Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: Inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis
Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: Inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis
复制标题
DOI：
10.1091/mbc.12.9.2629
复制
发表时间：
2001-09-01
影响因子：
3.3
通讯作者：
Dent, P
中科院分区：
文献类型：
--
作者：
Qiao, L;Studer, E;Dent, P
先前的研究认为，表皮生长因子受体（EGFR）和丝裂原活化蛋白激酶（MAPK）途径的活性增强可以促进肿瘤细胞在细胞毒性损伤下的存活。在这项研究中，我们检测了MAPK信号对暴露于低浓度脱氧胆酸（DCA, 50 muM）的原代肝细胞存活的影响。DCA处理肝细胞引起MAPK活化，而MAPK的活化依赖于与配体无关的EGFR活化以及Ras和PI3激酶的下游信号传导。无论是MEK1/2抑制剂单独抑制MAPK信号，还是单独暴露于DCA，都不会增强基底肝细胞凋亡，而抑制DCA诱导的MAPK激活在6小时内导致类似25%的凋亡。当使用显性阴性EGFR-CD533或显性阴性Ras N17来阻断MAPK激活时，也获得了类似的数据。dca诱导的细胞凋亡与procaspase 8、BID、procaspase 9和procaspase 3的顺序切割有关。抑制MAPK增强了胆汁酸诱导的FAS配体突变肝细胞的凋亡，但在FAS受体表达为零的肝细胞中没有增强。这些数据表明，DCA引起与配体无关的FAS受体激活，从而刺激凋亡反应，而这种反应被增强的与配体无关的EGFR/MAPK信号通路抵消。与fas介导的细胞杀伤一致，使用带有死亡结构域的显性阴性fas相关蛋白、caspase 8抑制剂（Ile-Glu-Thr-Asp-p-nitroanilide [IETD]）或显性阴性procaspase 8抑制caspase功能可阻断胆酸诱导的细胞凋亡。抑制胆汁酸诱导的MAPK信号可以增强BID的裂解和线粒体细胞色素c的释放，这些都被IETD阻断。尽管caspase 8被激活，但显性阴性procaspase 9的表达阻断了procaspase 3的裂解，并增强了dca诱导的细胞凋亡。用DCA处理肝细胞可瞬间增加caspase 8抑制剂蛋白c-FLIP-(S)和c-FLIP-(L)的表达，这些蛋白通过抑制MAPK或PI3激酶而降低。c-FLIP-(S)的组成性过表达可消除胆汁酸诱导的细胞凋亡。总的来说，我们的数据表明，dca诱导的EGFR/Ras/MAPK通路功能的丧失，通过减少c-FLIP亚型的表达，增强了dca刺激的fas诱导的肝细胞死亡。
Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 muM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI3 kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused similar to 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhiibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI3 kinase. Constitutive overexpression of c-FLIP-(S) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.