Poon Song Ling, An Beum-Soo, So Wai-Kin, Hammond Geoffrey L, Leung Peter C K
Department of Obstetrics and Gynaecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5.
Endocrinology. 2008 Oct;149(10):5162-71. doi: 10.1210/en.2008-0481. Epub 2008 Jul 3.
GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.
促性腺激素释放激素-II（GnRH-II）是一种强效的GnRH亚型，参与调节卵巢癌细胞系OVCAR-3细胞的增殖以及绒毛膜癌细胞系JEG-3细胞的侵袭特性，并且人GnRH-II启动子中的一个非典型环磷酸腺苷反应元件（CRE）影响其激活。我们证明，在包括αT3、TE671、JEG-3和OVCAR-3细胞在内的几种细胞系中，GnRH-II启动子被8-溴腺苷-环磷酸腺苷激活，并且环磷酸腺苷可提高JEG-3和OVCAR-3细胞中GnRH-II的mRNA水平。此外，8-溴腺苷-环磷酸腺苷可增加JEG-3和OVCAR-3细胞中环磷酸腺苷反应元件结合蛋白（CREB）的磷酸化，并以时间限定的方式增强CBP（CREB结合蛋白）和CCAAT/增强子结合蛋白（C/EBP）-β与核提取物中磷酸化CREB（p-CREB）的共免疫沉淀。当通过小干扰RNA敲低CREB、CBP和C/EBPβ水平时，这些转录因子中任何一种的减少都会降低环磷酸腺苷增强的JEG-3和OVCAR-3细胞中GnRH-II启动子活性和GnRH-II mRNA水平。重要的是，染色质免疫沉淀试验表明，p-CREB在1小时内结合内源性GnRH-II启动子内的CRE，并且p-CREB与C/EBPβ的结合在环磷酸腺苷刺激后2小时内发生，这与C/EBPβ首次出现在CRE处一致。相比之下，p-CREB与CBP之间的最大相互作用直到环磷酸腺苷刺激后至少4小时才发生，这通过染色质免疫沉淀证明为2-4小时内CBP在CRE处的逐步加载。综上所述，这些数据表明，p-CREB、C/EBPβ和CBP以时间限定的方式被招募到GnRH-II启动子的CRE，以响应环磷酸腺苷增强其在JEG-3和OVCAR-3细胞中的转录。
