ZO‐1‐Deficient Enterocytes Undergo Abortive Proliferative Expansion in Response to Mucosal Injury

Abstract

Intestinal damage can be caused by physical, infectious, and immune‐mediated injury. Rapid repair, which is essential for return to mucosal homeostasis, depends on integration of epithelial migration, as in restitution, proliferation and differentiation. These are, in turn, regulated by growth factors and other stimuli that promote expansion of stem and transit‐amplifying cells. Barrier restoration is essential to recovery, but it is not known if tight junction proteins also contribute to critical events prior to junctional repair.AIMTo determine the contributions of the intestinal epithelial tight junction protein zonula occludens‐1 (ZO‐1) to mucosal wound repair.RESULTSIntestinal epithelial ZO‐1 expression is reduced in biopsies from inflammatory bowel disease (IBD) patients and mice with experimental IBD. To determine if this ZO‐1 loss contributes to disease pathogenesis, we created intestinal epithelial ZO‐1‐deficient (Tjp1f/fx villin‐Cre) mice (ZO1IEC.KO). These mice are fertile, reproduce with Mendelian genetics, and, in the absence of stress, mice grow and gain weight normally and are indistinguishable from ZO‐1‐expressing (Tjp1f/f) mice (ZO1IEC.WT). However, 2% DSS induced greater mucosal damage and weight loss in ZO1IEC.KO, relative to ZO1IEC.WT, mice, likely reflecting competition between injury and repair, as 3% DSS induced similar weight loss in ZO1IEC.KO and ZO1IEC.WT mice. Consistent with inadequate repair, ZO1IEC.KO mice failed to recover, even 4 weeks after DSS discontinuation. Labeling with EdU (2h before sacrifice) demonstrated blunted proliferative responses in ZO1IEC.KO, relative toZO1IEC.WT, mice. More striking however, was the loss and nuclear fragmentation, suggestive of apoptosis, of BrdU‐labeled epithelial cells (24h before sacrifice) in ZO1IEC.KO mice. Partial rescue by the caspase‐3 inhibitor z‐DEVD‐FMK indicates that this abortive proliferation contributes to the enhanced severity of DSS colitis in ZO1IEC.KO mice. These defects were not limited to the colon, as ZO1IEC.KO mice also displayed delayed and abortive proliferative responses following T cell activation‐induced (anti‐CD3) small intestinal mucosal damage that were partially rescued by z‐DEVD‐FMK. ZO1IEC.KO mice demonstrated greater Olfm4+ stem cells loss than ZO1IEC.WT mice, and analyses of gene expression showed reduced expression of Lgr5, Ascl2, Oflm4, Bmi1, Lrig1, Wnt3, Dll1, Jag1, Dll4, Notch1, and Hes1, reflecting stem cell loss and defective wnt and notch signaling. In contrast, Tgfbr2 and Smad4 expression were relatively increased in ZO1IEC.KO mice.CONCLUSIONThese data indicate that ZO‐1 plays critical roles in stem cell activation and epithelial survival that are distinct from contributions to tight junction structure. We therefore conclude that non‐canonical ZO‐1 functions are central to stem cell survival, epithelial proliferation, and repair that are necessary for recovery after intestinal mucosal damage. These data further suggest that ZO‐1 loss may contribute to the defective wound healing observed in human and experimental IBD.Support or Funding InformationSupported by NIH R01 DK061931.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.