Growth hormone regulation of IGF-I gene expression

描述（由申请人提供）：生长激素（GH）是体细胞生长的主要调节因子。几十年来，人们已经知道GH主要通过刺激组织（包括肝脏）中胰岛素样生长因子-I（IGF-I）的基因表达来调节生长，但GH刺激IGF-I基因表达的分子机制知之甚少。理解这一机制的关键是鉴定IGF-I基因中的GH反应区，但由于缺乏合适的细胞系统和IGF-I基因结构的复杂性，直接鉴定这种顺式调节区一直很困难。最近的研究采用基因敲除小鼠模型和体内基因转移技术表明，信号转导和转录激活因子5（STAT 5），GH信号通路的一个既定的组成部分，是必不可少的GH刺激的IGF-I基因在肝脏中的表达。基于这一信息，并使用一种独特的方法，我们已经确定了一个远端IGF-I 5 '侧翼区，可以作为一个STAT 5响应增强子，包含多个共识STAT 5结合位点，是进化保守的。基于这些初步的观察，我们假设这个远端增强子介导了肝脏中IGF-I基因转录的GH-STAT 5激活。我们建议在小鼠系统中测试这一假设。在具体目标1中，我们将使用DNA酶I足迹法、凝胶移位和共转染测定来确认远端IGFI增强子含有功能性STAT 5结合位点。在具体目标2中，我们将确定GH是否导致STAT 5与该远端IGF-I增强子结合，以及在GH缺陷lit/lit小鼠的肝脏中，STAT 5结合是否发生在GH诱导的IGF-I转录之前。将通过染色质免疫沉淀试验（CHIP）结合实时PCR定量体内STAT 5-DNA结合。在具体目标3中，我们确定了IGF-I增强子是否足以在转基因小鼠中诱导GH基因表达的远端意愿。本研究结果为进一步阐明GH调控IGF-I基因表达的分子机制奠定了基础。

DESCRIPTION (provided by applicant): Growth hormone (GH) is a major regulator of somatic growth. It has been known for decades that GH regulates growth mainly through stimulating gene expression in tissues, including the liver, of insulin-like growth factor-i (IGF-I), but the molecular mechanism by which GH stimulates IGF-I gene expression is poorly understood. The key to understanding this mechanism is identification of GH-responsive regions in the IGF-I gene, but direct identification of such cis-regulatory regions has been difficult, due to the lack of appropriate cell systems and the complexity of IGF-I gene structure. Recent studies employing knockout mouse models and in vivo gene transfer technique demonstrated that signal transducer and activator of transcription 5 (STAT5), an established component of the GH signaling pathway, is essential for GH-stimulated IGF-I gene expression in the liver. Based on this information and using a unique approach, we have identified a distal IGF-I 5'-flanking region that can function as a STAT5-responsive enhancer, that contains multiple consensus STAT5 binding sites, and that is evolutionally conserved. Based on these preliminary observations, we hypothesize that this distal enhancer mediates GH-STAT5 activation of IGF-I gene transcription in the liver. We propose to test this hypothesis in the mouse system. In specific aim 1, we will confirm that the distal IGFI enhancer contains functional STAT5 binding sites using DNase I footprinting, gel-shift and co-transfection assays. In specific aim 2, we will determine whether GH causes binding of STAT5 to this distal IGF-I enhancer and whether the STAT5 binding occurs ahead of GH-induced IGF-I transcription in the liver of GH deficient lit/lit mice. The in vivo STAT5-DNA binding will be quantified by chromatin immunoprecipitation assays (CHIP) coupled with real-time PCR. In specific aim 3, we determine the distal will if IGF-I enhancer is sufficient for GH induction of gene expression in transgenic mice. The results of this research should provide a basis for further elucidating the molecular mechanism by which GH regulates IGF-I gene expression.