黄芪甲苷激活TGF-?茁1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究

摘要 目的：观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间充质干细胞为研究对象,采用全骨髓培养法,从SD大鼠乳鼠（5d-7d）股骨提取原代细胞,体外传代纯化,P4-P5代用于实验。实验分阴性对照组,大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum, FBS)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法：本研究以大鼠骨髓间培养基培养,不给予药物干预;阳性对照组,给予转化生长因子β1(TGF-β1) 5 ng/mL干预3天;黄芪甲苷(Astragaloside,Ast) 组,给予黄芪甲苷4 μg/mL分别干预1 d、2 d、3 d、5 d、7 d;阻断剂组,TGF-β1 受体阻断剂 sb431542 预处理后,再加入黄芪甲苷4 μg/mL分别诱导1 d、2 d、3 d。实时定量PCR( RT-PCR) 检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白( alpha smooth muscle actin,α-SMA )信使核糖核酸(mRNA)表达;印迹法(Western blot) 检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果：经黄芪甲苷诱导刺激后,大鼠骨髓间充质干细胞在mRNA和蛋白水平上,NG2、α-SMA 的表达量均升高;Ast 3d组,与阴性对照组和阳性对照组相比,NG2、?-SMA蛋白和mRNA的表达量均显著升高(p<0.05)。黄芪甲苷刺激细胞后,TGF-β1/Smad2信号传导通路被激活,从第一天开始,p-Smad2蛋白表达量升高,第3天到达顶峰,随后降低;其中第三天,p-Smad2蛋白表达量显著高于Ast 0d组(p<0.01) 。加入阻断剂后,黄芪甲苷受到 sb431542影响,对细胞的作用减弱,NG2、α-SMA蛋白和mRNA表达量均下调;sb431542+Ast 3d组,与Ast 3d组相比,NG2、α-SMA 蛋白和mRNA的表达量显著降低(p<0.001)。受阻断剂的影响,黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱,前三天p-Smad2蛋白的表达量均降低,sb431542+Ast 3d组,与Ast 3d组相比,p-Smad2蛋白的表达量显著降低(p<0.001)。结论：黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用,其机制与激活TGF-β1/Smad2信号传导通路相关。     

调节TGF-β1/Smad信号通路对内质网应激状态下肝癌HepG2细胞凋亡的影响

目的: 探讨TGF-β1/Smad信号通路对内质网应激(ERS)状态下肝癌HepG2细胞凋亡的影响机制。方法: 首先建立内质网应激模型:以3 μmol/L的衣霉素(TM)处理人肝癌HepG2细胞株24 h,诱导细胞发生ERS。实验分为6组,每组3个复孔,实验重复3次,6组分别为:Untreated组(未处理组)、TM组(3 μmol/L TM处理组)、TM+NC组(3 μmol/L TM+si-TGF-β1阴性对照组)、TM+si-TGF-β1组(3 μmol/L TM+si-TGF-β1组)、TM+pEX-3组(3 μmol/L TM+质粒对照组)及TM+TGF-β1 pEX-3组(3 μmol/L TM+TGF-β1过表达质粒组),利用脂质体的方法将TGF-β1小干扰RNA(si-TGF-β1)及TGF-β1过表达质粒(TGF-β1 pEX-3)转染入HepG2细胞,转染24 h后,利用RT-qPCR和Western blot检测各组HepG2细胞TGF-β1/Smad信号通路相关因子TGF-β1、p-Smad2表达的情况;CCK-8和流式细胞术分别检测各组HepG2细胞增殖抑制率和凋亡率变化情况。结果: 与Untreated组相比,TM组细胞的TGF-β1及p-Smad2的表达明显降低(P＜0.05);与TM组相比,TM+si-TGF-β1组细胞的TGF-β1及p-Smad2的表达和细胞的增殖抑制率、凋亡率显著降低(P＜0.01),而TM+TGF-β1 pEX-3组细胞的TGF-β1及p-Smad2的表达和细胞增殖抑制率、凋亡率显著升高(P＜0.01)。结论: TGF-β1/Smad信号通路在肝癌HepG2细胞发生ERS后受到抑制,当该通路被激活后,ERS状态下肝癌HepG2细胞的凋亡率显著升高。     

TGF-β1/Smad 2/3信号通路在椎间盘退变中的作用

临床实践表明,富含血小板的血浆(PRP)注射是延缓椎间盘退变的有效方法,但其作用机制尚不清楚。已有研究表明,活化的血小板可释放转化生长因子-β1 (TGF-β1),它可以正向调节髓核细胞的细胞外基质。本研究旨在揭示TGF-β1/Smad 2/3信号通路在PRP缓解椎间盘退变过程中的作用机制。本研究通过制备新西兰白兔PRP,并使用PRP和TGF-β1抑制剂(SB431542)处理白兔髓核细胞,检测PRP对髓核细胞增殖、软骨形成标志物(CollagenⅡ和Aggrecan) m RNA的表达、Smad 2/3及相关髓核细胞功能蛋白表达的影响。结果显示,用2%PRP处理的髓核细胞的增殖显著增强。PRP处理显著增加髓核细胞中的CollagenⅡ和Aggrecan的mRNA水平。Western blotting结果显示,TGF-β1信号通路的特异性抑制剂可显著抑制Smad 2/3和基质蛋白的表达。在兔椎间盘退变模型中,PRP+抑制剂注射组的Smad 2/3和CollagenⅡ的表达水平显著低于PRP处理组。这些结果表明,PRP中由于TGF-β1含量高,可激活TGF-β1/Smad 2/3途径,并促进CollagenⅡ和其他基质成分的合成和分泌,从而延缓了椎间盘退变。     

siRNA干扰PDGFRβ促进胃癌MGC-803/VCR细胞的凋亡

为研究血小板衍生生长因子受体β(PDGFRβ)在胃癌组织和长春新碱(vincristine, VCR)耐药胃癌细胞MGC-803/VCR中的表达,并探讨PDGFRβ沉默对MGC-803/VCR细胞增殖和凋亡的影响,本研究分别通过免疫组化和蛋白质印迹法(Western blotting)检测PDGFRβ蛋白在胃癌组织和耐药细胞株中的表达水平。通过Lipofectamine 2000将PDGFRβ小干扰RNA (si-PDGFRβ)转染到MGC-803/VCR细胞,Western blotting检测转染后PDGFRβ蛋白表达水平,Cell Counting Kit-8 (CCK-8)和流式细胞术分别检测si-PDGFRβ对VCR诱导人胃癌MGC-803细胞增殖和凋亡的影响。研究结果显示:PDGFRβ蛋白在胃癌组织中的表达显著高于正常胃组织;PDGFRβ蛋白在MGC-803/VCR细胞中的表达极显著高于MGC-803细胞,并且转染si-PDGFRβ后MGC-803/VCR细胞的PDGFRβ蛋白表达水平显著降低;1μmol/L、2μmol/L、4μmol/L、8μmol/L的VCR诱导MGC-803/VCR细胞后,si-PDGFRβ组细胞增殖抑制率分别为(21.97±0.84)%、(37.63±1.32)%、(55.77±1.39)%和(72.17±1.16)%,与对照组的(13.60±0.49)%、(22.33±1.01)%、(38.30±1.56)%和(52.90±1.08)%分别比较有极显著的差异(p<0.01);流式检测结果显示,与对照组的细胞凋亡率(13.61±0.49)%比较,发现si-PDGFRβ组胃癌MGC-803/VCR细胞凋亡率为(29.80±0.64)%,说明两者差异极显著(p<0.01)。本研究初步结论表明,si RNA干扰PDGFRβ能够促进VCR诱导的胃癌MGC-803/VCR细胞凋亡。     

不同浓度姜黄素对胃癌SGC-7901细胞增殖、自噬性凋亡和TGF-β/Smad信号通路的影响

摘要 目的：研究不同浓度姜黄素（Cur）体外对胃癌SGC-7901细胞增殖、自噬性凋亡和TGF-β/Smad信号通路的影响。方法：体外培养SGC-7901细胞,以不同浓度Cur作用于SGC-7901细胞。MTT法检测不同浓度的Cur对SGC-7901细胞增殖的影响。Hoechst 33258法观察不同浓度Cur对SGC-7901细胞凋亡影响,流式细胞仪检测细胞凋亡率。划痕实验检测不同浓度Cur对SGC-7901迁移能力的影响。免疫印迹法检测细胞凋亡相关蛋白NF-κB、自噬相关蛋白Beclin1、LC3Ⅱ及TGF-β/Smad信号通路蛋白TGF-β和p-smad2/3表达。结果：Cur能够抑制胃癌SGC-7901细胞的增殖和迁移,并且Cur对增殖和迁移的影响具有浓度依赖性。Cur能够促进胃癌SGC-7901细胞自噬性凋亡的发生,Cur浓度越高,SGC-7901细胞凋亡率越高（P<0.05）。Cur处理胃癌SGC-7901细胞后NF-κB、Beclin1、LC3Ⅱ表达明显升高,而TGF-β、p-smad2/3表达明显降低,且NF-κB、Beclin1、LC3Ⅱ、TGF-β和p-smad2/3的变化具有浓度依赖性。结论：Cur能够抑制胃癌SGC-7901细胞增殖和迁移并诱导自噬性凋亡的发生,其机制与促进NF-κB、Beclin1、LC3Ⅱ表达,抑制TGF-β/Smad信号通路激活有关。     

间充质干细胞来源的外泌体通过TGF-β1/Smad2/3信号通路抑制高糖诱导的成纤维细胞转分化

心脏纤维化是糖尿病患者心肌功能障碍的主要原因。成纤维细胞转分化为成肌纤维细胞是心脏纤维化过程中的一个关键性事件。该研究的目的是探究高糖诱导成纤维细胞转分化的分子机制,并找寻抑制成纤维细胞转分化的方法。结果显示,经高糖处理的BJ细胞(人皮肤成纤维细胞系)与正常BJ细胞相比,α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达明显上调。通过使用SB525334或转化生长因子-β1(transforming growth factor-β1,TGF-β1)si RNA抑制TGF-β1/Smad2/3信号通路的活化,发现α-SMA和胶原I的蛋白质水平及Smad2/3的磷酸化水平均降低。同时,SB525334也抑制了高糖诱导的BJ细胞增殖。大鼠骨髓间充质干细胞来源的外泌体(mesenchymal stem cell-derived exosome,MSC-Exo)通过降低Smad2/3磷酸化水平,抑制高糖诱导的α-SMA表达。综上所述,高糖通过激活TGF-β1信号通路导致BJ细胞的转分化,而MSC-Exo通过抑制该通路防止BJ细胞的转分化。     

黄芪甲苷激活TGF-β1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究

目的:观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间充质干细胞为研究对象,采用全骨髓培养法,从SD大鼠乳鼠(5d-7d)股骨提取原代细胞,体外传代纯化,P4-P5代用于实验。实验分阴性对照组,大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum,FBS)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间培养基培养,不给予药物干预;阳性对照组,给予转化生长因子β1(TGF-β1)5 ng/mL干预3天;黄芪甲苷(Astragaloside,Ast)组,给予黄芪甲苷4μg/mL分别干预1 d、2 d、3 d、5 d、7d;阻断剂组,TGF-β1受体阻断剂sb431542预处理后,再加入黄芪甲苷4μg/mL分别诱导1 d、2 d、3 d。实时定量PCR(RT-PCR)检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)信使核糖核酸(mRNA)表达;印迹法(Western blot)检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果:经黄芪甲苷诱导刺激后,大鼠骨髓间充质干细胞在mRNA和蛋白水平上,NG2、α-SMA的表达量均升高;Ast 3d组,与阴性对照组和阳性对照组相比,NG2、α-SMA蛋白和mRNA的表达量均显著升高(p0.05)。黄芪甲苷刺激细胞后,TGF-β1/Smad2信号传导通路被激活,从第一天开始,p-Smad2蛋白表达量升高,第3天到达顶峰,随后降低;其中第三天,p-Smad2蛋白表达量显著高于Ast 0d组(p0.01)。加入阻断剂后,黄芪甲苷受到sb431542影响,对细胞的作用减弱,NG2、α-SMA蛋白和mRNA表达量均下调;sb431542+Ast 3d组,与Ast 3d组相比,NG2、α-SMA蛋白和mRNA的表达量显著降低(p0.001)。受阻断剂的影响,黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱,前三天p-Smad2蛋白的表达量均降低,sb431542+Ast 3d组,与Ast 3d组相比,p-Smad2蛋白的表达量显著降低(p0.001)。结论:黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用,其机制与激活TGF-β1/Smad2信号传导通路相关。     

腺病毒介导转化生长因子β1 诱导结肠癌细胞凋亡的研究

目的:探讨腺病毒介导的转化生长因子β-1(TGF-β1)对结肠癌细胞凋亡的诱导作用。方法:结肠癌细胞系HCT116细胞培养后分实验组及对照组,实验组以腺病毒为载体将TGF-β1转染,逆转录聚合酶链反应(RT-PCR)以及免疫组织化学检测其m RNA及蛋白的表达,MTT法检测细胞生长抑制率,流式细胞仪检测细胞凋亡的情况。结果:实验组的TGF-β1 mRNA以及蛋白的表达明显增强;实验组细胞的吸光度波动较小,在低值区相对稳定,各时相点无明显变化,24 h的细胞增殖抑制率为50%,其后在70%-80%之间;对照组细胞吸光度显著升高,与实验组各时相点比较,差异有统计学意义(P0.01)。实验组24 h、36 h、48 h、60 h、72 h的凋亡率分别为(7.55±0.03)%、(8.53±0.11)%、(13.47±0.23)%、(15.51±0.26)%、(16.59±0.26)%,与对照组细胞各时相点比较差异有统计学意义(P0.01)。结论:TGF-β1能够显著地抑制HCT 116细胞的增值及诱导其凋亡。     

脂肪干细胞外泌体对子宫内膜癌HEC-251细胞增殖和凋亡的影响

为了探讨脂肪干细胞外泌体对子宫内膜癌HEC-251细胞增殖和凋亡的影响及分子机制,本研究从人体脂肪组织中分离脂肪干细胞并提取纯化其外泌体。实验分为3组:对照组、脂肪干细胞外泌体组和TGF-β阻断剂组。CCK8检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌HEC-251细胞活力;流式检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌细胞凋亡的影响;Western blotting检测脂肪干细胞外泌体及TGF-β阻断剂对子宫内膜癌细胞Smad2、p-smad2、Bcl2和TGF-β蛋白表达水平。CCK8结果显示,脂肪干细胞外泌体能够显著增强子宫内膜癌HEC-251细胞的增殖能力,TGF-β阻断剂能够显著抑制外泌体对子宫内膜癌的增殖促进作用,流式检测结果显示脂肪干细胞外泌体能够显著抑制子宫内膜癌HEC-251细胞的凋亡,TGF-β阻断剂能够显著抑制外泌体对子宫内膜癌的细胞凋亡的抑制作用,Western blotting检测显示脂肪干细胞外泌体能够显著抑制子宫内膜癌HEC-251细胞p-smad2、Bcl2和TGF-β蛋白表达。初步研究表明,脂肪干细胞外泌体通过促进TGF-β/smad通路促进子宫内膜癌HEC-251细胞增殖,抑制细胞凋亡。     

TGF-β1在应力介导的牙周膜成纤维细胞增殖中的作用及其机制

目的:探讨周期性张应力作用下人牙周膜成纤维细胞(HPDLF)转化生长因子β1(TGF-β1)对其细胞增殖的作用,及对其I型胶原基因表达的作用和影响.方法:在成功构建人牙周膜成纤维细胞体外培养力学刺激模型的基础上,利用多通道细胞牵张应力加载系统,对细胞分别施加2、6、12与24 h的周期性张应力,以不加力组为对照组,观察各组细胞形态变化,利用细胞计数试剂8检测细胞增殖活性,并利用ELISA试剂盒检测加力后各组TGF-β1的表达,并对加力12h组加入TGF-β1抑制剂SB431542,利用RT-PCR检测技术检测对I型胶原基因表达的影响.结果:与对照组比较,加力2h细胞增殖稍降低,6h增殖活性增强,12h达到峰值,24h增殖活性明显受到抑制;TGF-β1的表达与细胞增殖成正相关;TGF-β1受到抑制后细胞增殖受到影响,I型胶原的表达也受到影响.结论:人牙周膜成纤维细胞的增殖在一定时间的周期性张应力作用下先增加然后再抑制,其中TGF-β1参与细胞增殖,并且TGF-β1对人牙周膜成纤维细胞I型胶原的表达起促进作用.     

Resistance training-induced muscle hypertrophy is mediated by TGF-β1-Smad signaling pathway in male Wistar rats

The TGF-β1-Smad pathway is a well-known negative regulator of muscle growth; however, its potential role in resistance training-induced muscle hypertrophy is not clear. The present study proposed to determine whether and how this pathway may be involved in resistance training-induced muscle hypertrophy. Skeletal muscle samples were collected from the control, trained (RT), control + SB431542 (CI_TGF), and trained + SB431542 (RTI_TGF) animals following 3, 5, and 8 weeks of resistance training. Inhibition of the TGF-β1-Smad pathway by SB431542 augmented muscle satellite cells activation, upregulated Akt/mTOR/S6K1 pathway, and attenuated FOXO1 and FOXO3a expression in the CI_TGF group (all p < .01), thereby causing significant muscle hypertrophy in animals from the CI_TGF. Resistance training significantly decreased muscle TGF-β1 expression and Smad3 (P-Smad3^S423/425) phosphorylation at COOH-terminal residues, augmented Smad2 (P-Smad2-L^S245/250/255) and Smad3 (P-Smad3-L^Ser208) phosphorylation levels at linker sites (all p < .01), and led to a muscle hypertrophy which was unaffected by SB431542, suggesting that the TGF-β1-Smad signaling pathway is involved in resistance training-induced muscle hypertrophy. The effects of inhibiting the TGF-β1-Smad signaling pathway were not additive to the resistance training effects on FOXO1 and FOXO3a expression, muscle satellite cells activation, and the Akt/mTOR/S6K1 pathway. Resistance training effect of satellite cell differentiation was independent of the TGF-β1-Smad signaling pathway. These results suggested that the effect of the TGF-β1-Smad signaling pathway on resistance training-induced muscle hypertrophy can be attributed mainly to its diminished inhibitory effects on satellite cell activation and protein synthesis. Suppressed P-Smad3^S423/425 and enhanced P-Smad2-L^S245/250/255 and P-Smad3-L^Ser208 are the molecular mechanisms that link the TGF-β1-Smad signaling pathway to resistance training-induced muscle hypertrophy.     

The role of ERK and Smad2 signal pathways in the alternatively activated macrophages induced by TGF-β1 and high-ambient glucose

Macrophages can be alternatively activated by TGF-β1 and high-ambient glucose, in which the role of Smad2 and the crosstalk between ERK and Smad2 pathways are not fully understood. The activation of ERK and Smad2 pathways and the expression of arginase-1 were detected by Western blot. The role of Smad2 and the relationship between ERK and Smad2 pathways were investigated by using biochemical inhibitors. The protein of arginase-1 was significantly overexpressed in RAW264.7 cells stimulated by TGF-β1 and high-ambient glucose, which can be partially blocked by not only U0126 (ERK inhibitor) but also SB431542 (Smad2 inhibitor). Furthermore, simply inhibiting one pathway had no effect on the other pathway. In conclusion, both ERK and Smad2 signal pathways are involved in the activation of macrophages induced by TGF-β1 and high-ambient glucose, while there is no crosstalk shown in the process.     

Erythropoietin suppresses epithelial to mesenchymal transition and intercepts Smad signal transduction through a MEK-dependent mechanism in pig kidney (LLC-PK1) cell lines

Purpose

Tumor growth factor-β1 (TGF-β1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-β1-mediated EMT and fibrosis in kidney injury.

Methods

We examined apoptosis and EMT in TGF-β1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-β1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-β1 signal pathway proteins and EMT markers.

Results

We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-β1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-β1-mediated apoptosis and also partially inhibited TGF-β1-mediated EMT. We showed that EPO treatment suppressed TGF-β1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-β1-treated cells.

Conclusions

EPO inhibited apoptosis and EMT in TGF-β1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.     

TGF-β receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type β (TGF-β), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-β-receptors (TGF-β-Rs) during skeletal muscle differentiation. We found a decrease of TGF-β signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-β. No change in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-β-R type I (TGF-β-RI) and type II (TGF-β-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-β-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-β-RII lacking the cytoplasmic domain. The TGF-β-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-β-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-β receptors independent of Smad proteins are essential for skeletal muscle differentiation.     

Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-gamma

Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-γ compared to heterozygous control MEFs. Treatment with the PPAR-γ ligand 15d-PGJ₂ failed to down-regulate collagen gene expression in PPAR-γ null MEFs, whereas reconstitution of these cells with ectopic PPAR-γ resulted in their normalization. Compared to control MEFs, PPAR-γ null MEFs displayed elevated levels of the Type I TGF-β receptor (TβRI), and secreted more TGF-β1 into the media. Furthermore, PPAR-γ null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-β, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-γ null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-β responses. Taken together, these results indicate that loss of PPAR-γ in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-β stimulation.     

TGF-β1 inhibits the growth and metastasis of tongue squamous carcinoma cells through Smad4

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that regulates cell growth, differentiation, migration, apoptosis and extracellular matrix remodeling. TGF-β1 transduces signals from the cell membrane to the cell nucleus through serine/threonine kinase receptors and their downstream effectors, Smad molecules. Although many studies have been focused on TGF-β1-Smad signaling pathway, the role of TGF-β1/Smad in tongue squamous cell carcinoma is not fully understood. In the present study, we used a series of cell function assays to examine the role of TGF-β-Smad4 signaling in tongue squamous cell carcinoma. We observed the effects of TGF-β1 on the growth and metastatic potential of the tongue squamous cell carcinoma cell line Ts, which expresses lower level of Smad4 protein. We found that Smad4 could decrease TGF-β1-induced cell proliferation, and that Smad4 overexpression promoted Ts cell apoptosis. In Ts vector control cells, TGF-β1 increased the expression of TβRII, as well as MMP-2, and enhanced cell invasion through the basement membrane, and then induced cell metastasis. However in Ts cells stably expressing Smad4, Smad4 mediated TGF-β1-induced p21 expression promoted cell apoptosis and inhibited cell proliferation, delayed MMP-2 expression, and decreased cell metastasis. Therefore, TGF-β1 plays distinct roles in the Smad4-dependent and -independent signaling pathways.