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PubMed1997

Local estradiol metabolism in osteoblast- and osteoclast-like cells.

Bone is an estradiol-responsive tissue. Estrogen withdrawal during the menopause causes loss of bone mass and clinically relevant osteoporosis in a third of all women. Sufficient or impaired local production, as well as degradation of estradiol in cells present in the bone microenvironment might be an important mechanism of rescue or might contribute to the development of osteoporosis, respectively. We therefore investigated aromatase and 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV) expression in osteoblast- and osteoclast-like cells. Aromatase mRNA was increasingly expressed in myeloid THP 1 cells differentiated along the monocyte/phagocyte pathway exploiting vitamin D and either granulocyte-macrophage-stimulating factor (GMCSF) or macrophage-stimulating factor (MCSF). In long-term cultures, when sequentially exposed to vitamin D (days 0-21) and GMCSF (days 5-10) and plated on collagen, the amount of expression of aromatase mRNA steadily increased along with the increasing expression of osteopontin mRNA, alpha(v) integrin mRNA, c-fms (MCSF-receptor) mRNA and multinucleated cells developing. The conversion of estradiol from testosterone (10(-7) M/l) in the supernatants of dishes mirrored changes in aromatase mRNA expression and by day 21 rose to 30,000 ng/10(7) cells/24 h. 17Beta-HSD IV mRNA expression was abundant in undifferentiated THP 1 cells and was decreased to approximately 50% by day 21. Unstimulated SV-40 immortalized fetal osteoblasts did not express aromatase mRNA, but the expression was stimulated by the addition of the phorbol ester phorbol myristate acetate (PMA). Unstimulated osteoblasts from primary cultures did not express aromatase mRNA. Osteoblast-like osteosarcoma cells MG 63 expressed faint levels of aromatase mRNA in contrast to the osteosarcoma cell line HOS 58. 17Beta-HSD IV mRNA was expressed in fetal osteoblasts as well as in osteoblasts from primary culture, MG 63 and HOS 58 cells. In summary, we can show the expression of estradiol metabolizing enzymes in cells which are present in the bone microenvironment. Impaired aromatase expression and/or enhanced expression of 17beta-HSD IV may contribute to the pathogenesis of osteoporosis.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line.

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (approximately 4,000/nucleus) of androgen receptors (AR). Treatment with 5alpha-dihydrotestosterone (5alpha-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5alpha-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-beta1 (TGF-beta1) and TGF-beta-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5alpha-DHT, suggesting a role for the TGF-beta1-TIEG pathway in mediating 5alpha-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-beta1 (40 pg/ml) reversed the 5alpha-DHT-induced growth inhibition, whereas TGF-beta1 alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5alpha-DHT and testosterone (10(-8) M) inhibited basal and 1,25-(OH)2D3-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast.

Adrenal Glands↗

PubMed1999

Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides).

In vitro cell biocompatibility models are crucial in the study of any newly synthesized material. Our focus has been on the development of a new class of biocompatible, degradable, high-strength polymeric materials, the poly(anhydride-co-imides), for use in bone regeneration. This study examined osteoblast cell adherence, proliferation, viability, and phenotypic preservation on the surface of the poly(anhydride-co-imide) poly[pyromellitylimidoalanine (PMA-ala):1,6-bis(carboxyphenoxy) hexane (CPH)] over a period of time. Cell proliferation on PMA-ala:CPH degradable matrices over 21 days was examined. Throughout the 21-day period of study, osteoblast proliferation was similar on PMA-ala:CPH and on tissue culture polystyrene controls. Osteoblasts maintained their characteristic morphology as demonstrated by both scanning electron microscopy and immunofluorescence studies. Alkaline phosphatase activity for cells grown on PMA-ala:CPH was confirmed. Retention of the osteoblastic phenotype was demonstrated using immunofluorescence techniques and staining with antibodies against osteocalcin (an extracellular matrix protein of bone) and osteopontin (a marker of cell adhesion). Radioimmunoassay results provided evidence that levels of osteocalcin production by osteoblasts were similar when cells were cultured on PMA-ala:CPH and on tissue culture polystyrene controls. The present study provided evidence of normal osteoblast function on PMA-ala:CPH surfaces. PMA-ala:CPH may therefore be useful as a synthetic material for orthopedic applications.

Animals↗

PubMed1995

Osteoclast 121F antigen expression during osteoblast conditioned medium induction of osteoclast-like cells in vitro: relationship to calcitonin responsiveness, tartrate resistant acid phosphatase levels, and bone resorptive activity.

Osteoclast differentiation from hematopoietic precursors into multinucleated cells uniquely capable of removing the organic and inorganic components of bone matrix occurs in multistep process, during which osteoclasts acquire the specialized characteristics necessary for bone resorptive activity and physiological regulation. Among those traits is a novel plasma membrane glycoprotein, reactive with the anti-osteoclast monoclonal antibody 121F, which is expressed during the course of osteoclast differentiation, shares structural and functional homologies with Mn2+/Fe2+ superoxide dismutase, and has been hypothesized to protect the osteoclast from the damaging effects of superoxide radicals generated during active bone resorption. We have reported previously that the expression of this membrane antigen is induced on multinucleated giant cells when the prefusion marrow mononuclear cells are cultured in conditioned medium from avian calvaria. The studies reported here were designed to investigate the relationship between expression of the 121F antibody-reactive osteoclast membrane antigen and tartrate resistant acid phosphatase levels, bone resorptive activity, calcitonin responsiveness, and ultrastructural features of avian bone marrow-derived multinucleated giant cells formed either in the presence or absence of diffusible osteoblast secreted factors. Parallel analyses of in vivo formed osteoclasts isolated from the same animals were performed for direct comparisons. In this report we demonstrate: (1) that the 121F monoclonal antibody-reactive osteoclast membrane antigen is stably induced in giant cells by soluble osteoblast-derived factors in a species nonrestricted but concentration- and temporal-dependent manner; (2) that osteoblast-mediated antigen induction is reflected in both increased numbers of cells and elevated expression of individual cells that are reactive with the 121F antibody, as determined by ELISA and histomorphometry; (3) that osteoblast conditioned medium, in addition to inducing this antigen in bone marrow cells, also elevates other defining osteoclast characteristics in these avian giant cells including their TRAP activity, cell retraction from the bone surface in response to calcitonin, bone resorptive function, and expression of a series of additional osteoclast antigenic markers; and (4) that secreted osteoblast products alone do not raise the levels of these traits for in vitro formed marrow giant cells to the extent associated with in vivo formed osteoclasts. Therefore, osteoblast soluble factors alone appear unable to promote the full differentiation of bone marrow cells in vitro into mature bone-resorbing osteoclasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗

PubMed1991

Thrombin effects on osteoblastic cells. II. Structure-function relationships.

Thrombin has been shown to cause in vitro bone resorption and to stimulate osteoblastic cell proliferation, phosphoinositide turnover and cytosolic calcium levels. In the present study, the role of the active site of thrombin in its action on osteoblastic cells was investigated. Either hirudin or (4-amidinophenyl)methanesulfonyl fluoride inhibited, in a dose-dependent manner, the effects of thrombin on human osteoblast-like osteosarcoma cells (G292 and Saos-2 cell lines) and on normal rat calvarial osteoblastic cells. Thrombin-induced stimulation of cell proliferation, cytosolic calcium increases, and stimulation of phosphoinositide metabolism were concomitantly, and to a proportionally similar extent, inhibited. The inhibitors, when present in the absence of thrombin, did not affect the basal levels of cell functions. Both zeta-thrombin and gamma-thrombin, forms resulting from proteolytic cleavage of alpha-thrombin, were capable of stimulating the osteoblastic cells. These data indicate that thrombin's actions on osteoblast-like cells are dependent on the availability of its catalytic site.

Animals↗

PubMed2006

The soybean isoflavone genistein induces differentiation of MG63 human osteosarcoma osteoblasts.

A soybean-rich diet was shown to reduce the incidence of osteoporosis in Eastern countries; its effect on bone metabolism was ascribed to the action of the soybean isoflavones such as genistein. Although many studies have shown isoflavone-induced osteoblast differentiation, its preventative action on bone mass loss has not been clarified. Here, the osteogenetic effects of genistein on human cell line MG63 osteoblasts were elucidated using a variety of approaches. In particular, phalloidin-rhodamine staining revealed that genistein-treated osteoblasts possessed a more organized cytoskeleton, and genistein's inhibitory effect upon cell proliferation was associated with exposure of phosphatidylserines on the external plasmalemma surface. Although this phosphatidylserine exposure is considered a typical apoptotic marker, scanning and transmission electron microscopy revealed that genistein-treated osteoblasts released matrix vesicles and showed no evidence of chromatin condensation. Assays, stainings, and scanning electron microscopy showed that genistein-treated osteoblasts synthesized relatively high levels of collagen and alkaline phosphatase and, even in a nonosteogenic growth medium, formed mineralized bone noduli. A clear pattern of genistein-induced osteoblast activation therefore emerges, in which all of the essential components required for the rapid production of mineralized bone extracellular matrix are stimulated by this soybean isoflavone.

Apoptosis↗

PubMed2005

P53 and beta-catenin activity during estrogen treatment of osteoblasts.

BACKGROUND: This study was undertaken to examine the relationship between the tumor suppressor gene p53 and the nuclear signaling protein beta-catenin during bone differentiation. Cross talk between p53 and beta-catenin pathways has been demonstrated and is important during tumorigenesis and DNA damage, where deregulation of beta catenin activates p53. In this study, we used estrogen treatment of osteoblasts as a paradigm to study the relationship between the two proteins during osteoblast differentiation. RESULTS: We exposed osteoblast-like ROS17/2.8 cells to 17-beta estradiol (E2), in a short term assay, and studied the cellular distribution and expression of beta-catenin. We found beta-catenin to be up regulated several fold following E2 treatment. Levels of p53 and its functional activity mirrored the quantitative changes seen in beta-catenin. Alkaline phosphatase, an early marker of osteoblast differentiation, was increased in a manner similar to beta-catenin and p53. In order to determine if there was a direct relationship between alkaline phosphatase expression and beta-catenin, we used two different approaches. In the first approach, treatment with LiCl, which is known to activate beta-catenin, caused a several fold increase in alkaline phosphatase activity. In the second approach, transient transfection of wild type beta-catenin into osteoblasts increased alkaline phosphatase activity two fold over basal levels, showing that beta catenin expression can directly affect alkaline phosphatase expression. However increase in beta catenin activity was not associated with an increase in its signaling activity through TCF/LEF mediated transcription. Immunofluorescence analyses of p53 and beta-catenin localization showed that E2 first caused an increase in cytosolic beta-catenin followed by the accumulation of beta-catenin in the nucleus. Nuclear p53 localization was detected in several cells. Expression of p53 was accompanied by distribution of beta-catenin to the cytoplasm and cell borders. A sub population of cells staining strongly for both proteins appeared to be apoptotic. CONCLUSION: These results suggest that interactions between p53 and beta-catenin signaling pathways may play a key role in osteoblast differentiation and maintenance of tissue homeostasis.

Journal Article↗

PubMed2006

Direct effects of caffeine on osteoblastic cells metabolism: the possible causal effect of caffeine on the formation of osteoporosis.

BACKGROUND: Caffeine consumption has been reported to decrease bone mineral density (BMD), increase the risk of hip fracture, and negatively influence calcium retention. In this study, we investigated the influence of caffeine on the osteoblasts behaviour. METHOD: Osteoblasts derived from newborn Wistar-rat calvaria was used in this study. The effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay. Alkaline phosphatase (ALP) staining, von Kossa staining and biochemical parameters including ALP, lactate dehydrogenase (LDH), prostaglandin E2 (PGE2) and total protein were performed at day 1, 3, and 7. DNA degradation analysis under the caffeine influence was also performed. RESULTS AND DISCUSSION: The results showed that the viability of the osteoblasts, the formation of ALP positive staining colonies and mineralization nodules formation in the osteoblasts cultures decreased significantly in the presence of 10 mM caffeine. The intracellular LDH, ALP and PGE2 content decreased significantly, the LDH and PGE2 secreted into the medium increased significantly. The activation of an irreversible commitment to cell death by caffeine was clearly demonstrated by DNA ladder staining. CONCLUSION: In summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis.

Journal Article↗

PubMed2006

Canonical Wnt signaling in osteoblasts is required for osteoclast differentiation.

Inactivation of Lrp5, a gene encoding a likely Wnt co-receptor, results in low bone mass (osteopenia) by decreasing bone formation, suggesting that Wnt signaling in osteoblasts regulates bone formation. Here we show that Tcf1 and Tcf4 are expressed in osteoblasts during development and after birth; stabilization of beta-catenin, an essential component of canonical Wnt signaling, in differentiated osteoblasts results in high bone mass while its deletion from differentiated osteoblasts leads to osteopenia. Histological analysis showed that these mutations affect bone resorption. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates in osteoblasts the expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that, in differentiated osteoblasts, beta-catenin and presumably Wnt signaling are negative regulators of osteoclast differentiation; thus they broaden our knowledge about functions that Wnt proteins may have at various stages of skeletogenesis.

Animals↗

PubMed1985

Identification of a messenger ribonucleic acid fraction in human prostatic cancer cells coding for a novel osteoblast-stimulating factor.

Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.

Alkaline Phosphatase↗

PubMed2001

[A study of fluoride and dexamethasone affecting the expression of growth factors in osteoblast].

OBJECTIVES: Investigate the expression of growth factors or receptors in osteoblast, to understand the possible mechanism of low dose fluoride and dexamethasone on the development and differentiation of osteoblast. METHODS: Investigate the expression of platelet derived growth factors (PDGF-A) and insulin-like growth factors (IGF-I-Ralpha) in osteoblast cultured in vitro by immunohistochemistry, furthermore, to deal with the results by image analysis. RESULTS: There were no significant effects of PDGF-A both of low doses fluoride and dexamethasone treated on the MC3T3-E1 osteoblast during the various stages of the development. Otherwise, the expression of IGF-I-Ralpha might increase treated with them at the early stage of the cell culture, and dexamethasone did more significantly. CONCLUSION: Low doses of fluoride or dexamethasone may promote the expression of IGF-I-Ralpha in osteoblast precursors, which maybe contribute to the effects of fluoride and dexamethasone on the proliferation and differentiation of osteoblast.

English Abstract↗

PubMed2002

The role of signaling pathways in osteoblast gravity perception.

Bone loss is one of the major problems in long term spaceflight. This physiological consequence of microgravity is the rapid loss of weightbearing bone that is associated with skeletal unloading. Moreover, we have previously noted that sera deprived osteoblasts do not have a normal response to sera in microgravity. Where exercise (mechanical loading) has been shown to increase bone formation and stimulate osteoblastic function, the mechanisms underlying signal transduction of mechano-perception is yet to be fully understood. Osteoblasts have been shown to respond to mechanical stress such as fluid shear, bending, flexing and compression. The type of stress and amount of stress determine the osteoblast response Recently we have discovered that the isolated osteoblast responds to a very short pulse of g-force compression. The possible regulatory sensors include mechano-sensitive calcium channels, autrocrine responses to stress, response to FAK/integrin, alterations in the cytoskeleton as well as other known growth factor and cytokine receptors. The secondary signal may include growth factor related kinases such as ERK, p38 and JNK map kinase (MAPK) pathways. Experimental evidence suggests that normal osteoblast response to stress and sera requires normal earth gravity.

NASA Discipline Cell Biology↗

PubMed2001

[Relationship between periarticular osteoporosis and osteoblast senescence in patients with rheumatoid arthritis].

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life-span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential and life-span of osteoblasts have not so far been investigated to date. Here we present evidence that the cellular activity, telomere lengths and replicative life-span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age in patients with rheumatoid arthritis or osteoarthritis. Recently, human telomerase reverse transcriptase (hTERT) has been identified as a human teromerase catalytic subunit. We postulate that an expansion of the life-span of osteoblasts and their maintenance as differentiated bone matrix-producing cells may allow for autologus or allogenic cell and gene therapy in bone and joint diseases including osteoporosis. We therefore transfected human osteoblasts with a vector expressing hTERT cDNA, and investigated whether the replicative life-span can be expanded by the introduction of telomerase in human osteoblasts.

English Abstract↗

PubMed1997

Stimulation of interleukin-11 production from osteoblast-like cells by transforming growth factor-beta and tumor cell factors.

Bone is one of the most common sites of metastasis in melanoma and breast cancer cells. Human melanoma (A375M) and human breast cancer (MDA-MB-231) cells form osteolytic bone metastasis in vivo when these tumor cells are injected into the left ventricles of BALB/c nude mice. These tumor cells promote bone resorption in the in vitro neonatal murine calvaria organ culture system by indirectly stimulating the production of a bone resorption-inducing factor (or factors) from human osteoblast-like cells. This secreted factor was identified as interleukin-11 (IL-11). Although many cytokines and hormones were associated with IL-11 production from osteoblasts, transforming growth factor-beta (TGF-beta) was found to be involved in the promotion of IL-11 production from osteoblasts, because the addition of a neutralizing anti-TGF-beta antibody decreased the production of IL-11. However, these tumor cells did not produce TGF-beta by themselves. We found that they enhanced IL-11 production by activating latent TGF-beta produced from osteoblast-like cells. Our results indicate that metastatic tumor cells induce osteolysis by activating TGF-beta, which leads IL-11 production from osteoblasts to promote bone resorption.

Animals↗

PubMed1998

Osteoblast reaction at the interface between surface-active materials and bone in vivo: a study using in situ hybridization.

Osteoblast activation after implantation of two kinds of surface-active material in bone was investigated chronologically using in situ hybridization with digoxygenin-labeled procollagen alpha 1(I) complementary RNA probe. The bioactive materials used were hydroxyapatite (HA) and apatite- and wollastonite-containing glass-ceramic (A-W GC). A hole was drilled bilaterally in the distal epiphysis of rabbit femurs with subsequent implantation of HA or A-W GC cylinders in a press-fit manner. Specimens were collected at 3, 7, 14, and 28 days after operation and decalcified. Then the undecalcified implant cores were pushed out of the hole without causing damage to the bony side of the interface. In situ hybridization documented no qualitative differences in the expression of procollagen alpha 1(I) RNA between HA and A-W GC. Few osteoblasts at the bone-material interface showed a specific signal at day 3, whereas many osteoblasts were positive around the materials at days 7 and 14, indicative of active new bone formation. The positive osteoblasts seemed to originate from preexisting trabeculae and lined the trabeculae, newly formed bone, and material surface. At day 28, many osteoblasts lining material-surrounding bone were negative, whereas those in remodeling canals were positive, suggesting that the bone was in the remodeling stage after bone formation. These findings were comparable to those with beta-tricalcium phosphate in a previous study, thus suggesting osteoconductive bone formation on HA and A-W GC.

Animals↗

PubMed1997

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

Androgen Antagonists↗

PubMed1998

PTH-responsive osteoblast nuclear matrix architectural transcription factor binds to the rat type I collagen promoter.

In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen alpha1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression.

Animals↗

PubMed1998

Osteoblasts induce osteopontin expression in response to attachment on fibronectin: demonstration of a common role for integrin receptors in the signal transduction processes of cell attachment and mechanical stimulation.

Osteopontin is a predominant integrin binding protein of bone and its expression has been shown to be induced by mechanical stimuli within osteoblasts (Toma et al. [1997] J. Bone Miner. Res. 12:1626-1636). The present studies examined if the cell adhesion would mimic the mechano-transduction that stimulated opn mRNA expression and whether integrin receptors were involved in these processes. Osteopontin mRNA expression was induced three- to four-fold, 24 hours after embryonic chicken calvaria osteoblast attachment to fibronectin (FN), however no induction was observed if the cells were plated on tissue culture plastic alone. Osteopontin mRNA induction in response to cell attachment on FN was dependent on new protein synthesis and the activation of a tyrosine protein kinase(s) but unlike mechano-induction was independent of the maintenance of the cell's microfilament structure. Integrin receptor(s) were shown to be involved in mediating the signal transduction processes of both cell attachment and mechanical stimulation since incubation of osteoblasts with the integrin binding peptide RGDS partially blocked the induction of opn expression in response to both stimuli. Interestingly, incubation of the osteoblasts that were adherent on tissue culture plastic alone with the RGDS peptide lead to an induction in opn expression suggesting that integrin occupancy by itself was sufficient to initiate the signal transduction process that induced opn expression. In order to assess the role of integrin occupancy vs. focal adhesion complex formation that accompanies cell attachment, in the signal transduction process that induces opn expression, receptor clustering was stimulated pharmacologically with bombesin or lysophosphatidic acid in osteoblasts attached to tissue culture plastic. Neither compound in the absence of occupancy of the integrin receptors was capable of stimulating opn expression in attached cells, however if the cells were placed in suspension pharmacological mediation of receptor clustering and integrin occupancy were additive in their effect of inducing opn expression. These data demonstrate that induction of opn expression by mechanical stimuli and cell attachment are commonly mediated through integrin receptor(s). However, when cells are attached receptor clustering alone which accompanies focal adhesion formation was incapable of mediating signal transduction suggesting that receptor occupancy was a prerequisite to the signal transduction process that leads to the induction of opn mRNA expression.

Animals↗