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, the molecular mechanisms that control Flip in CD were examined. Methods: 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-LPLs) cultured with tosyl phenylalanyl chloromethyl ketone (TPCK; a nuclear factor-kappa B (NF-kappa B) inhibitor), AG490 (a Janus kinase 2 (Jak2)/signal transducer and activator of transcription (Stat) inhibitor) or 17-desmethoxy-17-N, N-dimethylamino-geldanamycin (DMAG; an inhibitor of heat shock protein 90). The rate of apoptosis was examined by flow cytometry. Results: In CD, upregulation of Flip occurred at both the RNA and protein level. Treatment of CD CD3(+) T-LPLs with TPCK or AG490 markedly reduced Flip RNA, suggesting a role for NF-kappa B and Jak/Stat pathways in the transcriptional control of Flip in this condition. Consistently, both TPCK and AG490 sensitised CD T-LPLs 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: The data demonstrate that Flip is regulated at both the 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.

Caprioli, F., Stolfi, C., Caruso, R., Fina, D., Sica, G., Biancone, L., et al. (2008). Transcriptional and post-translational regulation of Flip, an inhibitor of Fas-mediated apoptosis, in human gut inflammation. GUT, 57(12), 1674-1680 [10.1136/gut.2008.149286].

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

Stolfi,C;SICA, GIUSEPPE;BIANCONE, LIVIA;PALLONE, FRANCESCO;MONTELEONE, GIOVANNI
2008-01-01

Abstract

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, the molecular mechanisms that control Flip in CD were examined. Methods: 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-LPLs) cultured with tosyl phenylalanyl chloromethyl ketone (TPCK; a nuclear factor-kappa B (NF-kappa B) inhibitor), AG490 (a Janus kinase 2 (Jak2)/signal transducer and activator of transcription (Stat) inhibitor) or 17-desmethoxy-17-N, N-dimethylamino-geldanamycin (DMAG; an inhibitor of heat shock protein 90). The rate of apoptosis was examined by flow cytometry. Results: In CD, upregulation of Flip occurred at both the RNA and protein level. Treatment of CD CD3(+) T-LPLs with TPCK or AG490 markedly reduced Flip RNA, suggesting a role for NF-kappa B and Jak/Stat pathways in the transcriptional control of Flip in this condition. Consistently, both TPCK and AG490 sensitised CD T-LPLs 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: The data demonstrate that Flip is regulated at both the 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.
2008
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore MED/12 - GASTROENTEROLOGIA
English
Con Impact Factor ISI
17 desmethoxy 17 n,n dimethylamino geldanamycin; CD3 antigen; Fas antigen; FLICE inhibitory protein; heat shock protein 90 inhibitor; immunoglobulin enhancer binding protein; Janus kinase 2; n benzyl 2 cyano 3 (3,4 dihydroxyphenyl)acrylamide; proteasome; protein kinase B; RNA; STAT protein; tosylphenylalanyl chloromethyl ketone; ubiquitin; unclassified drug; apoptosis; article; clinical article; colon biopsy; controlled study; Crohn disease; flow cytometry; human; human cell; human tissue; lamina propria; lymphocyte culture; priority journal; protein degradation; protein expression; protein function; real time polymerase chain reaction; transcription regulation; translation regulation; ulcerative colitis; upregulation; Western blotting; Antigens, CD3; Apoptosis; Blotting, Western; Caspases; Colitis, Ulcerative; Crohn Disease; Fas Ligand Protein; Female; Humans; Male; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; T-Lymphocytes; Tosylphenylalanyl Chloromethyl Ketone; Up-Regulation
Caprioli, F., Stolfi, C., Caruso, R., Fina, D., Sica, G., Biancone, L., et al. (2008). Transcriptional and post-translational regulation of Flip, an inhibitor of Fas-mediated apoptosis, in human gut inflammation. GUT, 57(12), 1674-1680 [10.1136/gut.2008.149286].
Caprioli, F; Stolfi, C; Caruso, R; Fina, D; Sica, G; Biancone, L; Pallone, F; Monteleone, G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/57256
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