Li C, Locker J, Wan Y J
Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509, USA.
DNA Cell Biol. 1996 Nov;15(11):955-63. doi: 10.1089/dna.1996.15.955.
Retinoic acid (RA) is known to have potent effects on development and differentiation. RA exerts its effects on transcription through two distinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), that bind to specific RA-responsive elements (RARE) in target genes. alpha-Fetoprotein (AFP), a hepatocyte differentiation, maturation, and carcinogenesis marker, is transcriptionally upregulated by RA in McA-RH8994 hepatoma cells. Using deletion mapping analysis, we have identified a RARE-like sequence that is located between -2406 and -2378 of the transcription initiation site of the rat AFP gene. Sequence analysis demonstrated that this cis-acting element consists of three direct repeats and one inverted repeat of a GGGTCA-like half-site. The putative RARE can specifically bind to both RXR homodimers and RAR/RXR heterodimers as determined by gel mobility shift assays. A DR1 direct repeat was more efficient than a DR5 direct repeat oligonucleotide in competition for binding of the putative RARE to RXR and RAR/RXR. A mutagenesis study indicated that to have a full-strength induction, all the repeats were required. To further analyze the function of this element in vivo, a reporter gene construct of the putative RARE combined with the thymidine kinase promoter was cotransfected with RAR and RXR expression plasmids in CV1 cells. CAT assays demonstrated that overexpression of RXRalpha conferred the best RA response, consistent with our previous observation that 9-cis-RA is more potent than all-trans-RA for inducing the expression of the AFP gene. In addition, the RXR selective ligand LG100153 alone can stimulate the expression of the AFP gene. Our data suggest that an RXR-mediated pathway exists for modulation of AFP gene expression through a specific element.
维甲酸（RA）已知对发育和分化具有强大作用。RA 通过两类不同的核受体，即维甲酸受体（RAR）和类视黄醇 X 受体（RXR）发挥其对转录的作用，这些受体与靶基因中的特定维甲酸反应元件（RARE）结合。甲胎蛋白（AFP）是一种肝细胞分化、成熟和致癌作用的标志物，在 McA-RH8994 肝癌细胞中，RA 可使其转录上调。通过缺失定位分析，我们在大鼠 AFP 基因转录起始位点的 -2406 和 -2378 之间鉴定出一个类似 RARE 的序列。序列分析表明，这个顺式作用元件由一个 GGGTCA 样半位点的三个直接重复和一个反向重复组成。凝胶迁移率变动分析表明，推定的 RARE 能特异性地与 RXR 同二聚体和 RAR/RXR 异二聚体结合。在竞争推定的 RARE 与 RXR 和 RAR/RXR 的结合中，DR1 直接重复比 DR5 直接重复寡核苷酸更有效。诱变研究表明，要产生完全强度的诱导，所有重复都是必需的。为了进一步分析该元件在体内的功能，将推定的 RARE 与胸苷激酶启动子结合的报告基因构建体与 RAR 和 RXR 表达质粒共转染到 CV1 细胞中。CAT 分析表明，RXRα 的过表达赋予了最佳的 RA 反应，这与我们之前观察到的 9-顺式 RA 比全反式 RA 更有效地诱导 AFP 基因表达的结果一致。此外，单独的 RXR 选择性配体 LG100153 就能刺激 AFP 基因的表达。我们的数据表明，存在一条通过特定元件调节 AFP 基因表达的 RXR 介导途径。
