CYP2E1 induced by ethanol causes oxidative stress, proteasome inhibition and cytokeratin aggresome (Mallory body-like) formation

Abstract

The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory body-like cytokeratin inclusions in transformed human liver cells in vitro.

Introduction

When liver cells from drug primed mice are cultured to form Mallory bodies (cytokeratin aggresomes) in vitro, the expression of MAP kinase p38 is up regulated 17.1-fold compared with the control level (Nan et al., 2006). P38 kinase is activated by oxidative stress, osmotic shock, LPS, proinflammatory cytokines, UV light and growth factors. Mallory body formation in alcoholic hepatitis is associated with oxidative stress as evidenced by 4HNE adduct formation in the liver (Seki et al., 2003). Alcoholic-induced liver injury is characterized by oxidative stress where CYP2E1 induction is associated with lipid peroxidation, protein oxidation and MDA and 4HNE accumulation (French, 2001, Carmiel-Haggai et al., 2005, Sampey et al., 2003, Esterbauer et al., 1991). The formation of Mallory bodies is also associated with an increase in cytokeratin expression (Kachi et al., 1993), an increase in the ubiquitination of cytokeratin aggregates as seen on Western blots (Cadrin et al., 1992) and a decrease in proteasome activity (French et al., 2001a, French et al., 2001b). Drug primed mice develop Mallory bodies in their liver after ethanol ingestion (Zhang-Gouillon et al., 1998). Ethanol feeding causes inhibition of the 26S proteasome in the livers of rats (Fataccioli et al., 1999, Donohue et al., 1998, Bardag-Gorce et al., 2004c) and this inhibition of the proteasome is the result of oxidative stress generated by CYP2E1, which results in lipid peroxidation and 4HNE adduct formation (Bardag-Gorce et al., 2000, Albano et al., 1996, Albano et al., 1999, French et al., 2001a, French et al., 2001b).

All together, evidence supports the following hypothesis: ethanol induces CYP2E1; CYP2E1 increases the formation of free radicals during ethanol oxidation; the free radicals generate 4HNE, a product of lipid peroxidation; 4HNE adducts form with proteasomal subunits and this results in the inhibition of proteasome activity; inhibition of protein turnover, causing the accumulation of cytokeratins. Accumulation of ubiquitinated cytokeratins forms insoluble aggresomes, which coalesce to form Mallory bodies (Wu and Cederbaum, 2005, Chen and Cederbaum, 1998, Kessova and Cederbaum, 2005, Sakurai and Cederbaum, 1998).

To test this hypothesis the Cederbaum tissue culture model of HepG2 cells transfected with CYP2E1, primed with arachidonic acid and iron followed by ethanol exposure of 1 to 2 days was used. The formation of cytokeratin ubiquitin aggresomes is visualized by double label immunofluorescent microscopy. Colocalization of ubiquitin and cytokeratin in the formed aggresomes is visualized by confocal microscopy.