GnRH signaling in LbetaT2 gonadotrope cells

描述(申请人提供)：尽管GnRH脉冲频率对促性腺激素亚单位基因表达和分泌的调节已在整个动物和细胞培养系统中得到很好的证明，但这种差异调节的分子基础尚不清楚。我们在这项研究中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启动子上的转录激活因子和共抑制因子的平衡所决定的。我们还假设，激活物和抑制物的水平是由脉冲的频率通过不同的第二信使通路调制的。首先，我们将研究针对LHP和FSH|3启动子的转录因子和辅助抑制因子。我们有证据表明，激活剂(Egr-1，AP-1)和辅助抑制物(DAX-1，NAB-2，TGIF，SnoN)都是在特定的脉冲频率下诱导的。我们将通过慢病毒过表达或siRNA敲除来调节这些因子的表达，以测试它们在脉冲解码中的参与。其次，我们将验证以下假设：GnRH-R在低脉冲频率选择性地激活Gs和cAMP信号，但在高脉冲频率也激活GQ/11、DAG/钙和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度表达或siRNA耗尽来操纵这些信号级联中的单个蛋白质，以测试对信号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的元件的表达，从而改变促性腺激素对GnRH脉冲的反应。最后，我们将通过基因操作在小鼠中创建扰乱脉搏感应的遗传模型。这些遗传模型将包括细胞特异性转基因过表达选定的共抑制因子，以及使用cre-loxP技术特异性删除Gs和GQ/11通路的促性腺激素成分。

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.