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Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.

ABSTRACT: 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.

SUBMITTER: Lee YH

PROVIDER: S-EPMC2864711 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.

Lee Young H YH Mungunsukh Ognoon O Tutino Rebecca L RL Marquez Ana P AP Day Regina M RM

Journal of cell science 20100420 Pt 10

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 apop ...[more]

PMID: 20406888

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Project description:BackgroundNatural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).Methodology/principal findingsEndothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.Conclusion/significanceApoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

Project description:UnlabelledAngiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis).ConclusionAngiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.

Dataset Information

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.

Publications

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.

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