GnRH signaling in LbetaT2 gonadotrope cells

描述（由申请人提供）：虽然GnRH脉冲频率对促性腺激素亚基基因表达和分泌的调节在全动物和细胞培养系统中已经得到了很好的记录，但这种差异调节的分子基础尚不清楚。我们的目标是揭示促性腺激素细胞脉冲敏感性的分子机制。我们假设脉冲敏感性是由LHB和FSHB启动子上转录激活因子和共抑制因子的平衡所赋予的。我们还假设激活因子和抑制因子的水平是由脉冲频率通过不同的第二信使途径调节的。首先，我们将研究靶向LHP和FSH b|3启动子的转录因子和协同抑制因子。我们有证据表明，激活因子（Egr-1, AP-1）和辅抑制因子（DAX-1, NAB-2， TGIF, SnoN）在特定脉冲频率下都是诱导的。我们将使用慢病毒过表达或siRNA敲低来调节这些因子的表达，以测试它们在脉冲解码中的作用。其次，我们将验证GnRH-R在低脉冲频率下选择性激活Gs和cAMP信号，但在高脉冲频率下也激活Gq/11和DAG/钙和ERK信号的假设。这些研究将使用标准生化测量和最新的实时测量在活细胞中使用新开发的FRET报告相结合。我们将通过慢病毒过表达或siRNA消耗来操纵这些信号级联中的单个蛋白质，以测试对信号传导和促性腺激素表达和分泌的影响。我们进一步假设，类固醇激素环境将改变这些途径中元素的表达，从而改变促性腺激素对GnRH脉冲的反应。最后，我们将在小鼠中通过基因操作创建中断脉冲传感的遗传模型。这些遗传模型将包括选定的共抑制因子的细胞特异性转基因过表达，以及使用cre-loxP技术对Gs和Gq/11通路的成分进行促性腺激素特异性删除。

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.