Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.
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 SHP-2 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.
Lee
Young H
YH
Department of Pharmacology, C2023, 4301 Jones Bridge Road, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Mungunsukh
Ognoon
O
Tutino
Rebecca L
RL
Marquez
Ana P
AP
Day
Regina M
RM
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism*; D011651:Pulmonary Artery / Q000473:pathology; D011658:Pulmonary Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016601:RNA-Binding Proteins / Q000378:metabolism*; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics; D000097796:Nucleolin
D000804:Angiotensin II; D020169:Caspases; D045304:Cytochromes c; D010750:Phosphoproteins; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D016601:RNA-Binding Proteins; D044139:Receptor, Angiotensin, Type 2; D051020:bcl-X Protein; D000097796:Nucleolin
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