Functional interaction of CARD15/NOD2 and Crohn's disease-associated TNFα polymorphisms

Background and aims: Mutations/polymorphisms in the CARD15/NOD2 gene and in the promoter region of the TNF alpha gene are associated with susceptibility to and modulate the phenotype of Crohn's disease (CD). The molecular mechanisms for this genotype - phenotype correlation are yet to be elucidated. CARD15 is an intracellular receptor for bacterial muramyl dipeptide (MDP), and can elicit an inflammatory response via activation of the NF-kappa B pathway. MDP is also known to induce the expression of pro-inflammatory cytokines including TNF alpha, through a still poorly characterized signaling pathway. We sought to determine whether CARD15-mediated NF-kappa B activation can contribute to MDP-induced TNF alpha production and, consequently, if polymorphisms in both genes affect the control of such induction. Methods/results: Transfection and electrophoretic mobility shift assays ( EMSA) experiments in HEK293 cells demonstrated that MDP exposure stimulates TNF alpha gene transcription, as a result of CARD15-induced NF-kappa B activation and binding to TNFa promoter. When the CD-associated CARD15 1007fs variant was analyzed, induction of TNF alpha promoter activity was found to be defective. Different combinations of CARD15 and TNF alpha promoter polymorphisms gave rise to distinct TNF alpha transcription levels. Conclusions: CARD15 and TNF alpha promoter polymorphisms interact to exert a functional effect on MDP-induced TNF alpha production. This gene - gene interaction may contribute to interindividual variation in susceptibility to, and manifestation of, Crohn's disease.