Arachidonic acid stimulates cell growth in an osteoblastic cell line,MC3T3-E1, by noneicosanoid mechanism

Summary Arachidonic acid, added to α-minimum essential medium containing 10% fetal bovine serum at the final concentration of 10⁻⁴ M, significantly increased DNA content of an osteoblastic cell line, MC3T3-E1, along with an increase of DNA synthesis. No growth-stimulatory effect of arachidonic acid was observed under serum-free condition. α-Linolenic acid, which cannot be converted to arachidonic acid, also increased DNA content at 10⁻⁴ M. Additionally, the stimulatory effects of these fatty acids were not inhibited by simultaneous addition of 10⁻⁵ M of indomethacin. Indomethacin, when added to α-minimum essential medium with 10% fetal bovine serum, also significantly increased DNA content of MC3T3-E1 cells. These results suggest that arachidonic acid may potentiate the growth-stimulatory effect of serumderived growth factors probably via noneicosanoid mechanism. Rat osteogenic sarcoma cell line, UMR106, also showed an increase in DNA content with arachidonic acid treatment. Hence, it is suggested that arachidonic acid may stimulate proliferation of cells of osteoblastic lineage. It is also suggested that indomethacin, probably by blocking endogenous prostaglandin E₂ synthesis, stimulates cell growth in MC3T3-E1 cells.     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.     

Nitric oxide promotes infectious bone resorption by enhancing cytokine-stimulated interstitial collagenase synthesis in osteoblasts.

This experiment was undertaken to determine the role of macrophage-derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)-induced bone resorption by using an in vitro co-culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-a mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR-106 (rat) and MC3T3-E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS-stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3-E1 and a trivial effect in UMR-106. On the other hand, CM induced matrix metalloproteinase-1 (MMP-1) gene expression in both osteoblast cell lines. The NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) did not alter this effect in MC3T3-E1 and UMR-106, whereas TNF-a antibody diminished the CM-induced MMP-1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP-1 stimulator for UMR-106, augmented the TNF-alpha-stimulated MMP-1 mRNA production in UMR-106. In a J774/UMR-106 co-culture system, LPS stimulated significant MMP-1 gene expression in UMR-106, and this upregulation was abolished by L-NMMA and TNF-alpha antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co-distributions of iNOS+ macrophages and MMP-1+ osteoblasts around the osteolytic areas. Administration of L-NMMA markedly reduced the extent of bone loss and the percentage of MMP-1-synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine-induced MMP-1 production in osteoblasts.     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures. In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor beta (TGF-beta). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.     

Response of osteoblastic clonal cell line (MC3T3-E1) to [Asu1,7]Eel calcitonin at a specific cell density or differentiation stage

Summary Clone MC3T3-E1 cells isolated from newborn mouse calvaria is an osteogenic cell line which retains an ability to differentiate into osteo-blastic cellin vitro. The effect of [Asu^1,7]eel calcitonin (ECT) on clonal MC3T3-E1 cells was investigated at different stages of differentiation. ECT caused an increase in alkaline phosphatase (ALP) activity. The stimulative effect was demonstrated to be dependent upon cell density or differentiation stage. At a cell density of 1.18×10⁵/cm² cells were incubated with ECT for 2 days. The treatment by ECT caused an increase in ALP activity. A specific response to ECT dependent on the cell density was observed in a narrow range of cell density. Moreover this range of cell density responsible to ECT was found to be a rapid differentiation stage of MC3T3-E1 cells. These results suggest that calcitonin stimulates differentiation of osteoblast. In addition to these results, cellular adenosine-3′, 5′-cyclic monophosphate (cAMP) level was raised by ECT treatment at a cell density of about 1.4×10⁵ cell/cm² and this response was also specific for cell density. At cell density lower or higher than this density no stimulative effect by ECT was observed. On the other hand, N⁶,O₂-dibutyryl adenosine-3′,5′-cyclic monophosphate (db-cAMP) and theophylline caused an increase in ALP activity in wide cell density range. These results indicate that an increase in ALP activity by ECT is mediated by intracellular cAMP and that the specific response to ECT dependent on the cell density is regulated in the process of cAMP formation and/or in the preceding process of cAMP formation.     

唑来膦酸对小鼠胚胎成骨细胞增殖和分化的影响

目的研究唑来膦酸对小鼠胚胎成骨细胞体外增殖和成骨分化的影响。方法将小鼠胚胎成骨细胞体外传代培养,使用含不同浓度（1.0、0.1μmol/L）唑来膦酸的成骨诱导培养基干预细胞,不含唑来膦酸的培养基作对照,培养1、3、5 d采用CCK-8试剂盒检测唑来膦酸对成骨细胞增殖的影响;培养14 d行碱性磷酸酶（ALP）染色;培养21 d行茜素红染色;培养7 d,实时定量聚合酶链式反应（Real-time PCR）检测转录因子Runx2、成骨标志物Ⅰ型胶原（Collagen TypeⅠ）、ALP、骨钙素（OCN）基因的表达,免疫印迹法（Western Blotting）检测转录因子Runx2蛋白的表达。结果不同浓度的唑来膦酸干预细胞后,CCK-8实验检测吸光度值随天数增加而升高,各组之间差异无统计学意义（P〉0.05）。随着唑来膦酸浓度的升高,ALP染色和茜素红染色逐渐变浅,Runx2、Collagen TypeⅠ、ALP、OCN基因的表达量降低,Runx2蛋白的表达量降低,差异均有统计学意义（P〈0.05）。结论唑来膦酸在选定浓度（1.0、0.1μmol/L）下不影响成骨细胞的增殖,但对其分化功能的抑制作用随着浓度的增加而增强。     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

MicroRNA-196a靶向调节HDAC9对MC3T3-E1细胞成骨分化的影响

目的 探究微小RNA（miR）-196a靶向调节组蛋白去乙酰化酶9（HDAC9）对MC3T3-E1细胞成骨分化的影响。方法 将MC3T3-E1细胞分为对照组（Cont）组、诱导组、miR-196a-mimics-NC组、miR-196a-mimics组、miR-196a-inhibitor-NC组、miR-196a-inhibitor组、miR-196a-mimics+pCMV-HDAC9-NC组、miR-196a-mimics+pCMV-HDAC9组,根据分组转染后进行成骨诱导。定量荧光PCR检测MC3T3-E1细胞中miR-196a、HDAC9表达量;试剂盒检测碱性磷酸酶（ALP）活性;茜素红染色观察矿化程度;Western blot检测HDAC9、ALP、Runt相关转录因子2（Runx2）、胶原蛋白I（COL1）、骨桥蛋白（OPN）、Histone H3、Histone H3（acetyl K9、K14和K23）表达量。结果 与Cont组相比,诱导组MC3T3-E1细胞中miR-196a表达、ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3 K9、K14、K23位点乙酰化水平增高（P＜0.05）,HDAC9 mRNA和蛋白表达降低（P＜0.05）。转染miR-196a-mimics可明显增加miR-196a表达,降低HDAC9表达,并增加ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3乙酰化,转染miR-196a-inhibitor则作用相反。miR-196a可靶向下调HDAC9表达,过表达HDAC9可部分逆转miR-196a mimics对MC3T3-E1细胞成骨分化的促进效应。结论 miR-196a可靶向下调HDAC9表达,增加组蛋白乙酰化水平,促进MC3T3-E1细胞成骨分化。     

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation.

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.     

黄瓜籽总皂苷对MC3T3-E1细胞分化及SPARC、OPG/RANKL表达的影响

目的观察黄瓜籽总皂苷提取物(cucumber seed saponins,CSS)对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响,以及与骨质疏松相关的SPARC、OPG/RANKL/RANK信号通路的作用。方法通过MTT实验、碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色,考察不同浓度CSS对MC3T3-E1细胞增殖、分化及矿化的影响;采用RT-PCR方法检测SPARC、OPG/RANKL mRNA表达水平; Western blot检测SPARC、OPG/RANKL的蛋白表达量。结果与阳性对照组相比,CSS能明显促进MC3T3-E1细胞增殖(P0.05),CSS高、中剂量组能明显提高MC3T3-E1细胞ALP活性及钙化结节数量(P0.05);与空白组相比,CSS不同剂量组均明显上调SPARC、OPG/RANKL mRNA及蛋白表达水平(P0.05)。结论黄瓜籽总皂苷能够促进成骨细胞MC3T3-E1的增殖、分化及矿化能力,并通过上调SPARC、OPG/RANKL的表达水平提高MC3T3-E1细胞的成骨能力。     

Twisted gastrulation and chordin inhibit differentiation and mineralization in MC3T3-E1 osteoblast-like cells

Bone morphogenetic proteins (BMPs) are potent inducers of osteoblast differentiation. The accessibility of BMP ligands for binding to their receptors is regulated by secreted proteins Twisted gastrulation (Tsg) and Chordin (Chd). Tsg antagonizes BMP signaling by forming ternary complexes with Chd and BMPs, thereby preventing BMPs from binding to their receptors. In addition to the anti-BMP function, Tsg also has pro-BMP activity, partly mediated by cleavage and degradation of Chd, which releases BMPs from ternary complexes. The roles of Tsg and Chd in osteoblast differentiation are not known. Therefore, in the present study, we investigated the effect of exogenous Tsg and Chd on osteoblast differentiation and mineralization using a well-characterized subclone of MC3T3-E1 osteoblast-like cells. Our results show that Tsg and Chd are expressed in MC3T3-E1 osteoblast-like cells. While Tsg mRNA levels decrease during osteoblast differentiation, Chd levels are found to increase. Tsg and Chd proteins accumulate in the cell culture media as the osteoblasts differentiate. Exogenous Tsg and Chd inhibit osteoblast differentiation and mineralization. Osteocalcin (OCN) mRNA levels decrease following both Tsg and Chd treatment. Tsg and Chd also inhibit alkaline phosphatase (ALP) activity in a dose-dependent manner. To provide insight into the mechanism of Tsg and Chd action, we investigated the effect of Tsg and Chd on BMP activity by determining phosphorylated Smad1 (pSmad1) levels. We show that both Tsg and Chd can independently and in combination reduce pSmad1 levels in MC3T3-E1 cells treated with BMP4. Further, BMP2 partially reverses the inhibitory effect of Tsg and Chd on ALP activity. Taken together, these results suggest that Tsg and Chd are involved in osteoblast differentiation and mineralization by regulating BMP signaling.     

Modulation of thrombospondin gene expression during osteoblast differentiation in MC3T3-E1 cells.

The levels of expression of two related extracellular matrix protein genes, thrombospondins 1 and 2 (TSP1 and TSP2), were analyzed in the mouse osteogenic cell line, MC3T3-E1. To monitor differentiation, we also measured two potential markers of the osteoblastic phenotype, alkaline phosphatase (ALP) activity, and alpha 1(I) collagen mRNA levels. TSP1 mRNA levels increased 10- to 15-fold during the first nine days of osteoblastic conversion, and then dropped to a level still significantly above baseline values. This increase in TSP1 mRNA closely paralleled that observed in ALP activity. In contrast, TSP2 mRNA levels were unchanged throughout the 21-day time course. These findings suggest that TSP1 is a marker for osteoblast differentiation and could play a role in the cellular changes that accompany acquisition of the osteoblastic phenotype in MC3T3-E1 cells.     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

Prostaglandin E2 (PGE2) Induces the c-fos and c-jun Expressions via the EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

This study examined which subtype(s) of PGE receptors is involved in the induction of c-fos and c-jun by PGE₂ in MC3T3-E1 cells. We also investigated the possibility that the induction of these genes is involved in the growth and differentiation of this cell line. PGE₂ dose-dependently induced c-fos and c-jun mRNA expressions in MC3T3-E1 cells. Of the PGE analogs, 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₁/EP₃ agonist) were far more potent than butaprost (EP₂ agonist) and 11-deoxy PGE₁ (EP₂/EP₄ agonist) in inducing c-fos and c-jun mRNA expressions. Since MC3T3-E1 cells do not express the EP₃ subtype, these results suggest that PGE₂ induces c-fos and c-jun mRNA expressions through the EP₁ subtype of its receptor. In order to study the functional relevance of these protooncogenes, we then studied the effect of inhibition of their synthesis by the use of antisense oligonucleotide. Alkaline phosphatase (ALP) suppression by 17-phenyl-ω-trinor PGE₂ was reversed by antisense oligonucleotide for either c-fos or c-jun. These results suggest that PGE₂, via the EP₁ subtype of the PGE receptor, negatively modulates the transition from proliferation to the matrix maturation stage through the induction of c-fos and c-jun. However, antisense oligonucleotide for c-fos or c-jun did not alter the prostaglandin G/H synthase-2 mRNA expression induced by EP₁. Thus, it is possible that c-fos and c-jun inductions do not account for all the EP₁-mediated PGE₂ actions in MC3T3-E1 cells. Received: 7 October 1998 / Accepted: 30 September 1999     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000