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

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pat hway can promote tumor cell survival in response to cytotoxic insults. In t his study, we examined the impact of MAPK signaling on the survival of prim ary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 muM). Treatment of hepatocytes with DCA caused MAPK activation, which was d ependent upon ligand independent activation of EGFR, and downstream signali ng through Ras and PI3 kinase. Neither inhibition of MAPK signaling alone b y MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte a poptosis, whereas inhibition of DCA-induced MAPK activation caused similar to 25% apoptosis within 6 h. Similar data were also obtained when either do minant 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 po tentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand , but did not enhance in hepatocytes that were null for FAS receptor expres sion. These data argues that DCA is causing ligand independent activation o f the FAS receptor to stimulate an apoptotic response, which is counteracte d by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS -mediated cell killing, inhibition of caspase function with the use of domi nant negative Fas-associated protein with death domain, a caspase 8 inhibit or (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhiibition of b ile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite ac tivation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treat ment of hepatocytes with DCA transiently increased expression of the caspas e 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhib ition of MAPK or PI3 kinase. Constitutive overexpression of c-FLIP-(S) abol ished the potentiation of bile acid-induced apoptosis. Collectively, our da ta argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiate s DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the e xpression of c-FLIP isoforms.