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

Abstract

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.

Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells

“…On the other hand, anti-TRAIL antibody very weakly blocked the apoptosis (Figure 6b). Several studies have reported that death receptors such as Fas and TNF-R can induce apoptosis in a ligand-independent manner (Beltinger et al, 1999;Fumarola et al, 2001;Qiao et al, 2001). Furthermore, DR5 is also believed to be able to induce ligand-independent apoptosis (Sheikh and Huang, 2004).…”

Taurocholic acid metabolism by gut microbes and colon cancer

“…The detergent properties of DCA (but not CA) cause membrane perturbations leading to the release of arachidonic acid, which is converted by the enzymes cyclooxygenase 2 (COX-2) and lipooxygenase, to proinflammatory and pro-angiogenic prostaglandins, and reactive oxygen species which damage DNA and inhibit DNA repair enzymes. 87,88 DCA also induces COX-2 expression through transactivation of the epidermal growth factor receptor, 87,89 and activates the b-catenin cell-signaling pathway resulting in colon cancer cell proliferation and invasiveness. 90 Notably, a large body of published work demonstrates the effectiveness of non-steroidal anti-inflammatory drugs in significantly reducing polyp formation and reducing CRC risk by inhibiting the activity of COX-2.…”

EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells

“…Indeed, we and others have previously reported that EGF induces proliferation [20] and protects hepatocytes from various apoptotic insults, including TGF-β, Fas, and deoxycholic acid [25,35,36].…”