Promoter activity and regulation of the CYP4F2 leukotriene B(4) omega-hydroxylase gene by peroxisomal proliferators and retinoic acid in HepG2 cells.
Promoter activity and regulation of the CYP4F2 leukotriene B(4) omega-hydroxylase gene by peroxisomal proliferators and retinoic acid in HepG2 cells.
复制标题
HepG2 细胞中过氧化物酶体增殖物和视黄酸对 CYP4F2 白三烯 B(4) omega-羟化酶基因的启动子活性和调节。
DOI：
10.1006/abbi.2000.1836
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发表时间：
2000
影响因子：
3.9
通讯作者：
Hardwick,JP
中科院分区：
文献类型：
--
作者：
Zhang,X;Chen,L;Hardwick,JP
人肝脏CYP4F2基因AF221943)编码白三烯B4ω-羟化酶，将白三烯B4（LTB4）代谢为不太有效的促炎类二十烷，20-OH-LTB4。我们对人类CYP4F2基因的6.7 kb基因组片段进行了测序，该片段具有前5个外显子和500 bp的5 '侧区。主要转录起始位点位于1号外显子3′端上游49bp处，ATG翻译起始密码子位于2号外显子。除了- 39bp的TATA box和基础转录因子结合位点外，启动子区域和412bp的内含子1还有几个可能介导炎症反应和脂质稳态的核因子的推测结合位点。我们在5 ‘启动子中发现了两个DR1元件，在内含子1中发现了一个DR2元件，在内含子1和5 ’启动子中都发现了RXR/RAR结合位点。DNase I足迹显示了三个受保护的序列，该区域包含两个CAATT盒，位于- 71和- 111 bp，在CYP4F2基因表达中很重要。荧光素酶报告基因检测显示，上游500 bp序列具有较强的启动子活性。瞬时转染实验发现，5 '启动子和内含子1中的两个位点协同基因转录，而外显子1和外显子1两侧富含gc的区域抑制转录。反式维甲酸和9-顺式维甲酸分别刺激启动子活性3倍和6倍，而RXRα或RAR/RXRα共转染进一步增强了活性。过氧化物酶体增殖物抑制CYP4F2基因启动子活性，与PPARα或PPARα/RXRα共转染可轻微减弱这种抑制作用。饱和脂肪酸和12-羟基十二烷酸（12-OH-C12）均能刺激CYP4F2基因启动子活性。因此，CYP4F2基因被过氧化物酶体增殖剂抑制并被维甲酸诱导，其中RAR/RXRα介导诱导，而PPARα/RXR既不参与过氧化物酶体增殖剂或维甲酸的抑制也不参与诱导。
The human liver CYP4F2 gene (Accession No. AF221943) encodes a leukotriene B4ω-hydroxylase that metabolizes leukotriene B4(LTB4) to a less potent proinflammatory eicosanoid, 20-OH-LTB4. 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 RXRα or RAR/RXRα further enhanced activity. Peroxisome proliferators inhibit CYP4F2 gene promoter activity and cotransfection with PPARα or PPARα/RXRα can slightly attenuate this inhibition. Both saturated fatty acids and 12-hydroxydodecanoic acid (12-OH-C12) can stimulate CYP4F2 gene promoter activity. Therefore, the CYP4F2 gene is repressed by peroxisomal proliferators and induced by retinoic acid, with RAR/RXRα mediating the induction while PPARα/RXR functions neither in the repression nor in the induction by peroxisomal proliferators or retinoic acid.