Cytochrome P450 2E1 responsiveness in the promoter of glutamate-cysteine ligase catalytic subunit

Abstract

Previous studies have shown cytochrome P450 2E1 (CYP2E1)-dependent transcriptional up-regulation of glutamate-cysteine ligase (GCL). To identify sequences mediating constitutive and induced expression of the catalytic subunit of GCL (GCLC), a series of deletion mutants from the 5'-flanking region (−3,802 to +465) were transfected into control (C34) and CYP2E1-overexpressing (E47) HepG2 cells. Increased luciferase expression, both basal (2- to 3-fold) and following exposure to ethanol, arachidonic acid (AA), or AA plus iron, was detected in E47 cells with the full-length but not shorter reporter vectors. Basal induction was blocked by CYP2E1 inhibitors and catalase. Basal and inducible luciferase expression in E47 cells was blunted by the full-length construct mutated in the ARE4 site. Catalase and diallyl sulfide prevented basal and AA-induced messenger RNA (mRNA) levels of GCLC and the modulatory subunit of GCL (GCLM). Preincubation with low doses of AA increased glutathione (GSH) levels as well as GCLC and GCLM mRNAs, and this protected against H 2O 2 and menadione toxicity. Primary hepatocytes from pyrazole-injected rats with high levels of CYP2E1 showed an increase in GSH levels as well as GCLC and GCLM mRNAs compared with saline controls, and this was prevented by diallyl sulfide. In conclusion, redox-sensitive elements directing constitutive and induced expression of the GCLC in CYP2E1-expressing cells are present in the ARE4 distal portion of the 5'-flanking region, between positions −3,802 and −2,752, perhaps a reflection of metabolic adaptation to CYP2E1-generated oxidative stress. (H EPATOLOGY 2003;37:96-106.)