Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17β-estradiol in breast cancer cells
Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17β-estradiol in breast cancer cells
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DOI：
10.1210/en.2002-0149
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发表时间：
2003-06-01
期刊：
影响因子：
4.8
通讯作者：
Safe, S
中科院分区：
文献类型：
--
作者：
Khan, S;Abdelrahim, M;Safe, S
cad 基因具有三功能，表达嘧啶生物合成所需的氨基甲酰磷酸合成酶/天冬氨酸氨基甲酰转移酶/二氢乳清酶。 Cad 基因活性在 MCF-7 人乳腺癌细胞中被诱导，用 10 nm 17β-雌二醇 (E2) 处理 MCF-7 或 ZR-75 细胞会导致两种细胞系中 cad mRNA 水平增加 3 至 5 倍。使用含有 cad 基因启动子生长响应 -90 至 +115 (pCAD1) 区域的构建体进一步研究了激素诱导的 cad 基因表达的机制。 E2 在转染 pCAD1 的 MCF-7 和 ZR-75 细胞中诱导报告基因（荧光素酶）活性，其中包含三个上游富含 GC 的基序和两个下游 E-box 基序。 cad 基因启动子的缺失和突变分析表明，只有结合 Sp1 蛋白的 GC 盒才是 E2 反应所必需的。电泳迁移率变动和染色质免疫沉淀测定的结果表明，Sp1 和雌激素受体 α 都与 cad 基因启动子的富含 GC 的区域相互作用。此外，在 pCAD1 反式激活测定中，显性失活 Sp1 表达质粒和 Sp1 的小抑制性 RNA 共转染可抑制激素诱导的反式激活，从而沉默细胞中的 Sp1 表达。这些结果表明，与许多其他参与 E2 诱导的细胞增殖的基因一样，cad 基因也受到非经典 ERα/Sp1 介导的途径的调节。
The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nm 17beta-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor alpha interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ERalpha/Sp1-mediated pathway.