PTPN2 Gene Deficiency Leads to Increased Epithelial Permeability and Promotes STAT‐1‐dependent Claudin‐2 Expression

Abstract

Interferon gamma (IFN‐γ) is an inflammatory cytokine involved in inflammatory bowel disease (IBD), and is known to compromise the intestinal barrier. siRNA silencing of the IBD candidate gene protein tyrosine phosphatase non‐receptor type‐2 (PTPN2) in human intestinal epithelial cells (IEC) elevates expression of claudin‐2 causing increased cation permeability of tight junctions. The aim of this study was to identify the cellular and molecular mechanisms underlying claudin‐2 expression in the absence of PTPN2. We developed a HT‐29 IEC line stably expressing short hairpin RNA for PTPN2 using a lentiviral system to knockdown PTPN2 expression. RT‐PCR and densitometry analysis indicated that PTPN2 expression was significantly reduced (60 ± 4%, p<0.0001) in PTPN2‐depleted cells, paralleled with an increased expression in claudin‐2 protein (180 ± 4%, p<0.0005). Western blot and immunofluorescence studies revealed that basal phosphorylation of the PTPN2 substrate, STAT‐1, as well as claudin‐2 protein expression, were significantly increased in PTPN2 KD cells. This effect was potentiated following IFN‐γ treatment. siRNA silencing of STAT‐1 decreased claudin‐2 protein expression in PTPN2 KD cells. There was also a significant increase in in vivo intestinal permeability to FITC‐Dextran (4 kD) in 3‐week old Ptpn2 knockout (298 ± 5%, p<0.0001) and heterozygote mice (206 ± 12%, p<0.001) compared to wild‐type mice. STAT‐1 phosphorylation and claudin‐2 protein expression were increased in Ptpn2 KO mouse cecum vs. wild type. In summary, PTPN2 deficiency leads to increased intestinal permeability and elevated claudin‐2 expression through a STAT‐1‐dependent pathway.