Role of Cigarette Smoke–Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease–Emphysema Pathogenesis

Abstract

Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first-and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)-emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CSinduced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS-exposed murine model and human COPD-emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P , 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P , 0.001) colocalize with autophagy protein microtubule-associated protein 1 light chain-3B 1 autophagy bodies, whereas U.S. Food and Drug Administration-approved autophagy-inducing drug (carbamazepine) significantly (P , 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein-p62 protein-protein interaction (P , 0.0005) and p62 expression (aberrant autophagy marker; P , 0.0001), verifying CSimpaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P , 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P , 0.001), confirming involvement of aggresome bodies in COPD-emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD-emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD-emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD-emphysema pathogenesis.Keywords: cigarette smoke; chronic obstructive pulmonary disease; ubiquitin; aggresomes; autophagy Clinical RelevanceWe anticipate that these research findings will have a great impact on development of novel therapeutics for treatment of chronic obstructive pulmonary disease-emphysema and other diseases involving impaired proteostasis or autophagy. In addition to the therapeutic application, thes...

Airway Exposure to E-Cigarette Vapors Impairs Autophagy and Induces Aggresome Formation

“…In addition, we identified that clearance of these AB using an autophagy inducer carbamazepine not only reduces apoptosis but also controls cigarette smoke (CS)-induced emphysema (51). Hence, recent studies from our group and others (27,51) have identified the potential of autophagy inducer carbamazepine in rescuing both smoke-induced and alpha-1-antitrypsin Z mutation-associated emphysema in COPD.…”

“…We recently identified proteostasis/autophagy impairment as a novel central mechanism leading to aggresome formation and subsequent downstream effects such as apoptosis in COPDemphysema (41,51). We and others have identified the global impact of first-and second-hand smoke exposure on overall protein synthesis, ubiquitination, and aggregation of misfolded proteins in perinuclear spaces as aggresome bodies (AB) leading to a unique pathology associated with severity of emphysema in COPD subjects (21,23,35,51,56,58). In addition, we identified that clearance of these AB using an autophagy inducer carbamazepine not only reduces apoptosis but also controls cigarette smoke (CS)-induced emphysema (51).…”

“…We and others have identified the global impact of first-and second-hand smoke exposure on overall protein synthesis, ubiquitination, and aggregation of misfolded proteins in perinuclear spaces as aggresome bodies (AB) leading to a unique pathology associated with severity of emphysema in COPD subjects (21,23,35,51,56,58). In addition, we identified that clearance of these AB using an autophagy inducer carbamazepine not only reduces apoptosis but also controls cigarette smoke (CS)-induced emphysema (51). Hence, recent studies from our group and others (27,51) have identified the potential of autophagy inducer carbamazepine in rescuing both smoke-induced and alpha-1-antitrypsin Z mutation-associated emphysema in COPD.…”

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

“…71 Similar to our findings in the diacetyl-exposed lung, ubiquitinated protein aggregates and the corresponding human protein, SQSTM1, accumulate in humans with chronic obstructive pulmonary disease. 72,73 Cigarette smoke is implicated in these aggregates in humans with chronic obstructive pulmonary disease. 72 Because our study demonstrates that diacetyl alters proteostasis and cigarette smoke contains diacetyl, 74,75 the results suggest that diacetyl may play a role in chronic obstructive pulmonary disease in addition to its role in flavorings-related lung disease.…”

“…72,73 Cigarette smoke is implicated in these aggregates in humans with chronic obstructive pulmonary disease. 72 Because our study demonstrates that diacetyl alters proteostasis and cigarette smoke contains diacetyl, 74,75 the results suggest that diacetyl may play a role in chronic obstructive pulmonary disease in addition to its role in flavorings-related lung disease. 11,12 In addition, during autophagy, SQSTM1 is phosphorylated, a process that activates the Kelch-like ECHassociated protein 1 (Keap1)enuclear factor E2erelated factor 2 (Nrf2) pathway.…”