Transcriptional and post-translational regulation of Flip, an inhibitor of Fas-mediated apoptosis, in human gut inflammation

Objective. Defects in Fas-mediated apoptosis are supposed to contribute to the accumulation of T lymphocytes in the gut of patients with Crohn's disease (CD). This phenomenon has been functionally linked with the elevated expression of Flip, an inhibitor of Fas-mediated apoptosis. In this study, we examined the molecular mechanisms that control Flip in CD. Design. Paired colonic biopsies of patients with CD, patients with ulcerative colitis (UC), and normal controls were analysed for Flip by real-time PCR and Western blotting. Flip was also evaluated in CD3+ lamina propria lymphocytes (T-LPL) cultured with TPCK (NF-kB inhibitor), AG490 (JAK2/Stat inhibitor), or DMAG (inhibitor of heat shock protein 90). The rate of apoptosis was examined by flow-cytometry. Results. In CD, up-regulation of Flip occurred at both RNA and protein level. Treatment of CD CD3+ T-LPL with TPCK or AG490 markedly reduced Flip RNA, suggesting a role for NF-kB and Jak/Stat pathways in the transcriptional control of Flip in this condition. Consistently, both TPCK and AG490 sensitized CD T-LPL to FAS-mediated apoptosis. Flip protein in cells from normal gut was rapidly degraded by the proteasome pathway. In contrast, in inflamed gut of both CD and UC patients, there was a reduced degradation of Flip via the ubiquitin-proteasome-dependent pathway, but Flip expression can be decreased by DMAG. Conclusions. Data demonstrate that Flip is regulated at both transcriptional and post-translational level in CD, and indicate that in the normal but not inflamed gut Flip is degraded via the ubiquitin proteasome-dependent pathway.