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PubMed1992

Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture.

The extracellular matrix may be considered as an insoluble local mediator which plays an important role in regulating cell function. Communication between the cell and its matrix occurs via the integrins, a family of transmembrane proteins composed of non-covalently linked alpha and beta subunits. The aim of this study was to establish which integrins are present on human bone cells in situ and in culture, using cryostat sections of undecalcified human bone, osteoclastoma tissue and cultured human osteoblasts. Integrin subunit expression was identified indirectly using alkaline phosphatase anti-alkaline phosphatase conjugates and FITC-labelled secondary antibodies. Subunits expressed by cultured human osteoblast-like cells were then quantified by FACS analysis. Staining patterns observed in situ show that osteoblasts and osteoclasts possess different integrin subunits. Osteoblasts primarily express alpha 1, alpha 3 and beta 1 and weakly express alpha 2. Osteoclasts express alpha 2, alpha V, beta 1 and beta 3. Subunits alpha 4, alpha 5, alpha 6, alpha L, alpha M and beta 2 were not expressed by either of these cell types. Expression of beta 1 by all cells of the osteoblastic lineage was constitutive, but alpha 1 and alpha 3 subunits were expressed by osteoblasts actively synthesizing bone and some of the osteoblast lining cells. All integrin subunits identified on osteoblasts in situ were maintained on culture but there was an increased expression of alpha 2 and alpha V subunits were weakly positive. Expression of alpha 2, alpha 3, alpha V and beta 1 subunits was independent of cell density but expression of alpha 1 was much greater in confluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Matrix↗

PubMed1992

Tenascin in bone morphogenesis: expression by osteoblasts and cell type-specific expression of splice variants.

The extracellular matrix glycoprotein, tenascin, is associated in vivo with mesenchyme undergoing osteogenesis and chondrogenesis, but is absent from mature bone and cartilage matrix. The expression of tenascin by osteoblastic cells in vitro has been investigated by immunoblotting and immunocytochemistry. Tenascin was secreted into the medium and deposited in the matrix by human and rat osteoblast-like cell lines, as well as by primary osteoblast-enriched cultures from chick embryo calvarial bones. In primary osteoblast-enriched cultures, extracellular tenascin was found only in cell aggregates expressing the osteoblast marker alkaline phosphatase. Chicken osteoblast cultures synthesized almost exclusively the largest tenascin subunit, whereas fibroblast cultures from periostea of chicken calvariae synthesized approximately equal amounts of all three subunits. In situ hybridization studies of developing chicken bones, using a cDNA probe that hybridizes to all chicken tenascin splice variants, showed specific labelling of both osteogenic and chondrogenic regions of developing endochondral bones. In contrast, a cDNA probe specific for the large tenascin splice variant showed specific hybridization in osteogenic but not chondrogenic regions. Within osteogenic regions, tenascin mRNA was expressed by osteoblasts. A comparison of in situ hybridization and immunohistochemical studies demonstrated that tenascin mRNA and protein were codistributed in osteogenic regions of endochondral and membrane bones, whereas protein was retained in regions of differentiating cartilage where mRNA was no longer detectable. The results presented here demonstrate that tenascin is synthesized by osteoblasts. Moreover, within developing bones, there are at least three different cell type-specific patterns of expression of tenascin splice variants.

Animals↗

PubMed1996

Modulation of osteoblast behaviour by tenascin.

The extracellular matrix protein tenascin is secreted by osteoblasts but absent from mineralized bone matrix. The current study was undertaken to test the hypothesis that tenascin regulates osteoblast behaviour. Three osteoblast-like cell lines UMR-106, ROS-17/2.8 (rat) and SAOS-2 (human) were used to investigate the role of tenascin in osteoblast morphology, differentiation and proliferation. Two of three cell lines adhered specifically to tenascin, remaining round and failing to spread. Tenascin as a substratum stimulated alkaline phosphatase activity (a marker of osteoblast differentiation) in two of three cell lines. Moreover, anti-tenascin in the medium caused a reduction in alkaline phosphatase levels in all three cell lines. Anti-tenascin also inhibited collagen synthesis, an important osteoblast function. Since it seemed possible that tenascin may exert its effects on cell function through its ability to cause cell rounding, the ability of cell shape change alone to influence alkaline phosphatase levels was investigated. Cells were incubated in the presence of cytochalasin D and alkaline phosphatase levels assayed. Alkaline phosphatase activity was not elevated by cytochalasin D treatment, indicating that cell rounding alone is insufficient to mimic the effect of tenascin. Anti-tenascin caused a slight increase in proliferation of SAOS-2 cells, indicating that tenascin is itself inhibitory. In ROS 17/2.8 and UMR-106 cells, in contrast, proliferation was inhibited by anti-tenascin. The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenous tenascin helps to maintain the functional state of cultured osteoblast-like cells.

Alkaline Phosphatase↗

PubMed2004

Osteoclast deficiency results in disorganized matrix, reduced mineralization, and abnormal osteoblast behavior in developing bone.

UNLABELLED: Studies of the influence of the osteoclast on bone development, in particular on mineralization and the formation of the highly organized lamellar architecture of cortical bone by osteoblasts, have not been reported. We therefore examined the micro- and ultrastructure of the developing bones of osteoclast-deficient CSF-1R-nullizygous mice (Csf1r(-/-) mice). INTRODUCTION: Colony-stimulating factor-1 receptor (CSF-1R)-mediated signaling is critical for osteoclastogenesis. Consequently, the primary defect in osteopetrotic Csf1r(-/-) mice is severe osteoclast deficiency. Csf1r(-/-) mice therefore represent an ideal model system in which to investigate regulation by the osteoclast of osteoblast-mediated bone formation during development. MATERIALS AND METHODS: Bones of developing Csf1r(-/-) mice and their littermate controls were subjected to X-ray analysis, histological examination by light microscopy and transmission electron microscopy, and a three-point bending assay to test their biomechanical strength. Bone mineralization in embryonic and postnatal bones was visualized by double staining with alcian blue and alizarin red. Bone formation by osteoblasts in these mice was also examined by double-calcein labeling and in femoral anlagen transplantation experiments. RESULTS AND CONCLUSIONS: Frequent spontaneous fractures and decreased strength parameters (ultimate load, yield load, and stiffness) in a three-point bending assay showed the biomechanical weakness of long bones in Csf1r(-/-) mice. Histologically, these bones have an expanded epiphyseal chondrocyte region, a poorly formed cortex with disorganized collagen fibrils, and a severely disturbed matrix structure. The mineralization of their bone matrix at secondary sites of ossification is significantly reduced. While individual osteoblasts in Csf1r(-/-) mice have preserved their typical ultrastructure and matrix depositing activity, the layered organization of osteoblasts on the bone-forming surface and the direction of their matrix deposition toward the bone surface have been lost, resulting in their abnormal entrapment by matrix. Moreover, we also found that (1) osteoblasts do not express CSF-1R, (2) the bone defects in Csf1r(-/-) embryos develop later than the development of osteoclasts in normal embryos, and (3) the transplanted Csf1r(-/-) femoral anlagen develop normally in the presence of wildtype osteoclasts. These results suggest that the dramatic bone defects in Csf1r(-/-) mice are caused by a deficiency of the osteoclast-mediated regulation of osteoblasts and that the osteoclast plays an important role in regulating osteoblastic bone formation during development, in particular, in the formation of lamellar bone.

Animals↗

PubMed2005

Histone deacetylase inhibitors promote osteoblast maturation.

UNLABELLED: HDIs are potential therapeutic agents for cancer and neurological diseases because of their abilities to alter gene expression, induce growth arrest or apoptosis of tumors cells, and stimulate differentiation. In this report, we show that several HDIs promote osteoblast maturation in vitro and in calvarial organ cultures. INTRODUCTION: Histone deacetylase inhibitors (HDIs) are currently in phase I and II clinical trials as anticancer agents. Some HDIs are also commonly prescribed treatments for epilepsy and bipolar disorders. Although administered systemically, the effects of HDIs on osteoblasts and bone formation have not been extensively examined. In this study, we investigated the effect of histone deacetylase inhibition on osteoblast proliferation and differentiation. MATERIALS AND METHODS: MC3T3-E1 cells, calvarial-derived primary osteoblasts, and calvarial organ cultures were treated with various commercially available HDIs (trichostatin A [TSA], sodium butyrate [NaB], valproic acid [VPA], or MS-275). The effects of these inhibitors on cell proliferation, viability, cell cycle progression, Runx2 transcriptional activity, alkaline phosphatase production, and matrix mineralization were determined. Expression levels of osteoblast maturation genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in response to TSA were measured by quantitative PCR. RESULTS: Concentrations of HDIs that caused hyperacetylation of histone H3 induced transient increases in osteoblast proliferation and viability but did not alter cell cycle profiles. These concentrations of HDIs also increased the transcriptional activity of Runx2. TSA accelerated alkaline phosphatase production in MC3T3-E1 cells and calvarial organ cultures. In addition, TSA accelerated matrix mineralization and the expression of osteoblast genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in MC3T3-E1 cells. CONCLUSIONS: These studies show that histone deacetylase activity regulates osteoblast differentiation and bone formation at least in part by enhancing Runx2-dependent transcriptional activation. Therefore, HDIs are a potentially new class of bone anabolic agents that may be useful in the treatment of diseases that are associated with bone loss such as osteoporosis and cancer.

Acetylation↗

PubMed1997

Parathyroid hormone up-regulation of connexin 43 gene expression in osteoblasts depends on cell phenotype.

Accumulating evidence indicates that gap junctions, primarily composed of connexin 43 (Cx43), are distributed extensively throughout bone. We have previously reported that in osteoblastic cells parathyroid hormone (PTH) increases both the steady-state levels of transcripts for Cx43 and gap-junctional intercellular communication in a process involving cyclic adenosine monophosphate (cAMP). We now present data showing that the mechanism of stimulation of Cx43 gene expression by PTH involves an increased rate of Cx43 gene transcription without affecting Cx43 transcript stability in UMR 106 osteoblastic cells. Activation of the protein kinase C pathway is not involved in this process. Inhibiting translation consistently decreases the PTH-mediated stimulation of Cx43 gene expression at all the times we tested (1-3 h). However, this effect is only partial, demonstrating that de novo protein synthesis is required for full stimulation. PTH increases the steady-state levels of Cx43 mRNA in several osteoblastic cell lines, albeit to different levels. We were unable to detect PTH stimulation in ROS 17/2.8 osteoblastic cells, suggesting that the effect of PTH on Cx43 gene expression may depend on the developmental state of the cell along the osteoblastic differentiation pathway. In the MC3T3-E1 preosteoblastic cell line, we find that PTH increases Cx43 gene expression in proliferating and maturing osteoblastic cells, but not in nondividing, differentiated osteoblasts, where the basal level of Cx43 gene expression is elevated. Unlike PTH, the osteotropic hormones 1,25-dihydroxyvitamin D3 and 17beta-estradiol do not appear to affect Cx43 gene expression in UMR 106 osteoblastic cells.

3T3 Cells↗

PubMed1999

A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

Animals↗

PubMed1999

Thy-1 antigen expression by cells in the osteoblast lineage.

Identification of surface markers involved in osteoblast differentiation provides a method to isolate osteoblasts at various stages of maturation. In this study, we examined expression of the T lymphocyte differentiation antigen, Thy-1, by osteoblastic cells from different species. Murine skeletal progenitor, neonatal calvarial, and adult bone cells (ABCs) were selected to represent osteoblasts at distinct stages of maturation. Flow cytometric analysis showed that Thy-1 expression was undetectable on the progenitor cells (mouse limb bud clones 14 and 17), appeared on calvarial cells (45%+), and was decreased on ABCs (< 10%+). Thy-1 was also detected in situ on osteoblastic cells in mouse calvariae. Thy-1 mRNA expression correlated with cell surface expression. Antigen expression was markedly increased during the cells' proliferative phase in culture. Furthermore, examination of primary rat and human osteoblast-like cells revealed that significant levels of Thy-1 were also expressed on those cells derived from subconfluent culture. This study indicates that osteoblasts express Thy-1 antigen and that its expression is maximal at their earliest stage of maturation, during the proliferative phase, and then declines as the cells mature. In a role similar to the one it plays in the hematopoietic system, Thy-1 antigen may be useful as a differentiation marker in following the development of the osteoblast.

Animals↗

PubMed1999

Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines.

Whether the known calcium-sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti-CasR antisera (4637) and low stringency reverse transcriptase-polymerase chain reaction (RT-PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti-CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT-PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a approximately 165 kDa and approximately 140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild-type HEK 293 cells or Saos2, MG-63, or U-2 OS osteoblast-like cell lines. Standard two-step RT-PCR using CasR-specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT-PCR procedure amplified nonspecific products in wild-type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT-PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.

Amino Acid Sequence↗

PubMed2000

Human myeloma cells promote the recruitment of osteoblast precursors: mediation by interleukin-6 and soluble interleukin-6 receptor.

Multiple myeloma is associated with the development of osteolytic bone disease characterized by a disruption to normal bone resorption and bone formation. Although studies have shown that myeloma cells produce factors that promote bone resorption little data are available examining the mechanism of decreased bone formation or the factors that mediate this effect. In the present study we describe a novel in vitro coculture system in which to investigate the effect of myeloma cells on osteoblast recruitment and differentiation. Under appropriate conditions mesenchymal stem cells were shown to differentiate into colonies of cells, a proportion of which show characteristics of osteoblasts, in that they express alkaline phosphatase activity and stain positively for collagen and calcium. The addition of the human myeloma cells JJN-3, RPMI-8226, or NCI-H929 to these cultures stimulated a significant increase in the total number of colonies (p < 0.005) and the proportion of osteoblastic colonies (p < 0.005). Media conditioned by these cells also were able to promote the formation of both total and osteoblastic colonies (p < 0.005). The addition of an antibody against the interleukin-6 receptor (IL-6R) blocked myeloma cell and myeloma cell-conditioned media induced osteoblast recruitment (p < 0.01). Furthermore, media conditioned by myeloma cells incubated with phorbol ester, which promotes IL-6R shedding, or a metalloproteinase inhibitor, which inhibits IL-6R shedding, were able to stimulate (p < 0.005) and inhibit osteoblast recruitment (p < 0.005), respectively. In addition, soluble IL-6R (sIL-6R) and IL-6 together, but not alone, were able to promote osteoblastic colony formation (p < 0.01). Taken together these data show that myeloma cells promote osteoblast recruitment by release of sIL-6R from myeloma cells.

Alkaline Phosphatase↗

PubMed2000

The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

Animals↗

PubMed2001

Focal adhesion kinase activity is required for bone morphogenetic protein--Smad1 signaling and osteoblastic differentiation in murine MC3T3-E1 cells.

Cell-matrix interactions via integrins are essential for osteoblastic differentiation. We have shown that signals activated by aggregation of alpha2beta1-integrin with type I collagen are involved in the differentiation of osteoblastic MC3T3-E1 cells. Focal adhesion kinase (FAK) is an immediate downstream signal of the beta1-integrin, and inactivation of FAK has been shown to disrupt osteoblastic differentiation. To elucidate roles of FAK in osteoblastic cells, we examined MC3T3-E1 cells stably expressing antisense FAK (asFAK) messenger RNA (mRNA). Alkaline phosphatase (ALP) activity, an osteoblastic marker, did not increase in asFAK cells with a long-term culture until 21 days or in response to bone morphogenetic protein 2 (BMP-2). Treatment with BMP-2 also failed to stimulate the expression of osteocalcin in asFAK cells. In control MC3T3-E1 cells, BMP-2 induced translocation of Smad1 into nuclei to stimulate transcriptional activity of the Smad6 promoter gene that contains a Smad1 response element. In contrast, BMP-2 failed to increase transcriptional activity of Smad6 promoter gene in asFAK cells, although it induced nuclear translocation of Smad1. These results indicate that FAK was involved in Smad1-dependent transcriptional activity but not in nuclear translocation of Smad1 in osteoblastic cells. Hence, FAK activation by integrins might converge transcriptional activation by BMP of its target genes in osteoblastic cells. These observations suggest that FAK activity is essential for BMP-Smad signaling to stimulate osteoblastic differentiation.

Actin Cytoskeleton↗

PubMed2001

Endogenous prostaglandin E2 and insulin-like growth factor 1 can modulate the levels of parathyroid hormone receptor in human osteoarthritic osteoblasts.

Subchondral bone sclerosis may be important for the onset and/or progression of cartilage loss/damage in human osteoarthritis (OA). OA osteoblasts are resistant to parathyroid hormone (PTH) stimulation, which could explain bone sclerosis via the inhibition of PTH-dependent catabolism. Here, we investigated the molecular mechanism(s) responsible for reduced PTH-dependent cyclic adenosine monophosphate (cAMP) synthesis in OA subchondral osteoblasts. Although cholera toxin (CTX) increased basal cAMP formation in these cells, it failed to stimulate PTH-dependent cAMP synthesis, whereas pertussis toxin (PTX) did not inhibit basal cAMP, yet diminished PTH-dependent cAMP production. Binding of 125I-PTH indicated lower PTH receptor levels in OA than in normal osteoblasts (-50.5 +/- 9.5%). This could be attributed to either reduced expression of the PTH receptor (PTH-R) or altered recycling of existing pools of receptors. Reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated decreased PTH-R messenger RNA (mRNA) levels in OA cells that were highly variable (ranging from -10% to -60%), a situation that reflects disease severity. Interestingly, OA osteoblasts produced more prostaglandin E2 (PGE2) than normal osteoblasts, and using naproxen, a cyclo-oxygenase inhibitor, increased PTH-dependent cAMP formation to a level similar to normal osteoblasts. Because heterologous desensitization can explain a decrease in PTH binding but cannot account for reduced PTH-R expression, we looked at the possible effect of insulin-like growth factor 1 (IGF-1) on this parameter. Blocking IGF-1 signaling with a neutralizing receptor antibody increased 125I-PTH binding in both normal and OA osteoblasts. Conversely, treatments with IGF-1 receptor (IGF-1R) antibody only slightly increased the levels of PTH-R mRNA whereas the addition of IGF-1 significantly reduced PTH-R mRNA levels (-24.1 +/- 7.1%), yet neither PGE2 nor naproxen modified PTH-R levels. These results suggest that both IGF-1 signaling and PGE2 formation repress PTH-dependent response in OA osteoblasts, a situation that can contribute to abnormal bone remodeling and bone sclerosis in OA.

Aged↗

PubMed2001

Role of N-cadherin and protein kinase C in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in Apert craniosynostosis.

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N- and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N- and E-cadherin, but not N-cell adhesion molecule (N-CAM) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and alpha- and beta-catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase Cgamma (PLCgamma) and protein kinase Calpha (PKCalpha) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKCalpha-specific inhibitor Gö6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP, COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKCalpha signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.

Acrocephalosyndactylia↗

PubMed2002

Cell-type-dependent up-regulation of in vitro mineralization after overexpression of the osteoblast-specific transcription factor Runx2/Cbfal.

Functional expression of the transcriptional activator Runx2/Cbfal is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.

3T3 Cells↗

PubMed2003

Calcyclin mediates serum response element (SRE) activation by an osteoblastic extracellular cation-sensing mechanism.

UNLABELLED: The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression-cloning strategy, the calcium-binding protein calcyclin was found to mediate the response of MC3T3-E1 osteoblasts to extracellular cations, but not the calcimimetic NPS-568, indicating the presence of another cation-sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation. INTRODUCTION: Extracellular calcium and other cations seem to regulate the function of osteoblasts through a distinct calcium-sensing mechanism that is coupled to activation of c-fos gene transcription. The identity of this calcium-sensing mechanism is unknown. METHODS: To identify molecules that participate in this extracellular cation-sensing pathway, we developed an expression cloning strategy in COS-7 cells using cation stimulation of a serum response element (SRE) luciferase reporter derived from the c-fos promoter to screen a mouse MC3T3-E1 osteoblast cDNA library. RESULTS AND CONCLUSIONS: We identified calcyclin (S100A6), a calcium-binding protein of the EF-hand type belonging to the S100 family, as being responsible for transferring a cation-sensing response from osteoblasts to COS-7 cells. Transfection of the calcyclin cDNA into COS-7 and HEK-293 cells confirmed that the overexpression of calcylin caused these cells to gain the ability to sense extracellular cations, including aluminum, gadolinium, calcium, and magnesium. Conversely, we found that an antisense calcyclin construct reduced calcyclin expression and partially inhibited the cation-sensing response in MC3T3-E1 osteoblasts. These results implicate calcyclin in the activation of SRE and establish a role for calcyclin as an accessory protein involved in the cation-sensing pathway in osteoblasts.

Amino Acid Sequence↗

PubMed2003

Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells.

Runx2/Cbfa1 has been identified as a "master gene" controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2-15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2-15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2-15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.

Alkaline Phosphatase↗

PubMed2003

Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3T3-E1 osteoblast cultures.

UNLABELLED: The anti-glucocorticoid potential of BMP-2 in osteoblasts was tested in MC3T3-E1 cells using dexamethasone (1 microM) and rhBMP-2 (10 or 100 ng/ml). rhBMP-2 restored mineralization but not condensation or collagen accumulation. These results demonstrate the potential and limitations of BMPs in counteracting glucocorticoids. INTRODUCTION: Pharmacologic glucocorticoids (GCs) inhibit osteoblast function and induce osteoporosis. Bone morphogenetic proteins (BMPs) stimulate osteoblast differentiation and bone formation. Here we tested the anti-glucocorticoid potential of BMP-2 in cultured osteoblasts. MATERIALS AND METHODS: MC3T3-E1 cells were treated with dexamethasone (DEX; 1 microM) and/or recombinant human BMP-2 (rhBMP-2; 10 or 100 ng/ml). Culture progression was characterized by cell cycle profiling, biochemical assays for DNA, alkaline phosphatase (ALP), collagen, and calcium, and by reverse transcriptase-polymerase chain reaction (RT-PCR) of osteoblast phenotypic mRNAs. Mineralization was characterized by Alizarin red and von Kossa staining and by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). RESULTS: DEX inhibited differentiation-related cell cycle, nodule formation, collagen accumulation, osteocalcin, and BMP-2 gene expression as well as mineralization. Replenishment of GC-inhibited cultures with 10 or 100 ng/ml rhBMP-2 dramatically rescued mineral deposition. The rhBMP-2-rescued mineral was bone-like apatite nearly identical to the mineral of control cultures. The rhBMP-2 rescue was associated with increased mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. In contrast, rhBMP-2 did not rescue the GC-inhibited differentiation-related cell cycle, nodule formation, or collagen accumulation. When administered alone, rhBMP-2 also increased the mRNA levels for alpha1(I) collagen, osteocalcin, and Cbfa1 types I and II, as well as ALP activity. However, treatment with rhBMP-2 alone inhibited cell cycle progression, nodule formation, and collagen accumulation. Surprisingly, in contrast to its rescue of mineralization in DEX-treated cultures, rhBMP-2 inhibited mineralization in the absence of DEX. In parallel to its bimodal effect on mineralization, rhBMP-2 stimulated endogenous BMP-2 mRNA in the presence of DEX, but inhibited endogenous BMP-2 mRNA in the absence of DEX. CONCLUSIONS: Suppression of BMP-2 gene expression plays a pivotal role in GC inhibition of osteoblast differentiation. However, the inability of rhBMP-2 to rescue the entire osteoblast phenotype suggests BMP-2-independent inhibitory effects of CCs. BMP-2 exerts both positive and negative effects on osteoblasts, possibly depending on the differentiation stage and/or the existing BMP signaling.

Animals↗