198. (07/1987)
• Lau K-HW, Onishi T, Wergedal JE, Singer FR, and Baylink DJ (1987) Characteriza­tion and assay of tartrate-resistant acid phosphatase activity in serum: potential use to assess bone resorption. Clinical Chemistry 33 (4), 458-462. We improved the spectrophotometric assay of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. During development of the assay we found that human serum contains a dialyzable, mixed-type noncompetitive inhibitor(s) of TrACP activity, the effects of which on the assay were substantially lessened by diluting the serum sample with water before assay and increasing the substrate concentration. Hemolysis releases into serum a significant amount of TrACP activity from erythrocytes, which can be inactivated by incubating the serum at 37 degrees C for 1 h before assay. Our improved assay was reproducible (CV = 5%), and measured within 10% of the amount of added bovine skeletal TrACP activity. Preliminary application of the assay revealed that the amount of serum TrACP activity in patients with skeletal diseases differed from normal values and changed in the same direction as the expected change in bone turnover, suggesting that TrACP activity in serum could be useful clinically as a marker of bone metabolism, possibly of bone resorption. (04/1987) (link)
• Farley JR, Tarbaux NM, Lau K-HW, and Baylink DJ (1987) Monofluorophosphate is hydrolyzed by alkaline phosphatase and mimics the actions of NaF on skeletal tissues in vitro. Calcified Tissue International 40 (1), 35-42. These studies were intended to assess the osteogenic activity of monofluorophosphate (MFP) in vitro, and to identify the enzyme(s) responsible for MFP hydrolysis-alkaline phosphatase (ALP) and/or acid phosphatase (AcP). ALP and AcP activities were determined by hydrolysis of p-nitrophenylphosphate (PNPP) at pH greater than 8 and pH 5.5, respectively, and MFP hydrolysis was determined, between pH 5.5 and pH 9.0, from measurements of [fluoride ion], using an ion-specific electrode. We found (1) that MFP was an alternative substrate for purified ALP, but not for AcP; (2) that MFPase activity in the embryonic chick resembled ALP, but not AcP, with respect to pH-dependent hydrolysis, sensitivity to effectors (r = 0.98, P less than .001), and tissue distribution (r = 0.96, P less than .001); and (3) that intestinal MFPase activity in the embryonic chick co-purified with ALP activity (r = 0.93, P less than .01) and resembled ALP, but not AcP, in its distribution along the small intestine, being highest in the duodenum and lowest in the distal ileum (r = 0.96, P less than .001). We also found that in vitro exposure to MFP increased (1) the proliferation rate of embryonic chick calvarial cells in serum-free monolayer cultures (i.e., 3[H]-thymidine incorporation into DNA, P less than .001); (2) ALP activity in calvarial cells (P less than .005) and in intact calvaria (P less than .05); and (3) collagen production by intact calvaria (i.e., 3[H]-proline incorporation as 3[H]-hydroxyproline, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS) (01/1987) (link)
• Lau K-HW, Freeman TK, and Baylink DJ (1987) Purification and characterization of a tartrate-resistant acid phosphatase that displays neutral pH phosphotyrosyl protein phosphatase activity from bovine cortical bone matrix. Journal of Biological Chemistry 262 (3), 1389-1397. An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo. (01/1987) (link)
• McDonald GB, Lau K-HW, Schy AL, Wergedal JE, and Baylink DJ (1985) Intestinal metabolism and portal venous transport of 1,25(OH)2D3, 25(OH)D3, and vitamin D3 in the rat. American Journal of Physiology 248 (6 Pt 1), G633-G638. We compared the intestinal absorption of three vitamin D3 sterols and tested the hypothesis that the intestine hydroxylates absorbed vitamin D and transports polar metabolites in portal venous blood. Micellar solutions containing 50 nmol of a radiolabeled vitamin D3 sterol (1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or vitamin D3) were placed in closed jejunal segments of rats prepared with lymphatic and mesenteric venous fistulas. Venous blood loss was replaced by infusion of donor rat blood into the saphenous vein. After 1-2 h the rats were killed, and intestinal homogenates, mesenteric blood, and lymph were analyzed. The average rate of absorption of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was two- and fivefold higher than that of 25-hydroxyvitamin D3 [25(OH)D3] and vitamin D3 (D3), respectively. Transport of hydroxylated vitamin D sterols was primarily via the venous route; the average rate of venous transport of 1,25(OH)2D3 was 18.3 X 10(2) nmol X min-1 X g-1 compared with 8.8 X 10(2) for 25(OH)D3 and 0.13 X 10(2) for D3. High-performance liquid chromatography of intestinal and plasma extracts revealed that there was 25-hydroxylation of absorbed D3, 24- and putative 1-hydroxylation of absorbed 25(OH)D3, and prompt portal venous transport of all hydroxylated metabolites. When 1,25(OH)2D3 was infused into the lumen, the composition of radiolabeled sterols found in intestinal homogenates and mesenteric venous plasma was virtually identical to that of the infusate. These studies provide in vivo evidence for the intestinal metabolism of pharmacological quantities of absorbed vitamin D3 sterols and the prompt portal venous transport of more polar metabolites. (06/1985) (link)
• Lau K-HW, Lee MY, Linkhart TA, Mohan S, Vermeiden J, Liu CC, and Baylink DJ (1985) A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local prostaglandins production in bone. Biochimica et Biophysica Acta 840 (1), 56-68. Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro contained dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamin D metabolites or prostaglandins. However, breast tumor-conditioned medium stimulated production and release of prostaglandin E2 from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E2 production and release. The resorptive activity of parathyroid hormone (PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [3H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF-) like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a beta-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secrete into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E2 which in turn increases bone resorption in vitro. (05/1985) (link)
• Lau K-HW, Farley JR, and Baylink DJ (1985) Phosphotyrosyl specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme: comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase. Journal of Biological Chemistry 260 (8), 4653-4660. A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions. (04/1985) (link)
• Farley JR, Fitzsimmons R, Taylor AK, Jorch UM, and Lau K-HW (1985) Direct effects of ethanol on bone resorption and formation in vitro. Archives of Biochemistry and Biophysics 238 (1), 305-314. In vitro studies indicate that low concentrations of ethanol can have direct effects on bone formation and resorption. Bone resorption was increased when embryonic chick tibiae were exposed to ethanol at 0.03-0.3% (v/v), and bone formation was inhibited when tibiae were exposed to 0.2% ethanol in the presence of NaF or parathyroid hormone (P less than 0.01 for each). Ethanol also had direct effects on isolated bone cells in vitro, increasing both cAMP and PGE2 production (P less than 0.001 for each), and affecting cell proliferation in a biphasic, time- and dose-dependent manner. After 24 h of exposure, 0.03% ethanol increased bone cell proliferation (P less than 0.001), but 0.3% ethanol was inhibitory (P less than 0.01). Paradoxically, mitogenic doses of ethanol prevented the effects of two other mitogens, NaF and human skeletal growth factor, to increase bone cell proliferation (P less than 0.001). But how were these effects produced? Several observations suggest that these direct effects of ethanol on skeletal tissues in vitro were mediated by changes in bone cell membrane fluidity. (a) Dimethyl sulfoxide, ethylene glycol, and lecithin, which act, like ethanol, to increase membrane fluidity, mimicked the effects of ethanol on bone cell proliferation. Dimethyl sulfoxide also mimicked the effect of ethanol to increase cAMP (P less than 0.001). (b) Cholesterol, which decreases cell membrane fluidity, acted oppositely to ethanol and enhanced the mitogenic response to human skeletal growth factor (P less than 0.001). (c) Preincubation of calvarial cells with ethanol or with cholesterol altered the in situ reaction kinetics of the membrane-bound enzyme, alkaline phosphatase. Together, these data demonstrate that ethanol has direct effects on skeletal tissue in vitro, and suggest that those effects may be secondary to changes in bone cell membrane fluidity. (04/1985) (link)

Books and Chapters

• Lau K-HW and Baylink DJ (2001) Fluoride therapy for osteoporosis. In: Osteoporosis, 2nd ed, (Marcus R, Feldman D, and Kelsey J, eds.), Volume 2, Chapter 74, pp. 675-697, Academic Press, Inc., San Diego, California (2001)
• Lau K-HW (2000) Fluoride therapy of established osteoporosis. In: Management of Fractures in Severely Osteoporotic Bone: Orthopaedic and Pharmacological Strategies, (Obrant K, ed.), Chapter 32, pp. 443-461, Springer Verlag, London (2000)
• Lau K-HW and Baylink DJ (1999) Fluoride therapy of established osteoporosis. In: The Aging Skeleton, (Rosen C., Glowacki J, and Bilezikian JP, eds.), Chapter 48, pp. 587-612, Academic Press, San Diego (1999)
• Lau K-HW, Åkesson K, Libanati CR, and Baylink DJ (1998) Osteogenic actions of fluoride: its therapeutic use for established osteoporosis. In: Anabolic Treatments for Osteoporosis, (Whitfield, J. F., and Morley, P., eds.), Chapter 9, pp. 207-254, CRC Press, Inc., Boca Raton, Florida (1998)
• Lau K-HW, Libanati CR, and Baylink DJ (1997) Serum osteoclastic tartrate-resistant acid phosphatase: a potential bone resorption marker. In: Calcium regulating hormones and markers of bone metabolism: measurement and interpretation, (Schmidt-Gayk H, Blind E, and Roth H-J, eds), 2nd edition, Chapter 20, pp. 183-197, Clin Lab Publications, Heidelberg, Germany (1997)
• Libanati C, Lau K-HW, and Baylink D (1996) Fluoride therapy for osteoporosis. In: Osteoporosis, (Marcus R, Feldman D, and Kelsey J, eds), Chapter 66, pp. 1259-1277, Academic Press, Inc., San Diego, California (1996)
• Libanati CR, Lau K-HW, and Baylink DJ (1993) A strategy to develop an optimized therapeutic regimen for fluoride. In: Proceedings of the 4th Int Symp on Osteoporosis, (Christiansen C, Riis BJ, eds), pp. 424-427, Handelstrykkeriet Aalborg ApS, Aalborg, Denmark. (06/1993)
• Bell NH, Finkelman RD, Linkhart TA, Mohan S, Lau K-HW, and Baylink DJ (1991) Evidence of a role for vitamin D and transforming growth factor beta  in osteoinduction. In: Vitamin D. Gene Regulation, Structure-Function Analysis and Clinical Applica­tion, (Norman AW, Bouillon R, and Thomasset M, eds), pp. 559-560, Walter de Gruyter & Co., Berlin. (06/1991)
• Lau K-HW, Farley JR, and Baylink DJ (1987) Molecular mechanism of the mitogenic action of fluoride: inhibition of osteoblastic acid phosphatase-like phosphotyrosyl pro­tein phosphatase activity. In Osteoporosis 1987, (Christiansen C, Johansen JS, and Riis BJ, eds), Volume 1, pp. 215-221, Handelstrykkeriet Aalborg ApS, Aalborg, Denmark. (12/1987)
• Lau K-HW, Kraenzlin M, Onishi T, and Baylink DJ (1987) Development of an improved assay of tartrate-resistant acid phosphatase in serum: potential use to assess bone resorption. In: Osteoporosis 1987, (Christiansen C, Johansen JS, and Riis BJ, eds), Volume 2, pp. 682-687, Handelstrykkeriet Aalborg ApS, Aalborg, Denmark. (12/1987)