Glycochenodeoxycholic Acid Induces Transcription of CFOS/JUNB in a Human Hepatoma Cell Line

Abstract

Background & Aims: Glycochenodeoxycholic acid (GCDCA) is the major hydrophobic bile acid in human cholestasis and induces apoptosis in hepatocytes at low micromolar concentrations. The transcription factor activator protein 1 (AP–1) is a key regulator of cell cyle and modulates gene transcription associated with apoptosis. The effect of GCDCA on AP–1 expression in hepatocytes is unknown. Thus, the aim of this study was to investigate whether GCDCA affects transcription of AP–1 proteins cFos and JunB in hepatocytes. Methods: HepG2-Ntcp cells were stimulated with GCDCA (75µmol/L) for 4h or left untreated. Caspase 3/7 activity was determined to assess apoptosis. Total RNA from stimulated and control cells was isolated and analyzed by microarray and quantitative real-time PCR (RT-PCR). For microarray analysis, mRNA was labelled with Cy3 or Cy5 and hybridized on a cell death Piqor microarray. A two-step quantitative RT-PCR for cFos and JunB was performed using SYBR Green to determine dsDNA. A standard curve method with GAPDH as endogenous control was applied for precise quantification. Results: GCDCA at 75µmol/L effectively induced apoptosis in HepG2-Ntcp cells after 4h: caspase 3/7 activity was 27±6-fold over control. Analysis of total RNA with Bioanalyzer 2100 via gel electrophoresis and elution diagram demonstrated high quality and purity. Semi-quantitative read-out of the mircoarray showed enhanced transcription of AP–1 proteins in HepG2-Ntcp cells treated with 75µmol/L GCDCA: cFos was upregulated 4.0-fold and JunB 2.5-fold compared to controls. These results were confirmed using quantitative RT-PCR. In HepG2-Ntcp cells treated with 75µmol/L GCDCA for 4h, cFos mRNA was upregulated 26.3-fold (p<0.01 compared to controls). In addition, JunB mRNA in GCDCA-stimulated cells was 3.1-fold higher compared to unstimulated cells (p<0.02). Conclusions: GCDCA significantly enhanced transcription of AP–1 proteins cFos and JunB. GCDCA-induced upregulation of AP–1-dependent genes may contribute to the cytotoxicity of this bile acid.