Osteoblast-specific targeting of soluble colony-stimulating factor-1 increases cortical bone thickness in mice.

UNLABELLED: The soluble and membrane-bound forms of CSF-1 are synthesized by osteoblasts and stromal cells in the bone microenvironment. Transgenic mice, generated to selectively express sCSF-1 in bone, showed increased cortical thickness in the femoral diaphysis caused by new bone formation along the endosteal surface. The ability of sCSF-1 to enhance bone cell activity in vivo is potentially relevant for increasing cortical bone in a variety of disorders. INTRODUCTION: The soluble form of colony-stimulating factor-1 (sCSF-1) and the membrane-bound form of CSF-1 (mCSF-1) have been shown to support osteoclastogenesis in vitro; however, the effect of each peptide on bone remodeling in vivo is unclear. To determine the effect of sCSF-1, selectively expressed in bone, the skeletal phenotype of transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter was assessed. METHODS: At 5 and 14 weeks, mice were analyzed for CSF-1 protein levels, weighed, and X-rayed, and femurs were removed for peripheral quantitative computed tomography, histology, and histomorphometry. RESULTS: High levels of human sCSF-1 were detected in bone extracts and, to a lesser extent, in plasma. Adult transgenic mice showed normal body weight and increased circulating monocytic cells. At 5 weeks, the femoral diaphysis was similar in CSF-1T and wt/wt littermates. However, by 14 weeks, the femoral diaphysis in CSF-1T mice showed increased cortical thickness and bone mineral density. In contrast to the diaphysis, the femoral metaphysis of CSF-1T mice showed normal cancellous bone comparable with wt/wt littermates at each time point. Histological sections demonstrated increased woven bone along the endosteal surface of the diaphysis and intracortical remodeling. Fluorochrome-labeling analysis confirmed endocortical bone formation in CSF-1T, with a 3.1-fold increase in the percentage of double-labeled surfaces and a 3.6-fold increase in the bone formation rate compared with wt/wt mice. Although remodeling resulted in a slightly porous cortex, sCSF-1 preferentially stimulated endocortical bone formation, leading to increased cortical thickness. CONCLUSIONS: These findings indicate that sCSF-1 is a key determinant of bone cell activity in the corticoendosteal envelope.

Rescue of the osteopetrotic defect in op/op mice by osteoblast-specific targeting of soluble colony-stimulating factor-1.

Soluble colony-stimulating factor-1 (sCSF-1) and membrane bound CSF-1 are synthesized by osteoblasts and stromal cells. However, the precise role of each form in osteoclastogenesis is unclear. In the op/op mouse, absence of osteoblast-derived CSF-1 leads to decreased osteoclasts and osteopetrosis. To determine whether sCSF-1 gene replacement can cure the osteopetrotic defect, we took advantage of the osteoblast specificity of the osteocalcin promoter to selectively express sCSF-1 in the bone of op/op mice. Transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter were generated and cross-bred with heterozygous op/wt mice to establish op/op mutants expressing the transgene (op/opT). The op/op genotype and transgene expression were confirmed by PCR and Southern blot analysis, respectively. High levels of human sCSF-1 protein were selectively expressed in bone. At 2(1/2) wk, op/opT mice showed normal growth and tooth eruption. Femurs removed at 5 and 14 wk were analyzed by peripheral quantitative computed tomography and histomorphometry. The abnormal bone mineral density, cancellous bone volume, and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk, and this effect persisted at 14 wk, with measurements comparable with wt/wt mice at each time point. Correction of the skeletal abnormalities in the 5-wk-old op/opT mice correlated with a marked increase in the total osteoclast number, and their number per millimeter of bone surface compared with that of op/op mutants. Osteoclast number was maintained at 14 wk in op/opT mice and morphologically resembled wt/wt osteoclasts. These results indicate that sCSF-1 is sufficient to drive normal osteoclast development and that the osteocalcin promoter provides an efficient tool for delivery of exogenous genes to the bone. Moreover, targeting sCSF-1 to osteoblasts in the bone microenvironment may be a potentially useful therapeutic modality for treating bone disorders.

Ceramide blocks PDGF-induced DNA synthesis in mesangial cells via inhibition of Akt kinase in the absence of apoptosis.

The mechanism of action of ceramide in glomerular mesangial cells has not been studied. We investigated the effect of C2 ceramide on the mitogenic signal transduction pathways induced by PDGF in mesangial cells. Increasing concentrations of C2 ceramide inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 15 microM. This inhibition of DNA synthesis was associated with attenuation of PDGF-induced early response gene c-fos transcription. PDGF receptor beta immunecomplex kinase assay showed no inhibitory effect of C2 ceramide on PDGF receptor tyrosine kinase activity. We have recently shown that the mitogenic effect of PDGF is mediated by the enzyme phosphatidylinositol (PI) 3 kinase in mesangial cells. C2 ceramide had no effect on PDGF-induced PDGFR-associated PI 3 kinase activity. These data indicate that inhibitory effect of C2 on PDGF-induced DNA synthesis is likely due to post-receptor and post-PI 3 kinase events. To address the mechanism of C2-mediated inhibition of DNA synthesis, we investigated the downstream target of PI 3 kinase, Akt. PDGF time-dependently increased Akt kinase activity in a PI 3 kinase-dependent manner. Incubation of mesangial cells with C2 ceramide inhibited PDGF-induced Akt activity. Akt kinase inhibits apoptosis of cells via phosphorylation of multiple proapoptotic proteins. However, inhibition of Akt activity by C2 ceramide did not induce apoptosis in mesangial cells. These data provide the first evidence that in mesangial cells, ceramide cross-talks with PI 3 kinase-dependent Akt kinase to inhibit PDGF-induced DNA synthesis without inducing apoptosis.

Autoregulation of mouse BMP-2 gene transcription is directed by the proximal promoter element.

Bone morphogenetic protein-2 (BMP-2) stimulates the commitment and differentiation of precursor mesenchymal cells to mature bone. We have isolated and sequenced 2712 base pairs (bp) of the 5' flanking region of mouse BMP-2 gene. Using RNase protection assay we identified two transcription initiation sites within this 2712 bp region of the BMP-2 gene. The distal start site was mapped to -736 bp in relation to the proximal start site (+1). Recombinant BMP-2 preferentially stimulated transcription initiation from the proximal start site. To investigate the mechanism of transcription initiation from these two start sites, we identified two promoter elements upstream of the proximal and distal transcription initiation sites. Transfection of promoter-luciferase reporter constructs into cells of different organs demonstrated differential transcriptional activity of proximal and distal promoters, with highest activity in the osteoblast cell lineage. In osteoblasts, BMP-2 stimulated transcription from the proximal promoter only. Together our data provide the first evidence for the presence of two transcription initiation sites with two upstream promoter elements in mouse BMP-2 gene. Furthermore, we demonstrate for the first time that BMP-2 autoregulates its expression in osteoblasts through the proximal promoter-dependent transcriptional mechanism.

Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells.

The biologic effects and mechanisms by which bone morphogenetic proteins (BMPs) function in breast cancer cells are not well defined. A member of this family of growth and differentiation factors, BMP-2, inhibited both basal and estradiol-induced growth of MCF-7 breast tumor cells in culture. Flow cytometric analysis showed that in the presence of BMP-2, 62% and 45% of estradiol-stimulated MCF-7 cells progressed to S-phase at 24 h and 48 h, respectively. Estradiol mediates growth of human breast cancer cells by stimulating cyclins and cyclin-dependent kinases (CDKs). BMP-2 significantly increased the level of the cyclin kinase inhibitor, p21, which in turn associated with and inactivated cyclin D1. BMP-2 inhibited estradiol-induced cyclin D1-associated kinase activity. Also estradiol-induced CDK2 activity was inhibited by BMP-2. This inhibition of CDK activity resulted in hypophosphorylation of retinoblastoma protein thus keeping it in its active form. These data provide the first evidence by which BMP-2 inhibits estradiol-induced proliferation of human breast cancer cells.

Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation.

Bone morphogenetic protein-2 (BMP-2) has been shown to act as an antiproliferative agent for a number of different cell types. We show that BMP-2 dose-dependently inhibits growth of MDA MB 231 human breast cancer cells. Epidermal growth factor (EGF) stimulates DNA synthesis and entry of these cells into the S-phase. BMP-2 inhibits EGF-induced DNA synthesis by arresting them in G1 phase of the cell cycle. BMP-2 increases the level of cyclin kinase inhibitor p21. Furthermore, we show that exposure of MDA MB 231 cells to BMP-2 stimulates association of p21 with cyclin D1 and with cyclin E resulting in the inhibition of their associated kinase activities. Finally, BMP-2 treatment is found to cause hypophosphorylation of the retinoblastoma protein (pRb), a key regulator of cell cycle progression. Our data provide a mechanism for the antiproliferative effect of BMP-2 in the breast cancer cells.

Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line.

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Evaluation of 2 novel approaches for assessing the ability of demineralized freeze-dried bone allograft to induce new bone formation.

BACKGROUND: Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro. METHODS: We characterized the response of 2T9 cells, an immature osteoprogenitor cell line derived from the calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone morphogenetic protein (BMP)-2 promoter, to recombinant human BMP-2 by measuring alkaline phosphatase specific activity, osteocalcin production, and matrix mineralization. Responses were compared to those obtained with 1,25-(OH)2D3. In addition, 2T9 cells were cultured with active or inactive human DFDBA in the presence or absence of BMP-2. We also tested the hypothesis that radio-opacity of tissue following implantation of DFDBA in vivo correlates with the ability of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by age, were implanted subcutaneously in the thorax of 18 nude (nu/nu) mice. Tissue was harvested at 36 days postoperatively and examined histologically and biochemically for calcium and phosphorus uptake. RESULTS: 2T9 cells exhibited a dose- and time-dependent response to soluble BMP-2. Proliferation was decreased and alkaline phosphatase activity, osteocalcin production, and mineralized nodule formation were increased. The effects were dose- and time-dependent. Peak effects on alkaline phosphatase and osteocalcin were noted on day 8, whereas mineral deposition did not begin to occur until day 12. 1,25-(OH)2D3 did not regulate these effects unless used with BMP-2. When the cells were exposed to active or inactive DFDBA in the presence or absence of BMP-2, no effect on 2T9 cell differentiation was observed. This indicated that DFDBA released no soluble factors with bone inductive ability and that if any active factors were adsorbed to the DFDBA, they were inactivated. When DFDBA was implanted subcutaneously in the thorax of nude mice, there was no histologic evidence of new bone formation. However, there was a donor age-dependent decrease in Ca and P uptake of the implanted tissue, reflecting a donor age-dependent decrease in remineralization of DFDBA. CONCLUSIONS: These data indicate that cell culture assays like the one used in this study may not be appropriate indicators of bone induction ability by DFDBA since soluble factors may not be responsible for bone induction in vivo. Nonetheless, in vitro assays are still needed. While Ca and P uptake by DFDBA-implanted tissue in the present study correlated with the age-dependent decrease in bone induction at intramuscular sites in a previously reported study, these data show that early x-rays may actually detect remineralization and not new bone formation. Thus, assessment of bone induction ability may still depend on histologic analysis of animal models.

Bone morphogenetic protein-2 inhibits MAPK-dependent Elk-1 transactivation and DNA synthesis induced by EGF in mesangial cells.

Bone morphogenetic protein-2 (BMP-2) is a member of the TGFbeta superfamily of growth and differentiation factors. We investigated the effect of BMP-2 on epidermal growth factor (EGF)-induced mitogenic signaling in kidney glomerular mesangial cells. BMP-2 dose-dependently inhibits EGF-induced DNA synthesis. Maximum effect was obtained at a concentration of 100 ng/ml. BMP-2 had no inhibitory effect on the EGF receptor (EGFR)-associated tyrosine kinase activity indicating that inhibition of DNA synthesis is due to regulation of post-receptor signaling event(s). EGF stimulates MAPK activity in mesangial cells in a time-dependent manner. Inhibition of MAPK by the MEK inhibitor PD098059 blocks EGF-induced DNA synthesis indicating the requirement of this enzyme activity in EGF-mediated mitogenic signaling. Furthermore, we show that exposure of mesangial cells to BMP-2 blocks EGF-induced MAPK activity which leads to phosphorylattion of Elk-1 transcription factor. Using a GAL-4 DNA binding-domain-Elk-1 transactivation domain fusion protein-based reporter assay, we demonstrate that BMP-2 inhibits EGF-induced Elk-1-mediated transcription. These data provide the first evidence that BMP-2 signaling in mesangial cells initiates a negative regulatory cross-talk with MAPK-based transcription to inhibit EGF-induced DNA synthesis.

Bone morphogenetic protein 2 inhibits platelet-derived growth factor-induced c-fos gene transcription and DNA synthesis in mesangial cells. Involvement of mitogen-activated protein kinase.

Bone morphogenetic proteins (BMPs) play an important role in nephrogenesis. The biologic effect and mechanism of action of these proteins in the adult kidney has not yet been studied. We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells in vitro and in vivo. Incubation of mesangial cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF receptor beta immunoprecipitates from lysates of mesangial cells treated with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine phosphorylation. This indicates that the inhibition of DNA synthesis is likely due to postreceptor events. However, BMP2 significantly inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK) activity that phosphorylates the Elk-1 transcription factor, a component of the ternary complex factor. Using a fusion protein-based reporter assay, we also show that BMP2 blocks PDGF-induced Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2 inhibits PDGF-induced transcription of c-fos gene, a natural target of Elk-1 that normally forms a ternary complex that activates the serum response element of the c-fos gene. These data provide the first evidence that in mesangial cells, BMP2 signaling cross-talks with MAPK-based transcriptional events to inhibit PDGF-induced DNA synthesis. One target for this inhibition is the early response gene c-fos.

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor.

PDGF stimulates tyrosine phosphorylation of Janus kinase 1 (JAK1) and the signal transducer and activator of transcription 1 (STAT1alpha). However, it is not known whether JAKs are required for STAT1alpha phosphorylation or if the PDGF receptor itself can directly tyrosine phosphorylate and activate STAT1alpha. In vitro immunecomplex kinase assay of PDGF beta receptor (PDGFR) or STAT1alpha immunoprecipitates from lysates of mesangial cells treated with PDGF showed phosphorylation of a 91- and an 185-kD protein. Incubation of lysates prepared from quiescent mesangial cells with purified PDGFR resulted in STAT1alpha activation. Immunodepletion of Janus kinases from the cell lysate before incubation with the purified PDGFR showed no effect on STAT1alpha activation. Moreover, lysates from mesangial cells treated with JAK2 inhibitor, retained significant STAT1alpha activity. To confirm that STAT1alpha is a substrate for PDGFR, STAT1alpha protein was prepared by in vitro transcription and translation. The addition of purified PDGFR to the translated STAT1alpha resulted in its phosphorylation. This in vitro phosphorylated and activated protein also forms a specific protein-DNA complex. Dimerization of the translated STAT1alpha protein was also required for its DNA binding. Incubation of pure STAT1alpha with autophosphorylated PDGFR resulted in physical association of the two proteins. These data indicate that activated PDGFR may be sufficient to tyrosine phosphorylate and thus directly activate STAT1alpha.

Lovastatin induces apoptosis by inhibiting mitotic and post-mitotic events in cultured mesangial cells.

Lovastatin, an inhibitor of protein prenylation, was reported to inhibit DNA synthesis and induce apoptosis in cultured cells. This report describes the morphological consequences of lovastatin treatment. Lovastatin (50 microM) induced mesangial cell rounding and disassembly of actin stress fibers within 24 to 48 h. After 48 to 72 h of lovastatin treatment, the cells detached from the substratum and underwent apoptotic cell death as evidenced by condensed nuclear chromatin, nuclear fragmentation, cell blebbing and decrease in cell size. Time lapse cinematography revealed that lovastatin caused cell rounding by either inhibiting cytokinesis or cell spreading following cytokinesis. Lovastatin-induced cell rounding, detachment, and apoptosis were dependent upon cell proliferation. These effects were prevented by serum deprivation to inhibit cell proliferation or by plating cells at densities which resulted in contact inhibition of cell growth. Lovastatin-induced mesangial cell rounding and apoptosis were also prevented by the inclusion of the isoprenoids all-trans-farnesol or all-trans-geranylgeraniol in the incubation medium. These results indicate that the effects of lovastatin were mediated by inhibition of protein isoprenylation because exogenous all-trans-geranylgeraniol can be used only in protein prenylation. The small GTP-binding protein RhoA, which may be important for cell spreading and cytokinesis, accumulated in the cytosol following treatment with lovastatin, suggestive of its inactivation. This effect was also prevented by the inclusion of either farnesol or geranylgeraniol in the incubation medium. Thus, lovastatin-induced apoptosis in mesangial cells occurs by interfering with prenylation dependent mitotic and post-mitotic events.

Role of Rho and myosin phosphorylation in actin stress fiber assembly in mesangial cells.

Treatment of renal glomerular mesangial cells with adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents induces actin stress fiber disassembly, myosin light chain (MLC) dephosphorylation, loss of adhesion to the substratum and cell shape change [J. I. Kreisberg and M. A. Venkatachalam. Am. J. Physiol. 251 (Cell Physiol. 20): C505-C511, 1986]. Thrombin and vasopressin block the effects of cAMP. Because these agents are known to promote stress fiber formation via the small GTP-binding protein Rho, we investigated the effect of an activated variant of Rho on the response to cAMP elevation. Microinjecting V14-Rho completely blocked the effect of cAMP elevation on cell shape and the actin cytoskeleton, whereas inactivating Rho with botulinum C3 exoenzyme induced stress fiber disruption and cell retraction that was indistinguishable from that caused by elevations in intracellular levels of cAMP. Disruption of actin stress fibers by cAMP has previously been ascribed to MLC dephosphorylation; however, both C3 and cytochalasin D also caused dephosphorylation of MLC, whereas blocking MLC dephosphorylation failed to block the cAMP-induced loss of actin stress fibers. We conclude that Rho can modulate the effects of cAMP elevation and suggest that MLC dephosphorylation may be a consequence of actin stress fiber disassembly.

Clonal osteoblastic cell lines from p53 null mouse calvariae are immortalized and dependent on bone morphogenetic protein 2 for mature osteoblastic phenotype.

p53 protein regulates cell cycle progression and its absence will result in unlimited cell divisions required for immortalization of cells. Immortalized osteoblastic cell lines were established from p53 null mouse calvariae of normal phenotype. The clonal murine cell lines demonstrated osteoblastic phenotype as exemplified by alkaline phosphatase enzyme activity. They also express bone morphogenetic protein 2 (BMP2) mRNA. Addition of recombinant BMP2 to these cells dramatically increased the alkaline phosphatase activity in a dose dependent manner. In the absence of BMP2 these cells do not undergo osteoblastic differentiation. Treatment of these cells with recombinant bone morphogenetic protein 2 stimulated differentiated osteoblast formation, as determined by mineralized nodule formation. Thus, these immortalized cells in culture represent osteoblast progenitors that lack p53 protein and respond to osteogenic stimuli. These cell lines offer a model system to study the role of p53 in osteoblastic differentiation and programmed cell death. Also these cells will be useful in studying the effects of p53 on transcriptional regulation of osteoblast specific gene expression.

Bone morphogenetic protein 2 transcripts in rapidly developing deer antler tissue contain an extended 5' non-coding region arising from a distal promoter.

To understand the regulation of the BMP-2 gene expression, we recently isolated the BMP-2 gene from a mouse genomic library and characterized the exon-intron structure and promoter. RNase protection assay using poly (A)+ RNA of mouse osteoblasts demonstrates that two regions in BMP-2 gene are protected by antisense mouse BMP-2 RNA probes. These results demonstrate that BMP-2 gene utilizes two alternative promoters, a distal and a proximal promoter. In the present study we demonstrate that BMP-2 mRNA from rapidly growing deer antler tissue has an extended 5' non-coding region compared with the human and rat BMP-2 mRNA. The extended 5' non-coding region in the deer mRNA represents transcripts from the upstream distal promoter. This is the first evidence of a natural BMP-2 mRNA from a bone-forming tissue that most likely initiated from the distal transcription start site.

Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice.

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.

The effects of cytokines and growth factors on osteoblastic cells.

In this short review, some regulatory mechanisms that are involved in the control of normal bone formation are proposed, based on several in vivo and in vitro models our group has utilized recently to study osteoblast differentiation and mineralized bone matrix formation. Of course, these proposals must be assessed in the light of the limitations of the models, which probably represent a simplification of the complex and different ways in which normal mammalian bone is formed at different sites. Nevertheless, it is likely that the same general types of control mechanisms are active in each of the different types of bone formation. In adult humans, bone formation predominantly occurs by remodeling, the process by which bone which has recently been resorbed by osteoclasts is replaced by teams of osteoblasts. Other types of bone formation such as endochondral bone formation and appositional bone formation are also important, particularly during growth and adolescence. The end results of each of these processes are the same, namely a complex mineralized proteinaceous bone matrix. These processes are modulated by systemic hormonal influences, which are particularly important with respect to pituitary hormones and sex steroids during growth and adolescence, and by local cellular microenvironmental differences. The former will not be discussed here. Rather, we will concentrate on the local events and factors which are likely involved in the bone formation process occurring during normal bone remodeling.

Structure and sequence of mouse bone morphogenetic protein-2 gene (BMP-2): comparison of the structures and promoter regions of BMP-2 and BMP-4 genes.

Screening of a mouse spleen genomic DNA library with mouse BMP-4 and BMP-2 cDNA probes led to the isolation of mouse BMP-2 gene. This gene contains approximately 11 kb transcription unit and 3 exons. The deduced protein has 394 amino acids. The C-terminal amino acid mature coding region is identical to that of the human BMP-2. Comparison of mouse BMP-2 and BMP-4 gene shows little similarity in exon/intron structure, although the mature coding peptide regions of both genes share over 90% identity at the amino acid level.

Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts.

Transforming growth factor beta (TGF-beta) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-beta is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes proliferation of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-beta 1 on first subcultures of fetal rat osteoblasts obtained from calvarial bones and cultured from confluence with ascorbic acid and beta-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear by day 8. Similar to experiments described by Antosz et al., TGF-beta added to confluent cultures inhibited the formation of bone nodules. Both the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-beta 1. TGF-beta also impaired the expression of genes associated with bone formation, including type I collagen, alkaline phosphatase, osteopontin, and osteocalcin. TGF-beta also inhibited the expression of mRNA for the bone morphogenetic protein 2 (BMP-2). These results, showing suppression of markers representative of osteoblast differentiation, suggest that the effects of TGF-beta to stimulate bone formation in vivo are not likely a result of effects on differentiated mineralizing osteoblasts but, as suggested by previous studies, more likely are caused by effects on osteoblast precursors. These results also suggest that endogenous BMP-2 expression in fetal rat calvaria cells is important for bone cell differentiation.

Effect of tunicamycin and monensin on the transport to the cell surface and secretion of a viral membrane glycoprotein containing both N- and O-linked sugars.

Most membrane glycoproteins contain either N-linked or O-linked oligosaccharides, which play important roles in correct folding, stability, and intracellular transport. Some glycoproteins, however, contain both the N- and O-linked sugars. To study the roles of the two types of glycosylation in intracellular transport we have used as a model the glycoprotein gC-1 of herpes simplex virus type 1 (HSV-1), which contains both N- and O-linked oligosaccharides. Cloned gene of gC-1 was expressed constitutively in mammalian cells to produce the gC-1 glycoprotein containing both types of glycosylation. Only a fraction of the gC-1 glycoprotein was secreted into the medium. Addition of tunicamycin blocked N-glycosylation and the gC-1 protein of reduced size containing only O-linked sugars was formed. This O-glycosylated gC-1 protein was transported to the cell surface and secreted into the medium, indicating that glycoprotein transport to and across the cell surface occurs in the absence of N-glycans. The data suggest either that O-glycans may contribute to this process or that transport can occur in the absence of both N- and O-glycans.