GNRH MODULATION OF GONADOTROPIN GENE TRANSCRIPTION

促性腺激素基因转录的 GNRH 调节

• 批准号: 6590766
• 负责人: MARGARET A SHUPNIK
• 金额: $ 17.42万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6590766
• 关键词: DNA footprinting; biological signal transduction; calcium flux; calmodulin dependent protein kinase; cell line; enzyme activity; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic transcription; genetically modified animals; gonadotropin releasing factor; gonadotropins; hormone regulation /control mechanism; laboratory mouse; laboratory rat; mitogen activated protein kinase; nuclear runoff assay; protein kinase C; reporter genes; southern blotting; steroid metabolism; tissue /cell culture; transfection; western blottings

GnRH is released in pulses and acts on the pituitary to stimulate the synthesis and release of LH and FSH. This pulsatility is crucial to maintain normal fertility. We previously demonstrated frequency-dependent, subunit-specific effects of GnRH pulses on rat gonadotropin gene transcription in vivo and in vitro, and now have GnRH-responsive luciferase (LUC) reporter gene constructs for the rat alpha and LHbeta genes. Frequency-dependent effects on transcription could arise from differential activation of Ca+2 and protein C(PCK)/mitogen-activated protein kinase (MAPK) cascade and/or divergent GnRH-responsive gene regions and transcription factors. The role of different intracellular signals in GnRH-stimulated endogenous gonadotropin gene transcription will be assessed by nuclear run-off assays in gonadotropes after treatment with specific activators of inhibitors of Ca+2 influx, PKC, and MAPK, in the absence or presence of GnRH. Pituitary cells from transgenic mice expressing LHbetaLUC will be similarly treated and Lhbeta promotor activity measured. Finally, alphaLUC and LHbetaLUC constructs will be transfected into clonal gonadotrope cell lines, and GnRH-stimulated activity measured in the absence or presence of inhibitors of calcium influx, MAPK, and with co-transfected dominant negative or constitutively active forms of MAPK and calcium-calmodulin kinase (CAMK). MAPK and CAMK enzyme activity will be measured directly in treated cells. GnRH- sensitive elements of the alpha and LHbeta genes will be identified by transfection analysis using deletion/mutation constructs of the reporter genes. Activity of the isolated DNA elements will be demonstrated on heterologous promoters, and DNA-nuclear protein binding performed by gels shift and footprinting assays and southwestern blot analysis. The ability of steroids to directly influence the response to GnRH, and binding of nuclear proteins to GnRH-sensitive gene regions will be determined. These studies will help define the underling mechanisms of frequency-dependent regulation of gonadotropin synthesis, and begin to identify specific cellular targets which confer the GnRH response.

GnRH以脉冲形式释放，作用于脑垂体， LH和FSH的合成和释放。这种脉动对于 保持正常的生育能力。我们以前证明了频率依赖性， 促性腺激素释放激素脉冲对大鼠促性腺激素基因亚单位特异性的影响 在体内和体外转录，现在有GnRH反应 大鼠α和LH β的荧光素酶（LUC）报告基因构建体 基因.转录的频率依赖效应可能来自于 Ca+2和蛋白C（PCK）的差异活化/丝裂原活化 蛋白激酶（MAPK）级联和/或趋异GnRH应答基因 区域和转录因子。不同细胞内 GnRH刺激的内源性促性腺激素基因转录中的信号将 在促性腺激素治疗后通过核径流试验进行评估 Ca+2内流、PKC和MAPK抑制剂的特异性激活剂， GnRH的存在或不存在。转基因小鼠的骨髓细胞 将类似地处理表达LHbetaLUC的小鼠， 活动测量。最后，alphaLUC和LHbetaLUC构建体将被 转染到克隆促性腺激素细胞系中，并且GnRH刺激 在不存在或存在钙抑制剂的情况下测量的活性 内流，MAPK，并与共转染显性阴性或组成性 MAPK和钙-钙调蛋白激酶（CAMK）的活性形式。MAPK和CAMK 酶活性将在处理的细胞中直接测量。 GnRH- α和LH β基因的敏感元件将通过 使用报告基因的缺失/突变构建体的转染分析 基因.分离的DNA元件的活性将在 异源启动子和DNA-核蛋白结合 移位和足迹分析以及西南印迹分析。的能力 类固醇直接影响对GnRH的反应， 将确定GnRH敏感基因区域的核蛋白。这些 研究将有助于确定频率依赖性的潜在机制 促性腺激素合成的调节，并开始确定具体的 赋予GnRH反应的细胞靶点。