GnRH signaling in LbetaT2 gonadotrope cells

LbetaT2 促性腺激素细胞中的 GnRH 信号传导

基本信息

批准号：
7615096
负责人：
NICHOLAS J WEBSTER
金额：
$ 26.09万
依托单位：
VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7615096
关键词：
Adenoviruses Animals Arts Binding Biochemical Biological Assay Calcium Cell Culture System Cells Complex Coupled Cyclic AMP Dependence Development Dominant-Negative Mutation Elements Environment Equilibrium Family member Fluorescence Resonance Energy Transfer Frequencies Gene Expression Genes Genetic Genetic Models Goals Gonadal Steroid Hormones Gonadotrope Cell Gonadotropins Hormonal In Vitro Individual JUN gene Knock-out Life Measurement Measures Modeling Molecular Mus Mutation Neurosecretory Systems Pathway interactions Physiologic pulse Pituitary Gland Process Proteins Rattus Regulation Reporter Reproduction Research Personnel Role Second Messenger Systems Signal Pathway Signal Transduction Small Interfering RNA Steroids Techniques Technology Testing Time Transcription Coactivator Transcription Factor AP-1 Transcriptional Activation Transfection Transgenic Organisms base desensitization genetic manipulation mouse model novel overexpression prevent programs promoter research study response second messenger steroid hormone transcription factor vector

项目摘要

DESCRIPTION (provided by applicant): Although the regulation of gonadotropin subunit gene expression and secretion by GnRH pulse frequency has been well documented in whole animals and cell culture systems, the molecular basis for this differential regulation is not understood. Our goal in this proposal is to uncover the molecular mechanisms underlying the pulse sensitivity of the gonadotrope cell. We hypothesize that the pulse sensitivity is conferred by the balance of transcriptional activators and co-repressors on the LHB and FSHB promoters. We also hypothesize that the levels of the activators and repressers are modulated by the frequency of pulses via distinct second messenger pathways. Initially, we will investigate the transcription factors and co-repressors that target the LHP and FSH|3 promoters. We have evidence that both activators (Egr-1, AP-1) and co-repressors (DAX-1, NAB-2, TGIF, SnoN) are induced at specific pulse frequencies. We will modulate the expression of each of these factors using lentiviral overexpression or siRNA knockdown to test their involvement in pulse decoding. Secondly, we will test the hypothesis that the GnRH-R activates Gs and cAMP signaling selectively at low pulse frequencies, but also activates Gq/11 and DAG/calcium and ERK signaling at high pulse frequencies. These studies will use a combination of standard biochemical measurements and state-of-the-art real-time measurements in live cells using newly developed FRET reporters. We will manipulate individual proteins in these signal cascades by lentiviral overexpression or siRNA depletion to test the effect on both signaling and gonadotropin expression and secretion. We furthermore hypothesize that the steroid hormone milieu will alter the expression of elements of these pathways and, consequently, the response of the gonadotrope to pulses of GnRH. Lastly, we will create genetic models of disrupted pulse sensing through genetic manipulation in the mouse. These genetic models will include cell-specific transgenic overexpression of selected co-repressors as well as gonadotrope-specific deletion of components of the Gs and Gq/11 pathways using cre-loxP technology.

描述（由申请人提供）：尽管在整个动物和细胞培养系统中对促性腺激素亚基基因表达和分泌的促性腺激素亚基基因表达和分泌已得到充分证明，但这种差异调节的分子基础尚不清楚。我们在此提案中的目标是揭示促性腺细胞脉冲敏感性的分子机制。我们假设脉冲灵敏度是由LHB和FSHB启动子上转录激活因子和共抑制剂的平衡所赋予的。我们还假设激活剂和阻遏物的水平是通过脉冲的频率通过不同的第二信使途径调节的。最初，我们将研究针对LHP和FSH | 3启动子的转录因子和共抑制剂。我们有证据表明，激活剂（EGR-1，AP-1）和共抑制剂（DAX-1，NAB-2，TGIF，SNON）均在特定的脉搏频率下诱导。我们将使用慢病毒过表达或siRNA敲低调节这些因素的表达，以测试其参与脉冲解码。其次，我们将检验以下假设：GNRH-R在低脉搏频率下选择性地激活GS和CAMP信号，但也激活GQ/11，以及在高脉冲频率下的DAG/Calcium and Calcium and Calcium and Calcium and Clacium and Clacem and Clastium and Calcium and Clacemium and Claceium和ERK信号。这些研究将结合使用新开发的FRET记者在活细胞中进行标准生化测量和最先进的实时测量。我们将通过慢病毒过表达或siRNA耗竭来操纵这些信号级联反应中的单个蛋白质，以测试对信号传导和促性腺激素表达和分泌的影响。我们还假设类固醇激素环境将改变这些途径的元素的表达，从而改变促性腺激素对GnRH脉冲的反应。最后，我们将通过小鼠中的遗传操纵创建脉搏传感的遗传模型。这些遗传模型将使用CRE-loxp技术包括选定的共抑制剂的细胞特异性转基因过表达以及GS和GQ/11途径的促性腺素特异性缺失。