Boulanger Ana, McLemore Pamela, Copeland Neal G, Gilbert Debra J, Jenkins Nancy A, Yu Shirley S, Gentleman Susan, Redmond T Michael
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2740, USA.
FASEB J. 2003 Jul;17(10):1304-6. doi: 10.1096/fj.02-0690fje. Epub 2003 May 20.
Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.
β-胡萝卜素15,15'-单加氧酶（BCM）催化从维生素A原类胡萝卜素生物合成维生素A的第一步。我们希望确定该基因转录调控的潜在因素。在克隆了40千碱基对（kbp）的小鼠Bcm基因并确定其基因组结构后，对2 kb的5'-侧翼区域进行分析，发现了几个推定的转录因子结合位点，包括TATA盒、过氧化物酶体增殖物反应元件（PPRE）、AP2和bHLH。该2 kb片段仅在表达BCM的细胞系（TC7、PF11和猴视网膜色素上皮细胞）中驱动体外特异性荧光素酶基因表达。核苷酸-41至+163和-60至+163分别驱动基础和特异性Bcm转录活性。定点诱变和凝胶迁移实验表明，PPRE对Bcm启动子特异性至关重要，并且过氧化物酶体增殖物激活受体（PPAR）γ（PPARγ）特异性结合该元件。此外，体外共转染实验和药物处理，使用特异性PPARγ激动剂LY17883和吡格列酮，表明PPRE元件通过PPARγ和视黄酸X受体-α异二聚体赋予过氧化物酶体增殖物反应性。用PPARα/γ激动剂WY14643处理小鼠可增加肝脏中BCM蛋白的表达。因此，PPAR是Bcm基因转录调控的关键转录因子，这表明PPAR在类胡萝卜素代谢调控中具有更广泛的功能，这与其在中性脂质代谢和转运中已确立的作用一致。
