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MC3T3-E1 Cells (mc3t3-e1 + cell)
Kinds of MC3T3-E1 Cells Selected AbstractsLysyl Hydroxylase-2b Directs Collagen Cross-Linking Pathways in MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2004Suchaya Pornprasertsuk Abstract To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. Introduction: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. Materials and Methods: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. Results: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced (,0.3 mol/mol of collagen) in AS clones. Conclusions: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues. [source] Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002Hideaki Sowa Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source] Bone Morphogenetic Protein-6-loaded Chitosan Scaffolds Enhance the Osteoblastic Characteristics of MC3T3-E1 CellsARTIFICIAL ORGANS, Issue 1 2010Abdullah C. Akman Abstract The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications. [source] The proteasome inhibitor bortezomib inhibits FGF-2-induced reduction of TAZ levels in osteoblast-like cellsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2010Homare Eda Abstract Objectives:,Bortezomib (PS-341; VelcadeÔ), a proteasome inhibitor, is used as a therapeutic agent for multiple myeloma. Bortezomib has been shown to strongly induce osteoblast differentiation and elevate the levels of osteoblast-related differentiation markers in the serum of patients with myeloma. Bortezomib also reportedly increases the activity of the transcription factor, Runx2. However, the mechanism of action by which bortezomib-elevated Runx2 activity mediates osteoblast differentiation remains unclear. On the other hand, fibroblast growth factor 2 (FGF-2) is found at high levels in patients with multiple myeloma. We previously reported that FGF-2 reduces the levels of the transcriptional coactivator with PDZ-binding motif (TAZ). We therefore investigated the effects of bortezomib on TAZ protein levels in the presence of FGF-2. Methods: Osteoblastic MC3T3-E1 cells were treated with different concentrations of bortezomib in the presence or absence of FGF-2 and various biologic responses were investigated by immunoblotting, RT-PCR, quantitative PCR, and alizarin red staining. Results: We found that bortezomib inhibited FGF-2-induced reduction of TAZ levels through a pathway other than that used for proteasome inhibition, while maintaining TAZ function, which in turn, enhanced the expression of Runx2-transcribed osteogenic differentiation markers. Bortezomib also suppressed the antimineralization effect of FGF-2. Conclusions: These findings suggest that bortezomib inhibited FGF-2-induced reduction of TAZ and consequently stimulated osteogenic differentiation independently of proteasome inhibition. These findings may contribute to elucidate the osteolytic mechanism in multiple myeloma, and to the development of new drugs for multiple myeloma and other osteolytic diseases. [source] Cyclin D1 as a Target for the Proliferative Effects of PTH and PTHrP in Early Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Nabanita S Datta MS Abstract PTHrP induced a proliferative cyclin D1 activation in low-density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP-1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts. Introduction: The mechanisms underlying the actions of PTH and its related protein (PTHrP) in osteoblast proliferation, differentiation, and bone remodeling remain unclear. The action of PTH or PTHrP on the cell cycle during osteoblast proliferation was studied. Materials and Methods: Mouse calvarial MC3T3-E1 clone 4 cells were synchronized by serum starvation and induced with 100 nM PTHrP for 2,24 h under defined low serum conditions. Western blot, real-time PCR, EMSAs, and promoter/luciferase assays were performed to evaluate cyclin D1 expression. Pharmacological inhibitors were used to determine the relevant signaling pathways. Ectopic ossicles generated from implanted bone marrow stromal cells were treated with acute (a single 8- or 12-h injection) or intermittent anabolic PTH treatment for 7 days, and RNA and histologic analysis were performed. Results: PTHrP upregulated cyclin D1 and CDK1 and decreased p27 expression. Cyclin D1 promoter/luciferase assays showed that the PTHrP regulation involved both activator protein-1 (AP-1) and cyclic AMP response element binding protein (CRE) sites. AP-1 and CRE double mutants completely abolished the PTHrP effect of cyclin D1 transcription. Upregulation of cyclin D1 was found to be protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) dependent in proliferating MC3T3-E1 cells. In vivo expression of cyclin D1 in ectopic ossicles was upregulated after a single 12-h PTH injection or intermittent anabolic PTH treatment for 7 days in early developing ossicles. Conclusions: These data indicate that PTH and PTHrP induce cyclin D1 expression in early osteoblastic cells and their action is developmental stage specific. [source] Negative Regulation by p70 S6 Kinase of FGF-2,Stimulated VEGF Release Through Stress-Activated Protein Kinase/c- Jun N-Terminal Kinase in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007Shinji Takai Abstract To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2,stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2,activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2,stimulated VEGF release and VEGF mRNA expression. The FGF-2,induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2,induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2,stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2,stimulated VEGF release and phosphorylation of SAPK/JNK. Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2,stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts. [source] Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Daichi Chikazu Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source] Overexpression of Lysyl Hydroxylase-2b Leads to Defective Collagen Fibrillogenesis and Matrix Mineralization,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2005Suchaya Pornprasertsuk Abstract Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. Introduction: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. Materials and Methods: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. Results: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. Conclusions: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone. [source] Lysyl Hydroxylase-2b Directs Collagen Cross-Linking Pathways in MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2004Suchaya Pornprasertsuk Abstract To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. Introduction: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. Materials and Methods: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. Results: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced (,0.3 mol/mol of collagen) in AS clones. Conclusions: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues. [source] Identification of Novel Target Genes of the Bone-Specific Transcription Factor Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Michael Stock Abstract Fifteen putative transcriptional target genes regulated by the osteogenic transcription factor Runx2 were identified by cDNA microarray and differential hybridization techniques. Expression pattern and regulation of one gene, Pttg1ip, was analyzed in detail. Introduction: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. Materials and Methods: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10T1/2 cells with constitutive and inducible overexpression of Runx2. Results and Conclusions: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecm1, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esx1, Osr1, and Sox9, whereas others were Ptn, Npdc-1, Hig1, and Tem1. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg1ip promoter in MC3T3-E1 cells. Therefore, Pttg1ip is likely to be a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation. [source] Ascorbic Acid Induces Collagenase-1 in Human Periodontal Ligament Cells but Not in MC3T3-E1 Osteoblast-Like Cells: Potential Association Between Collagenase Expression and Changes in Alkaline Phosphatase Phenotype,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2003Momotoshi Shiga Abstract Ascorbic acid (AA) enhances osteoblastic differentiation by increasing collagen accumulation, which in turn, results in increased alkaline phosphatase (AP) expression in some osteogenic cells. However, in other cells, including human periodontal ligament (PDL) cells, additional osteoinductive agents are required for this response. To understand the potential basis for the maintenance of the AP phenotype of PDL cells exposed to AA, we examined the modulation of the tissue-degrading matrix metalloproteinases (MMPs) and their inhibitors by AA in short-term cell cultures. Early passage PDL cells in serum-free medium were exposed to AA for 5 days. The samples were analyzed for MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), AP, collagen I(,1), and osteocalcin. We found that AA dose-dependently increased the expression of collagenase-1, and minimally TIMP-1, but not stromelysin-1 or TIMP-2. Additionally, AA caused substantial increases in levels of type I collagen. AA was unable to increase AP activity or osteocalcin messenger RNA in PDL cells. However, the cells retained the ability to show a significantly greater AP expression in high- versus low-density cultures, and increased osteocalcin as well as AP levels when cultured in the presence of dexamethasone. Moreover, in cells exposed to dexamethasone, increases in AP and osteocalcin were accompanied by a repression of collagenase-1 expression. In contrast to PDL cells, AA did not induce collagenase but produced a significant increase in AP expression in MC3T3-E1 cells. These findings provide the first evidence that AA, by modulating both collagen and collagenase-1 expression in PDL cells, most likely contributes to a net matrix remodeling response in these cells. Furthermore, the relationship between changes in collagenase expression and alterations in AP activity in PDL and MC3T3-E1 cells suggests a potential role for collagenase in modulating the AP phenotype of cells with osteoblastic potential. [source] Strong Static Magnetic Field Stimulates Bone Formation to a Definite Orientation In Vitro and In Vivo,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2002Hiroko Kotani Ph.D. Abstract The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects. [source] Smad3 Promotes Alkaline Phosphatase Activity and Mineralization of Osteoblastic MC3T3-E1 Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2002Hideaki Sowa Abstract Transforming growth factor (TGF) , is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-, signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-, on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-, inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L -azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs. [source] Fluid Flow Induction of Cyclo-Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal-Regulated Kinase Signaling Pathway,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2002Sunil Wadhwa Abstract Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with ,371/+70 base pairs (bp) of the COX-2 5,-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm2, induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at ,8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by ,80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway. [source] A Role for G Protein-Coupled Lysophospholipid Receptors in Sphingolipid-Induced Ca2+ Signaling in MC3T3-E1 Osteoblastic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2001Jeremy M. Lyons Abstract Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3-E1 osteoblastic cells to sphingosine (SPH), sphingosine-1-phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic-free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP- and SPC-induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3-E1 cells were found to express transcripts encoding Edg -1 and Edg -5 but not Edg -3, Edg -6, or Edg -8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA-induced Ca2+ signaling was blocked in MC3T3-E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2-aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels in the ER. No effect of 2-APB was observed on SPH- or LPA-induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg -1 and/or Edg -5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3 -gated channels. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Insulin-Like Growth Factor I Production Is Essential for Anabolic Effects of Thyroid Hormone in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2000Bill K. Huang Abstract Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both ,IR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses. (J Bone Miner Res 2000;15:188,197) [source] ,-cryptoxanthin stimulates cell differentiation and mineralization in osteoblastic MC3T3-E1 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Satoshi Uchiyama Abstract The effect of ,-cryptoxanthin, a kind of carotenoid, on cell differentiation and mineralization in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in a minimum essential medium containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or ,-cryptoxanthin (10,8 to 10,6 M) without FBS. Cells were cultured for 3 to 21 days. Gene expression in osteoblastic cells was determined using reverse transcription-polymerase chain reaction (RT-PCR). Culture with ,-cryptoxanthin (10,7 or 10,6 M) for 3 days caused a significant increase in Runx2 type 1, Runx2 type 2, ,1 (I) collagen, and alkaline phosphatase mRNA levels in osteoblastic cells. These increases were completely blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-,- D -ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. Meanwhile, vitamin A (10,6 M) did not have a significant effect on Runx2 type 1 mRNA expression in the cells. The effect of ,-cryptoxanthin (10,6 M) in stimulating Runx2 type 1 and ,1 (I) collagen mRNA levels, protein content, and alkaline phosphatase activity in the cells was also seen in the presence of vitamin A (10,6 M), suggesting that the mode of ,-cryptoxanthin action differs from that of vitamin A. Prolonged culture with ,-cryptoxanthin (10,6 M) for 3 to 21 days caused a significant increase in cell number, deoxyribonucleic acid (DNA) content, protein content, and alkaline phosphatase activity in osteoblastic cells, suggesting that ,-cryptoxanthin stimulates cell proliferation and differentiation. Moreover, culture with ,-cryptoxanthin (10,7 or 10,6 M) for 5 to 21 days caused a remarkable increase in mineralization. This study demonstrates that ,-cryptoxanthin has a stimulatory effect on cell differentiation and mineralization due to enhancing gene expression of proteins, which involve in bone formation in osteoblastic MC3T3-E1 cells. © 2005 Wiley-Liss, Inc. [source] Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2004Meenal Mehrotra Abstract Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24,48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation. © 2004 Wiley-Liss, Inc. [source] Presence of a functional receptor for GLP-1 in osteoblastic cells, independent of the cAMP-linked GLP-1 receptorJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Bernardo Nuche-Berenguer Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [125I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30,min at 25°C; in these conditions, [125I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70,kDa band, almost undetectable in the presence of 10,6,M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor. J. Cell. Physiol. 225: 585,592, 2010. © 2010 Wiley-Liss, Inc. [source] Impaired cell cycle regulation of the osteoblast-related heterodimeric transcription factor Runx2-Cbf, in osteosarcoma cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Inga A. San Martin Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbf, heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G1. Runx2 levels are modulated during the cell cycle, which are maximal in G1 and minimal beyond the G1/S phase transition (S, G2, and M phases). It is not known whether Cbf, gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbf, are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbf, gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G1 (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbf, protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbf, levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G1 and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma. J. Cell. Physiol. 221: 560,571, 2009. © 2009 Wiley-Liss, Inc. [source] cAMP activation by PACAP/VIP stimulates IL-6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2009Azusa Nagata The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/vasoactive intestinal peptide (VIP) superfamily, stimulates cyclic AMP accumulation initiating a variety of biological processes such as: neurotropic actions, immune and pituitary function, learning and memory, catecholamine biosynthesis and regulation of cardiopulmonary function. Both osteoclasts and osteoblasts have been shown to express receptors for PACAP/VIP implicated in their role in bone metabolism. To further understand the role of PACAP/VIP family in controlling bone metabolism, we investigated differentiation model of MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria. Quantitative RT-PCR analysis demonstrated that MC3T3-E1 cells expressed only VPAC2 receptor and its expression was upregulated during osteoblastic differentiation, whereas VPAC1 and PAC1 receptors were not expressed. Consistent with expression of receptor subtype, both PACAP and VIP stimulate cAMP accumulation in a time- and dose-dependent manner with the similar potency in undifferentiated and differentiated cells, while Maxadilan, a specific agonist for PAC1-R, did not. Furthermore, downregulation of VPAC2-R by siRNA completely blocked cAMP response mediated by PACAP and VIP. Importantly, PACAP/VIP as well as forskolin markedly suppressed the induction of alkaline phosphatase mRNA upon differentiation and the pretreatment with 2,,5,-dideoxyadenosine, a cAMP inhibitor, restored its inhibitory effect of PACAP. We also found that PACAP and VIP stimulated IL-6 release, a stimulator of bone resorption, and VPAC2-R silencing inhibited IL-6 production. Thus, PACAP/VIP can activate adenylate cyclase response and regulate IL-6 release through VPAC2 receptor with profound functional consequences for the inhibition of osteoblastic differentiation in MC3T3-E1 cells. J. Cell. Physiol. 221: 75,83, 2009. © 2009 Wiley-Liss, Inc [source] Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2008Tatsuya Miyazaki Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N -acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization. J. Cell. Physiol. 217: 769,777, 2008. © 2008 Wiley-Liss, Inc. [source] Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2007Kyosuke Uno Activation of particular glutamate (Glu) receptors is shown to promote cellular differentiation toward maturation during osteoblastogenesis. In the present study, we have evaluated the possible modulation by Glu of cellular proliferation in osteoblastic cells endowed to proliferate for self-renewal and to differentiate toward matured osteoblasts. Exposure to Glu significantly suppressed the proliferation activity at a concentration over 500 µM without inducing cell death in osteoblastic MC3T3-E1 cells before differentiation. The suppression by Glu occurred in a manner sensitive to the prevention by either cystine or reduced glutathione. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits in these undifferentiated osteoblastic cells. A significant decrease was seen in intracellular total glutathione levels in undifferentiated MC3T3-E1 cells cultured with Glu, indeed, whereas the cellular proliferation activity was drastically decreased by the addition of the glutathione depleter cyclohexene-1-one and the glutathione biosynthesis inhibitor L -buthionine-[S,R]-sulfoximine, respectively. Exposure to Glu led to a significant increase in mRNA expression of nuclear factor E2 p45-related factor 2 (Nrf2) together with the generation of reactive oxygen species, while a significant decrease was seen in the proliferation activity in MC3T3-E1 cells with stable overexpression of Nrf2. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the upregulation of Nrf2 expression in association with the depletion of intracellular glutathione after promoting the retrograde operation of the cystine/Glu antiporter in undifferentiated MC3T3-E1 cells. J. Cell. Physiol. 213: 105,114, 2007. © 2007 Wiley-Liss, Inc. [source] The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Chirag B. Khatiwala Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance. J. Cell. Physiol. 211: 661,672, 2007. © 2007 Wiley-Liss, Inc. [source] Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralizationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Akemichi Ueno Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322,332, 2006. © 2006 Wiley-Liss, Inc. [source] Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Takahito Yuasa The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 ,g) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1,5 ,g) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP. © 2002 Wiley-Liss, Inc. [source] Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, osterix, and Dlx5JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2010Richard Chiu Abstract Polymethylmethacrylate (PMMA) particles have been shown to inhibit the differentiation of osteoprogenitor cells, but the mechanism of this inhibitory effect has not been investigated. We hypothesize that the inhibitory effects of PMMA particles involve impairment of osteoprogenitor viability and direct inhibition of transcription factors that regulate osteogenesis. We challenged MC3T3-E1 osteoprogenitors with PMMA particles and examined the effects of these materials on osteoprogenitor viability and expression of transcription factors Runx2, osterix, Dlx5, and Msx2. MC3T3-E1 cells treated with PMMA particles over a 72-h period showed a significant reduction in cell viability and proliferation as indicated by a dose- and time-dependent increase in supernatant levels of lactate dehydrogenase, an intracellular enzyme released from dead cells, a dose-dependent decrease in cell number and BrdU uptake, and the presence of large numbers of positively labeled Annexin V-stained cells. The absence of apoptotic cells on TUNEL assay indicated that cell death occurred by necrosis, not apoptosis. MC3T3-E1 cells challenged with PMMA particles during the first 6 days of differentiation in osteogenic medium showed a significant dose-dependent decrease in the RNA expression of Runx2, osterix, and Dlx5 on all days of measurement, while the RNA expression of Msx2, an antagonist of Dlx5-induced osteogenesis, remained relatively unaffected. These results indicate that PMMA particles impair osteoprogenitor viability and inhibit the expression of transcription factors that promote osteoprogenitor differentiation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:571,577, 2010 [source] Exposure of mouse preosteoblasts to pulsed electromagnetic fields reduces the amount of mature, type I collagen in the extracellular matrixJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2006Yoshitada Sakai Abstract We tested the hypothesis that exposure of a mouse preosteoblast cell line to pulsed electromagnetic fields (PEMF) would affect components of the extracellular matrix. We report that exposure of MC3T3-E1 cells to a single PEMF waveform significantly reduced the amount of mature, ,1(I) collagen in the extracellular matrix (ECM) and the conditioned medium, without affecting the amount of total ECM protein. This decrease was not due to changes in the steady-state level of Col1A1 mRNA or to degradation of mature collagen. We then tested the effect of three distinct PEMF waveforms, two orthogonal coil orientations, and two waveform amplitude levels on the amount of ,1(I) collagen in the conditioned medium. A sequence of factorial ANOVAs and stepwise regression modeling revealed that the period (duration) of the individual pulses accounted for a significant proportion of the variance associated with the amount of ,1(I) collagen in the conditioned medium. The total variance accounted for, however, was small (R2,=,0.155, p,<,0.001 and R2,=,0.172, p,<,0.001, in the horizontal and vertical orientations, respectively). The positive and negative regression coefficients for the coil orientations revealed that the influence of pulse period was significantly different for the orthogonal coil orientations (p,<,0.001). The findings imply that the dominant influence of PEMF on the amount of mature, ,1(I) collagen in the ECM is related to variables other than those expressed in the time-amplitude domain. The results provide objective direction toward identifying waveform characteristics that contribute to the observed between-waveform differences with regard to collagen. Advances in this area may lead toward improving waveforms and waveform delivery protocols. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] | |||||||||||||