BACKGROUND/AIMS: The effect of CD95 ligand (CD95L) and cycloheximide (CHX) on CD95 membrane targeting and apoptosis was studied in activated, cultured rat hepatic stellate cells (HSC). METHODS: CD95 membrane targeting was analysed by fluorescence staining. Protein and mRNA expression were determined by Western blotting and real time PCR. Apoptosis was detected by TUNEL, caspase-3 and -8 assay. RESULTS: Neither CD95L nor CHX alone induced CD95 membrane trafficking and HSC apoptosis. When, however, both compounds were added simultaneously, CD95 was targeted within one hour to the plasma membrane and apoptosis was induced. CHX induced a transient and CD95L a delayed activation of c-Jun-N-terminal kinase (JNK), but when added together initial JNK activation was enhanced and prolonged. Inhibition of JNK abolished the CD95 membrane targeting in response to CD95L/CHX, however had little effect on the apoptotic response. Likewise, 8-CPT-cAMP inhibited CD95 membrane targeting, but did not prevent apoptosis induction by CD95L/CHX. Neither CHX nor CD95L affected protein kinase B phosphorylation, but when added together a marked dephosphorylation of PKB was observed. CONCLUSION: Sensitization of HSC towards CD95L-induced apoptosis by cycloheximide is accompanied by a JNK-dependent CD95 membrane targeting, which, however, has little impact for the apoptotic response. Copyright 2002 S. Karger AG, Basel
BACKGROUND/AIMS: The effect of CD95 ligand (CD95L) and cycloheximide (CHX) on CD95 membrane targeting and apoptosis was studied in activated, cultured rat hepatic stellate cells (HSC). METHODS: CD95 membrane targeting was analysed by fluorescence staining. Protein and mRNA expression were determined by Western blotting and real time PCR. Apoptosis was detected by TUNEL, caspase-3 and -8 assay. RESULTS: Neither CD95L nor CHX alone induced CD95 membrane trafficking and HSC apoptosis. When, however, both compounds were added simultaneously, CD95 was targeted within one hour to the plasma membrane and apoptosis was induced. CHX induced a transient and CD95L a delayed activation of c-Jun-N-terminal kinase (JNK), but when added together initial JNK activation was enhanced and prolonged. Inhibition of JNK abolished the CD95 membrane targeting in response to CD95L/CHX, however had little effect on the apoptotic response. Likewise, 8-CPT-cAMP inhibited CD95 membrane targeting, but did not prevent apoptosis induction by CD95L/CHX. Neither CHX nor CD95L affected protein kinase B phosphorylation, but when added together a marked dephosphorylation of PKB was observed. CONCLUSION: Sensitization of HSC towards CD95L-induced apoptosis by cycloheximide is accompanied by a JNK-dependent CD95 membrane targeting, which, however, has little impact for the apoptotic response. Copyright 2002 S. Karger AG, Basel
Authors: E Novo; F Marra; E Zamara; L Valfrè di Bonzo; L Monitillo; S Cannito; I Petrai; A Mazzocca; A Bonacchi; R S M De Franco; S Colombatto; R Autelli; M Pinzani; M Parola Journal: Gut Date: 2006-01-19 Impact factor: 23.059