Mueller M D, Vigne J L, Minchenko A, Lebovic D I, Leitman D C, Taylor R N
Center for Reproductive Sciences, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA 94143-0556, USA.
Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10972-7. doi: 10.1073/pnas.200377097.
Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17beta-estradiol (E(2)) resulted in a 3.8-fold increase in luciferase activity, whereas a 3. 2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the alpha- or the beta-form of the human ER. In cells cotransfected with ERalpha, E(2) induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERbeta. Through specific deletions, the E(2) response was restricted to a single 385-bp PvuII-SstI fragment in the 5' flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E(2) response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E(2)-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E(2)-induced VEGF gene expression.
血管内皮生长因子（VEGF）介导多种雌激素靶组织中的血管生成活性。为了确定雌激素是否对VEGF基因表达有直接转录作用，我们构建了一个模型系统，将人VEGF启动子-荧光素酶报告基因构建体瞬时转染到人原代子宫内膜细胞和源自高分化人子宫内膜腺癌的 Ishikawa 细胞中。在原代子宫内膜上皮细胞中，用 17β-雌二醇（E₂）处理导致荧光素酶活性增加 3.8 倍，而基质细胞中则显示出 3.2 倍的诱导。我们的 Ishikawa 细胞每个细胞的功能性雌激素受体（ER）少于 100 个，因此与编码人 ER 的α或β形式的表达载体共转染。在与 ERα共转染的细胞中，E₂诱导 VEGF 启动子荧光素酶活性增加 3.2 倍。在与 ERβ共转染的细胞中观察到增加 2.3 倍。通过特定缺失，E₂反应仅限于 5'侧翼 DNA 中的单个 385 bp PvuII-SstI 片段。该上游区域与 DNA 结合域 ER 突变体共转染，或该片段内变体雌激素反应元件（ERE）的定点诱变，导致 E₂反应丧失。电泳迁移率变动分析表明，相同的 ERE 序列特异性结合雌二醇-ER 复合物。这些研究表明，E₂调节的 VEGF 基因转录需要位于转录起始位点上游 1.5 kb 的变体 ERE。该 ERE 的定点诱变消除了 E₂诱导的 VEGF 基因表达。
