P115 THE IBD CANDIDATE GENE, PTPN2, RESTRICTS INTESTINAL BARRIER DEFECTS AND CLAUDIN-2 EXPRESSION VIA STAT INHIBITION

Loss-of-function mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene locus increase IBD susceptibility. We reported that PTPN2 knockdown (PTPN2-KD) in intestinal epithelial cells (IECs): (i) increased IFN-g activation of STAT signaling; (ii) reduced IEC barrier function; (iii) increased expression of the tight junction (TJ) protein, claudin-2, by IFN-g; and that the claudin-2 promoter contains a STAT binding sequence. Increased claudin-2 expression occurs in IBD and increases permeability to cations thus reducing transepithelial electrical resistance (TER). To confirm PTPN2 regulation of barrier function in vivo and identify the mechanisms of STAT regulation of claudin-2 expression. In Ussing chamber studies, TER was significantly reduced in cecum from Ptpn2 knockout (KO) and heterozygous (Het) mice vs. wild-type (WT) littermates indicating increased electrolyte permeability. Immunostaining and western blotting showed progressive increases in claudin-2 expression and STAT-1 phosphorylation (Y701) in Het and KO mice vs. wild-type intestine. Claudin-2 was also elevated in intestinal enteroids from Ptpn2-deficient mice. STAT-1 siRNA significantly decreased the elevated claudin-2 expression in PTPN2-KD IECs. Chromatin immunoprecipitation (ChIP) analysis showed increased STAT-1 binding to the claudin-2 promoter in PTPN2-KD vs. control cells. Claudin-2 promoter-luciferase reporter construct activity was significantly elevated in PTPN2-KD vs. Con-shRNA IEC and was further increased by IFN-g (1000 U/ml). This effect was lost by two different mutations of the STAT binding site thus confirming the requirement of STAT binding for increased claudin-2 transcription in PTPN2-KD IECs. Loss of functional PTPN2 increases susceptibility to inflammation-induced tight junction reorganization, providing an explanation for the association of PTPN2 mutants with UC and CD.