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 microM). Treatment of 
hepatocytes with DCA caused MAPK activation, which was dependent upon ligand 
independent activation of EGFR, and downstream signaling through Ras and PI(3) 
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 approximately 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. Inhibition 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 PI(3) 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.