RESEARCH ARTICLE Autophagy and the unfolded protein response promote profibrotic effects of TGF- 1 in human lung fibroblasts Saeid Ghavami, 1,2 Behzad Yeganeh, 2,3,4,5 Amir A. Zeki, 6 Shahla Shojaei, 1 Nicholas J. Kenyon, 6 Sean Ott, 6 Afshin Samali, 7 John Patterson, 8 Javad Alizadeh, 1,2 Adel Rezaei Moghadam, 1,2 Ian M. C. Dixon, 3,9 Helmut Unruh, 10 Darryl A. Knight, 11 Martin Post, 4,5 Thomas Klonisch, 1 and X Andrew J. Halayko 2,3,9 1 Department of Human Anatomy and Cell Science, University of Manitoba, Manitoba, Canada; 2 Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, Manitoba, Canada; 3 Department of Physiology and Pathophysiology, University of Manitoba, Manitoba, Canada; 4 Department of Physiology and Experimental Medicine, University of Toronto, Toronto, Canada; 5 Hospital for Sick Children Research Institute, Toronto, Canada; 6 Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, University of California, Davis, California; 7 Apoptosis Research Centre, National University of Ireland, Galway, Ireland; 8 Mannkind Corporation, Westlake Village, California; 9 St. Boniface Research Centre, Winnipeg, Canada; 10 Department of Internal Medicine, University of Manitoba, Manitoba, Canada; and 11 School of Biomedical Science and Pharmacy, University of Newcastle, Newcastle, Australia Submitted 16 August 2017; accepted in final form 24 October 2017 Ghavami S, Yeganeh B, Zeki AA, Shojaei S, Kenyon NJ, Ott S, Samali A, Patterson J, Alizadeh J, Moghadam AR, Dixon IM, Unruh H, Knight DA, Post M, Klonisch T, Halayko AJ. Autophagy and the unfolded protein response promote profibrotic effects of TGF-1 in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 314: L493–L504, 2018. First published October 26, 2017; doi:10.1152/ajplung.00372.2017.—Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), funda- mental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-1 (TGF- 1 ) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3II and Atg5-12), UPR (BIP, IRE1, and cleaved XBP1), and fibrosis (collagen 12 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3II) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-1 -induced collagen 12 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-1 induced the accumulation of LC3II in parallel with collagen 12 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-1 - induced collagen and fibronectin biosynthesis was significantly re- duced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-1-induced collagen 12 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells. IRE1; pulmonary fibrosis; spliced XBP1; transforming growth fac- tor-1 INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive restric- tive lung disease with a high mortality rate and median survival of 2.5 yr after initial diagnosis (39, 52). Precise triggers are unknown but IPF may be initiated by repetitive damage to the alveolar epithelium that can lead to uncontrolled wound repair orchestrated by lung fibroblasts. Recent reports sug- gest that the homeostatic cellular pathways, autophagy, and the unfolded protein response (UPR) may modulate IPF pathogenesis (3, 43). Autophagy mitigates the effects of cellular stress, delivering damaged or improperly processed proteins and organelles to lysosomes for degradation, thereby supplying metabolic fuel, in particular during periods of insufficient energy supply (16, 25). Excessive autophagy can initiate programmed cell death. The endoplasmic reticulum (ER) orchestrates protein folding, but when the need for proteins exceeds the capacity for protein synthesis, the UPR is induced; this triggers ER stress cascades that foster effective cellular processing of newly translated proteins and restore ER homeostasis (32). If the UPR fails to restore protein processing homeostasis, it can drive signaling that leads to apoptotic cell death that prevents excessive tissue damage (56, 60). Araya et al. (3) recently showed that insufficient activation of autophagy may underpin cyclin-dependent kinase inhibitor 1 (p21)-regulated senescence in airway epithelial cells from IPF donors. Autophagy inhibition has also been associated with myofibroblast phenoconversion (21), a response associated with increased synthesis of extracellular matrix (ECM) pro- teins in response to transforming growth factor- 1 (TGF- 1 ) (3). Reduced levels of autophagy markers are evident in whole lung cell from IPF patients, although no specific mechanism for this has been definitively identified (41, 43). Interestingly, Address for reprint requests and other correspondence: A. J. Halayko, John Buhler Research Centre (Rm. 605), Univ. of Manitoba, 715 McDermot Ave., R3P 3E4 Manitoba, Canada (e-mail: andrew.halayko@umanitoba.ca). Am J Physiol Lung Cell Mol Physiol 314: L493–L504, 2018. First published October 26, 2017; doi:10.1152/ajplung.00372.2017. 1040-0605/18 Copyright © 2018 the American Physiological Society http://www.ajplung.org L493 Downloaded from journals.physiology.org/journal/ajplung (023.022.042.100) on July 11, 2022.