c-Jun-N-terminal kinase dependent membrane targeting of CD95 in rat hepatic stellate cells
c-Jun-N-terminal kinase dependent membrane targeting of CD95 in rat hepatic stellate cells
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DOI：
10.1159/000066277
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发表时间：
2002-01-01
影响因子：
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通讯作者：
Häussinger, D
中科院分区：
文献类型：
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作者：
Cariers, A;Reinehr, R;Häussinger, D
背景/目的：研究CD95配体（CD95L）和环己亚胺（CHX）对活化培养的大鼠肝星状细胞（HSC） CD95膜靶向及凋亡的影响。方法：荧光染色法检测CD95膜靶向性。Western blotting和real - time PCR检测蛋白和mRNA的表达。TUNEL、caspase-3、caspase- 8检测细胞凋亡。结果：CD95L和CHX均未诱导CD95膜转运和HSC凋亡。然而，当两种化合物同时加入时，CD95在1小时内被靶向到质膜上并诱导细胞凋亡。CHX诱导c- jun - n末端激酶（c-Jun-N-terminal kinase， JNK）的瞬时激活和CD95L的延迟激活，但当CHX和CD95L加在一起时，JNK的初始激活增强并延长。抑制JNK可消除CD95L/CHX对CD95膜的靶向作用，但对凋亡反应影响不大。同样，8-CPT-cAMP抑制CD95膜靶向，但不阻止CD95L/CHX诱导的细胞凋亡。CHX和CD95L都不影响蛋白激酶B的磷酸化，但当它们加在一起时，可以观察到PKB的显著去磷酸化。结论：环己亚胺对cd95l诱导的细胞凋亡的增敏作用伴随着jnk依赖性CD95膜靶向作用，但对细胞凋亡反应影响不大。版权所有(C) 2002 S, Karger AG，巴塞尔。
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 HISC 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 (C) 2002 S, Karger AG, Basel.