Osteogenic role of endosomal chloride channels in MC3T3-E1 cells

PHR protein family consists of C. elegan Rpm-1/Drosophila Highwire/Zebrafish Esrom/Mouse Phr-1/Human Pam. Esrom is required for correct neurites exiting the paused state at intermediate targets as well as pteridine synthesis. This study reports the identification and characterization of two novel Esrom splice variants, named splice variants 2 (splicing out 5′ 24 bp of exon 17) and 3 (splicing out 5′ 24 bp of exons 17 and 18). Polypeptides encoded by 5′ 24 bp of exons 17 and 18 are part of basic amino-acid-rich region inside Esrom RCC1-like domain (RLD). These two splice variants maintain the whole protein reading frame and alternative exons usage patterns are conserved with mammal. At different developmental stages and adult zebrafish tissues, abundances of these splice variants are different. Importantly, by yeast two-hybrid screen and confocal colocalization analysis, it was found that alternative splicing of exon 18 regulates Esrom RLD interaction with kinesin family member 22 and G protein beta-subunit 1. Taken together, these results suggest that Esrom RLD functions are regulated by alternative splicing at temporal and spatial-specific manner.     

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.     

Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.     

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.     

Apocynin stimulates osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, the effect of apocynin on the function of osteoblastic MC3T3-E1 cells was studied. Apocynin caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells (P < 0.05). Antimycin A (AMA), which inhibits complex III of the electron transport system, has been used as a reactive oxygen species (ROS) generator in biological systems. We exposed cultured osteoblastic MC3T3-E1 cells to AMA with or without pretreatment with apocynin. Apocynin significantly (P < 0.05) increased cell survival, calcium deposition, and osteoprotegerin release and decreased the production of ROS and osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of AMA. These results demonstrate that apocynin can protect osteoblasts from mitochondrial dysfunction-induced toxicity and may have positive effects on skeletal structure.     

小鼠前成骨细胞MC3T3-E1在自组装多肽水凝胶支架中的成骨分化

目的研究MC3T3-E1细胞在自组装多肽水凝胶支架上的生长和成骨分化.方法在多肽水凝胶支架RADA16上接种MC3T3-E1细胞,荧光染色观察细胞形态和存活情况;组织化学染色检测MC3T3-E1细胞碱性磷酸酶活性以及细胞外钙质沉积;RT-PCR分析成骨特异性基因的表达.结果 MC3T3-E1细胞在水凝胶支架RADA16上粘附铺展良好,呈纺锤样形态.诱导培养后支架上的细胞有较高水平的碱性磷酸酶表达和矿化基质沉积.此外,骨分化特异性基因骨桥蛋白和骨涎蛋白也有表达,且表达量随培养时间的延长而增多.结论 在自组装水凝胶内MC3T3-E1细胞可向成骨方向分化,并能在凝胶内产生矿化的细胞外基质.     

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells

Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4–10), bone matrix formation/maturation (days 10–16), and mineralization stages (days 16 –30). During the proliferation period (days 4–10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10–16), type I collagen expression and biosynthesis, fibronectin, TGF-β1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-β1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16–30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation. © 1996 Wiley-Liss, Inc.     

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.     

Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells

We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 microM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

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.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells

Background  

The main morphological features of primitive cells, such as stem and progenitor cells, are that these cells consists only one nucleus. The main purpose of this study was to determine the differentiation capacities of stem and progenitor cells. This study was performed using mononucleated cells originated from murine peripheral blood and MC3T3-E1 cells. Three approaches were used to determine their differentiation capacities: 1) Biochemical assays, 2) Gene expression analysis, and 3) Morphological observations.     

体外培养所形成的细胞外基质对MC3T3-E1细胞生长和分化的影响

细胞外基对组织细胞起支持、保护、营养作用,对细胞的增殖、分化有重要影响,在细胞和组织工程中,应该充分考虑细胞外基质的作用。本研究首先脱去培养板中融合培养的原代小鼠心肌成纤维细胞和成骨细胞,获得两种体外形成的细胞外基质包被的培养板,其中成骨细胞细胞外基质中含有骨形成蛋白2。然后将MC3T3-E1成骨前体细胞接种在这种培养板中,发现成纤维细胞胞外基质包被的培养板中的细胞增殖活性最高,而成骨细胞胞外基质包被的培养板中细胞的碱性磷酸酶活性、骨形成蛋白2和骨桥蛋白的相对蛋白表达量最高,细胞外钙沉积量比其他组高1倍左右。结果表明：包被在培养板上的这两种细胞外基质有不同的生物活性,成纤维细胞胞外基质可促进成骨前体细胞增殖,成骨细胞胞外基质可促进成骨前体细胞骨向分化。     

Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation

In this in vitro study, the hypothesis that the beneficial effects of dietary genistein on bone are through the modulation of the bone marker synthesis by osteoblastic MC3T3-E1 cells was tested, and the possible roles of estrogen receptors in the actions of genistein on osteoblastic cells were also examined. Interleukin-6 production was decreased 40% to 60% in osteoblastic cells treated with genistein from either day 8-16 or day 12-16, at dietarily achievable concentrations (10(-10) to 10(-8) M) (P<0.05). The mRNA expression of osteoprotegerin increased about 140% in cells treated from with genistein day 4-8 at a concentration of 10(-8) M (P<0.05). The ratio of estrogen receptor-alpha to beta expression increased 10-fold from day 0 to 12 of culture (P<0.05). Correlating with this time-dependent variation in estrogen receptor expression, treatments of 17beta-estradiol and genistein had opposite dose patterns on the ratio of estrogen receptor-alpha to beta expression following treatment from day 4 to 6 compared to from day 0 to 2. The addition of ICI-182,780, an estrogen receptor blocker, reduced the inhibitory effect of genistein on IL-6 production by 30-50%. In summary, these findings suggest that the beneficial skeletal effects of genistein, at dietarily achievable levels, appear to be mediated, at least in part, by interleukin-6 and osteoprotegerin, and estrogen receptors play important roles in the inhibition of interleukin-6 synthesis by genistein in osteoblastic MC3T3-E1 cells.     

Osteogenin inhibits proliferation and stimulates differentiation in mouse osteoblast-like cells (MC3T3-E1)

Osteogenin, a novel bone differentiation factor, was recently purified and characterized. We examined its effect on the proliferation and differentiation of MC3T3-E1 osteoblast-like cells. Cell proliferation was inhibited the first 48 h after addition of osteogenin, and this effect was independent of serum. Osteogenin did not influence the cell morphology. Alkaline phosphatase promptly increased in a dose and time-dependent manner and appeared to be specific. Treatment with TGF-beta 1 resulted in inhibition of alkaline phosphatase activity, and was reversed by osteogenin within 48 h. Cell cultures treated with osteogenin for 72 h after confluence became responsive to parathyroid hormone. Synthesis of collagenous proteins was stimulated by osteogenin. The present results demonstrate a significant influence of osteogenin on the differentiation of osteogenic phenotype in MC3T3-E1 cells in vitro.