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The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB(fl/fl)) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1(-/-) mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1(-/-) mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1(-/-) mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification. Refereed/Peer-reviewed

The TWIST-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor important in mediating skeletal and head mesodermal tissue development. Bone marrow-derived mesenchymal stem/stromal cells (BMSC), express high levels of TWIST-1, which is down regulated during ex vivo expansion. Cultured BMSC over-expressing TWIST-1 display decreased capacity for osteogenic differentiation and an enhanced capacity to undergo adipogenesis, suggesting that TWIST-1 is a mediator of lineage commitment. However, little is known regarding the mechanism(s) by which TWIST-1 mediates cell fate determination. In this study, microarray analysis was used to identify a novel downstream TWIST-1 target, tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1), which was down regulated in TWIST-1 over-expressing BMSC. Chromatin immunoprecipitation analysis showed that TWIST-1 directly bound to two E-box binding sites on the proximal C-ROS-1 promoter. Knock-down of C-ROS-1 in human BMSC and cranial bone cells resulted in a decreased capacity for osteogenic differentiation in vitro. Conversely, suppression of C-ROS-1 in BMSC resulted in an enhanced capacity to undergo adipogenesis. Furthermore, reduced C-ROS-1 levels led to activation of different components of the PI3K/AKT/mTORC1 signalling pathway during osteogenic and adipogenic differentiation. Collectively, these data suggest that C-ROS-1 is involved in BMSC fate switching between osteogenesis and adipogenesis, mediated via PI3K/AKT/mTORC1 signalling.

Bone marrow derived mesenchymal stem/stromal cells (MSC) contribute to skeletal tissue formation and the regulation of haematopoiesis. The Eph/ephrin family of receptor tyrosine kinases is potentially important in the maintenance of the stem cell niche within neural, intestinal and dental tissues and has recently been shown to play a role in regulating bone homeostasis. However, the contribution of EphB/ephrin-B molecules in human MSC function remains to be determined. In the present study, EphB and ephrin-B molecules were expressed by ex vivo expanded human MSC populations and within human bone marrow trephine samples. To elucidate the contribution of EphB/ephrin-B molecules in MSC recruitment, we performed functional spreading and migration assays and showed that reverse ephrin-B signalling inhibited MSC attachment and spreading by activating Src-, PI3Kinase- and JNK-dependent signalling pathways. In contrast, forward EphB2 signalling promoted MSC migration by activating the Src kinase- and Abl-dependent signalling pathways. Furthermore, activation of ephrin-B1 and/or ephrin-B2 molecules expressed by MSC was found to increase osteogenic differentiation, while ephrin-B1 activation promoted chondrogenic differentiation. These observations suggest that EphB/ephrin-B interactions may mediate the recruitment, migration and differentiation of MSC during bone repair. Refereed/Peer-reviewed

Circulating 1 alpha,25-dihydroxyvitamin D(3) (1,25D) derives from renal conversion of 25-hydroxyvitamin D(3) (25D), by the 25D 1 alpha-hydroxylase (CYP27B1). Blood 25D levels, but not 1,25D levels, are the best indicator of vitamin D status and predict fracture risk in the elderly. We examined the extent to which osteoblasts can metabolize 25D. Well-characterized human primary osteoblasts and osteosarcoma (OS) cell lines were examined for the expression and regulation of genes associated with vitamin D metabolism, using real-time PCR. Primary osteoblasts and OS cell lines were found to express CYP27B1 mRNA and secreted detectable 1,25D in response to 25D. Of the OS cell lines tested, HOS expressed the most CYP27B1 mRNA and secreted the highest levels of 1,25D. All osteoblastic cells examined up-regulated expression of the catabolic regulator of 1,25D, the 25-hydroxyvitamin D-24-hydroxylase (CYP24), when incubated with either 1,25D or 25D. Exposure to physiological levels of 25D resulted in up-regulated transcription of the 1,25D responsive genes, osteocalcin (OCN), osteopontin (OPN) and RANKL. Specific knockdown of CYP27B1 in HOS cells using siRNA resulted in up to 80% reduction in both 1,25D secretion and the transcription of OCN and CYP24, strongly implying that the 25D effect in osteoblasts is preceded by conversion to 1,25D. Incubation with 25D, like 1,25D, inhibited primary osteoblast proliferation and promoted in vitro mineralization. Finally, we detected expression by osteoblasts of receptors for vitamin D binding protein (DBP), cubilin and megalin, suggesting that osteoblasts are able to internalize DBP-25D complexes in vivo. Together, our results suggest that autocrine, and perhaps paracrine, pathways of vitamin D(3) metabolism may regulate key osteoblast functions independently of circulating, kidney derived 1,25D. Our results are therefore consistent with the reported benefits of maintaining a healthy vitamin D status in the elderly to reduce the risk of fractures.

Fragility fractures, including neck of femur fractures, result from reductions in the amount, quality and architecture of bone. The aim of this study was to compare the cancellous bone structure, and static indices of bone turnover, in female patients, who had sustained fragility fracture at the femoral neck, with age-matched females without fragility fracture. Bone samples were taken from the intertrochanteric region of the proximal femur of female patients undergoing hip arthroplasty surgery for a subcapital fragility fracture of the femoral neck (#NOF) or from age-matched female control individuals at routine autopsy. Contiguous bone samples were analyzed for undecalcified histomorphometry and for mRNA expression. The histomorphometric data, which were normally distributed, indicated no difference between the mean values for any of the structural parameters in control and fracture samples. In particular, the bone volume (BV/TV) values were not different and did not change significantly with age in these cohorts of individuals aged65 years. The static indices of bone turnover, eroded surface (ES/BS) and osteoid surface (OS/BS), were positively correlated with age in the65-year-old control group (p0.055 and p0.03, respectively). The median values for these indices were not different between the fracture and control groups. However, both the median and the range of OS/BS values were increased for65-year-old controls compared with a group of younger females aged65 years, suggesting an increase in bone formation surface in older females in the proximal femur after 65 years of age. When the data were further interrogated, a reduction in the percentage osteoid surface to eroded surface quotient (OS/ES) was found for the fracture group compared with the age-matched control group suggesting a reduced adaptive modeling drift capability in the fracture group. In contiguous bone samples, increased median values for receptor activator of nuclear factor kappa beta (RANK) and interleukin-6 (IL-6) mRNA expression were observed in the fracture group. Study of cultured human osteoblasts showed that recombinant human IL-6 (rhIL-6) inhibited osteoblast differentiation, as measured by an increase in the immature osteoblast marker, STRO-1 and concomitantly decreased expression of the osteoblast maturation marker, alkaline phosphatase. Importantly, cells cultured in the presence of IL-6 showed significantly less mineral deposition in vitro compared with control cultures. These data suggest that perturbations in bone formation surface, relative to resorption surface, are potentially important in producing bone in the proximal femur with increased propensity to fracture.

Previous studies have attributed the increase in bone mass observed following bisphophonate (BP) therapy to their effects on bone-resorbing osteoclasts (OCs). However, recent evidence suggests that BPs can also act directly on bone forming osteoblasts (OBs) to increase their anabolic activity. Using an established model of in vitro OB differentiation, we found that the potent nitrogen-containing BP, zoledronic acid (ZOL), may enhance the bone forming potential of human adult OB-like cells in vitro by inducing their differentiation. ZOL dose dependently induced both cytostasis and cell death in OB-like cells at concentrations of 0.5 μM or greater. Cells expressing high levels of the osteoprogenitor antigen, STRO-1, exhibited a greater proliferative potential than STRO-1 negative/dim cells, and were more susceptible to the cytostatic and apoptotic effects of ZOL. ZOL was also found to promote bone cell differentiation, as evidenced by an increase in the number of cells exhibiting a more differentiated (STRO-1 − /AP + and STRO-1 − /AP − ) phenotype. Analysis of gene expression, using semi-quantitative RT-PCR, demonstrated that ZOL treatment resulted in a significant upregulation of osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2) gene expression. Furthermore, in vitro mineralisation studies revealed that ZOL enhanced mineralised matrix formation at concentrations between 5 and 25 μM. These results show that, in addition to its direct effects on OCs, ZOL also directly affects the proliferation and differentiation of human OB-like cells in vitro and may enhance bone formation in vivo.

Apo2 ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. Apo2L/TRAIL can selectively induce programmed cell death in transformed cells, although its wide tissue distribution suggests potential physiological roles. We have investigated the expression, in human osteoblast-like cells (NHBC), of Apo2L/TRAIL and the known Apo2L/TRAIL death receptors, DR4 and DR5, and the Apo2L/TRAIL decoy receptors, DcR-1, DcR-2, and osteoprotegerin (OPG). NHBC expressed abundant mRNA corresponding to each of these molecular species. Immunofluorescence staining demonstrated that Apo2L/TRAIL protein was abundant within the cytoplasm of NHBC and OPG was strongly expressed at the cell surface. DR5 and DcR-2 were present in the cell membrane and cytoplasm and DcR-1 was confined to the nucleus. DR4 staining was weak. Neither Apo2L/TRAIL alone, nor in combination with chemotherapeutic agents of clinical relevance to treatment of osteogenic sarcoma, induced cell death in NHBC, as assessed morphologically and by activation of caspase-3. In contrast, the human osteogenic sarcoma cell lines, BTK-143 and G-292, were sensitive to exogenous Apo2L/TRAIL alone, and to the combined effect of Apo2L/TRAIL/cisplatin and Apo2L/TRAIL/doxorubicin treatments, respectively. In NHBC, we observed strong associations between the levels of mRNA corresponding to the pro-apoptotic molecules, Apo2L/TRAIL, DR4, and DR5, and those corresponding to pro-survival molecules, DcR-1, DcR-2, OPG, and FLIP, suggesting that the balance between pro-survival and pro-apoptotic molecules is a mechanism by which NHBC can resist Apo2L/TRAIL-mediated apoptosis. In contrast, osteogenic sarcoma cells had low or absent levels of DcR-1 and DcR-2. These results provide a foundation to explore the role of Apo2L/TRAIL in osteoblast physiology. In addition, they predict that therapeutic use of recombinant Apo2L/TRAIL, in combination with chemotherapeutic agents to treat skeletal malignancies, would have limited toxic effects on normal osteoblastic cells.

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of κB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Injured growth plate cartilage is often repaired by bony tissue resulting in impaired bone growth in children. Using a rat injury model, our previous studies show that following the injury-induced initial inflammatory response, an influx of mesenchymal-like cells occurs within the growth plate injury site prior to formation of bony tissue. As platelet-derived growth factor (PDGF-BB) is a potent chemotactic factor of mesenchymal cells during skeletal tissue repair, we examined its role during the early fibrogenic response and the subsequent bony repair of injured growth plate. Following growth plate injury, rats received daily injection of the PDGF receptor (PDGFR) inhibitor, Imatinib, for 7 days. Immunohistochemical analysis of injured growth plate at day 1 showed the presence of PDGF-BB expression in some inflammatory cells, while at day 4 PDGFR was expressed by a proportion of the infiltrating mesenchymal cells at the injury site. By day 4, PDGFR inhibition reduced mesenchymal infiltrate (P < 0.05); by day 14, Imatinib-treated rats exhibited less bony trabeculae and cartilaginous repair tissues, fewer osteoclasts and less bone marrow (BM) at the injury site, compared to vehicle controls (P < 0.01). In vitro “scratch” migration assays with rat BM mesenchymal cells revealed that recombinant PDGF-BB increased cell migration into the “wound” (P < 0.05), while Imatinib inhibited this chemotactic response. Quantitative RT-PCR analysis showed that Imatinib treatment decreased expression of the cartilage and bone related genes, Col2a1 and osteocalcin, respectively. These results suggest that PDGF-BB contributes to growth plate injury repair by promoting mesenchymal progenitor cell infiltration, the chondrogenic and osteogenic responses, and remodelling of the repair tissues. Refereed/Peer-reviewed