IGF-1、GM-CSF和EGF通过MAPK途径上调MC3T3-E1及C2C12细胞中核心结合因子α1的表达(英文)

目的:研究生长因子胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、巨噬一粒细胞集落刺激因子(GM-CSF)和表皮生长因子(EGF)对核心结合因子α1(Cbfα1)的调节作用及其与有丝分裂原激活蛋白激酶(MAPK)信号转导通路的关系。方法:采用报告基因方法和RT-PCR技术检测Cbfα1基因启动子活性及mRNA表达的变化。结果:在MC3T3-E1细胞中,IGF-Ⅰ(1 nmol/L 1μmol/L)、GM-CSF(100 nmol/L)和EGF(1μmol/L)作用24小时能够增加Cbfα1基因启动子活性(P<0.05),加入PD98059(10μmol/L)后不但抑制了Cbfα1基因启动子活性的基础表达,而且完全阻断了IGF-1、GM-CSF和EGF所诱导的Cbfα1基因启动子活性的增加(P<0.01),在C2C12细胞中得到了类似结果,另外,MC3T3-E1和C2C12细胞分别经IGF-Ⅰ(1,100 nmol/L)、GM-CSF (100 nmol/L,1μmol/L)和EGF(1 μmol/L,100 nmol/L)处理后均使Cbfα1基因mRNA表达水平提高(P<0.05),而加入PD98059 可抑制上述因子的刺激作用。结论:IGF-Ⅰ、GM-CSF和EGF能够刺激MC3T3E1和C2C12细胞中小鼠Cbfα1基因启动子活性及mRNA的表达,此作用可能经MAPK信号传导通路介导。     

五灵胶囊有效成分对TGF-β1诱导HSC—T6表达Ras／ERK、TGF-β／Smad信号通路蛋白的影响

目的：研究五灵胶囊有效成分对转化生长因子（TGF—B1）诱导HSC—T6细胞表达Ras／ERK、TGF-β／Smad信号通路蛋白的作用,探寻产生药效的主要化学成分。方法：HSC-T6细胞分组：对照组、TGF-β1（10ng·ml^-1）组、PD98059组、PT组、五味子醇甲、五味子乙素、柴胡皂苷、柴胡多糖、丹参新醌乙、X1、二氢丹参酮I（各100μg·ml^-1）、丹参酚酸B（10ng·ml^-1）组,培养24h（10ng·ml^-1TGF-β1于最后12h加入）;ELISA检测培养上清中COL—I,Western Blot检测COL—I、α-SMA、Ras、ERK、p—ERK、TβRⅠ、TβRⅡ、Smad2／3蛋白表达。结果：与TGF—β1组相比,各有效成分组能明显降低COL-Ⅰ分泌,其中以X1、丹参新醌乙、二氢丹参酮Ⅰ、五味子醇甲、丹参酚酸B为显著（P〈0．05）,同时各药物组能显著下调COL—I、α—SMA、ERK、p-ERK、T8RI、TβRII蛋白表达（P〈0．05）;五味子醇甲、X1、二氢丹参酮I能显著下调表达Ras蛋白（P〈0．01）;柴胡皂苷对TβRI表达无作用;X1、醇甲、乙素、二氢丹参酮I、柴胡多糖能显著下调Smad2／3蛋白表达（P〈0．01）。结论：五灵胶囊有效成分对Ras／ERK和TGF—β1／Smad信号通路蛋白的作用不尽相同,强度也不尽一致。抗纤维化作用的最强的成分是X1、二氢丹参酮 I、柴胡多糖、五味子醇甲,丹参新醌乙的作用较弱。     

雷公藤内酯醇减少放射性肺纤维化中肌成纤维细胞活化与抑制TGF-β1/ERK/Smad3通路相关

目的通过体内外实验,观察雷公藤内酯醇(TPL)减少放射性肺纤维化中肌成纤维细胞(MFBs)活化与TGF-β1/ERK/Smad3通路的相关性。方法以TGF-β1刺激成纤维细胞建立体外MFBs活化模型,C57BL/6小鼠胸部照射形成体内MFBs活化、放射性肺纤维化模型。MFBs活化状态通过检测小鼠成纤维细胞中α-SMA(RT-PCR、Western blot方法)和ColⅠ(RT-PCR、ELISA方法)的表达,通路活性采用Western blot法检测p-ERK、p-Smad3(Ser208)、p-Smad3(Ser423)的水平。ERK siRNA及Smad3 siRNA观察ERK、Smad3在MFBs活化中的地位。结果 TGF-β1激活p-ERK/p-Smad3(Ser208)及p-Smad3(Ser423),诱导成纤维细胞表达α-SMA,活化为MFBs,合成Col I明显增多;使用ERK siRNA及Smad3 siRNA确定了ERK及Smad3均参与α-SMA、ColⅠ的表达,其中ERK可能通过磷酸化Smad3连接区(Ser208)起相关作用。小鼠胸部照射可致肺组织中p-ERK、p-Smad3(Ser208)、p-Smad3(Ser423)的水平上调,α-SMA表达增加,显示多量MFBs活化。TPL对体内和体外实验中ERK、Smad3(Ser208)、Smad3(Ser423)的磷酸化活化均有明显抑制作用,可明显下调α-SMA表达及ColⅠ合成,减少MFBs的活化。结论 TPL可通过抑制TGF-β1/ERK/Smad3通路,减少肺部照射后MFBs活化,从而抑制放射性肺纤维化进展。     

氟伐他汀对糖基化终末产物诱导肾小管细胞转分化及ERK1/2信号通路的影响

目的观察氟伐他汀对糖基化终末产物(AGEs)诱导肾小管上皮细胞(HKC)转分化及ERK1/2信号通路的影响。方法将肾小管上皮细胞(HKC)分为对照组、AGEs刺激组、AGEs加氟伐他汀组和AGEs加ERK1/2阻断剂PD98059组,采用免疫细胞化学检测平滑肌肌动蛋白(α-SMA)的表达情况;Western blot检测α-SMA、E-钙黏着糖蛋白(E-cadher-in)、Ⅰ型胶原(collagenⅠ,ColⅠ)、细胞外信号调节酶1和2(ERK1/2)及其磷酸化蛋白(p-ERK1/2)的表达;酶联免疫吸附实验(ELISA)测定细胞上清液中转化生长因子-β1(TGF-β1)的分泌;逆转录-聚合酶链反应(RT-PCR)检测α-SMA和E-cadherin mRNA的表达。结果与对照组相比,AGEs组肾小管细胞α-SMA和Col I蛋白表达明显上调,细胞培养上清中TGF-β1含量增加,α-SMA mRNA的表达增加,而E-cadherin蛋白和mRNA表达下调;AGEs刺激细胞15minp-ERK1/2表达明显增强,1h达到高峰。PD98059和氟伐他汀能够抑制AGEs刺激引起的HKC细胞α-SMA和ColI的表达及ERK1/2的磷酸化;减少TGF-β1的含量;下调AGEs刺激引起的HKC细胞α-SMA mRNA的表达;同时能够逆转AGEs刺激引起的HKC细胞E-cadherin蛋白和mR-NA的下调表达。结论氟伐他汀抑制AGEs诱导肾小管上皮细胞转分化和胶原I的合成可能是通过抑制ERK1/2信号通路活化实现的。     

TGF-β3与熊果酸抑制人结肠癌细胞增殖作用的研究

目的研究TGF-β3与熊果酸(ursolic acid,UA)抑制结肠癌细胞增殖作用的关系及可能的分子机制。方法采用结晶紫染色法和流式细胞术分析不同浓度UA对HCT116细胞增殖的作用。利用Western blot和流式细胞术等方法分析UA对HCT116细胞凋亡的影响。通过RT-PCR和(或)Western blot实验分析UA在HCT116细胞中对TGF-β3、Smad2/3和β-catenin表达及磷酸化水平的影响。采用TGF-β3重组腺病毒和特异性抑制剂以及萤光素酶报告质粒分析TGF-β3介导UA抑制结肠癌细胞增殖的可能分子机制。结果UA能明显抑制HCT116细胞增殖,诱导G1期阻滞,并促进其凋亡。UA能明显降低TGF-β3的mRNA和蛋白表达水平;UA对Smad2/3的总蛋白水平无明显影响,但能明显降低Smad2/3的磷酸化水平;外源性过表达TGF-β3可部分逆转UA对HCT116细胞增殖的抑制作用,TGF-β3特异性抑制剂则增强UA对HCT116细胞增殖的抑制作用;UA降低β-catenin蛋白水平,并明显抑制Wnt/β-catenin信号转导;外源性过表达TGF-β3促进β-catenin蛋白表达,并部分逆转UA对β-catenin蛋白表达的抑制效应;TGF-β3特异性抑制剂增强UA对β-catenin蛋白表达的抑制作用。结论 UA能抑制结肠癌细胞HCT116增殖并诱导凋亡,其作用可能是通过下调TGF-β3表达,进而抑制Wnt/β-catenin信号转导。     

卡托普利对MC3T3-E1细胞增殖、分化及Ⅰ型胶原mRNA表达的影响

目的观察卡托普利对MC3T3-E1细胞增殖、分化和Ⅰ型胶原基因表达水平影响。方法在MC3T3-E1细胞中加入不同浓度的卡托普利,应用MTT法、对硝基苯磷酸盐法、RT-PCR的方法观察卡托普利对MC3T3-E1细胞的增殖、碱性磷酸酶活性和Ⅰ型胶原mRNA表达的影响。结果 10-11 mol.L-1浓度的卡托普利作用24 h能明显促进MC3T3-E1细胞增殖,作用3 d能明显促进MC3T3-E1细胞表达碱性磷酸酶。卡托普利可刺激MC3T3-E1细胞表达Ⅰ型胶原,以10-11 mol.L-1作用最强。结论卡托普利有促进MC3T3-E1细胞增殖、促进碱性磷酸酶表达和增加Ⅰ型胶原mRNA表达的作用。     

西红花酸在AngⅡ诱导的大鼠肺成纤维细胞中的作用

目的:通过观察西红花酸(CRT)对血管紧张素Ⅱ(AngⅡ)诱导的肺成纤维细胞(LFB)增殖及转化生长因子β1(TGF-β1)、Ⅰ型胶原(COL-Ⅰ)、α-平滑肌肌动蛋白(α-SMA)、ERK1/2和P-ERK1/2基因蛋白水平的影响,探讨其抗肺纤维化的作用机制.方法:酶消化法提取原代LFB,采用四氮唑盐(MTT)比色法测定细胞增殖,RT-PCR法相对定量检测TGF-β1和COL-Ⅰ mRNA表达,Western Blot法检测α-SMA、ERK1/2和P-ERK1/2蛋白的表达.结果:10-7、10-6 mol· L-1西红花酸可显著减少AngⅡ诱导的LFB细胞增殖,降低TGF-β1和COL-Ⅰ mRNA的表达,降低α-SMA和P-ERK1/2的蛋白表达.结论:西红花酸可显著减少AngⅡ诱导的LFB细胞增殖,并呈剂量依赖关系,其作用机制可能与调节TGF-β1-ERK1/2信号通路有关.     

缬沙坦对人肾近端小管上皮细胞TGF-β/Smad信号途径的影响

目的观察缬沙坦对TGF-β刺激人肾近端小管上皮细胞表达Smad的影响。方法以人肾小管上皮细胞(HKC)为对象,分为正常对照组;TGF-β1刺激组;缬沙坦组。Western blot检测p-Smad2/3、smad2/3、Smad7以及细胞上清ColⅠ蛋白含量;RT-PCR检测Smad2 mRNA和Smad7 mRNA水平。MTT法检测刺激组及缬沙坦组在不同时间、不同药物浓度对细胞增殖状态的影响。结果①Western blot显示,与TGF-β1刺激组相比,缬沙坦使Smad2/3、p-Smad2/3蛋白的表达下降,ColⅠ分泌显示相同趋势,而Smad7蛋白的表达没有变化。②半定量RT-PCR显示,与TGF-β1刺激组相比,缬沙坦组Smad2 mRNA降低,Smad7 mRNA无变化。③MTT法检测显示不同浓度缬沙坦对TGF-β1刺激所引起的细胞增殖受抑制现象均有改善,并且随药物浓度的增加该作用增强。结论提示缬沙坦的肾脏保护作用可能部分是通过抑制TGF-β/Smads信号途径实现的。     

扶肾方调节各种细胞因子干预腹膜间皮细胞 EMT的实验研究

摘要：目的 探讨扶肾方对大鼠腹膜间皮细胞转化生长因子-β受体Ⅰ（TGF-βRⅠ）、TGF-βRⅡ、Smad泛素化相 关因子2（Smurf2）、E-钙黏蛋白（E-cadherin）、紧密连接蛋白-1（ZO-1）的影响及抑制上皮-间充质转分化（EMT）的作 用机制。方法 原代培养大鼠腹膜间皮细胞,传至第2代并鉴定后,分为空白对照（C）组,含药血清（B）组,模型（T） 组（40 μg/L TGF-β1）,模型+含药血清（T+B）组,模型+MG132（T+M）组。培养24 h后收集细胞和培养液上清液,采用 蛋白印迹法检测Smurf2蛋白水平,qPCR检测各组TGF-βRⅠ、TGF-βRⅡ、E-cadherin、ZO-1的mRNA转录水平。结果 与C组比较,T组E-cadherin、ZO-1、TGF-β1RⅡ mRNA、Smurf2蛋白表达明显上调（P＜0.05）;与T组比较,B组 E-cadherin、ZO-1 mRNA 表达均上调,T+B 组E-cadherin、ZO-1、TGF-β1RⅡ mRNA 表达均上调,Smurf2蛋白表达下 调,T+M组E-cadherin、ZO-1、TGF-β1RⅠ mRNA、Smurf2蛋白表达均下调,TGF-β1RⅡ mRNA表达上调（P＜0.05）;与 B组比较,T+B组和T+M组E-cadherin mRNA表达下调,T+M组ZO-1、TGF-β1RⅠ mRNA表达下调（P＜0.05）;与T+B 组比较,T+M组E-cadherin、ZO-1、TGF-β1R,TGF-β1RⅡ mRNA表达均下调（P＜0.05）。结论 扶肾方抑制大鼠腹膜 间皮细胞EMT可能与上调TGF-βRⅡ mRNA转录,下调Smurf2蛋白含量,促进E-cadherin、ZO-1 mRNA的转录相关。     

转化生长因子-β1对绒毛膜癌JEG-3细胞增殖及Smad3、Smad7表达的影响

目的:观察转化生长因子(TGF)-β1对绒毛膜癌JEG-3细胞增殖能力及其Smad3、Smad7蛋白表达的影响.方法:体外培养人绒毛膜癌JEG-3细胞,分别给予不同剂量及不同作用时间的TGF-β1进行诱导,四甲基氮唑比色法检测细胞增殖能力变化,蛋白印迹法检测Smad3、Smad7蛋白的表达变化.结果:JEG-3细胞对TGF-β1的诱导作用表现为良好的浓度效应和时间效应.100、200μg/L TGF-β1作用后,细胞增殖率显著增加(P＜0.01),Smad3蛋白的相对表达量增加(P＜0.05或P＜0.01),Smad7蛋白无明显改变(P＞0.05);同时100μg/L TGF-β1作用12 h后,细胞增殖率显著增加,Smad3蛋白相对表达量增加(P＜0.05或P＜0.01),Smad7蛋白表达无改变(P＞0.05).结论:外源性TGF-β1能够促进绒毛膜癌JEG-3细胞的增殖,具有良好的浓度效应和时间效应;绒毛膜癌JEG-3细胞逃逸TGF-β1介导的生长抑制作用,可能是由于TGF-β1/Smads信号通路中的Smad3、Smad7蛋白发生异常而致.     

Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways.     

A synergetic role of 1,25-dihydroxyvitamin D(3) in 17β-estradial induced-proliferation and differentiation of osteoblastic MC3T3-E1 cells

Although the effect of 17β-estradial, a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models, the synergetic role of 1, 25-dihydroxyvitamin D(3) on 17β-estradial induced-proliferation and differentiation of osteoblastic cells, and the underlying mechanism are obscure. Here, we investigated the in vitro effect of 17β-estradial on cell proliferation and osteoblastic maturation in MC3T3-E1 cells. 17β-estradial could promote the proliferation and viability of MC3T3-E1 cells, associated with upregulation of cyclin E and proliferation cell nuclear antigen (PCNA) mRNA expression, and downregulation of cyclin-dependent kinase inhibitor 2b (Cdkn2b) mRNA expression. Moreover, 17β-estradial also could stimulate osteoblastic differentiation and bone formation as assessed by alkaline phosphatase (ALP) and Alizarin Red S staining, through induction of the expression of osteoblastic markers, including ALP, osteopontin and type I collagen in MC3T3-E1 cells. However, 1,25-dihydroxyvitamin D(3) treatment alone showed no effect on proliferation and differentiation of MC3T3-E1 cells, but could coordinately augment effects of 17β-estradial on MC3T3-E1 cells. The mechanism conducted demonstrated that 17β-estradial activated ERK1/2 but not JNK and p38, and U0126, an ERK1/2 pathway inhibitor, significantly downregulated vitamin D receptor expression induced by 17β-estradial in MC3T3-E1 cells. Thus, our data demonstrated a synergistical role of 1,25-dihydroxyvitamin D(3) and 17β-estradial in proliferation and differentiation of osteoblasts, and this coordinated regulation might depend on the upregulation of vitamin D receptor in osteoblasts by 17β-estradial. Moreover, during the process of vitamin D receptor upregulation by 17β-estradial, ERK1/2 signaling is involved.     

Rho-kinase inhibitors decrease TGF-β-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We have previously reported that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In order to investigate whether Rho-kinase is involved in the TGF-β-stimulated VEGF synthesis in these cells we examined the effects of Rho-kinase inhibitors on the VEGF synthesis. TGF-β time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1) which is a well known substrate of Rho-kinase. Y27632 and fasudil, Rho-kinase inhibitors, significantly reduced the TGF-β-stimulated VEGF synthesis as well as the MYPT-1 phosphorylation. Y27632 and fasudil failed to affect the TGF-β-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or Smad2. On the contrary, Y27632 as well as fasudil markedly suppressed the TGF-β-induced phosphorylation of SAPK/JNK. Taken together, our results strongly suggest that Rho-kinase regulates TGF-β-stimulated VEGF synthesis via SAPK/JNK activation in osteoblasts.     

染料木素促成骨样细胞增殖作用及其机制研究

目的观察染料木素(genistein)对成骨样细胞MC3T3-E1细胞增殖及骨形成蛋白-2(BMP-2)、β-连环素(β-catenin)表达的影响。方法培养成骨样细胞MC3T3-E1,加入不同浓度的染料木素(1×10-10、1×10-9、1×10-8、1×10-7、1×10-6和1×10-5mol·L-1),以1×10-9mol·L-1雌激素(β-estradiol,E2)作为阳性对照组,采用MTT比色试验法和碱性磷酸酶(ALP)活性检测法,观察染料木素作用后MC3T3-E1的增殖及分化情况,以Westernblot方法检测成骨样细胞中BMP-2和β-catenin蛋白的表达。结果染料木素可促进MC3T3-E1细胞增殖,染料木素作用后细胞的ALP值也明显升高,呈浓度依赖性;染料木素可增加BMP-2和β-catenin表达,且呈浓度依赖性。结论染料木素促进MC3T3-E1细胞增殖与分化,可通过调节BMP-2和Wnt/β-catenin信号通路而影响成骨样细胞的活性。     

Ubiquitination and regulation of Smad7 in the TGF-β1/Smad signaling of aristolochic acid nephropathy

Abstract

Aristolochic acid I (AAI) affects TGF-β1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-β1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10?µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TβRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TβRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-β1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-β1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TβRI.     

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4?h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH₂-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.