转化生长因子β_1通过Smad_2途径调节足细胞结缔组织生长因子表达

Transforming growth factor β_1 modulates connective tissue growth factor expression via Smad_2 signaling pathway in podocyte in vitro

目的 研究肾脏足细胞是否表达结缔组织生长因子 (CTGF)以及TGFβ1对CTGF表达调控的信号途径。方法 以肾小球足细胞为对象 ,应用Western印迹分析技术 ,观察了 3种促进肾脏纤维化的蛋白因子 ,转化生长因子 β1(TGFβ1)、血小板源生长因子 (PDGF)和血管紧张素Ⅱ (AngⅡ )对体外培养的足细胞CTGF蛋白表达的影响 ,以及ERK、Smad两条信号途径在TGFβ1调节CTGF蛋白表达中的影响 ;逆转录 聚合酶链反应 (RT PCR)检测CTGFmRNA的变化。结果 体外培养的足细胞表达基础水平CTGF蛋白 ,2 0ng/mlPDGF和 10 -6mol/LAngII刺激 2 4小时后细胞内CTGF蛋白水平与对照相比差异无显著意义 (P >0 0 5 ) ,而 1ng/mlTGFβ1刺激 2 4h足细胞CTGF蛋白水平显著高于对照 (P <0 0 5 ) ,且增加呈TGFβ1剂量依赖趋势 ;1ng/mlTGFβ1刺激 12h可以使细胞CTGFmRNA表达增加。1ng/mlTGFβ1使足细胞Smad2 和细胞外信号调节激酶 (ERK1/ 2 )磷酸化 ,在刺激 30min达高峰 ;应用丝 /苏氨酸激酶抑制剂Staurosporine抑制Smad2 磷酸化可以消减TGFβ1刺激的CTGF蛋白增加 ,但ERK1/ 2 活化抑制剂PD 980 5 9阻断ERK1/ 2 磷酸化不能减弱TGFβ1刺激CTGF蛋白表达的效应。结论 在足细胞上 ,TGFβ1刺激CTGF表达依赖于Smad2 信号通路的活化 ,而不依赖于ERK1/

Objective To assess the expression of connective tissue growth factor(CTGF), and the signaling pathway for the regulation of CTGF by transforming growth factorβ1(TGFβ_1) in podocytes. Methods In this study, we observed the effects of three potent profibrotic growth factors- TGFβ_1, Platelet-derived growth factor(PDGF), and Angiotensin Ⅱ(AngⅡ) on the expression of CTGF protein by Western blot analysis in cultured mouse podocytes, which is one of the most important cell construction of glomerular filter barrier, and we also investigated the underlying ERK and Smads signaling pathway through which TGFβ_1 regulates CTGF expression. The levels of CTGF mRNA were assayed by RT-PCR. Results Basal levels of CTGF protein were observed in cultured podocytes, treatment with 20 ng/ml PDGF and 10 -6 mol/L Ang. II for 24 h did not stimulate the expression of CTGF protein compared with control( P >0.05), but significantly increase in levels of CTGF protein were seen in 1 ng/ml TGFβ_1 treated cells compared with with control( P <0.05), and the levels of CTGF were up-regulated in a TGFβ_1 dose-dependent manner;The level of CTGF mRNA was also stimulated by 1 ng/ml TGFβ_1 at 12 h. 1 ng/ml TGFβ_1 induced phosphorylation of Smad_2 and ERK_ 1/2 , and both reached the peak at 30 min;suppression of phosphorylation of Smad_2 with Staurosporine, a Serine/Threonine kinase inhibitor, diminished TGFβ_1-triggered expression of CTGF protein, while blockade of phosphorylation of ERK_ 1/2 with PD98059, a specific ERK_ 1/2 activation inhibitor, did not decrease the TGFβ_1-triggered expression of CTGF protein. Conclusion TGFβ_1 stimulated the expression of CTGF protein via Smad_2-dependent and ERK_ 1/2 -independent signaling pathway in podocyte in vitro.