High MUC2 Biosynthesis Induce ROS Production which Increases Goblet Cell Susceptibility to ER stress and Apoptosis

MUC2 is a large glycoprotein, produced by goblet cells, which forms the protective mucus blanket overlying the intestinal epithelium. Apart from its role as a physical barrier, other functions of MUC2 have not been explored. During pathological conditions such as inflammatory bowel disease (IBD), MUC2 biosynthesis and secretion are accelerated. There is however little known on how MUC2 production is regulated and whether goblet cells undergo increased endoplasmic reticulum (ER) stress and apoptosis following high MUC2 biosynthesis and secretion. We studied the role of MUC2 in goblet cell ER stress and apoptosis using a high MUC2‐producing goblet cell line (HT29‐H) and a clone of HT29‐H (HT29‐L) in which MUC2 has been silenced using lentivirus shRNA technology. Cells were treated with ER stress‐inducing agents, and markers for ER stress (GRP78, ATF4, sXBP1 and CHOP) and apoptosis (caspase 3 cleavage, DNA fragmentation and PARP cleavage) were quantified by western blotting, confocal microscopy and transmission electron microscopy. Compared to HT29‐L, HT29‐H cells showed significant increase in ER stress and apoptosis in response to ER stress and apoptosis‐inducing agents. HT29‐H cells also exhibited higher mitochondrial activity and reactive oxygen species (ROS) production than HT29‐L although the cells had similar mitochondrial mass. Inhibition of ROS abrogated basal ER stress levels, tunicamycin and thapsigargin‐induced ER stress, and apoptotic events including cytoplasmic calcium influx, loss of mitochondrial membrane potential, and caspases 3 and 4 activation. This indicates that high MUC2 production specifically increases ROS production, which drives ER stress and apoptosis. These findings were corroborated in isolated colonic epithelial cells and colonic tissues from Muc2 +/+ and Muc2 −/− mice that showed significant increase in ER stress in Muc2 +/+ mice as compared to Muc2 −/− mice. Finally, goblet cells in Muc2 +/+ mice exhibited severe ER stress and apoptosis during progressive development of DSS‐induced colitis leading to disruption of the mucus barrier. We conclude that high ROS output, as a consequence of high MUC2 production, induces ER stress and apoptosis in goblet cells that could subsequently impair mucus barrier function in disease pathogenesis. Support or Funding Information Canadian Institutes of Health Research (CIHR)