Promoter activity and regulation of the CYP4F2 leukotriene B(4) omega-hydroxylase gene by peroxisomal proliferators and retinoic acid in HepG2 cells.

The human liver CYP4F2 gene (Accession No. AF221943) encodes a leukotriene B(4) omega-hydroxylase that metabolizes leukotriene B(4) (LTB(4)) to a less potent proinflammatory eicosanoid, 20-OH-LTB(4). We sequenced a 6.7-kb genomic fragment of the human CYP4F2 gene that has the first five exons and 500 bp of the 5'-flanking region. The major transcription start site was found to be 49 bp upstream of the 3' end of exon 1 and the ATG translation initiation codon was located in exon 2. Besides the TATA box at -39 bp and basal transcription factor binding sites, the promoter region and 412-bp intron 1 have several putative binding sites for nuclear factors that may mediate the inflammatory response and lipid homeostasis. We found two DR1 elements in the 5' promoter, a DR2 element in intron 1, and RXR/RAR binding sites in both intron 1 and the 5' promoter. DNase I footprinting revealed three protected sequences, with the region containing two CAATT boxes at -71 and -111 bp important in CYP4F2 gene expression. Luciferase reporter assays showed that the 500-bp upstream sequence has strong promoter activity. Transient transfection experiments identified two sites in the 5' promoter and intron 1 that cooperate in gene transcription while exon 1 and a GC-rich region flanking exon 1 inhibit transcription. trans-Retinoic acid and 9-cis-retinoic acid stimulate promoter activity 3- and 6-fold, respectively, while cotransfection with RXRalpha or RAR/RXRalpha further enhanced activity. Peroxisome proliferators inhibit CYP4F2 gene promoter activity and cotransfection with PPARalpha or PPARalpha/RXRalpha can slightly attenuate this inhibition. Both saturated fatty acids and 12-hydroxydodecanoic acid (12-OH-C(12)) can stimulate CYP4F2 gene promoter activity. Therefore, the CYP4F2 gene is repressed by peroxisomal proliferators and induced by retinoic acid, with RAR/RXRalpha mediating the induction while PPARalpha/RXR functions neither in the repression nor in the induction by peroxisomal proliferators or retinoic acid.