The Combination of Alcohol and Cigarette Smoke Induces Endoplasmic Reticulum Stress and Cell Death in Pancreatic Acinar Cells

Abstract

Background & Aims Smoking, an independent risk factor for pancreatitis, accelerates the development of alcoholic pancreatitis. Alcohol feeding of mice induces upregulation of spliced X-box binding protein 1 (XBP1s), which regulates the endoplasmic reticulum (ER) unfolded protein response and promotes cell survival upon ER stress. We examined whether smoking affects the adaptive mechanisms induced by alcohol and accelerates disorders of the ER in pancreatic acinar cells. Methods We studied the combined effects of ethanol and cigarette smoke extract (CSE) on ER-stress and cell death responses in mouse and human primary acini and the acinar cell line AR42J. Cells were incubated with ethanol (EtOH, 50 mM), CSE (20–40 μg/ml), or both (CSE+EtOH), and analyzed by immunoblotting, quantitative reverse transcription PCR, and cell death assays. Some cells were incubated with MKC-3946, an inhibitor of endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1) that blocks XBP1s formation. Male Sprague-Dawley rats were fed isocaloric amounts of an ethanol-containing (Lieber-DeCarli) or control diet for 11 weeks and exposed to cigarette smoke or room air in an exposure chamber for 2 hours each day. During the last 3 weeks, a subset of rats received intravenous injections of lipopolysaccharide (LPS, 3 mg/kg per weeks) to induce pancreatitis or saline (control). Pancreatic tissues were collected and analyzed by histology and immunostaining techniques. Results In AR42J and primary acini, CSE+EtOH induced cell death (necrosis and apoptosis), but neither agent alone had this effect. Cell death was associated with a significant decrease in expression of XBP1s. CSE+EtOH, but neither agent alone, slightly decreased ATP levels in AR42J cells, but induced oxidative stress and sustained activation (phosphorylation) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, also called PERK) and increased protein levels of DNA damage inducible transcript 3 (DDIT3, also called CHOP). CHOP regulates transcription to promote apoptosis. Incubation of AR42J or primary mouse or human acinar cells with MKC-3946 reduced expression of XBP1s, increased levels of CHOP and induced cell death. In rats fed ethanol diet, exposure to cigarette smoke increased ER stress in acinar cells and sensitized the pancreas to LPS-induced pathology. Conclusions Cigarette smoke promotes cell death and features of pancreatitis in ethanol-sensitized acinar cells by suppressing the adaptive unfolded protein response signaling pathway. It also activates ER stress pathways that promote acinar cell death.

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“…Dispersed pancreatic acini were isolated from mouse or rat pancreas and cultured as described previously (Lugea et al 2017a). Briefly, the pancreas was digested with chromatographically purified collagenase (CLSPA) and the resulting acini preparation was suspended in Medium 199 supplemented with 50 μg ml −1 soybean trypsin inhibitor.…”

“…NFAT-luciferase and NF-κB-luciferase adenoviruses constructs were constructed as described previously (Muili et al 2013b). Freshly isolated mouse pancreatic acini or AR42J rat acinar cells (ATCC CRL1492; ATCC, Rockville, MD, USA, cultured and treated with 100 nM dexamethasone for 72 h as described in Lugea et al 2017a) were acutely infected with adenovirus encoding Ad-NFAT-luciferase gene expression reporter construct for 30 min, then stimulated for 5 h with CCK (100 nM) in the presence or absence of 3 μM CM4620. At the end of the incubation period, NFAT-luciferase activity was measured using a Luciferase Assay System (no.…”

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Critical Role of TFEB-Mediated Lysosomal Biogenesis in Alcohol-Induced Pancreatitis in Mice and Humans

“…14 Chronic alcohol feeding causes mild pancreatic damages in rodents but can promote more severe pancreatitis in the presence of endotoxin, smoking, or cholecystokinin. 13,[33][34][35][36] Although genetic factors such as mutations of cationic trypsinogen gene (PRSS1) are strongly associated with pancreatitis, 37 acinar cells may develop adaptive response to protect against insults from alcohol abuse and other environmental factors. These genetic factors together with the cellular adaptive response may explain why only a small portion of alcohol abusers develop severe pancreatitis.…”