A264 AUTOPHAGY IS CRITICAL FOR GOBLET CELLS TO MAINTAIN HOMEOSTASIS UNDER HIGH METABOLIC STRESS. (1st March 2018)

• Record Type: Journal Article
• Authors: Tiwari, S; Moreau, F; Chadee, K
• Is Part Of: Journal of the Canadian Association of Gastroenterology. Volume 1(2018)Supplement 1
• Page Start: 459
• Page End: 460
• Publication Date: 2018-03-01
• Subjects: Gastroenterology -- Periodicals; 616.33005
• DOI: 10.1093/jcag/gwy008.265
• Languages: English
• ISSNs: 2515-2084

Abstract

Abstract: Background: Cellular stress induced by external or internal agents lead to the activation of several processes to maintain homeostasis. These processes involve the unfolded protein response (UPR), apoptosis and autophagy. Autophagy is a catabolic pathway that mediates the degradation of cellular components into double-membrane vesicle fused with lysosomes forming autolysosomes that lead to the removal of non-functional proteins and organelles. Goblet cells in the gut secrete MUC2 mucin to form the mucus layers which serves as a protective barrier against invading microbes and noxious substances. Autophagy has been suggested to play a role in the regulation of mucin secretion and cell survival by an unknown mechanism. AMPK has been shown to be an essential mediator to activate autophagy through mTOR inhibition to maintain metabolic homeostasis and cell survival. As goblet cells undergo increased ER stress to produce MUC2 mucin under inflammatory and disease conditions we hypothesize that autophagy plays a role in MUC2 degradation and/or cell survival. Aims: To quantify the autophagy response and AMPK signaling in high and low MUC2 producing HT29 cells. Methods: High MUC2-producing human HT29-H cells and a clone of HT29-H silenced for MUC2 (HT29-L) by lentivirus shRNA was used in the study. Autophagy proteins were evaluated in HT29H/L cells by Western blot using monoclonal antibodies for ULK1 and their phospho-antibodies. AMPK signaling was quantified by anti-AMPKα antibody. Autophagy and AMPK genes were quantified transcriptionally by RT-PCR. Acadesine (AICAR) and Torin1 was used to activate AMPK signaling and autophagy respectively. Results: High MUC2 producing HT29-H cells under metabolic stress accumulated misfolded MUC2 proteins and impaired basal autophagy protein expression (LC3-II and ULK1) as compared to HT29-L cells. Basal phosphorylation of pULK1(S555) was upregulated through an AMPK-mediated mechanism in HT29-H cells whereas pAMPKα (T172) was downregulated in HT29-L cells. Cells stimulated with the AMPK agonist, acadesine (AICAR), enhanced pAMPKα (T172) and ULKI phosphorylation in HT29-H but not HT29-L cells. Similarly, stimulation with Torin1 (an autophagy inducer) increased the expression of AMPKα and ULK1 in HT29-H but not HT29-L cells. These results suggest that the impaired phosphorylation events in autophagy and AMPK signaling in HT29-H may lead to perturbed homeostasis. Conclusions: The interplay between autophagy with AMPK signaling helps to maintain homeostasis in intestinal epithelial cells. This study demonstrates that impaired autophagy in high MUC2 mucin-producing cells might be a cellular defense to enhance cell survival. Funding Agencies: CCC