The cytochrome P450 (CYP) 2J2 arachidonic acid epoxygenase gene was down-regulated at a pre-translational level in human hepatoma-derived HepG2 cells incubated in a hypoxic environment; under these conditions, the expression of c-Jun and c-Fos mRNA and protein was increased. The 5′-upstream region of the CYP2J2 gene was isolated by amplification of a 2341 bp fragment and putative regulatory elements that resembled activator protein-1 (AP-1)-like sequences were identified. From transient transfection analysis, c-Jun was found to strongly activate a CYP2J2–luciferase reporter construct, but co-transfection with plasmids encoding c-Fos or c-Fos-related antigens, Fra-1 and −2, abrogated reporter activity. Using a series of deletion-reporter constructs, a c-Jun-responsive module was identified between bp −152 and −50 in CYP2J2: this region contained an AP-1-like element between bp −56 and −63. The capacity of this element to interact directly with c-Jun, but not c-Fos, was confirmed by electromobility-shift assay analysis. Mutagenesis of the −56/−63 element abolished most, but not all, of the activation of CYP2J2 by c-Jun, thus implicating an additional site within the c-Jun-responsive region. The present results establish an important role for c-Jun in the control of CYP2J2 expression in liver cells. Activation of c-Fos expression by hypoxia promotes the formation of c-Jun/c-Fos heterodimers, which decrease the binding of c-Jun to the CYP2J2 upstream region, leading to gene down-regulation.
- activator protein-1
- cytochrome P450
- human gene regulation
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AF039089.
Abbreviations used: ANP, atrial natriuretic peptide; AP-1, activator protein-1; CYP, cytochrome P450; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EET, epoxyeicosatrienoic acid; EMSA, electrophoretic mobility-shift assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; RT-PCR, reverse transcription-PCR.
- The Biochemical Society, London ©2003