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.

A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation.

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB36₋321) was demonstrated to bind to and partially activate the HGF receptor Met. InlB36₋321 has a stable ß-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB36₋321 (1×InlB36₋321) and engineered a head-to-tail repeat of InlB36₋321 with a linker peptide (2×InlB36₋321); 1×InlB36₋321 and 2×InlB36₋321 were purified from E. coli. Both 1× and 2×InlB36₋321 activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB36₋321 activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB36₋321 activated only STAT3 and ERK1/2. The 2×InlB36₋321 promoted improved motility compared with 1×InlB36₋321 and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB36₋321 prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB36₋321 to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.

Characterization of the bovine angiotensin converting enzyme promoter: essential roles of Egr-1, ATF-2 and Ets-1 in the regulation by phorbol ester.

The protease angiotensin converting enzyme (ACE) is a key regulator of blood pressure homeostasis, and is responsible for proteolytic activation of angiotensin I to angiotensin II (Ang II), a potent vasoconstrictor, and proteolytic inactivation of bradykinin, a vasodilator. Recent studies have also implicated ACE and Ang II dysregulation in the progression of fibrotic tissue diseases. Although many studies have utilized bovine tissues and cells for investigating the regulation of ACE gene expression, the bovine ACE promoter has not been previously characterized. Here we present the analysis of the bovine ACE promoter. We investigated cis elements regulated by phorbol 12-myristate 13-acetate (PMA). Sequence analysis shows that the bovine ACE promoter contains several putative binding sites for the transcription factors ATF-2, Ets-1, Egr-1 and SP1/SP3. Chromatin immunoprecipitation (ChIP) indicated that the activation of the bovine ACE promoter by PMA involves histone H4 acetylation, and that PMA induced Egr-1 and ATF-2 binding to the ACE promoter, whereas Ets-1 binding was suppressed by PMA. The regulatory roles of these transcription factors in the bovine ACE gene regulation were confirmed by co-expression of either wild type or dominant negative transcription factors with the luciferase reporter constructs. The bovine and human ACE promoters share similarities in binding sites for transcription factors and PMA regulation within the core regions but contain significant differences in the distal promoter regions.

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants.

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.