Role of Phosphoinositide-3 Kinase and Epidermal Growth Factor Signaling in the Cytoprotective Effect of Ursodeoxycholate
Cholestatic liver disorders lead to the retention of endogenous cytotoxic compounds, such as bile acids. Certain bile acids, glycochenodeoxycholate (GCDC) induce hepatocyte apoptosis, while others, tauroursodeoxycholic acid (TUDC), are cytoprotective.
The aim of this study was to determine the role of phosphoinositide-3 kinase (PI3K) and transactivation of the epidermal growth factor receptor (EGFR) in TUDC's cytoprotective effect in hepatocytes.
Primary rat hepatocyte cultures (4 hrs or 24 hrs post-isolation) were exposed to GCDC (50 or 100 uM) alone or with TUDC (50 or 100 uM) followed by GCDC treatment. Some cultures were pretreated with 20 uM LY294002, a PI3K inhibitor, or 5 uM AG1478, an inhibitor of EGFR phosphorylation, prior to bile acid treatment. Apoptosis was determined by morphological evaluation of Hoechst stained cells. EGFR activation was measured by immunoblotting with EGFR-PTY1173 antibodies or by direct immunoprecipitation of tyrosine phosphorylated proteins with a phosphotyrosine antibody, A4G10, followed by immunoblotting with a total EGFR antibody.
TUDC protected 4 and 24 hr cultures from GCDC induced apoptosis (80% and 60% of control values, respectively). PI3K inhibition alone induced a 3.5 and 7.5 fold increase in apoptosis in 4 hr and 24 hr hepatocytes, respectively. However, only in 24 hr hepatocyte cultures did PI3K inhibition reverse the protective effect of TUDC in GCDC mediated apoptosis. TUDC treatment increased EGFR phosphorylation in 4 hr, but not in 24 hour cultures. Inhibition of the EGFR phosphorylation had no effect on the cytoprotective effect of TUDC.
TUDC is cytoprotective in GCDC induced apoptosis in 4 hr and 24 hr hepatocyte cultures. The anti-apoptotic effect of TUDC is EGFR independent but PI3K dependent in 24 hr cultures and EGFR and PI3K independent in 4 hr cultures. Increasing time in culture promotes a greater dependent of rat hepatocytes on PI3K activation for survival.