Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells
Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells
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DOI：
10.1242/jcs.063545
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发表时间：
2010-05-15
影响因子：
4
通讯作者：
Day, Regina M.
中科院分区：
文献类型：
--
作者：
Lee, Young H.;Mungunsukh, Ognoon;Day, Regina M.
血管紧张素II(Ang II)是纤维组织疾病中的一个关键的促凋亡因子。然而，Ang-II诱导内皮细胞死亡的机制尚未阐明。利用中性彗星试验和特异性受体拮抗剂和激动剂，我们发现Ang-II介导的原代肺内皮细胞凋亡需要AT2受体。Ang II诱导线粒体释放细胞色素c的同时，caspase-3激活和DNA片段化，细胞凋亡被Bax蛋白通道形成抑制剂抑制，提示线粒体介导的细胞凋亡。没有证据表明外源性的凋亡途径参与了这一过程，因为血管紧张素转换酶II激活的是caspase-9，而不是caspase-8。细胞凋亡需要磷酸蛋白磷酸酶的激活，抑制SHP-2磷酸酶(由Ptpn11编码)可以阻止细胞死亡。抗细胞凋亡的Bcl2家族成员表达水平降低可启动内源性细胞凋亡，我们发现Ang-II处理可降低胞浆Bclx(L)蛋白水平。由于核仁蛋白已被证明能结合Bcl一xmRNA(L)并阻止其降解，我们研究了核仁素在血管紧张素-II诱导的Bcl一x丢失中的作用(L)。核糖核酸免疫沉淀实验表明，Ang II减少了核仁素与Bc l-x(L)m RNA的结合，这可能与B l-x(L)m RNA的不稳定性有关。抑制SHP2可阻止血管紧张素Ⅱ诱导的Bclx(L)基因的降解。综上所述，我们的发现提示核仁素是Ang-II信号转导的主要靶点，Ang-II激活的Shp-2抑制核仁素与Bc l-x(L)基因的结合，从而影响Bc l-2家族促凋亡和抗凋亡成员之间的平衡。
Angiotensin II (Ang II) is a key proapoptotic factor in fibrotic tissue diseases. However, the mechanism of Ang-II-induced cell death in endothelial cells has not been previously elucidated. Using the neutral comet assay and specific receptor antagonists and agonists, we found that Ang-II-mediated apoptosis in primary pulmonary endothelial cells required the AT2 receptor. Ang II caused cytochrome c release from the mitochondria concurrent with caspase-3 activation and DNA fragmentation, and apoptosis was suppressed by an inhibitor of Bax-protein channel formation, implicating mitochondrial-mediated apoptosis. There was no evidence that the extrinsic apoptotic pathway was involved, because caspase-9, but not caspase-8, was activated by Ang-II treatment. Apoptosis required phosphoprotein phosphatase activation, and inhibition of the SHP-2 phosphatase (encoded by Ptpn11) blocked cell death. Reduced levels of anti-apoptotic Bcl-2-family members can initiate intrinsic apoptosis, and we found that Ang-II treatment lowered cytosolic Bcl-x(L) protein levels. Because the protein nucleolin has been demonstrated to bind Bcl-x(L) mRNA and prevent its degradation, we investigated the role of nucleolin in Ang-II-induced loss of Bcl-x(L). RNA-immunoprecipitation experiments revealed that Ang II reduced the binding of nucleolin to Bcl-x(L) mRNA in an AU-rich region implicated in instability of Bcl-x(L) mRNA. Inhibition of SHP2 prevented Ang-II-induced degradation of Bcl-x(L) mRNA. Taken together, our findings suggest that nucleolin is a primary target of Ang-II signaling, and that Ang-II-activated SHP-2 inhibits nucleolin binding to Bcl-x(L) mRNA, thus affecting the equilibrium between pro-and anti-apoptotic members of the Bcl-2 family.