Hypoxia disrupts intestinal gap junctions through activation of a PP1-dependent phosphatase and the de-phosphorylation of connexin43 in the pathogenesis of necrotizing enterocolitis

Abstract

Introduction: Necrotizing enterocolitis (NEC) develops after a period of global hypoxia, and is characterized by a disruption in the intestinal barrier. The mechanisms by which hypoxia leads to barrier dysfunction remain largely unknown. We have shown that intestinal barrier function requires gap junctions comprised of connexin43 (Cx43) that must be phosphorylated to function. We now hypothesize that hypoxia leads to an impairment in intestinal gap junction function in vitro and in vivo, and sought to determine the mechanisms involved. Methods: IEC-6 cells were subjected to hypoxia in a flow regulated, vacuum sealed hypoxia chamber (1% O2, 37°C) for 1-12h. The expression and phosphorylation of Cx43 were assessed by SDS-PAGE and confocal immunofluorescence. To induce NEC, newborn rats were subjected to global hypoxia (10mins 5% O2) and formula gavage, and mucosal scrapings were obtained from the terminal ileum and subjected to SDS-PAGE. To restore Cx43 phosphorylation, IEC-6 cells were pre-treated with a variety of tyrosine phosphatase inhibitors, including pervanadate(10uM,1h) and calyculin A(2nM,30nM). Results: Hypoxic treatment caused a time-dependent decrease in the phosphorylation of Cx43, which correlated with a decrease in gap junction communication between adjacent enterocytes. There were no effects of hypoxia on the expression of total Cx43, or on cell viability. To verify the in vivo significance of these findings, phosphorylation of Cx43 was also significantly decreased in ileal mucosal scrapings from newborn rats subjected to global hypoxia and formula gavage as compared to control, breast fed animals. Strikingly, pre-treatment of IEC-6 cells with the protein phosphatase 1 (PP1) dependent phosphatase inhibitor calyculin A led to the restoration of phosphorylation of Cx43 and enhanced gap junction connectivity, indicating that a hypoxia-induced phosphatase mediated the effect. Conclusion: Hypoxia inhibits gap junction function in enterocytes through the activation of a protein phosphatase 1 dependent phosphatase resulting in the de-phosphorylation of Cx43. These findings suggest that further targeting of this hypoxia-induced phosphatase may provide a novel therapeutic strategy in the management of diseases of intestinal inflammation like NEC.