Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD

International Journal of Chronic Obstructive Pulmonary Disease Dovepress open access to scientific and medical research International Journal of Chronic Obstructive Pulmonary Disease downloaded from https://www.dovepress.com/ on 30-Mar-2023 For personal use only. Open Access Full Text Article ORIGINAL RESEARCH Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD Yun-Jeong Jeong 1, *, Kyoung-Hee Lee2,*, Jisu Woo2, Ji Yeon Kim2, Chang-Hoon Lee 2,3 , Chul-Gyu Yoo 2,3 1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Dongguk University Ilsan Hospital, Ilsan Dong-gu, Goyang-si, Gyeonggi-do, Korea; 2Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea; 3Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea *These authors contributed equally to this work Correspondence: Chul-Gyu Yoo, 101 Daehakno, Jongno-gu, Seoul, 03080, Korea, Tel +82-2-2072-3760, Fax +82-2-762-9662, Email Background: Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. Methods: We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. Results: We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, upregulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Conclusion: Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis. Keywords: COPD, LAMP-2A, lung epithelial cells, aging, apoptosis Introduction Chronic obstructive pulmonary disease (COPD) affects more than 210 million people,1 thus representing a major health and economic burden worldwide. The most important identifiable risk factor for COPD is cigarette smoke (CS). CS causes inflammation, oxidative stress, and an imbalance between proteases and anti-proteases, which have been suggested as the pathogenic triad in the pathogenesis of COPD.2–4 Recent evidence supports the role of cellular senescence, apoptosis, and autophagy in the development of COPD.5 Autophagy is a lysosomal degradation pathway for cytoplasmic materials,6 which is induced by diverse stimuli including nutrient starvation, cytokines, and oxidative stress. Three main types of autophagy have been identified in mammals: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). These 3 forms of autophagy differ in their delivery methods to lysosomes. In macroautophagy, cargo is sequestered inside autophagosomes for delivery to lysosomes through vesicular fusion. In microautophagy, cytosolic material is internalized for degradation in International Journal of Chronic Obstructive Pulmonary Disease 2023:18 289–303 Received: 23 June 2022 Accepted: 27 February 2023 Published: 14 March 2023 289 © 2023 Jeong et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Dovepress Jeong et al single-membrane vesicles that form through invaginations on the surface of lysosomes or late endosomes. In contrast, vesicles are not necessary in CMA, in which a cytosolic chaperone identifies substrate proteins and delivers them to the surface of lysosomes for internalization through a translocation complex. This complex is formed by the multimerization of the CMA receptor protein, lysosome-associated membrane protein-2A (LAMP-2A).7 LAMP-2A is a critical compo nent of CMA that functions as a receptor for cytosolic substrate proteins. The level of LAMP-2A is known to correlate with CMA activity. The essential function of autophagy is to maintain cellular homeostasis and adapt to adverse environments. However, when not regulated, persistence of inefficient autophagy may be detrimental to lung epithelial cells, leading to apoptosis through a cell-autodigestive process. Dysfunction of the autophagy-lysosomal pathway has recently been implicated in respiratory diseases such as interstitial lung disease, asthma, cystic fibrosis, and COPD.8 Numerous reports have documented aberrant activation of autophagy in lung epithelial cells and lung tissues of COPD patients, murine models, and cell culture model systems.9–15 Moreover, knockout of light chain-3B (LC3B, a marker of macroautophagy) reduces lung apoptosis and airspace enlargement in mice and inhibits the cleavage of poly ADP-ribose polymerase (PARP), indicating that macroautophagy contributes to apoptosis in human bronchial epithelial cells, causing emphysematous changes.16,17 Thus, it is likely that macroautophagy is involved in the pathogenesis of COPD. However, it has not been clear if and how CMA contributes to the pathogenesis of COPD. In the present study, we first investigated the aberrant expression of LAMP-2A in lung parenchyma and bronchial epithelial cells in COPD, and then the contribution of CMA to CS-induced bronchial epithelial cell senescence and death. Methods Cells and Human Lung Tissues Normal human bronchial epithelial cells (BEAS-2B, from American Type Culture Collection, Manassas, VA, USA) were maintained in defined keratinocyte serum-free medium (GIBCO) at 37°C and 5% CO2. Primary human bronchial epithel (...truncated)