Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells
Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells
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DOI：
10.1074/jbc.274.45.32099
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发表时间：
1999-11-05
影响因子：
4.8
通讯作者：
Safe, S
中科院分区：
文献类型：
--
作者：
Dong, LA;Wang, WL;Safe, S
T47D和MCF-7人类乳腺癌细胞中的bcl-2基因表达受17β-雌二醇（E2）诱导，通过分析bcl-2基因启动子进一步研究了E2反应机制。启动子的-1602至-1534远端区域（bcl-2j）具有E2反应性；然而，在凝胶迁移试验中，雌激素受体α（ERalpha）并不与[P-32]bcl-2j结合，而Spl蛋白则形成了迟缓带复合物。进一步分析表明，bcl-2 基因启动子的上游区域（-1603 至-1579）包含两个富含 GC/GA 的位点，分别位于-1601（5'-GGGCTGG-3'）和-1588（3'-GGAGGG-5'），这两个位点与 Spl 蛋白结合。随后的研究证实，E2 的反式激活依赖于 ERalpha/Sp1 与这两个富含 GC 的位点的相互作用，这一点通过体外足印法得到了证实。相反，21 个碱基对的 E2 反应下游区域（-1578 至 -1534）没有与 Spl 或 ER 蛋白结合；这些数据以及瞬时转染研究的结果表明，E2 对 bcl-2 基因启动子 -1578 至 -1534 区域的转录激活依赖于 cAMP 的诱导以及随后通过 cAMP 反应元件的激活，因此，激素对乳腺癌细胞中 bcl-2 基因表达的调控涉及多个增强子元件，E2 介导的转录激活并不需要雌激素受体与启动子 DNA 直接结合。
bcl-2 gene expression is induced by 17 beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ERalpha) did not bind [P-32]bcl-2j, whereas Spl protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Spl protein. Subsequent studies confirmed that transactivation by E2 was dependent on ERalpha/Sp1 interactions with both GC-rich sites, and this was confirmed by in uitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Spl or ER, protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif, These data coupled,with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element, Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.