Park-Sarge O K, Sarge K D
Department of Physiology, University of Kentucky, Lexington 40536-0084, USA.
Endocrinology. 1995 Dec;136(12):5430-7. doi: 10.1210/endo.136.12.7588292.
We have previously shown that both pituitary gonadotropins and forskolin induce progesterone receptor (PR) messenger RNA expression at the level of transcription in granulosa cells of the rat ovary. To determine the DNA regulatory elements that are important for cAMP-induced transcription of the PR gene in the ovary, we examined the cAMP-induced activity of promoter sequences in rat granulosa cells transfected with various fusion constructs containing PRB promoter sequences linked to the luciferase reporter gene. When cells were transfected with a luciferase fusion construct containing the 1375-base pair 5'-flanking region of the rat PRB gene, forskolin treatment substantially increased luciferase activity. Analysis of a series of 5'-deletion mutants indicated that a minimal PRB promoter containing 116 base pairs of upstream sequence (-116/3) was sufficient to increase luciferase activity in response to forskolin in transfected rat granulosa cells. This promoter contains a consensus CCAAT site in reverse orientation (5'-ATTGG-3') and a consensus GC box (5'-GGGGCGGGCC-3'), but no known cAMP-responsive element. Site-specific mutation of the GC box notably decreased both basal and cAMP-induced activity of this minimal PRB promoter. In addition, site-specific mutation of the CCAAT binding site within this proximal promoter of the PRB gene substantially decreased cAMP-induced activity, but did not significantly affect the basal activity of this promoter. Either mutation alone failed to abolish cAMP inducibility. In contrast, double mutation of both the GC box and the CCAAT box completely abolished cAMP inducibility, suggesting that the GC box and the CCAAT box act together to mediate cAMP-induced transcription of the PRB gene. Gel shift analysis shows that the minimal PRB promoter sequences form multiple complexes with nuclear proteins of granulosa cells, all of which are specifically competed by oligonucleotides containing the GC box and the CCAAT box. Taken together, our results suggest a functional role for transcription factors binding the GC box and the CCAAT box in mediating cAMP-induced transcription of the rat PRB promoter in rat granulosa cells.
我们先前已经表明，垂体促性腺激素和福斯高林均可在大鼠卵巢颗粒细胞中转录水平上诱导孕激素受体（PR）信使核糖核酸的表达。为了确定对卵巢中cAMP诱导的PR基因转录重要的DNA调控元件，我们检测了用各种融合构建体转染的大鼠颗粒细胞中启动子序列的cAMP诱导活性，这些融合构建体包含与荧光素酶报告基因相连的PRB启动子序列。当用含有大鼠PRB基因1375个碱基对5'-侧翼区的荧光素酶融合构建体转染细胞时，福斯高林处理显著增加了荧光素酶活性。对一系列5'-缺失突变体的分析表明，包含116个碱基对上游序列（-116/3）的最小PRB启动子足以在转染的大鼠颗粒细胞中响应福斯高林增加荧光素酶活性。该启动子含有一个反向的共有CCAAT位点（5'-ATTGG-3'）和一个共有GC框（5'-GGGGCGGGCC-3'），但没有已知的cAMP反应元件。GC框的位点特异性突变显著降低了该最小PRB启动子的基础活性和cAMP诱导活性。此外，PRB基因近端启动子内CCAAT结合位点的位点特异性突变显著降低了cAMP诱导活性，但对该启动子的基础活性没有显著影响。单独的任何一种突变都未能消除cAMP诱导性。相反，GC框和CCAAT框的双重突变完全消除了cAMP诱导性，表明GC框和CCAAT框共同作用介导PRB基因的cAMP诱导转录。凝胶迁移分析表明，最小PRB启动子序列与颗粒细胞核蛋白形成多个复合物，所有这些复合物都被含有GC框和CCAAT框的寡核苷酸特异性竞争。综上所述，我们的结果表明，结合GC框和CCAAT框的转录因子在介导大鼠颗粒细胞中大鼠PRB启动子的cAMP诱导转录中具有功能性作用。
