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.     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα

Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα.     

The combination of high glucose and advanced glycation end-products (AGEs) inhibits the mineralization of osteoblastic MC3T3-E1 cells through glucose-induced increase in the receptor for AGEs.

Type 1 diabetes mellitus is known to be associated with reduced bone mass and increased bone fractures. This is thought to be due to a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption, but the precise mechanism is unknown. In this study, we examined whether or not high glucose or advanced glycation end-products (AGEs), which play key roles in the pathogenesis and complications of diabetes, affect the differentiation of osteoblastic MC3T3-E1 cells. First, MC3T3-E1 cells were incubated in media containing either 22 mM glucose, 22 mM mannitol, 300 microg/ml AGE2, or 300 microg/ml AGE3. Each of these agents alone did not affect the mineralization of the cells by von Kossa staining and Alizarin red staining. However, high glucose but not mannitol or AGEs markedly increased mRNA expression of AGE receptor (RAGE) by real-time PCR. Next, we examined the combined effects of high glucose and AGEs on the differentiation of MC3T3-E1 cells. The combination of 22 mM glucose and 300 microg/ml AGE2 significantly inhibited the mineralization of MC3T3-E1 cells, and 22 mM glucose in combination with either 300 microg/ml AGE2 or AGE3 apparently decreased osteocalcin mRNA expression. These results suggest that high glucose or AGEs alone might have no effect on osteoblastic differentiation, but their combination could additionally or synergistically inhibit osteoblastic mineralization through glucose-induced increase in RAGE expression.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway

Extracellular matrix proteins (ECMs) serve as both a structural support for cells and a dynamic biochemical network that directs cellular activities. ECM proteins such as those of the SIBLING family (small integrin-binding ligand glycoprotein) could possess inherent growth factor activity. In this study, we demonstrate that exon 5 of dentin matrix protein 3 (phosphophoryn (PP)), a non-collagenous dentin ECM protein and SIBLING protein family member, up-regulates osteoblast marker genes in primary human adult mesenchymal stem cells (hMSCs), a mouse osteoblastic cell line (MC3T3-E1), and a mouse fibroblastic cell line (NIH3T3). Quantitative real-time PCR technology was used to quantify gene expression levels of bone markers such as Runx2, Osx (Osterix), bone/liver/kidney Alp (alkaline phosphatase), Ocn (osteocalcin), and Bsp (bone sialoprotein) in response to recombinant PP and stably transfected PP. PP up-regulated Runx2, Osx, and Ocn gene expression. PP increased OCN protein production in hMSCs and MC3T3-E1. ALP activity and calcium deposition was increased by PP in hMSC. Furthermore, an alpha(v)beta(3) integrin-blocking antibody significantly inhibited recombinant PP-induced expression of Runx2 in hMSCs, suggesting that signaling by PP is mediated through the integrin pathway. PP was also shown to activate p38, ERK1/2, and JNK, three components of the MAPK pathway. These data demonstrate a novel signaling function for PP in cell differentiation beyond the hypothesized role of PP in biomineralization.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells

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 microM 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.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells

Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCmu.