GNRH GONADOTROPIN TRANSCRIPTION AND STEROID FEEDBACK

促性腺激素释放激素和性激素作用于脑下垂体，调节黄体生成素和卵泡刺激素的合成和分泌。在过去的资助期间，我们克隆了大鼠α亚单位启动子，鉴定了大鼠LHβ和α亚单位启动子的GnRH反应DNA元件和转录因子，并研究了介导GnRH反应的细胞内信号通路。LHbeta启动子在体内需要GnRH的脉动刺激，并且有两个协同作用的复杂DNA元件，一个是具有重叠的Sp1和Carg盒位点的远端元件，另一个是近端元件 Sf-1和Egr-1具有两个二分位点的元件。这两个元件都能与Sp1家族锌指蛋白结合，从而对转录产生不同的影响。这些转录因子在GnRH刺激中的相对作用将通过DNA蛋白结合、染色质免疫沉淀(ChIP)和实时RT-PCR以及在LbetaT2细胞中转染缺失/突变的荧光素酶结构来评估。初步数据显示，类固醇既增强(17β-雌二醇(E)或PM双氢睾酮，DHT)又抑制(NM DHT)GnRH转录反应，而不是 类固醇受体与DNA结合。共激活/整合子蛋白SNURF和CBP将在共转染和蛋白质-蛋白质结合研究中进行测试，以调节GnRH反应。平行的生化和功能策略将被用来研究类固醇对正常促性腺激素和LbetaT2细胞的刺激和抑制作用。这些测试包括芯片检测中Sp1/Egr-1家族成员改变启动子占有率的测试、救援 通过转录因子的过表达抑制，以及通过聚焦微阵列分析和实时RT-PCR改变基因表达。将定义介导类固醇反应的启动子区域和转录因子。核受体的要求将用类固醇拮抗剂和TFM雄激素受体突变小鼠进行测试。在没有或存在促性腺激素释放激素的情况下，将测量非基因组类固醇对细胞内信号通路的影响。这些研究将进一步加深我们对GnRH对促性腺激素的调节的理解，以及 这一反应的类固醇调节。这项研究对理解多囊卵巢综合征等生育障碍具有重要意义，在这些疾病中，循环中的雄激素升高伴随着GnRH和LH分泌的改变。

GnRH pulses and sex steroids act on the pituitary to regulate the synthesis and secretion of LH and FSH. In the past funding period, we cloned the rat alpha-subunit promoter, identified GnRH-responsive DNA elements and transcription factors for rat LHbeta and alpha-subunit promoters, and characterized intracellular signaling pathways mediating GnRH responses. The LHbeta promoter requires pulsatile GnRH stimulation in vivo, and has two cooperating complex DNA elements, a distal element with overlapping Sp1 and CArG box sites, and a proximal element with two bipartite sites for SF-1 and Egr-1. Both elements bind Sp1 family zinc finger proteins that could differentially affect transcription. Relative roles of these transcription factors in GnRH stimulation will be assessed by DNA-protein binding, chromatin immunoprecipitation (CHIP) and real-time RT-PCR, and transfection ot deletion/mutation luciferase constructs in LbetaT2 ceils. Preliminary data show that steroids both enhance (17beta- estradiol (E) or pM dihydrotestosterone, DHT) and suppress (nM DHT) the GnRH transcriptional response, without steroid receptor binding to DNA. Coactivator/integrator proteins SNURF and CBP will be tested in cotransfection and protein-protein binding studies for modulation of the GnRH response. Parallel biochemical and functional strategies will be used to investigate both stimulatory and suppressive effects of steroids in normal gonadotropes and LbetaT2 cells. These include tests of altered promoter occupancy by Sp1/Egr- 1 family members in ChIP assays, rescue of suppression by overexpression of transcription factors, and alteration of gene expression by focused microarray analysis and real-time RT-PCR. Promoter regions and transcription factors mediating steroid responses will be defined. Nuclear receptor requirements will be tested with steroid antagonists, and the Tfm androgen receptor mutant mouse. Non-genomic steroid effects on intracellular signaling pathways will be measured in the absence or presence of GnRH. These studies will further our understanding of GnRH regulation of the gonadotropins, and steroid modulation of this response. This research has important implications for understanding fertility disorders such as PCOS, in which elevated circulating androgens are accompanied by altered GnRH and LH secretion.