The Met196Arg Variation of human TNFR2 confers increased apoptotic signaling and impaired TNF-alpha responses: implication for inflammatory bowel disease

Abstract

Background & Aims: Tumor necrosis factor-a (TNF-a)-induced signalling is pivotally involved in the pathogenesis of human chronic inflammatory bowel disease (IBD). Selective upregulation of TNF receptor 2 (TNFR2) in colonic epithelial cells has been described to play an important role in epithelial migration (Corredor et al., 2003) and colonic inflammation-associated alteration in the intestinal epithelium (Mizoguchi et al., 2002). A polymorphism in the TNF receptor 2 (TNFR2) gene resulting in an inversion from methionine (TNFR2196MET) to arginine (TNFR2196ARG) at the amino acid 196 has been genetically associated with an increased risk for ulcerative colitis (Mierik et al., 2004). The aim of the present study was to elucidate the molecular effect of the variant on TNFR2-mediated signalling. Methods: Stable transfectants carrying either variant of TNFR2 were generated in epithelial cell lines and embryonic fibroblasts from tnfr1/tnfr2 double deficient mice. Apoptosis was assessed using MTT assays and AnnexinV FACS. NF-kB activity was investigated by reporter gene assays. TNFR2/TRAF2 association was determined using co-immunoprecipitation and confocal-laser-microscopy. Results: The present study provides evidence that the mutation results in a significantly lower capability to induce NF-kB signalling downstream of TNFR2. Pre-triggering of TNFR2 with a receptor specific mutein leads to an enhancement of TNFR1-induced apoptosis, which is further increased in cells carrying the TNFR2196ARG variant. In parallel, a diminished induction of NF-kB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, TRAF1, IL–6 or IL–8 is observed (real-time PCR, Western blot, ELISA). The mutated form TNFR2196ARG shows a lack of inducible TRAF2 recruitment upon TNF-a stimulation. Conclusions: Our data suggest a profound impairment of TNF-a signalling via the mutated TNFR2 variant at a level of the complexation of the adaptor protein TRAF2. The findings gain insight into basic mechanisms of TNFR2 signalling and suggest a molecular principle for the involvement of the Met196Arg TNFR2 mutation in the etiopathogenesis of inflammatory bowel disease via increased epithelial cell death.