Osteoblast-specific Transcription Factor Research Articles

The transcriptional factor Osterix directly interacts with RNA helicase A

Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation.

Extracellular Signal‐Regulated Kinase 1/2 Is Involved in Ascorbic Acid–Induced Osteoblastic Differentiation in Periodontal Ligament Cells

Periodontal ligament (PDL) cells possess osteoblast-like properties and play key roles in periodontal regeneration. Previously, we have reported that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating the type I collagen-integrin interaction. However, the signaling pathway activated following collagen-integrin interaction is still unclear. In this study, we examined the involvement of extracellular signal-regulated kinase (ERK)1/2 in the expression of osteoblastic marker genes such as the osteoblast-specific transcriptional factor runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) in PDL cells. PDL cells were cultured on a conventional or type I collagen-coated dish in the presence or absence of ascorbic acid and examined for ALP activity and osteoblastic marker genes. For detection of ERK1/2, cells were plated on a petri (non-adhesive) dish or type I collagen-coated dish, and Western blot analysis was performed. The effect of the ERK1/2 inhibitor on osteoblastic marker gene expression was examined. Ascorbic acid increased gene expression of Runx2, ALP, and OCN. A combination of ascorbic acid and type I collagen remarkably upregulated Runx2, ALP, and OCN gene expression and ALP activity. Western blot analysis revealed an increased level of ERK1/2 phosphorylation in cells plated on type I collagen. An ERK1/2 inhibitor suppressed ascorbic acid-induced ALP and OCN gene expression, whereas Runx2 was not affected in PDL cells. These results indicate that ERK1/2 is involved in ascorbic acid-induced osteoblastic differentiation during PDL cell attachment to type I collagen.

HGF Inhibits BMP-Induced Osteoblastogenesis: Implications for the Bone Disease in Multiple Myeloma.

Multiple myeloma is a hematological malignancy characterized by accumulation of malignant plasma cells in the bone marrow. Most patients develop osteolytic lesions, caused by an uncoupling of bone formation from bone resorption. In patients with osteolytic lesions the osteoblasts are fewer and less active. HGF (Hepatocyte Growth Factor) is produced by the malignant plasma cells and is found at high concentrations in the bone marrow of most patients with multiple myeloma. In this study we show that HGF inhibited BMP-induced expression of alkaline phosphatase and mineralization in human mesenchymal stem cells (MSC) and the murine myoid cell line C2C12. Furthermore, the expression of the osteoblast-specific transcription factors Runx2 and Osterix was reduced by HGF treatment. HGF inhibited BMP-induced differentiation of MSC partly by keeping the cells in a proliferative, undifferentiated state. The in vitro data was supported by the observation of a negative correlation between HGF and a marker of osteoblast activity, bone specific alkaline phosphatase (rho=−0,45, p=0,008), in sera from 34 myeloma patients. These observations suggest that HGF inhibits bone formation in multiple myeloma.

Parathyroid Hormone Controls Receptor Activator of NF-κB Ligand Gene Expression via a Distant Transcriptional Enhancer

RANKL, a protein essential for osteoclast development and survival, is stimulated by parathyroid hormone (PTH) via a PTH receptor 1/cyclic AMP (cAMP)/protein kinase A (PKA)/CREB cascade, exclusively in osteoblastic cells. We report that a bacterial artificial chromosome-based transcriptional reporter construct containing 120 kb of RANKL 5'-flanking region was stimulated by dibutyryl-cAMP in stromal/osteoblastic cells, but not other cell types. Full cAMP responsiveness was dependent upon a conserved 715-bp region located 76 kb upstream from the transcription start site, which we identified by sequential deletion analysis and by comparison of human and mouse genomic sequences in silico. This region contained conserved consensus sequences which bound CREB and the osteoblast-specific transcription factor Runx2, and when mutated blunted cAMP responsiveness. Overexpression of Runx2 potentiated cAMP responsiveness of the endogenous RANKL gene in a cell-type-specific manner. Lastly, PTH responsiveness of the endogenous RANKL gene was abrogated in mice from which we deleted this conserved upstream region. Thus, PTH responsiveness of the RANKL gene is determined by a distant regulatory region that responds to cAMP in a cell-type-specific manner and Runx2 may contribute to such cell-type specificity.

Atm-deficient mice: an osteoporosis model with defective osteoblast differentiation and increased osteoclastogenesis

Atm is a Ser/Thr kinase involved in DNA damage response and is required for genome integrity and stem cell renewal. Here, we report an additional role for Atm in bone remodeling. Atm-/- mice showed reduced bone mass, especially at the trabecular bones, accompanied by a decrease in bone formation rate and defective differentiation of osteoblasts, but normal numbers of osteoprogenitor cells and osteoblasts. Atm might affect osteoblast differentiation by modulating the expression of osterix, a lineage-specific transcription factor essential for osteoblast maturation, likely via the bone morphogenetic proteins pathway. Atm-/- mice also displayed a marked increase in osteoclastogenesis and bone resorption, although Atm had no cell-autonomous effect on osteoclast differentiation and resorption. Increased osteoclastogenesis could be caused by a substantial reduction in testosterone and estradiol levels in male and female mice, respectively. The steroid hormone deficiency is a result of gonad developmental defects, which led to an increase in serum gonadotrophic hormone, FSH via a feedback regulation. Overall, these results indicate that Atm deficiency leads to osteoporosis mainly as a result of hypogonadism-induced bone resorption together with compromised osteoblast differentiation, and that Atm plays a positive role in regulating expression of osteoblast-specific transcription factor, osterix.

Runx2 phosphorylation induced by fibroblast growth factor‐2/protein kinase C pathways

Runx2 is a key transcription factor in osteoblast differentiation, and its activity is regulated by fibroblast growth factors (FGFs). Craniosynostosis, characterized by premature suture closure, results from mutations that generate constitutively active FGF receptors (FGFRs). We previously showed that FGF/FGFR-activated protein kinase C (PKC) is involved in the expression and activity of Runx2. Activated PKCdelta physically interacts with Runx2 in FGF2-stimulated MC3T3-E1 preosteoblastic cells. Immunopurified Runx2 protein reacted with PKCdelta kinase, and a phosphorylated 1460-Da peptide fragment (amino acids 241-252, 1380-Da) derived from Runx2 was also detected in MS analysis. Computer analysis predicted that Ser247 in this Runx2 can be a possible phosphorylation site by PKCdelta. We also showed that Runx2 activity after FGF stimulation correlates with the presence of the Runx2 Ser247 residue. The S247A (Ser --> Ala) mutation confers decreased transcriptional activity on a Runx2-responsive promoter after FGF treatment.

Tumor Necrosis Factor Promotes Runx2 Degradation through Up-regulation of Smurf1 and Smurf2 in Osteoblasts

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.

Elastin Calcification in the Rat Subdermal Model Is Accompanied by Up-Regulation of Degradative and Osteogenic Cellular Responses

Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with fibroblast and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2 and MMP-9) and transforming growth factor-beta1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding factor alpha-1, an osteoblast-specific transcription factor, increased in parallel with elastin calcification and attained approximately 9.5-fold higher expression at 21 days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontin and alkaline phosphatase also increased progressively, but osteocalcin levels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.

Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells

Runt-related transcription factor 2 (Runx2) and osterix are osteoblast-specific transcription factors essential for the development of osteoblastic cells and bone formation. PTH given intermittently has anabolic effects on bone; however, the exact role remains to be understood completely. The purpose of this study was both to investigate whether PTH regulates Runx2 as well as osterix expression and to identify the signaling used. Using RT-PCR, we confirmed that PTH (1-34) regulated Runx2 and osterix mRNA expression, in rat osteoblast-like cell line UMR 106, in a dose- and time-dependent manner. PTH in low concentrations stimulated both Runx2 and osterix mRNA expression while that in high concentrations did not. Forskolin, an adenylate cyclase activator, also enhanced Runx2 and osterix transcription, and the stimulatory effects of PTH and forskolin were blocked by the pre-treatment of the cells with H-89, a protein kinase A (PKA) inhibitor. In contrast, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) had no effect on Runx2 transcription, but induced an increase in osterix mRNA level at the concentration of 500 nM at 12 h after treatment. Moreover, pre-treatment of the cells with calphostin C, a PKC-specific inhibitor, reduced the increase in osterix transcripts enhanced by PTH and PMA 12 h after treatment. However, these inhibitory effects were not sustained for longer terms. These observations demonstrate that PTH stimulates Runx2 and osterix expression in vitro, at least in part, at transcriptional level. Induction of Runx2 mRNA is mediated through the activation of cAMP/PKA signal transduction. In the case of osterix, although the increase in mRNA level is predominantly mediated via cAMP/PKA signaling, PKC activation might also be involved in this process.

CD99 Acts as an Oncosuppressor in Osteosarcoma

CD99 was recently reported to be under control of the osteoblast-specific transcription factor Cbfa1 (RUNX2) in osteoblasts, suggesting a role in the phato-physiology of these cells. No extensive information is available on the role(s) of this molecule in malignant phenotype, and osteosarcoma, in particular, has never been studied. We report that in 11 different cell lines and 17 clinical samples CD99 expression is either undetectable or very low. Being expressed in the normal counterpart, we tested the hypothesis that CD99 down-regulation may have a role in osteosarcoma development and progression. CD99-forced expression in two osteosarcoma cell lines significantly reduced resistance to anoikis, inhibited growth in anchorage independence as well as cell migration, and led to abrogation of tumorigenic and metastatic ability. Therefore, the molecule acts as a potent suppressor of malignancy in osteosarcoma. CD99 gene transfection induces caveolin-1 up-regulation and the two molecules were found to colocalize on the cell surface. Treatment with antisense oligonucleotides to caveolin-1 abrogates the effects of CD99 on migration. The findings point to an antioncogenic role for CD99 in osteosarcoma, likely through the regulation of caveolin-1 and inhibition of c-Src kinase activity.

A New Role for p53

The tumor suppressor p53, which is mutated in many cancers, activates or inhibits transcription of various target genes and thereby initiates apoptosis or cell cycle arrest. Wang et al. found that mice lacking p53 ( p53 –/– mice) showed increased bone mass and bone formation compared with wild-type littermates. Primary cultures of osteoblasts from newborn p53 –/– mice showed accelerated differentiation (assayed by alkaline phosphatase activity, osteocalcin expression, and mineralization) and enhanced proliferation compared with osteoblasts from wild-type mice. Reverse transcription polymerase chain reaction (RT-PCR) analysis of osteoblast-specific transcription factors in p53 –/– osteoblasts revealed a specific increase in osterix expression. Further, an increase in osterix expression was greater in p53 –/– than wild-type mouse embryonic fibroblasts when cells were treated with bone morphogenetic protein 2 (BMP2), whereas osterix expression was decreased in wild-type osteoblasts in which p53 was up-regulated. Coexpression of p53 inhibited transcription of an osterix gene reporter, as did a p53 mutant that lacked DNA binding, whereas p53 mutants that attenuate the ability of p53 to repress the transcription of other target genes (which, in some cases, is independent of DNA binding) did not. Wild-type osteoblasts infected with retroviruses that expressed osterix showed increased alkaline phosphatase activity and osteocalcin expression, whereas osterix knockdown decreased alkaline phosphatase activity and osteocalcin expression in p53 –/– osteoblasts. Osteoblasts synthesize various substances that regulate osteoclast differentiation, including macrophage colony-stimulating factor (M-CSF), and coculture experiments indicated that p53 –/– osteoblasts, which showed increased expression of M-CSF, stimulated the differentiation of osteoclasts from bone marrow monocytes more effectively than did wild-type osteoblasts. Thus, in addition to acting as a tumor suppressor, p53 appears to play a role in regulating osteoblast differentiation, osteoclast differentiation, and the formation of bone. X. Wang, H.-Y. Kua, Y. Hu, K. Guo, Q. Zeng, Q. Wu, H.-H. Ng, G. Karsenty, B. de Crombrugghe, J. Yeh, B. Li, p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling. J. Cell Biol . 172 , 115-125 (2006). [Abstract] [Full Text]

Type II/III Runx2/Cbfa1 is required for tooth germ development

Runx2/Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. Runx2/Cbfa1 knockout mice showed both a complete lack of ossification and the developmental arrest of tooth germ. We here report Runx2/Cbfa1 isoform-type specific functional roles in the development of tooth germ by the administration of antisense phosphorothioate oligodioxynucleotides (S-ODNs) into cultured mouse mandibles. The administration of type II/III Runx2/Cbfa1 antisense S-ODNs into the culture media resulted in an arrest of tooth germ growth at the bud-like stage in cultured mandible taken from the 11-day-old embryos, while also causing the inhibition of the differentiation of odontogenic cells into ameloblast and odontoblast in cultured tooth germs taken from the 15-day-old embryos. The expression of dentin matrix protein 1, dentin sialophosphoprotein, amelogenin, and ameloblastin was shown to be markedly suppressed in cultured tooth germ by the semi-quantitative RT-PCR. Meanwhile, no developmental arrest of tooth germ, no inhibition of gene expression, or differentiation of odontogenic cells was observed in samples treated with the type I Runx2/Cbfa1 antisense S-ODNs. The same findings were also observed in either the control or the sense and random sequence S-ODNs-treated samples. These data indicate that the type II/III Runx2/Cbfa1 isoform is closely related to the development and differentiation of tooth germ.

Big bones in p53 mutants

On page 115, a famous tumor suppressor is uncovered as a suppressor of bone development. Wang et al. show that p53 curbs bone growth by inhibiting osteoblast differentiation and proliferation.Mice deficient in p53 have a high incidence of tumor formation and often die prematurely from lymphomas. Few developmental abnormalities had been found, however. The authors suspected a bone defect might exist, since they had shown that c-Abl, a kinase that interacts with p53, is required for osteoblast differentiation. By looking beyond bone morphology, they indeed found abnormalities in the p53 mutant skeletons. The deficiencies were able to rescue the differentiation defect of mice lacking c-Abl. Figure Osterix mRNA (white) is increased in the bones of p53 mutant mice (right). Mice lacking only p53 had increased bone mass. The effects were due to accelerated differentiation of bone-forming osteoblasts. Mutants also had more osteoblasts, suggesting that p53 slows both differentiation and proliferation of these precursors. The effects are due to increased expression of an osteoblast-specific transcription factor called osterix. p53 has the ability to repress osterix transcription directly, although in this case it did not need to bind to the osterix promoter. It is possible that p53 might sequester the p300 core transcription factor, with which it has been shown to associate, and thereby repress transcription. The bone defects are partially corrected by a negative feedback pathway that increases the number of bone-destroying osteoclasts. The increased osterix in the p53 mutant osteoblasts induced increased M-CSF expression; this M-CSF then induced osteoclast differentiation. Early embryos have high levels of p53, which might help to block premature bone mineralization. The mineralization occurs later, after p53 levels have dropped, but osteoblasts up-regulate p53 again later during differentiation. Its return might put on the brakes before bone formation gets out of hand.

Functional analysis of a novel RUNX2 missense mutation found in a family with cleidocranial dysplasia

Mutations of the RUNX2 gene result in dominantly inherited cleidocranial dysplasia (CCD). RUNX2 encodes for an osteoblast-specific transcription factor, which recognizes specific DNA sequences by the runt domain. DNA binding is stabilized by the interaction with the protein CBFbeta, which induces structural modifications of the runt domain. A novel 574G > A RUNX2 missense mutation has been found in members of a family clinically diagnosed with CCD. This mutation causes the glycine at position 192 to change to arginine (G192R), in loop 9 of the runt domain. Unlike other residues of loop 9, G192 does not establish DNA contacts. Accordingly, the G192R mutant showed a 50% reduction in binding activity compared to the wild-type runt domain. However, the mutation completely abolished the activating properties of the protein on osteocalcin promoter. Moreover, the G192R mutant exerts a dominant-negative effect when overexpressed. Computer modeling indicated that the G192R mutation perturbs not only loop 9, but also other parts of the runt domain, suggesting impairment of the interaction with CBFbeta.

Holoprosencephaly and cleidocranial dysplasia in a patient due to two position‐effect mutations: case report and review of the literature

Holoprosencephaly (HPE) is a genetically heterogeneous developmental field defect in which midline cleavage of the forebrain and craniofacial structures is impaired. Based on the analysis of HPE patients with chromosome rearrangements, at least six loci for the disorder have been assigned. The sonic hedgehog gene (SHH) at 7q36 has been identified as the HPE3 locus. Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal disorder characterized by clavicular, pelvic and dental anomalies. It is caused by mutations in the osteoblast-specific transcription factor CBFA1/RUNX2, which maps to 6p21. We report a 20-year-old female with premaxillary agenesis (part of the HPE spectrum), as well as skeletal abnormalities and impacted teeth reminiscent of CCD. She carries a de novo 6;7 reciprocal translocation, with breakpoints at 6p21.1 and 7q36. We have shown previously that the 7q36 breakpoint maps 15 kb telomeric to the 5' end of SHH, which explains the patient's HPE phenotype. Now, using fluorescence in situ hybridization, we have identified a P1 artificial chromosome clone 800 kb upstream of CBFA1/RUNX2 that spans the 6p breakpoint. We propose that the proband's complex phenotype is due to two position-effect (PE) mutations, one at each translocation breakpoint, which have altered the expression of the SHH and CBFA1/RUNX2 genes. The role of PE mutations in human disease is also reviewed.

Differential Regulation of Dentin Sialophosphoprotein Expression by Runx2 during Odontoblast Cytodifferentiation

Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was down-regulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.

Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC)

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular bone cells showed 17.2% of apoptosis, significantly lower than 24.2% of UBC cells ( p-value = 0.007). With the various zoledronate concentrations, mean apoptotic fractions of trabecular bone cells was 19.2%, significantly lower than 27.8% of UBC cells ( p-value = 0.040). With GGOH co-treatment in various zoledronate concentrations, 15.1% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells ( p-value = 0.076). This data suggests that zoledronate causes apoptosis in both UBC and trabecular bone cells by inhibition of the mevalonate pathway. In addition to the known anti-osteoclastogenic effect of bisphosphonates, the GGOH inhibitory effects of zoledronate were more prominent in UBC cells than trabecular bone cells, indicating their potential therapeutic role in UBC.

Expression of transcription factors Osterix and Core binding factor alpha1 in two differentiating models of primary human osteoblasts in culture

Osterix (OSX) and Core binding factor alpha 1 (Cbfa1) are osteoblast specific transcription factors which activate osteoblast marker genes in vivo during the process of bone formation.

βig-h3 mediates osteoblast adhesion and inhibits differentiation

βig-h3 is an extracellular matrix (ECM) protein induced by TGF-β, and it has motifs interacting with the α3β1, αvβ5, and αvβ3 integrins. Our previous study shows the role of βig-h3 in osteoblast differentiation and its involvement in melorheostosis, a rare bone disease. Here we demonstrate that βig-h3 expression is down-regulated during the early stage of differentiation of the murine preosteoblastic cell line, KS483. The recombinant βig-h3 and its FAS1 domain significantly inhibited in vitro osteoblast differentiation as evaluated by matrix mineralization/bone nodule formation. Furthermore, inhibition of expression of osteoblast differentiation marker genes [such as type I collagen, alkaline phosphatase, and osteocalcin (OC)] was accompanied by suppression of osteoblast-specific transcription factors, Cbfa1/Runx2 and osterix. Flow cytometric analyses, cell adhesion, and inhibition assays disclosed αvβ3 and αvβ5 as the principal integrins mediating the adhesion of osteoblastic cells to βig-h3. The disruption of interactions between βig-h3 and osteoblasts by a function-blocking antibody specific for αvβ3 but not for αvβ5 abolished the inhibitory effect of βig-h3 on osteoblast differentiation. We suggest that these interacting integrins may play an important role in βig-h3-mediated inhibition of osteoblast differentiation.

Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and osterix in ROS 17/2.8 cells

FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor α1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H 2O 2 (100 μM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10 −9 M) and 1,25 (OH) 2D 3 (Vitamin D 3) (10 −7 M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.