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.
Zhang
X
X
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272, USA.
Chen
L
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Hardwick
J P
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D000072470:Cytochrome P450 Family 4; D018983:DNA Footprinting; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D007975:Leukotriene B4 / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020025:Peroxisome Proliferators / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism*
D020025:Peroxisome Proliferators; C000607071:RARA protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014157:Transcription Factors; D007975:Leukotriene B4; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000072470:Cytochrome P450 Family 4; C117480:CYP4F2 protein, human; D042926:Cytochrome P-450 CYP4A