Abstract
The human cytochrome P450 2J2 (CYP2J2) generates cytoprotective epoxyeicosatrienoic acids from arachidonic acid. Expression of CYP2J2 is decreased in hypoxia, and the resultant decrease in CYP2J2-derived epoxyeicosanoids may contribute to the pathogenesis of cardiac ischaemia. Recent studies have indicated that AP-1 (activator protein-1) regulates CYP2J2 expression in normoxia and hypoxia. Down-regulation of CYP2J2 in hypoxic HepG2 cells was closely associated with the up-regulation of c-fos and transient transfection analysis demonstrated that c-Fos abolishes the activation of CYP2J2 by the AP-1 protein c-Jun. Deletion of the region between nt -122 and -50 upstream of the start codon in CYP2J2 prevented c-Jun transactivation. In this study we demonstrate that the sequence at -105/-95 is a major regulatory element that binds c-Jun and has a prominent role in CYP2J2 gene transactivation. Mutagenesis of both the -105/-95 region and the previously identified element at -56/-63 was required for complete loss of transactivation by c-Jun; separate mutagenesis of the -105/-95 element or, to a lesser extent, the -56/-63 element resulted in a partial loss of gene activation. In contrast to the behaviour of the -56/-63 element, c-Jun homodimers and c-Fos/c-Jun heterodimers bound to the -105/-95 element. These findings demonstrate that the c-Jun-responsive module between -122 and -50 in the CYP2J2 proximal promoter contains an atypical AP-1 element at -105/-95 that has a major role in c-Jun transactivation and acts in conjunction with the -56/-63 element to regulate expression.