582 Dexamethasone Stimulates Tight Junction Sealing in Intestinal Epithelial Cells by up Regulating Claudin-4 Expression via a Pathway Involving MKP-1 and p38

Abstract

Background and aims: Tight junctions within the intestinal epithelium constitute pivotal elements of the intestinal barrier, perturbations of which have been associated with the pathogenesis and progression of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We previously demonstrated that glucocorticoid hormones stimulate intestinal tight junction sealing In Vitro by increasing the expression of claudin-4. The aim of this study was to identify the signaling pathways responsible. Methods: Caco-2 monolayers grown on semipermeable filter supports were treated with dexamethasone, the p38 inhibitor SB203580 or the MKP-1 inhibitor triptolide and tight junction function was assessed by measuring transepithelial electrical resistance (TEER). Activation of the claudin-4 promoter was analyzed in Caco-2 cells stably expressing the human claudin-4 promoter fused to a luciferase reporter. In addition, quantitative rt-PCR, Western Blotting and immunofluorescence were employed to analyze changes in the expression, activation and localization of claudin-4, MKP-1 and p38. Results: Treatment of Caco-2 cells with dexamethasone resulted in increased TEERs, increased expression of claudin-4 on the RNA and protein level and activation of the claudin-4 promoter. This was accompanied by decreased phosphorylation of the MAPK p38 as well as increased expression of the MAPK phosphatase MKP-1. Inhibition of p38 by SB203580 was sufficient to mimic the effect of dexamethasone both on TEER and claudin-4 expression as well as claudin-4 promoter activation. Conversely, inhibition of MKP-1 by triptolide prevented the dephosphorylation of p38 by dexamethasone and reversed its effect on TEER, claudin-4 expression and activation of the claudin-4 promoter. Localization of claudin-4 to the tight junction or cell viability was not affected by either of the compounds. Conclusions: Our data provide evidence that glucocorticoid induced up regulation of claudin-4 is mediated by dephosphorylation of p38 resulting from increased expression of theMAPK phosphatase MKP-1.We thereby identified a novel signaling pathway mediating an epithelial specific effect of glucocorticoids on intestinal tight junctions. Given the central role of barrier dysfunction in inflammatory bowel diseases, components of this pathway might represent promising new pharmacological targets for the treatment of these disorders.