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PubMed2001

The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair.

We investigated the effects of transplantation of osteoblastic cells with a bone morphogenetic protein (BMP)/carrier complex on bone repair by in vitro and in vivo experiments. Poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS) was used as a carrier for cell transplantation. In the in vitro experiments, three cell types, C3H10T1/2 cells, MC3T3-E1 cells, and primary osteoblastic cells, isolated from newborn rat calvariae (ROB cells), were cultured for 2 weeks on PGS alone or PGS containing BMP-2 (PGS/BMP). C3H10T1/2 cells cultured on PGS/BMP expressed several markers related to differentiation of both osteoblasts and chondrocytes, such as alkaline phosphatase (ALP) activity and mRNAs for osteocalcin and aggrecan, whereas the cells cultured on PGS alone expressed no such markers. MC3T3-E1 cells cultured on PGS/BMP exhibited a more ALP-positive cells than those cultured on PGS alone. PGS/BMP promoted ROB cell differentiation into both osteoblasts and chondrocytes. In the in vivo experiments, we transplanted ROB cells, which had been cultured on PGS alone or PGS/BMP in vitro for 2 weeks, into bone defects created in rat calvariae. Transplantation of ROB cells cultured on PGS alone generated little new bone. Transplantation of ROB cells cultured on PGS, which absorbed a low dose (10 ng) of rhBMP-2,; induced significantly higher bone mineral content than PGS/BMP alone, although application of a high dose (1 microg) of rhBMP-2 induced no difference in bone mineral content between transplantation of PGS/BMP with or without ROB cells. These results show that transplantation of osteoblastic cells after induction of osteoblast maturation in vitro by cultivation on PGS/BMP is a potent technique for cell therapy of bone repair.

Aggrecans↗

PubMed1997

Absence of androgen-mediated transcriptional effects in osteoblastic cells despite presence of androgen receptors.

Androgen excess and deficiency affect skeletal maturation and bone cell function. Understanding the molecular basis for these androgen effects could improve therapy/prevention of short stature and osteoporosis. Androgens act through binding to androgen receptors (ARs), which modulate gene transcription via interactions with DNA response elements on target genes. Because osteoblasts contain ARs at levels just below certain androgen-sensitive tissues, we sought to define the function of AR in a number of commonly used osteoblastic cell lines. Presence and quantification of AR protein and mRNA were evaluated by ligand binding assay, western blotting, and RNAse protection assay. AR-containing osteoblastic cell lines were exposed to nonaromatizable androgens and effects on gene expression were assessed. We found no evidence for direct effects of androgen on endogenous genes nor was androgen involved in modulation of parathyroid hormone effects on early gene activation. Androgen-sensitive reporter gene constructs were stimulated by androgen only when AR cDNA expression vectors were introduced into cells by cotransfection. We conclude that, in commonly used osteoblastic cell lines, the presence of AR at the levels described here does not guarantee androgen transcriptional activity. The effects of androgen on bone in vivo may involve direct stimulation of osteoblastic cells in a different setting or stage of differentiation. Alternatively, androgen may act on bone cells other than osteoblasts, or through metabolic conversion to estrogens.

Androgens↗

PubMed1997

Monocyte chemoattractant protein-1 induces monocyte recruitment that is associated with an increase in numbers of osteoblasts.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of cytokines. The principal function of MCP-1 is thought to be the stimulation of monocyte recruitment. Monocyte products are potential regulators of bone cell activity. Growth factors produced by monocytes may stimulate bone formation, while cytokines such as IL-1 and IL-6 can induce bone resorption. To determine whether MCP-1 enhances recruitment of monocytes during bone healing, studies were carried out in which MCP-1 was applied to osseous sites in vivo. Changes in monocyte number were determined by immunohistochemistry using the antibody ED-1 specific for peripheral monocytic cells. The effect of MCP-1 on osteoblast number was determined by counting the number of alkaline phosphatase positive cells in close proximity to bone. For comparison, osteoblast number was also determined following stimulation with platelet-derived growth factor (PDGF)-BB plus IGF-1 in vivo. Results indicate that MCP-1 stimulated a large increase in monocyte recruitment compared to vehicle alone. An increase in monocytes induced by MCP-1 was associated with an increase in the number of osteoblasts lining the bone surface, although not to the same magnitude as a positive control, PDGF-BB, and IGF-1. These results indicate that MCP-1 induces the recruitment of monocytes to bone and suggest that the recruitment is associated with an increase in osteoblast number. This is likely to occur via indirect mechanisms, because MCP-1 did not directly enhance DNA synthesis in osteoblastic cells in vitro. Thus, activated mononuclear phagocytes may play an important role in osseous wound healing by stimulating proliferation of osteoblastic cells, presumably through the elaboration of growth factors.

Alkaline Phosphatase↗

PubMed1998

Formoterol and isoproterenol induce c-fos gene expression in osteoblast-like cells by activating beta2-adrenergic receptors.

Formoterol, a beta2-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of beta3-adrenergic receptors (betaARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that betaAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. beta1AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. beta2ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that beta3AR expression was found in one of three human primary osteoblast cDNAs tested. beta3AR expression was not found in any of the osteoblast-like cell lines. The nonspecific betaAR agonist, isoproterenol, and the beta2AR-specific agonist, formoterol, induced c-fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the beta2AR-specific antagonist, ICI 118551, but not by the beta1AR-specific antagonist, CGP 20712, indicating that induction of c-fos gene expression is specifically mediated by beta2ARs. c-fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c-fos gene expression. Thus, betaARs are expressed in osteoblast-like cells and are coupled to c-fos gene expression via the beta2AR, increases in cAMP levels and activation of a PKA-dependent pathway.

Adrenergic beta-Agonists↗

PubMed1998

Morphological changes induced by prostaglandin E in cultured rat osteoblasts.

Prostaglandin E (PGE)-induced morphological changes of osteoblasts and its possible mechanisms were investigated in cultured calvaria and isolated osteoblasts from long bone fragments of neonatal rats. The control osteoblasts, either on the calvaria or isolated from the long bone fragments, were flat, polygonal in shape, and arranged in a monolayer under scanning electron microscopy (SEM) or phase contrast microscopy. Treatment with 1 mumol/L of prostaglandin E2 (PGE2, 2 h) caused these bone cells to contract a soma, whereas 10 and 100 mumol/L PGE2 (2 h) caused 18%-30% of the bone cells to elongate and expose the undersurface. Incubation of the cultured osteoblasts with PGE2 at different time periods showed a bell-shaped pattern with the optimal response at 2 h of incubation. A similar reaction can be induced by treatment with prostaglandin E1 (PGE1) or dibutyryl cyclic adenosine monophosphate (DBcAMP) in combination with 3-isobutyl-1-methylxanthine (IBMX). Furthermore, we assessed the percentage of responsive isolated bone cells to investigate interactions with other agents. The morphological changes induced by PGEs were inhibited by H-8, a protein kinase inhibitor. On the other hand, elevated intracellular calcium enhanced the PGE-induced morphological changes. Fluorescence labeling showed that PGEs caused the breakdown of the actin microfilaments, but spared the microtubules and vimentin filaments in the isolated osteoblast-like cells. These results suggest that the morphological changes of osteoblasts induced by PGEs may be related to the intracellular cAMP and calcium levels.

1-Methyl-3-isobutylxanthine↗

PubMed1999

Effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of decorin and biglycan by cultured osteoblastic cells.

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta (TGF-beta) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24 h with graded concentrations of TGF-beta1 (0, 0.1, 1.0, 5.0, and 10 ng/ml). TGF-beta1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-beta1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-beta1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.

Alkaline Phosphatase↗

PubMed2000

Protein production by osteoblasts: modulation by breast cancer cell-derived factors.

Breast cancer cells (BCC) frequently metastasize to bone where they may cause tumor-induced osteolysis (TIO). While the important eroding role of the osteoclasts in TIO is well admitted, the possibility that BCC and/or osteoblasts activated by tumoral factors could also directly degrade bone matrix in this pathology has been much less investigated. We show here that the net collagen amount produced in vitro by normal human osteoblasts and osteoblast-like cells was significantly reduced by culture medium conditioned by several BCC lines, including three newly isolated ones. There was no evidence for a decrease in collagen synthesis, as assessed by the production of the carboxyterminal propeptide of type I collagen. In contrast, the effect of BCC-derived medium on collagen amount was attenuated by inhibitors of matrix metalloproteinases (MMPs) as well as by tranexamic acid, an inhibitor of the plasminogen conversion to plasmin, while it was abolished in presence of the two kinds of proteinase inhibitors. This osteoblastic protein degradation activity appeared to be attributable to factors secreted by the osteoblasts as well as by BCC. These factors had molecular weights lower as well as higher than 10 kD. Our data suggest that besides the eroding action of osteoclasts, BCC- and osteoblast-derived MMPs and serine proteinases might play a direct role in bone collagen degradation in TIO.

Adenocarcinoma↗

PubMed2003

The effects of enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect.

OBJECTIVE: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. MATERIALS AND METHODS: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2'-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. RESULTS: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. CONCLUSION: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.

Alkaline Phosphatase↗

PubMed2003

Involvement of FGF-2 in the action of Emdogain on normal human osteoblastic activity.

OBJECTIVE: The present study was designed to evaluate the pharmacological characteristics of Emdogain (EMD) on cell growth and cell activity in human osteoblasts. METHODS: Cell proliferation as well as several gene and protein expressions were examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) procedures in human osteoblastic cells (SaM-1) treated with EMD (30 microg ml(-1)). RESULTS: Treatment of osteoblasts with EMD significantly stimulated cell proliferation and fibroblast growth factor (FGF)-2 expression but decreased alkaline phosphatase expression. In addition, increases in cyclooxygenase (COX)-2 expression and decreases in matrix metalloproteinases (MMP)-1 expression were observed in osteoblasts treated with EMD. The effects of EMD on FGF-2 and MMP-1 expressions were not observed in osteoblasts treated with NS-398, an inhibitor of COX-2. The decrease in MMP-1 mRNA by EMD was prevented by treatment with antisense oligodeoxynucleotide (AS-ODN) for FGF-2. CONCLUSION: Emdogain showing both stimulation of cell proliferation and inhibition of cell differentiation has been shown to increase FGF-2 expression in the mediation of prostaglandin E2 and to decrease MMP-1 mRNA expression through the activation of FGF-2. FGF-2 may underlie in the action of EMD on osteoblasts during periodontal regeneration.

Adult↗

PubMed2005

Fhl2 deficiency results in osteopenia due to decreased activity of osteoblasts.

Osteoporosis is one of the major health problems today, yet little is known about the loss of bone mass caused by reduced activity of the bone-forming osteoblasts. Here we show that mice deficient for the transcriptional cofactor four and a half LIM domains 2 (Fhl2) exhibit a dramatic decrease of bone mass in both genders. Osteopenia is caused by a reduced bone formation rate that is solely due to the diminished activity of Fhl2-deficient osteoblasts, while their number remains unchanged. The number and activity of the bone-resorbing cells, the osteoclasts, is not altered. Enforced expression of Fhl2 in differentiated osteoblasts boosts mineralization in cell culture and, importantly, enhances bone formation in transgenic animals. Fhl2 increases the transcriptional activity of runt-related transcription factor 2 (Runx2), a key regulator of osteoblast function, and both proteins interact in vitro and in vivo. In summary, we present Fhl2-deficient mice as a unique model for osteopenia due to decreased osteoblast activity. Our data offer a novel concept to fight osteoporosis by modulating the anabolic activity of osteoblasts via Fhl2.

Animals↗

PubMed2003

Induction of interleukin-8 gene expression by black-pigmented Bacteroides in human pulp fibroblasts and osteoblasts.

AIM: To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts. METHODOLOGY: The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. RESULTS: Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P < 0.05). CONCLUSIONS: Black-pigmented Bacteroides are capable of amplifying the local immune response and promoting pulpal/periapical tissue inflammation by stimulating pulp fibroblasts and osteoblasts to express IL-8.

Analysis of Variance↗

PubMed2001

Effect of asymmetric dimethylarginine on osteoblastic differentiation.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS) that accumulates in renal insufficiency and may be a uremic toxin. To determine whether ADMA inhibits bone metabolism, we investigated the in vitro effect of ADMA on osteoblastic differentiation in mouse bone marrow-derived mesenchymal stem cells (BMSCs). METHODS: The effect of ADMA on nitric oxide (NO) production was determined by measuring the stable end product of NO, nitrite, in the culture medium using commercial NO kit. The temporal sequence of osteoblastic differentiation in BMSCs was assessed in the presence and absence of ADMA by measuring alkaline phosphatase (ALP) activity, mineralization, and osteoblast gene expression at 0, 4, 8, 12 days of culture. RESULTS: ADMA (5, 50, 500 micromol. L-1) resulted in a dose-dependent decrease in nitrite formation in conditioned media of BMCS cultures, consistent with inhibition of NOS. ADMA treatment was associated with reduced ALP activity, calcium deposition and osteoblast-related gene expression in BMSCs cultures. Concurrent treatment with l-arginine (3600 micromol. L-1) reversed the ADMA (500 micromol. L-1)-mediated decrease in NO production, restored the differentiation potential of BMSCs, and significantly attenuated the down-regulation of Cbfa1 and osteocalcin gene expression by ADMA. CONCLUSIONS: ADMA inhibition of the NO-NOS pathway in BMSCs impairs osteoblastic differentiation of mouse BMSC cultures. These studies further support a role of NO in the local regulation of bone metabolism and the possibility that ADMA may act as uremic toxin on bone through its effect to inhibit NO actions in osteoblasts.

Alkaline Phosphatase↗

PubMed2001

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6.

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.

Alkaline Phosphatase↗

PubMed2006

Smad3 differently affects osteoblast differentiation depending upon its differentiation stage.

Smad3, a critical component of the TGF-beta signaling pathways, plays an important role in the regulation of bone formation. However, how Smad3 affects osteoblast at the different differentiation stage remains still unknown. In the present study, we examined the effects of Smad3 on osteoblast phenotype by employing mouse bone marrow ST-2 cells and mouse osteoblastic MC3T3-E1 cells at the different differentiation stage. Smad3 overexpression significantly inhibited bone morphogenetic protein-2 (BMP-2)-induced ALP activity in ST-2 cells, indicating that Smad3 suppresses the commitment of pluripotent mesenchymal cells into osteoblastic cells. Smad3 increased the levels of COLI and ALP mRNA at 7 day cultures in MC3T3-E1 cells, and its effects on COL1 were decreased as the culture periods progress, although its effects on ALP were sustained during 21 day cultures. Smad3 overexpression enhanced the level of Runx2 and OCN mRNA at 14 day and 21 day cultures. Smad3 increased the levels of MGP and NPP-1 mRNA, although the extent of increase in MGP and NPP-1 was reduced and enhanced during the progression of culture period, respectively. Smad3 did not affect the level of ANK mRNA. On the other hand, Smad3 enhanced the level of MEPE mRNA at 14 and 21 day cultures, although Smad3 decreased it at 7 day cultures. In conclusion, Smad3 inhibits the osteoblastic commitment of ST-2 cells, while promotes the early stage of differentiation and maturation of osteoblastic committed MC3T3-E1 cells. Also, Smad3 enhanced the expression of mineralization-related genes at the maturation phase of MC3T3-E1 cells.

3T3 Cells↗

PubMed1988

Enhanced osteoblast proliferation and collagen gene expression by estradiol.

Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17 beta-estradiol on bone-forming cells, we used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphology. 17 beta-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17 alpha-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17 beta-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro alpha 1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17 beta-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by [3H]proline pulse) that was digestible by collagenase was increased, indicating that 17 beta-estradiol acts at pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17 beta-estradiol.

Animals↗

PubMed1996

An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.

Animals↗

PubMed1997

Reconstitution of stretch-activated cation channels by expression of the alpha-subunit of the epithelial sodium channel cloned from osteoblasts.

Osteoblasts respond to repetitive strain by activating stretch-activated, nonselective cation channels (SA-CAT) and increasing matrix protein production. SA-CAT channels are thought to be responsible for mechano-transduction in osteoblasts, although the molecular identity of the SA-CAT channel has previously been unknown. We have demonstrated that both the UMR-106 osteoblast-like cell line and human osteoblasts in primary culture express the alpha-subunit of the epithelial sodium channel (alpha-ENaC). The ENaC gene product is closely related to a class of proteins that confer touch sensitivity to Caenorhabditis elegans and are referred to as degenerins. A cDNA clone was obtained of the entire coding region of rat alpha-ENaC (alpha-rENaC). Sequence analysis indicated that the osteoblast clone's sequence was identical to that originally cloned from rat colon. The alpha-rENaC cDNA was cloned into an expression plasmid and transfected into LM(TK-) cells, a null cell for SA-CAT activity. Stable transfectants expressed mRNA and the expected 74-kDa protein corresponding to alpha-rENaC. Reconstitution of alpha-rENaC resulted in the expression of a 24.2 +/- 1.0 psec SA-CAT channel (P(Na):P(K) = 1.1 +/- 0.1). The channel is calcium permeable (P(Na):P(Ca) = 1.4 +/- 0.1) and highly selective for cations over anions (P(Na):P(Cl) >> 20). The channel is only active after negative pressure is applied to cell attached patches, cell swelling, or patch excision. These results represent the first heterologous expression of an SA-CAT channel in a mammalian cell system and provide evidence that the ENaC/degenerin family of proteins are capable of mediating both transepithelial sodium transport and are involved in signal transduction by mechano-sensitive cells such as osteoblasts.

Amiloride↗

PubMed1996

Differentiation and cell surface expression of transforming growth factor-beta receptors are regulated by interaction with matrix collagen in murine osteoblastic cells.

Although transforming growth factor (TGF)-beta enhances bone formation, it inhibits the differentiation of osteoblasts. To clarify the regulatory mechanism of osteoblastic differentiation and TGF-beta actions, the relationship among differentiation, TGF-beta actions, and matrix protein synthesis was examined using murine osteoblast-like MC3T3-E1 cells. Alkaline phosphatase (ALP) activity continued to increase during long-term cultures, and the increase was closely associated with a reduction in cell surface TGF-beta receptors competent to bind TGF-beta. Both the stimulation of proteoglycan synthesis and the inhibition of ALP activity by TGF-beta were also suppressed. Collagen synthesis inhibitors and an anti-alpha2beta1 integrin blocking antibody blocked the changes in ALP activity and TGF-beta receptors, and a DGEA peptide that interferes binding of collagen to alpha2beta1 integrin also blocked the increase in ALP activity. Furthermore, when MC3T3-E1 cells were cultured on extracellular matrix layers obtained from these cells, all the differentiation-associated changes could be observed without collagen production, and the extracellular matrix-induced differentiation was also blocked by an anti-alpha2beta1 integrin antibody. These results demonstrate that the interaction of cell surface alpha2beta1 integrin with matrix collagen synthesized by osteoblasts themselves is involved in the osteoblastic differentiation and the reduction in cell surface receptors and actions of TGF-beta. It is suggested that matrix collagen synthesized under the stimulation by TGF-beta plays an important role in the regulation of osteoblastic differentiation and TGF-beta actions by differentiation-associated down-regulation of TGF-beta receptors.

3T3 Cells↗