Reinehr Roland, Graf Dirk, Fischer Richard, Schliess Freimut, Häussinger Dieter
Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
Hepatology. 2002 Sep;36(3):602-14. doi: 10.1053/jhep.2002.35447.
The effect of hyperosmolarity on CD95 membrane targeting and CD95 ligand (CD95L)-induced apoptosis was studied in rat hepatocytes. CD95 showed a predominant intracellular localization in normoosmotically exposed rat hepatocytes, whereas hyperosmotic exposure induced, within 1 hour, CD95 trafficking to the plasma membrane followed by activation of caspase-3 and -8. Hyperosmotic CD95 membrane targeting was sensitive to inhibition of c-Jun-N-terminal kinase (JNK), protein kinase C (PKC), and cyclic adenosine monophosphate, but not to inhibition of extracellular regulated kinases (Erks) or p38 mitogen activated protein kinase (p38(MAPK)). Hyperosmotic CD95 targeting to the plasma membrane was dose-dependently diminished by glutamine or taurine, probably caused by an augmentation of volume regulatory increase. Despite CD95 trafficking to the plasma membrane and caspase activation, hyperosmolarity per se did not induce apoptosis. Hyperosmolarity, however, sensitized hepatocytes toward CD95L-induced apoptosis, as assessed by annexin V staining and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) assay. This sensitization was abolished when hyperosmotic CD95 membrane trafficking was prevented by cyclic adenosine monophosphate, PKC, or JNK inhibition, whereas these effectors had no effect on CD95L-induced apoptosis in normoosmotically exposed hepatocytes. CD95L addition under normoosmotic conditions caused CD95 membrane trafficking, which was sensitive to JNK inhibition, but not to cyclic adenosine monophosphate or inhibition of PKC, Erks, and p38(MAPK). In conclusion, multiple signaling pathways are involved in CD95 membrane trafficking. Hyperosmotic hepatocyte shrinkage induces CD95 trafficking to the plasma membrane, which involves JNK-, PKA-, and PKC-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis.
在大鼠肝细胞中研究了高渗对CD95膜靶向及CD95配体（CD95L）诱导的细胞凋亡的影响。在等渗暴露的大鼠肝细胞中，CD95主要定位于细胞内，而高渗暴露在1小时内诱导CD95转运至质膜，随后激活半胱天冬酶-3和-8。高渗诱导的CD95膜靶向对c-Jun氨基末端激酶（JNK）、蛋白激酶C（PKC）和环磷酸腺苷的抑制敏感，但对细胞外调节激酶（Erks）或p38丝裂原活化蛋白激酶（p38(MAPK)）的抑制不敏感。谷氨酰胺或牛磺酸可剂量依赖性地减少高渗诱导的CD95向质膜的靶向，这可能是由于容积调节性增加增强所致。尽管CD95转运至质膜且半胱天冬酶被激活，但高渗本身并未诱导细胞凋亡。然而，通过膜联蛋白V染色和末端脱氧核苷酸转移酶介导的X-dUTP缺口末端标记（TUNEL）分析评估，高渗使肝细胞对CD95L诱导的细胞凋亡敏感。当通过环磷酸腺苷、PKC或JNK抑制阻止高渗诱导的CD95膜转运时，这种敏感性消失，而这些效应物对等渗暴露的肝细胞中CD95L诱导的细胞凋亡没有影响。在等渗条件下添加CD95L会导致CD95膜转运，其对JNK抑制敏感，但对环磷酸腺苷或PKC、Erks和p38(MAPK)的抑制不敏感。总之，多种信号通路参与CD95膜转运。高渗性肝细胞收缩诱导CD95转运至质膜，这涉及JNK、PKA和PKC依赖性机制，并使肝细胞对CD95L介导的细胞凋亡敏感。
