ERK和Smad通路在TGF-β_1抑制大鼠血管平滑肌细胞增殖中的作用

Interaction of Smad and ERK pathway in the process of antiproliferation induced by TGF-β_1 in rat vascular smooth muscle cells

目的:探讨ERK通路是否参与TGF-β/Smad通路诱导的血管平滑肌细胞增殖及可能的分子机制。方法:原代培养大鼠胸主动脉平滑肌细胞,细胞分4组:(1)对照组;(2)TGF-β1组;(3)ERK阻断剂(PD98059)组;(4)TGF-β1+ERK阻断剂(PD98059)组。MTT法测细胞的增殖活性,Western blotting法检测VSMCs中细胞内核增殖抗原(PCNA)、Smad2/3、ERK1/2、Smad7蛋白表达及相关磷酸化蛋白含量,RT-PCR方法测VSMCs中Smad2、3、7mRNA的表达。结果:(1)各组PCNA蛋白表达和MTT法测得A值均低于对照组(P<0.05),TGF-β1+ERK阻断剂组与TGF-β1组相比无显著差异。(2)与对照组相比,TGF-β1组p-Smad2/3、p-ERK1/2蛋白含量和Smad7蛋白表达增高(P<0.05),ERK阻断剂组则明显降低(P<0.05);与TGF-β1组比,TGF-β1+ERK阻断剂组降低(P<0.05)。各组间Smad2/3、ERK1/2蛋白表达无显著差异。(3)与对照组相比,TGF-β1组Smad7 mRNA表达增高(P<0.05),ERK阻断剂组和TGF-β1+ERK阻断剂组降低(P<0.05);与TGF-β1组比,TGF-β1+ERK阻断剂组表达降低(P<0.05)。各组内VSMCs的Smad2、Smad3 mRNA表达无显著差异。结论:(1)TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但对TGF-β1的抑制平滑肌细胞增殖的作用无影响,可能有其它信号通路参与此过程。(2)ERK通路可通过蛋白和mRNA水平促进Smad7表达,反过来其又可促进ERK通路激活。(3)ERK通路对Smad2/3蛋白和mRNA水平无影响。

AIM： To investigate whether extracellular signal regulated kinase （ERK） pathway participates the proliferation of vascular smooth muscle ceils （VSMCs） that is regnlated by transforming growth factorβ（TGF- β）/Smad pathway. METHODS： The rat＇s aorta were removed. The primary VSMCs were isolated and cultured in vitro, then the VSMCs were divided into four groups： （ 1 ） control group; （2） TGF - β1 group; （3） ERK blocking agent group; （4） TGF- β1 ＋ ERK blocking agent group. The prollferation of VSMCs was detected by MTT, the expressions of PCNA, Smad2/3, ERK1/2, Smad7 proteins, the content of phosphorylated ERK1/2 and Smad2/3 proteins were determined by Western blotting and the expressions of Smad2/3, Smad7 mRNA were measured by reverse transcription - polymerase chain reaction （RT -PCR）. RESUILTS ： （1） The expression of PCNA proteins and A value in other groups were decreased obviously compared with control group （P 〈 0. 05 ）, and no difference between the TGF- β1 group and .the TGF-β1 ＋ ERK blocking agent group was observed. （2） The content of phosphorylated Smad2/3, ERK1/2 proteins and the expression of Smad7 proteins in TGF - β1 group were increased （ P 〈 0. 05 ）, and decreased in ERK blocking agent group （ P 〈 0. 05 ）, compared with control group. The content of phosphorylated Smad2/3, ERK1/2 proteins and the expression of Smad7 proteins in TGF - β1 ＋ ERK blocking agent group were decreased compared with TGF - β1 group （P 〈0. 05）. No difference in the expression of Smad2/3, ERK1/2 proteins among different groups was found. （3） The expression of Smad7 mRNA in TGF - β1 group was increased compared with control group （P 〈 0. 05 ）, and decreased in ERK blocking agent and TGF -β1 ＋ ERK blocking agent groups （P 〈0. 05）. Compared with TGF -β1 group, the expression of Smad7 mRNA in TGF - β1 ＋ ERK blocking agent group was decreased （P 〈 0. 05 ）. There was no difference in Smad2 and Smad3 mRNA among different groups. CONCLUSION： （ 1 ） The results suggest that TGF - β1 induces Smad2/3 proteins to phosphorylate dependent on the activated ERK pathway, which has no effect on the inhibition of VSMCs proliferation by TGF -β1 and the other pathway may be involved in this process. （2） In VSMCs, ERK pathway promotes the expression of Smad7 at the level of protein and gene. Conversely, Smad7 can activate ERK pathway. （3） ERK pathway does not affect the expression of Smad2/3 at the level of protein and gene.