Apoptosis of alveolar epithelial cells (AECs) is a critical event in the pathogenesis of pulmonary fibrosis. Recent studies showed that the precursor SP-C contains a BRICHOS domain which has chaperone-like properties that protect the peptide from aggregation. SP-C BRICHOS mutants have been shown to induce endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR), leading to cell death and subsequent lung fibrosis. Therefore, understanding the pathogenesis of pulmonary fibrosis requires us to understand the regulation of AEC apoptosis. Previous studies from our laboratory have shown that angiotensin-converting enzyme 2 (ACE-2) plays a protective role against fibrosis by degrading the pro-apoptotic peptide angiotensin II (ANGII) to generate the anti-apoptotic peptide angiotensin 1-7 (ANG1-7), which inhibits AEC apoptosis. We also showed that the generation of ANGII, which is mediated by cathepsin D, is important for the progression of lung fibrosis. Our laboratory was the first to show that ER stress-induced apoptosis of AECs is regulated by the autocrine ANGII/ANG1-7 system. I examined the role of the angiotensin system in response to ER stress by using the proteasome inhibitor MG132 as a chemical inducer of human AECs. MG132-induced ER stress upregulated ANGII-generating enzyme cathepsin D and downregulated ANGII-degrading enzyme ACE-2 in human epithelial cells (Chapter 2). Human AECs in response to ER stress were assessed for apoptosis by measuring mitochondrial function, caspase activation and nuclear fragmentation. It was demonstrated that ER stress-induced apoptosis of AECs was significantly prevented by the ANG receptor blocker, saralasin or drastically inhibited by ANG1-7. Moreover, blockade of ANG1-7 by the specific mas antagonist, A779 could enhance the ER stress-induced apoptosis of AECs. These data suggest that ER stress-induced apoptosis of AECs might be regulated by the autocrine ANGII/ANG1-7 system, and modulation of the ACE-2/ANG1-7/mas axis could provide support for new therapeutic treatment of fibrotic diseases. It was hypothesized that ER stress-induced apoptosis of human AECs might be mediated by the unfolded protein response (UPR) via the autocrine ANGII/ANG1-7 system (Chapter 3). A549 cells were challenged with MG132 or SP-C BRICHOS domain mutant G100S to induce ER stress and UPR activation. MG132 or G100S SP-C mutation activated all 3 canonical pathways of the UPR (IRE1/XBP1, ATF6, and PERK/eIF2α), which led to an increase in cathepsin D or ADAM17/ACE (an ACE-2 ectodomain shedding enzyme) - and eventually caused AEC apoptosis. However, ER stress-induced UPR activation of AECs could be prevented by a chemical chaperone (4-PBA) or by UPR specific blockers (4μ8C, GSK2656157). It also suggested that ATF6 and IRE1 pathways might play important role in regulation of angiotensin system. These data demonstrate that ER stress induces apoptosis of human AECs through mediation of UPR pathways, which in turn regulate the autocrine ANGII/ANG1-7 system.Collectively, the results in these studies suggest the mechanisms by which ER stress and UPR activation induce apoptosis of human AECs. It provides evidence that manipulation of the angiotensin system might lead to therapeutic strategies to limit lung fibrosis.
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