Cloning and characterization of the 5 '-flanking region of the rat glutamate-cysteine ligase catalytic subunit

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione sy nthesis, is made up of two subunits, a catalytic (heavy) subunit (GCLC) and a modifier (light) subunit (GCLM), which are differentially regulated. Inc reased hepatic GCLC expression occurs during rapid growth, oxidative stress and after ethanol treatment. To facilitate studies of GCLC transcriptional regulation, we have cloned and characterized a 1.8 kb 5 ' -flanking region of the rat GCLC (GenBank accession number AF218362). A consensus TATA box and one transcriptional start site are located at 302 and 197 nucleotides u pstream of the translational start site, respectively. The promoter contain s consensus binding sites for many transcription factors including nuclear factor kappaB (NF-kappaB) and activator protein I (AP-1). The rat GCLC prom oter was able to efficiently drive luciferase expression in H4IIE cells. Se quential deletion analysis revealed that three DNA regions, -595 to -111, - 1108 to -705 and -705 to -595, are involved in positive (the first two regi ons) and negative (the latter region) gene regulation. Specific protein bin ding to these regions was confirmed by DNase I footprinting and electrophor etic mobility-shift assays (EMSAs). Ethanol-fed livers exhibit increased pr otein binding to region -416 to -336 on DNase I footprinting analysis, whic h was found to be NF-kappaB and AP-1 on EMSA and supershift analysis. Aceta ldehyde treatment of H4IIE cells led to a time- and dose-dependent increase in GCLC mRNA levels, binding of NF-kappaB and AP-1 to the GCLC promoter, a nd luciferase activity driven by the GCLC promoter fragment containing thes e binding sites.