AUTOPHAGY AMELIORATES ER STRESS-MEDIATED INTESTINAL TIGHT JUNCTION BARRIER DYSFUNCTION

Background: Endoplasmic reticulum (ER) stress which undermines cellular homeostasis and survival has been linked with the pathogenesis of inflammatory bowel disease. During ER stress, induction of unfolded protein response (UPR) restores ER homeostasis and enable cell survival. ER stress is known to disrupt the TJ barrier function in intestine, however, the underlying mechanisms are not completely clear. In our previous study we have shown that the cell survival mechanism of autophagy promotes TJ barrier. The aim of this study was to investigate the role of autophagy in preventing the ER stress-induced intestinal TJ barrier disruption and cell death.
Methods: Tunicamycin (Tn) and rapamycin were used for induction of ER stress and autophagy, respectively, in human intestinal Caco-2 cells and mice. Western blot and immunofluorescence analysis were performed to determine the level of TJ proteins and autophagy.
Results: Treatment with Tn (24 μM) (and other ER stress inducers thapsigargin and brefeldin) induced increased expression of UPR genes including CHOP, GRP78, and ATF4 decreased the transepithelial resistance (TER) and increased paracellular flux of small solutes in Caco-2 cells. Similar stress response and TJ barrier dysfunction was observed in the colon of wild type (WT) mice injected with Tn. Channel-forming TJ protein claudin-2 levels were found to be significantly increased with ER stress while claudin-1, -3, and -4 did not show change in protein levels. Although, autophagy is induced following ER stress, we found that persistent ER stress (48-60 hours) inhibited autophagy, as indicated by the reduction in LC3-II/I ratio and an increase in p62 levels. ER stress also led to lysosomal damage as seen by the cytoplasmic presence of cathepsin-B. Rapamycin (1μM), a potent activator of autophagy, prevented the Tn-induced TJ barrier dysfunction, increase in claudin-2 levels and the lysosomal damage, and ameliorated the ER stress in Caco-2 cells. Rapamycin also ameliorated the ER stress and TJ barrier disruption in WT mouse colon and human colonic explants. Furthermore, autophagy deficient ATG7 KO Caco-2 cells and intestinal epithelium specific Atg7^IECΔ/Δ mice showed exaggerated ER stress, TJ barrier disruption, and apoptosis in response to Tn. Induction of autophagy with enterically-coated rapamycin diet also ameliorated ER stress and TJ barrier disruption in chronic dextran sodium sulfate (DSS) model of colitis. Among the three major arms of UPR, PERK, IRE1, and ATF6, inhibition of IRE1α kinase activity prevented ER stress-induced inhibition of autophagy, claudin-2 trafficking defects, and increase in claudin-2 levels.
Conclusions: Our data indicate that persistent ER stress-induced IRE1α activation and downstream signaling pathway inhibits autophagy and escalates TJ barrier defects during chronic inflammatory conditions.