Loss of PTPN2 in Intestinal Epithelial Cells Upregulates a Novel STAT‐Dependent Mechanism of Claudin‐2 Expression

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Inflammatory bowel disease (IBD) is a multi‐factorial condition characterized by elevated levels of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Genome‐wide‐association‐studies (GWAS) revealed that individuals with single nucleotide polymorphic mutations in the locus of the protein tyrosine phosphatase non‐receptor type 2 (PTPN2) gene that encodes T‐cell protein tyrosine phosphatase (TCPTP), have an increased susceptibility to IBD. We have reported that reduced TCPTP expression increased effects of IFN‐γ on Signal Transducer and Activator of Transcription (STAT) signaling and led to a greater reduction in barrier function in intestinal epithelial cells (IEC). The protein composition of tight junctions (TJ) determines the tightness of the barrier. Claudin‐2 is a TJ protein that is upregulated in IBD and increases paracellular permeability to cations thus reducing transepithelial electrical resistance (TER). We have previously shown that transient knockdown of TCPTP permits increased expression of claudin‐2 through Janus Kinase (JAK)‐STAT signaling. The aim of the present study was to identify the molecular mechanisms underlying STAT regulation of claudin‐2 expression in PTPN2 (TCPTP)‐knockdown (PTPN2‐KD) HT‐29 IEC. Western blot and immunofluorescence studies revealed that basal STAT1 phosphorylation (Y701) and claudin‐2 expression were increased in unchallenged PTPN2‐KD cells compared to control shRNA transfected cells, and this effect was greatly enhanced by IFN‐γ treatment. Protein analysis of soluble and insoluble fractions showed increased claudin‐2 in the insoluble fraction in KD cells compared to control cells. siRNA silencing of STAT1 mRNA levels significantly decreased claudin‐2 protein and mRNA expression. To confirm STAT binding to the claudin‐2 promoter region, we used Chromatin immunoprecipitation (ChIP) assay to immunoprecipitate STAT1 from IFN‐γ‐treated PTPN2‐KD and control IEC with anti‐STAT1 antibody, and probed the associated DNA with primers against the claudin‐2 promoter. ChIP analysis showed increased STAT1 binding to the claudin‐2 promoter region in KD cells compared to control cells. Next we determined if STAT1 activates claudin‐2 promoter activity through the STAT binding site. We performed a luciferase reporter assay using a human claudin‐2 promoter‐luciferase construct expressed in PTPN2‐KD or Con‐shRNA IEC. Luciferase activity was significantly increased (214 ± 9%, p<0.0001; n=3) in PTPN2‐KD cells over control IEC. In further studies using two different constructs of the claudin‐2 promoter region with mutations of the STAT binding site, there was a significant decrease in promoter activity (43±15%, p<0.0001; n=3) when compared to full length claudin‐2 promoter activity in KD cells, but no significant difference with control cells. In summary, we have demonstrated that STAT1 regulates claudin‐2 expression through binding to its promoter region under conditions of decreased activity of the IBD candidate gene, PTPN2.