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 (LTB4) to a less pot ent proinflammatory eicosanoid, 20-OH-LTB4. We sequenced a 6.7-kb genomic f ragment of the human CYP4F2 gene that has the first five exons and 500 bp o f the 5'-flanking region. The major transcription start site was found to b e 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 tran scription factor binding sites, the promoter region and 412-bp intron 1 hav e several putative binding sites for nuclear factors that may mediate the i nflammatory response and lipid homeostasis. We found two DR1 elements in th e 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 protecte d 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 trans fection experiments identified two sites in the 5' promoter and intron 1 th at cooperate in gene transcription while exon 1 and a GC-rich region flanki ng exon 1 inhibit transcription. trans-Retinoic acid and 9-cis-retinoic aci d stimulate promoter activity 3- and 6-fold, respectively, while cotransfec tion with RXR alpha or RAR/RXR alpha further enhanced activity. Peroxisome proliferators inhibit CYP4F2 gene promoter activity and cotransfection with PPAR alpha or PPAR alpha/RXR alpha 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 repr essed by peroxisomal proliferators and induced by retinoic acid, with RAR/R XR alpha mediating the induction while PPAR alpha/RXR functions neither in the repression nor in the induction by peroxisomal proliferators or retinoi c acid. (C) 2000 Academic Press.