Ethanol upregulates pro-fibrogenic connective tissue growth factor (CTGF) gene expression in HepG2 cells via cytochrome P450 2E1-mediated ethanol oxidation

Abstract

Connective tissue growth factor (CTGF) is a pro-fibrogenic molecule involved in several human fibrotic disorders. CTGF overexpression in the liver, through upregulation of CTGF mRNA in hepatic stellate cells (HSCs), is reported to correlate with the degree of fibrosis. Although alcohol is a major cause of hepatic fibrosis, the role of CTGF in alcoholic hepatic fibrogenesis, has been poorly understood. Oxidative stress, mediated by ethanol-inducible Cytochrome P-4502E1(CYP2E1), has been implicated as a crucial factor in alcoholic hepatic fibrogenesis. Therefore, we investigated the contribution of CYP2E1-mediated ethanol oxidation to CTGF mRNA expression by using a well-established HepG2 cell line constitutively expressing CYP2E1 (E9 cells). CTGF mRNA quantitation by a real-time reverse polymerase chain reaction (RT-PCR) showed a significant increase in CTGF mRNA levels in ethanol-treated E9 cells. Phorbol myristate acetate (PMA), which enhances CYP2E1 expression, increased CTGF mRNA levels. This increase was diminished after co-incubation with 4-methylpyrazole (4MP), a CYP2E1 inhibitor, or an antioxidant, N -acetylcysteine. We visualized the state of oxidative stress only in ethanol-treated E9 cells by a newly-established anti-acrolein-modified antibody. In conclusion, our study has first identified a possible role of CYP2E1-mediated ethanol oxidation in CTGF gene upregulation, suggesting a considerable role of CTGF in alcoholic hepatic fiberogenesis.