Active site and frameshift mutants of Protein Tyrosine Phosphatase non‐receptor type 2 inhibit STAT1 dephosphorylation and compromise epithelial barrier function

Abstract

Mutations in Protein Tyrosine Phosphatase non-receptor type 2 (PTPN2) have been linked to Inflammatory Bowel Disease (IBD) in a subset of patients. Moreover, loss of PTPN2 promotes intestinal epithelial cell (IEC) barrier dysfunction, itself a feature of IBD. We investigated whether loss of function mutations in PTPN2 influence IEC barrier function. HCA7 clone 29 IECs were transiently transfected with constructs encoding for wild type (wt), active site (ast), or deletion frameshift mutations (fs) of PTPN2, all fused to EGFP and under the control of the CMV promoter. Western blotting and densitometry indicated that EGFP-ast or EGFP-fs transfected cells treated with the IBD cytokine, IFNγ (100U/ml, 24hrs), show increased phosphorylation of the signaling molecule, STAT1 when compared to cells transfected with EGFP-wt + IFNγ. TER measurements revealed further decreases of 20% with EGFP-ast and 19% with EGFP-fs, respectively, following IFNγ treatment (days 1–9 post-IFNγ, n=2), compared to cells transfected with EGFP alone + IFNγ. Interestingly, EGFP-wt protected cells from TER loss. These observations were further supported by FITC-dextran permeability measurements. Our data indicate that both PTPN2 mutants act as dominant-negatives, and support our hypothesis that PTPN2 dysfunction plays a role in the intestinal epithelial barrier defects associated with IBD. Supported by the CCFA and NIH T32 DK07202