P119 THE ROLE OF PTPN2 SNP IN THE PATHOGENESIS OF FIBROSIS IN CROHN’S DISEASE

Abstract

Genome-wide association studies (GWAS) have identified >200 risk loci in Crohn’s disease and ulcerative colitis or as shared loci. Pathway analysis of these risk loci identified several variants in the Jak-STAT candidate network and of protein tyrosine phosphatase non-receptor type 2 (PTPN2, TC-PTP). PTPN2 dephosphorylates not only receptor protein tyrosine kinases but also non-receptor protein tyrosine kinases like JAK1, JAK2, JAK3, the Src family kinases, STAT3. In subepithelial myofibroblasts from strictured ileum of patient with fibrostenotic Crohn’s disease we identified a unique signaling pathway, of JAK-mediated STAT3(S727) and STAT3(Y705) phosphorylation regulated increased TGF-β1 expression and increased collagen I production. This pathway also led to activation of Erk1/2 in Rab5+ endosomes andthe elicited increased cell proliferation and migration. PTPN2 gene variants occur in patients with Crohn’s disease with SNP rs7234029 associated with apparent ‘loss-of-function’ and stricturing disease. The functional genomics of these SNPs on cytokine signaling and development of fibrosis in Crohn’s remains largely unknown. In this study we investigated the role of PTPN2 variants on the pathogenesis of intestinal fibrosis in patients with stricturing Crohn’s disease. SEMF isolated from the strictured ileum and normal ileum in the same patient were used to initiate primary cell culture. The presence of the Rs7234029 SNP and the PTPN2 haplotype were determined in each subject. SEMF expressing Rs7234029 were transfected with wtPTPN2 and in those from subjects not expressing Rs7234029 CRISPR/Cas9-mediated PTPN2 gene deletion was performed. Our data showed that PTPN2 expression was increased in SEMF of affected ileum compared to normal ileum of the same patient with Rs7234029. This was phenocopied by treatment of SEMF isolated from normal ileum with IL-6. STAT3(Y705) and Erk1/2 phosphorylation, and proliferation were also increased despite higher levels of PTPN2 in SEMF from strictured ileum compared to SEMF from normal ileum in the same patient. Erk1/2 phosphorylation and proliferation were inhibited by either the JAK inhibitor, tofacitinib, by the STAT3 inhibitor, Stattic, or by expression of dnSTAT3(Y705F). In SEMF from patients with Rs7234029 transfected with wtPTPN2 STAT3(Y705) and Erk1/2 phosphorylation, and proliferation were decreased in response to IL-6. In contrast CRISPR/Cas9 mediated PTPN2 deletion in SEMF isolated from subject without Rs7234029 resulted in higher levels of STAT3(Y705) and Erk1/2 phosphorylation, and proliferation. Our results suggest that Rs7234029 does confer a loss of PTPN2 phosphatase activity. This loss of PTPN2 phosphatase function is associated with higher levels of activity in its non-receptor tyrosine kinase targets and leads to greater activity in the JAK-STAT3 pathway that lead to increased proliferation and TGF-β1-mediated collagen production.