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PubMed2002

Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Several genes, such as alkaline phosphatase, osteocalcin, and Cbfa1/Osf2, are known to be regulated during osteoblastic differentiation and are commonly used as "osteoblast markers" for in vitro or in vivo studies. The number of these genes is very limited, however, and it is of major interest to identify new genes that are activated or repressed during the process of osteoblast differentiation and bone formation as well as to extend the available information on gene families relevant to this particular differentiation pathway. To identify such genes, we have implemented a genome-wide analysis by determining changes in expression levels of 27,000 genes during in vitro differentiation of primary osteoblasts isolated from mouse calvaria. This study focuses on the description of the analytical and filtering process applied; on the transcriptional analysis of well-established "bone," "adipocyte," and "muscle" pathway markers; and on a description of the regulation profiles for genes recently described in the Skeletal Gene Database. We also demonstrate that new array technologies constitute reliable and powerful tools to monitor the transcription of genes involved in osteoblastic differentiation, allowing a more integrated vision of the biological pathways regulated during osteoblast commitment, differentiation, and function.

Adipocytes↗

PubMed1996

Phase-Dependent effects of transforming growth factor beta 1 on osteoblastic markers of human osteoblastic cell line sV-HFO during mineralization.

A human osteoblastic cell line (SV-HFO) established in our laboratory expresses osteoblastic markers, including mineralization in vitro, in response to differentiation-inducing agents such as dexamethasone. In this study, we examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the mineralization of SV-HFO cells and show that TGF-beta 1 inhibited the mineralization of the cells via down regulation of tetranectin and alkaline phosphatase without influencing other osteoblastic markers. To examine precisely the effects of TGF-beta 1 on the process of mineralization, we tentatively divided the whole process of mineralization into four phases: induced ALP activity (days 0-5), maximal ALP activity (days 5-10), early mineralization (days 10-15), and progressive mineralization (days 15-20). These inhibitory effects of TGF-beta 1 on the expression of tetranectin and alkaline phosphatase, like that on mineralization, were observed only when TGF-beta 1 was applied in the early phase of the process of mineralization. On the other hand, the other osteoblastic markers were not influenced by treatment with TGF-beta 1. These results suggest that TGF-beta 1 may inhibit mineralization of osteoblasts by the downregulation of tetranectin and alkaline phosphatase expression in the early phase. Thus, TGF-beta 1 has phase-dependent effects on a human osteoblastic cell line during the process of mineralization.

Alkaline Phosphatase↗

PubMed1999

Influence of skeletal site of origin and donor age on 1,25(OH)2D3-induced response of various osteoblastic markers in human osteoblastic cells.

Age-related bone loss may be a consequence of a lack of osteoblastic formation and/or function. In vitro, the osteoblastic response to 1,25(OH)2D3, an important regulator of osteoblastic function, appears to depend on the stage of osteoblastic maturation. In this study, we examined the response to 1,25(OH)2D3 of C-terminal type I procollagen (PICP), alkaline phosphatase (ALP), and osteocalcin (OC) secretion in primary cultures of osteoblastic cells from human trabecular bone (hOB). Forty-four bone samples were obtained from subjects undergoing knee arthroplastia, 20 aged 50-70 (64 +/- 5), and 24 >70 (73 +/- 2) years. Another 33 bone samples were obtained from subjects undergoing hip arthroplastia, 21 were aged 50-70 (64 +/- 4) and 12 >70 (75 +/- 5) years. Pooling knee and hip hOB cell cultures, we found that PICP secretion decreased after 1,25(OH)2D3 in hOB cells from the older group (>70 years). Treatment with 1,25(OH)2D3 increased ALP secretion in these cells only in the younger group (50-70 years), whereas it increased OC secretion in hOB cells in both age groups. By pooling hOB cell cultures from both age groups we found that knee hOB cells increased OC secretion, and decreased PICP secretion, after 1,25(OH)2D3. This metabolite also increased OC secretion in hip hOB cells. Considering the influence of donor age at the same skeletal site, 1,25(OH)2D3 was found to stimulate ALP secretion only in knee hOB cells in the younger group. In contrast, this metabolite decreased ALP secretion in hip hOB cells in the older group. PICP secretion decreased after 1,25(OH)2D3 only in hOB cells in the older group, at both skeletal sites. In age-matched cultures, OC secretion was lower in hip hOB cells compared with those from the knee in the older group, but was similar in these cell cultures from both skeletal sites in the younger group. OC secretion after 1,25(OH)2D3 stimulation did not show age differences in knee hOB cells, but was lower in hip hOB in the older group. In summary, our results demonstrate that the response of various osteoblastic markers to 1,25(OH)2D3 in primary cultures of hOB cells depends on the donor age and skeletal site of origin.

Aged↗

PubMed1999

Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions.

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Amino Acid Sequence↗

PubMed2006

Up-regulation 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.

C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-beta, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading.

Animals↗

PubMed2000

Osteoblast-like cells of the hypophysectomized rat: a model of aberrant osteoblast development.

In a previous work, we demonstrated that the osteoprogenitors derived from the marrow stroma of the hypophysectomized (HX) rat demonstrate enhanced proliferative and differentiation capacities when placed in an optimal microenvironment. In this study, we sought to investigate the potential of the trabecular osteoblast-like cells of the HX rat. These cells represent a more mature pool of osteoblasts than the progenitors derived from the marrow stroma. We examined all three stages of osteoblast development using trabecular osteoblast-like cells derived from age-matched intact rats as a control. Using thymidine incorporation and cell number as indicators of proliferation, we found that these cells, like the osteoprogenitors derived from the HX rat, demonstrate augmented proliferation when placed in culture. Additionally, type I collagen expression remained at significant levels past the end stages of proliferation, at which point it is expected to be downregulated. Matrix maturation markers, such as alkaline phosphatase activity and bone sialoprotein expression, however, were significantly lower than in the controls. Mineralization potential, as measured by mineralized nodule formation, Ca(2+) content, and OPN and OCN expression, was also significantly reduced. Our results have uncovered an aberrant model of osteoblast development in which proliferation is deregulated, resulting in a minimal capacity of these cells to develop into fully differentiated mineralizing osteoblasts.

Animals↗

PubMed2001

1-(5-oxohexyl)-3,7-Dimethylxanthine, a phosphodiesterase inhibitor, activates MAPK cascades and promotes osteoblast differentiation by a mechanism independent of PKA activation (pentoxifylline promotes osteoblast differentiation).

We have investigated the effect of 1-(5-oxohexyl)-3,7-dimethylxanthine or pentoxifylline (PeTx), a nonselective phosphodiesterase inhibitor, on osteoblastic differentiation in vitro by using two mesenchymal cell lines, C3H10T1/2 and C2C12, which are able to acquire the osteoblastic phenotype in the presence of bone morphogenetic protein-2 (BMP-2). PeTx induced the osteoblastic markers, osteocalcin and Osf2/Cbfa1, in C3H10T1/2 and C2C12 cells and enhanced BMP-2-induced expression of osteocalcin, Osf2/Cbfa1, and alkaline phosphatase. This activity was partially attributed to the fact that PeTx is able to enhance BMP-2-induced Smad1 transcriptional activity. Although PeTx clearly stimulates PKA in these cells, neither pretreatment of cells with the PKA inhibitor H89 nor transfection with the specific PKA inhibitor PKI prevented the induction or enhancement of osteoblast markers by PeTx, demonstrating that these effects were independent of PKA activation. On the other hand, PeTx induced the activation of ERK1/2 and p38 kinase pathways independently of the activation of PKA. Selective inhibitors of these MAPK cascades prevented the induction of osteoblastic markers in cells treated with PeTx, suggesting that the activation of these two pathways plays a role in the effect of PeTx on osteoblastic differentiation.

Animals↗

PubMed1993

Relationship between the expression of the gap junction protein and osteoblast phenotype in a human osteoblastic cell line during cell proliferation.

We examined i) the kinds of connexins, component proteins of gap junctions, that are expressed in osteoblasts and ii) the relationship between the expression of gap junctions and osteoblastic phenotype during cell proliferation and after the treatment with 1 alpha,25-dihydroxyvitamin D3. Human osteoblastic cells with (SV-HFO) or without (HFO) transformation by simian virus 40 and mouse osteoblast-like cells (MC3T3-E1) expressed connexin 43 (Cx43), but not Cx26 or Cx32, as revealed by Northern blot analysis and immunocytochemistry. The expression of Cx43 was significantly higher in SV-HFO cells in the confluent phase than in the proliferative phase. Similarly, the expression of alkaline phosphatase (ALP) and osteocalcin in SV-HFO cells in the confluent phase were higher than those in the proliferative phase. On the other hand, treatment of 1 alpha,25-dihydroxy-vitamin D3 did not change the expression of Cx43 in SV-HFO cells, but significantly induced the expression of ALP and osteocalcin. These results showed that the expression of gap junction protein in osteoblastic cells was coupled with cell differentiation in association with the expression of osteoblastic phenotype, but that the connexin expression is regulated in a way different from that of ALP and osteocalcin.

Alkaline Phosphatase↗

PubMed2001

Acidic fibroblast growth factor signaling inhibits peroxynitrite-induced death of osteoblasts and osteoblast precursors.

After trauma injury to the musculoskeletal system, conditions such as ischemia and inflammation involve excess production of superoxide (O2*), nitric oxide (*NO), and their reaction product, peroxynitrite (ONOO-). Exposure of murine osteoblasts and rat-derived primary osteoblast precursors to ONOO- resulted in a dose- and time-dependent delayed cell death that was more characteristic of apoptosis than necrosis. Exposure of both cell populations to ONOO- immediately enhanced phosphorylation and nitration of tyrosine residues within several polypeptides. Treatment of osteoblasts and osteoblast precursors with exogenous acidic fibroblast growth factor (FGF-1) enhanced cellular growth, increased endogenous levels of tyrosine phosphorylation, and significantly induced expression of both osteopontin and osteocalcin messenger RNA (mRNA) as well as osteopontin protein. Pretreatment of both cell populations with exogenous FGF-1 prevented ONOO(-)-mediated death. Cell signaling induced by FGF-1 pretreatment had no major effect of total levels of tyrosine nitration after ONOO- treatment. Collectively, these in vitro efforts show that FGF-1 signaling renders osteoblasts and osteoblast precursors resistant to the cytotoxic effects of ONOO-. Consequently, results presented here predict the therapeutic use of this growth factor for promoting the progression of bone repair mechanisms after fracture trauma.

Animals↗

PubMed2001

Enhanced activity of osteoblast differentiation factor (PEBP2alphaA2/CBFa1) in affected sutural osteoblasts from patients with nonsyndromic craniosynostosis.

OBJECTIVE: Nonsyndromic craniosynostosis is characterized by premature closure of one or more cranial sutures in infants. The purpose of this investigation was to evaluate cellular and molecular events that lead to pathogenesis of nonsyndromic craniosynostosis. DESIGN: This study utilized discarded samples of normal and affected cranial sutures from 12 patients (7 boys, 5 girls) with nonsyndromic craniosynostosis. RESULTS: Histological evaluation of affected sutures revealed complete osseous obliteration instead of a zone of connective tissue and osteogenic cells as seen in normal sutures. Although proliferation of normal and affected osteoblasts did not vary substantially, elevated osteocalcin production and increased in vitro bone nodule formation indicated that the differentiation and the bone-forming potential of affected osteoblasts was significantly higher than that of normal cells. We therefore investigated the levels and activity of Cbfa1, a transcription factor that plays an integral role in osteoblast differentiation. Northern blot analysis of messenger RNA from both normal and affected sutural osteoblasts revealed a twofold increase in the expression of Cbfa1 in affected cells. This increase in the level of Cbfa1 transcript correlated with an increase in its transcriptional activity on the osteocalcin gene promoter, as assessed using gene transfer methods. CONCLUSION: Our results indicated that osteoblasts from synostosed sutures exhibit an increased potential for differentiation and bone formation. The increased level and activity of Cbfa1 could play a vital role in the aberrant function of these affected osteoblasts and may explain their altered behavior compared to the normal cells.

Alkaline Phosphatase↗

PubMed2002

In vitro response of primary rat osteoblasts to titania/hydroxyapatite coatings compared with transformed human osteoblast-like cells.

The biocompatibility of titania/hydroxyapatite (TiO(2)HA) composite coatings, at different ratio obtained by sol-gel process, was investigated studying the behavior of primary cultures of rat osteoblastic cells, isolated by femoral trabecular bone tissue. Moreover, the results have been compared with the response of human osteoblast-like MG63 cell line. Cytotoxicity of coatings was assessed by lactate dehydrogenase activity (LDH). The cellular behavior was analyzed by the cell proliferation (MTT test), cell morphology (SEM) and the biochemical markers evaluation of osteoblastic phenotype, such as alkaline phosphatase activity (ALP) and osteocalcin production. The results showed that TiO(2)/HA coatings have no toxic effects and seemed to be a good support for cell adhesion and proliferation. Moreover, these materials allowed the differentiation of osteoblasts, stimulating the expression of alkaline phosphatase activity. The responses of the primary rat osteoblasts and human osteoblast-like MG63 cell line grown onto these coatings were similar in terms of proliferation and ALP activity. Differences were found considering the osteocalcin production. The results show that these coatings, thanks to their chemical composition and the deposition technique, are very promising for the potential orthopedic and dental applications.

Journal Article↗

PubMed1997

The response of primary rat and human osteoblasts and an immortalized rat osteoblast cell line to orthopaedic materials: comparative sensitivity of several toxicity indices.

When studying the biocompatibility of orthopaedic biomaterials it is often necessary to discriminate between responses which show mild cytotoxicity. It is therefore essential to use a very sensitive index of toxicity. We have compared the sensitivity of four well-established indices of toxicity: total cell protein content, leakage of lactate dehydrogenase (LDH), reduced glutathione content and the MTT assay, with that of a novel index, alkaline phosphatase (ALP) activity. Comparisons were made by detecting nickel chloride toxicity in osteoblasts. ALP activity, the novel method, proved the most sensitive index of toxicity and it provides a convenient automated assay for assessing the interactions of materials with osteoblasts. The responses to nickel chloride and to aqueous extracts prepared from carbon fibre reinforced epoxy and polyetheretherketone (peek), two candidate materials for orthopaedic implants, were compared in primary and immortalized rat osteoblasts, and in primary human osteoblasts. Although the immortalized rat osteoblast cell line, FFC, was consistently the most sensitive cell type, the responses of the human cells and the FFC cell line were similar in terms of ALP activity throughout the range of nickel concentrations studied. Neither peek nor epoxy material extracts showed a significant decrease in the MTT or ALP responses in any of the three cell types. Our data suggest that immortalized rat osteoblasts may provide an in vitro model system for screening the biocompatibility of orthopaedic polymers.

Journal Article↗

PubMed1999

Estradiol formation by human osteoblasts via multiple pathways: relation with osteoblast function.

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.

17-Hydroxysteroid Dehydrogenases↗

PubMed2003

IGF-I and TGF-beta 1 incorporated in a poly(D,L-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture.

Previous in vivo studies revealed a stimulating effect of locally applied IGF-I and TGF-beta1 released from poly(D,L-lactide)-coated titanium implants on rat and porcine fracture healing. The purpose of the present study was to evaluate the effect of IGF-I (5% w/w) and TGF-beta1 (1% w/w) and the carrier PDLLA on osteoblasts in cell culture to improve the understanding of these growth factors. The well-characterized human osteoblast cell line hFOB 1.19 was used in the study. The implants and cells were cocultured in a noncontact manner. The cells were incubated for 10 days in total, and the implants (n = 6 each group and time point) were added for 1 h, 12 h, 24 h, 2 d, 4 d, or 10 d. To analyze a possible effect of the growth factors or the coating, cell proliferation, metabolism, and differentiation were investigated. As an indicator for differentiation the production of collagen I was chosen. All experimental groups showed comparable cell vitality. No change in the pH of the medium was detectable between the analyzed groups. When the effect of the titanium implant and the PDLLA coating were compared with the control culture, no differences in proliferation, metabolic activity, and collagen I production were detectable. The osteoblasts treated with IGF-I and TGF-beta1 released from PDLLA revealed a significantly enhanced collagen I production with a decrease in proliferation and metabolic activity compared to the other groups. No significant differences in collagen I production were seen due to the incubation time points. None of the experimental groups evoked an immunological response on mouse macrophages. In conclusion, the PDLLA-carrier showed no negative effect on osteoblasts, whereas the incorporated growth factors stimulated osteoblast differentiation.

Bone Substitutes↗

PubMed1990

Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

Alkaline Phosphatase↗

PubMed2001

A new protein expressed in bone marrow cells and osteoblasts with implication in osteoblast recruitment.

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail.

Amino Acid Sequence↗

PubMed1990

Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralization: mineralization by betaglycerophosphate in the absence of osteoblasts.

It has been shown in several laboratories that addition of beta-glycerophosphate (beta GP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM beta GP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone explants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from beta GP in all wells. beta GP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in beta GP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed2006

Glucocorticoids induce the differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and osteoblasts, but fail to induce terminal osteoblast differentiation.

To clarify the effects of glucocorticoids (GCs) on osteoblast and adipocyte differentiation, we investigated the effects of dexamethasone (Dex), a GC analogue on transcription factors for osteoblasts (Runx2, Dlx5 and Osterix) and adipocytes (C/EBPs such as C/EBPalpha, C/EBPbeta and C/EBPdelta, and PPARgamma2), late osteoblastic markers, bone sialoprotein (BSP) and osteocalcin (OC), and adipocyte differentiation-dependent protein, aP2 in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). C26 cells were dose- and time-dependently responsive to Dex in terms of an increase in not only mRNA and protein expressions of the C/EBPs, PPARgamma2 and aP2, but also Runx2, Dlx5, BSP and OC with no induction of Osterix, which is considered to act mainly on terminal osteoblast differentiation. Cycloheximide pretreatment indicated that Dex signaling immediately increases expressions of the C/EBPs and Dlx5, while expressions of the rest of the genes require de novo protein synthesis. Continuous Dex treatment stimulated adipocyte formation, but failed to induce Osterix expression and mineralized matrix formation. However, BMP-2 treatment of Dex-treated cells induced Osterix expression and subsequent mineralized matrix formation. These results indicate that Dex up-regulates the C/EBPs followed by increasing PPARgamma2 and aP2 expressions in C26 cells to induce adipocyte differentiation, while Dex enhances Dlx5 followed by increasing Runx2, BSP and OC expressions at gene and protein levels, but cannot induce Osterix expression, suggesting that Dex does not promote their terminal osteoblast differentiation.

Adipocytes↗