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A6206 - Inducible Alveolar Epithelial Type II (AT2) Cell -Specific Grp78 Knockout Mice Develop Endoplasmic Reticulum (ER) Stress and Reversible Pulmonary Fibrosis
Author Block: B. Zhou1, H. Wang1, Y. Liu1, P. Flodby1, E. D. Crandall1, A. Lee2, Z. Borok1; 1Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of Southern California, Los Angeles, CA, United States, 2USC Norris Comprehensive Cancer Center, and Department of Biochemistry and Molecular Biology, University of Southern Califor, Los Angeles, CA, United States.
Endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation are associated with pulmonary fibrosis. GRP78 is a master regulator of ER homeostasis that regulates UPR activation by binding to ER stress sensor proteins (e.g., protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase 1α (IRE1α) and activating transcription factor 6 (ATF6)). Using lung-specific Grp78 knockout (KO) mice, we showed that GRP78 regulates ER homeostasis and distal lung epithelial cell survival during development. To elucidate the role of ER stress in pathogenesis of lung fibrosis in the adult, we examined alveolar epithelial injury and fibrosis in mice with tamoxifen (Tmx)-inducible KO of Grp78 specifically in AT2 cells. Cre-mediated recombination (~60% efficiency) following injection of Sftpc+/creERT2;Grp78f/f mice (age 2-4 months) with Tmx (100-140 mg/kg intraperitoneally on two consecutive days) led to reduced Grp78 mRNA (~50%) and GRP78 protein (~30%), accompanied by increased expression of ER stress markers GRP94 (~3-fold) and CHOP (~2 fold), one week following Tmx injection compared to AT2 cells from control mice (Sftpc+/creERT2;Grp78f/f mice without Tmx). Grp78 KO mice demonstrated increased weight loss (~20%) and mortality (~24%) compared to control mice two weeks following Tmx injection. Grp78 KO effect on mortality was more severe in male (M) than female (F) mice (F=6% vs M=38%). In Grp78 KO mice two weeks following Tmx treatment, histology showed increased alveolar wall thickness, disorganized lung structure and appearance of structures resembling fibroblastic foci. Trichrome staining demonstrated increased collagen deposition and Sircol assay showed ~2-fold increase in soluble collagen with concomitant reductions in lung compliance (~22%). Western analysis showed increased expression of vimentin, fibroblast-specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) and immunostaining demonstrated increased α-SMA expressing-cells. TUNEL assay demonstrated an increase in apoptotic cells, and Western analysis showed increased cleaved caspase 3 and total caspase 3 suggesting that ER stress-induced apoptosis contributes to fibrosis. Histologic examination of the lungs of Grp78 KO mice at three months following Tmx administration surprisingly revealed resolution of fibrosis. Resolution of fibrosis at three months despite evidence of severe fibrosis by two weeks may be consistent with the hypothesis that, following deletion of Grp78 in ~60% of AT2 cells, residual uninjured AT2 cells can overcome injurious effects of ER stress in Grp78 KO cells and effectively repair the epithelium. These data support the concept that a critical threshold of alveolar epithelial injury is required for development of sustained pulmonary fibrosis.