P-212 A Role for Hypoxia-Inducible Factor (HIF) in Intestinal Epithelial Junctional Assembly and Barrier Function

The gastrointestinal mucosa constitutes the largest mucosal surface found in multicellular organisms, and provides the primary physical barrier against gut luminal contents. Intestinal epithelial cells that line the GI tract selectively permit flux of nutrients and water, while protecting against omnipresent luminal antigens. In mucosal diseases such as inflammatory bowel disease (IBD), hypoxia and inflammation occur coincidentally at the level of the epithelium. Adaptive transcriptional responses to oxygen deprivation are mediated primarily through the hypoxia-inducible factor complex (HIF), comprising an oxygen-labile “alpha” subunit and constitutively expressed “beta” subunit. A role for HIF has been implicated in orchestrating a signaling program that promotes intestinal epithelial function and homeostasis. Disruption of the epithelial barrier by proinflammatory cytokines such as IFN-gamma defines a major pathophysiological consequence of intestinal inflammation in IBD. We previously demonstrated that HIF activity and expression in intestinal epithelial cells (IECs) is prominently repressed at the transcriptional level by IFN-gamma. Given the concurrent expression of both HIF-1alpha and HIF-2alpha isoforms of the HIF transcriptional complex in IECs, we sought to define the relative contribution of HIF-1 and HIF-2 to epithelial barrier function. Human intestinal epithelial cell lines T84 and Caco-2 were used throughout. Stable knockdown lines targeting individual HIF subunits were generated using shRNA lentiviral transduction particles (MISSION TRC). To monitor apical junction assembly after calcium switch, T84 cells plated on transwell inserts were incubated for 16h in low Ca2+ medium (LCM) or 5 mins in HBSS with 2mM EDTA before switching to normal Ca2+ conditions (1.8 mM). Transepithelial resistance (TER) was measured over time using a voltohmeter, and expressed in ohm.cm2. Stable shRNA-mediated knockdown of HIF-1alpha, HIF-2alpha and HIF-1beta in model T84 epithelial cells resulted in significantly increased transcellular and paracellular permeability, as measured by transepithelial resistance and FITC-dextran flux assays. Immunolocalization studies of tight junction and adherens junction markers ZO-1 and E-cadherin, respectively, revealed non-uniform, undulating junctions in HIF-1alpha depleted T84 monolayers, consistent with attenuated intercellular tension and apical junction formation under conditions of altered HIF signaling. IFNgamma treatment of shControl IECs resulted in decreased barrier function and a junctional morphology similar to that observed in HIF-1alpha knockdown epithelia. Moreover, western blot analyses of shControl and knockdown lysates revealed increased myosin light chain (MLC) phosphorylation in HIF-1alpha knockdown IECs, indicative of an activated myosin II motor and increased perijunctional actomyosin contractility. Finally, inhibition of myosin light chain kinase (MLCK) or the Rho-associated protein kinases (ROCK) in HIF knockdown epithelia attenuated barrier defects and improved junctional morphology. These results define a role for HIF signaling in maintenance of apical IEC junctions at the level of the perijunctional actomyosin ring, and may in part explain the pathomechanism of IFN-gamma mediated loss of junctional integrity.