JAK-STAT参与血管平滑肌细胞血管紧张素原和心钠素基因的转录激活

Transcriptional Activation of Angiotensinogen Gene Mediated by JAK-STAT Pathway in Vascular Smooth Muscle Cells

为探讨Janus蛋白酪氨酸激酶2-转导及转录激活因子5(JAK2-STAT5)途径在介导血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(VSMC)血管舒-缩肽表达调节的作用及分子机制,以AngⅡ为诱发因素刺激培养的大鼠VSMC,用免疫共沉淀、Western印迹分析和激光共聚焦显微镜观察STAT5磷酸化及其核转位,用电泳迁移率改变分析(EMSA)确定STAT5与血管活性肽基因调控区顺式调控元件的结合活性.结果显示,STAT5磷酸化水平分别于AngⅡ刺激10 min和12 h出现两个高峰,增加的磷酸化STAT5主要分布在细胞核内.AngⅡ诱导的STAT5活化与核转位可被JAK2的特异抑制剂AG490所抑制.EMSA结果显示,用AngⅡ刺激VSMC后,核蛋白与含有血管紧张素原基因启动子STAT5识别序列的探针结合活性显著升高;而核蛋白与含有心钠素(ANF)基因启动子STAT5识别序列的探针结合活性则呈下降趋势,核蛋白与两种探针的结合活性均可被JAK2抑制剂AG490所消除,并且加入抗STAT5抗体后均可出现滞后的超迁移带.结果提示,AngⅡ通过激活JAK2-STAT5介导信号向胞核内传递,STAT5与相应的顺式元件结合是启动血管紧张素原和心钠素基因表达所必需的转录调控机制之一.

To explore the role of janus protein tyrosine kinase and signal transducers and activators of transcription （JAK-STAT） pathway in regulation of angiotensinogen gene in vascular smooth muscle cell （VSMC）, the cultured rat VSMCs were treated by angiotensin Ⅱ （Ang Ⅱ ） at final concentration 10-6 mol/L. Phosphorylation and nuclear translocation of STATS were detected by immunoprecipitate, Western blotting and confocal microscopy, respectively. The binding activity of STATS to cis-acting regulatory element located in the vasoactive peptide gene promoters was detected by electrophoretic mobility shift assay （EMSA）. The results showed that the phosphorylated STATS levels reach to peak at 10 min and 12 hours after treated with Ang Ⅱ , respectively. High level phosphorylated STATS was observed mainly in nucleus of VSMC at 30 minutes following Ang Ⅱ treatment. The phosphorylation and nuclear translocation of STATS were inhibited by JAK2 specific inhibitor AG490. EMSA showed that following the treatment with Aug Ⅱ the binding activity of nuclear extracts from VSMC to cis-acting regulatory element located in the angiotensinogen gene promoter was enhanced significantly, While the binding activity to cis-acting regulatory element located in the atrial natriuretic factor （ANF） gene promoter was down-regulated. The shift band of Ang Ⅱ-inducible complex was supershifted with anti-STATS antibody, and the binding activity to cis-element was inhibited by AG490. The results suggested that signal transduction of Ang Ⅱ was mediated by JAK-STAT pathway, and the binding activity of STATS to the regulatory regions was one of the molecular mechanisms of transcription activation of Ang Ⅱ gene.