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, 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-kappaB (NF-kappaB) 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-kappaB 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
F
Department of Internal Medicine, University Tor Vergata of Rome, Rome, Italy.
Stolfi
C
C
Caruso
R
R
Fina
D
D
Sica
G
G
Biancone
L
L
Pallone
F
F
Monteleone
G
G
D017209:Apoptosis / Q000276:immunology; D015153:Blotting, Western; D017252:CD3 Complex / Q000302:isolation & purification; D020169:Caspases / Q000276:immunology / Q000378:metabolism*; D003093:Colitis, Ulcerative / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D003424:Crohn Disease / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D053222:Fas Ligand Protein / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000378:metabolism; D015854:Up-Regulation
D017252:CD3 Complex; D053222:Fas Ligand Protein; D014108:Tosylphenylalanyl Chloromethyl Ketone; D020169:Caspases