Autophagy alleviates intestinal Tight Junction barrier dysfunction induced by ER stress

Background: Endoplasmic reticulum (ER) has pivotal role in cellular homeostasis and its dysfunction has been linked with the pathogenesis of inflammatory bowel diseases (IBD). During ER stress, induction of unfolded protein response (UPR) activates various signaling pathways to restore ER homeostasis and enable cell survival. Based on the extent of activation, UPR signaling can either exacerbate or alleviate the severity of the loss of cellular homeostasis. ER stress is known to disrupt the tight junction (TJ) barrier function in intestine. Autophagy plays a crucial role in cell survival after ER stress and our previous studies showed that autophagy promotes TJ barrier. Present study aims at understanding the impact of ER stress in autophagy and TJ axis in intestinal homeostasis. Methods: Human intestinal Caco-2 cells and mice were treated with tunicamycin (Tn) and rapamycin for induction of ER stress and autophagy, respectively. Level of TJ proteins and autophagy were determined by western blot and immunofluorescence. Permeability was measured by Transepithelial electrical resistance (TER) and flux of inulin. Results: Induction of ER stress with Tn (and other ER stress inducers thapsigargin and brefeldin) decreased the TER and increased the expression of UPR genes including ATF4, GRP78 and CHOP and the paracellular flux of small molecular solute, inulin in Caco-2 cells. Similar effect on TJ barrier was observed in the colon of wild type (WT) mice injected with Tn. Examination of TJ composition after ER stress induction showed a significant increase in channel-forming TJ protein claudin-2 levels while no alteration in claudin-1, -3, and -4 levels. Confocal immunofluorescence analysis displayed redistribution of claudin-2 and occludin to cytoplasm in the presence of Tn. Importantly, in contrast to acute ER stress which induces autophagy, persistent ER stress hindered autophagy. Rapamycin, a mTOR inhibitor and potent activator of autophagy alleviated the ER stress markers and prevented ER stress-induced lysosomal damage, as detected by cytoplasmic leakage of cathepsin B. Rapamycin also prevented the Tn induced defects in TJ permeability in Caco-2 cells and surgically resected human colon. Deletion of autophagy related gene, ATG7 in Caco-2 cells and intestinal epithelium of mice (Atg7 IEC Δ / Δ ) increased ER stress and associated TJ barrier disruption in the presence of Tn. Autophagy induction with enterically-coated rapamycin diet reduced ER stress, TJ barrier disruption, and inflammation in experimental colitis (chronic DSS) mice model. Rescuing effect of rapamycin was not observed in ATG7 conditional KO mice as the autophagy machinery was defective. Conclusion: Our data specifies that ER stress induces loss of epithelial TJ barrier and causes increased paracellular permeability. Additionally, Persistent ER stress inhibits autophagy and escalate TJ barrier defects during chronic inflammatory conditions. National Institute of Diabetes and Digestive and Kidney Diseases Grant DK114024 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.