Growth hormone regulation of IGF-I gene expression

描述(申请人提供)：生长激素(GH)是身体生长的主要调节剂。几十年来，生长激素主要通过刺激组织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却知之甚少。了解这一机制的关键是识别IGF-I基因中的GH反应区域，但由于缺乏合适的细胞系统和IGF-I基因结构的复杂性，直接识别此类顺式调控区域一直是困难的。最近利用基因敲除小鼠模型和体内基因转移技术的研究表明，信号转导和转录激活因子5(STAT5)是生长激素信号通路的既定组成部分，对于生长激素刺激的肝脏IGF-I基因的表达是必不可少的。基于这些信息并使用一种独特的方法，我们已经确定了一个远端的IGF-I 5‘侧翼区，它可以作为STAT5反应的增强子，包含多个共识的STAT5结合位点，并且在进化上是保守的。基于这些初步观察，我们推测该末端增强子介导了肝脏中GH-STAT5对IGF-I基因转录的激活。我们建议在小鼠系统中测试这一假设。在特定的目标1中，我们将通过DNase I足迹、凝胶移位和共转染实验来证实IGFI远端增强子含有功能STAT5结合位点。在特定的目标2中，我们将确定GH是否导致STAT5与这个远端的IGF-I增强子结合，以及STAT5结合是否先于GH诱导的IGF-I转录在GH缺陷的LIT/LIT小鼠的肝脏中发生。体内STAT5与DNA的结合将通过染色质免疫沉淀分析(CHIP)与实时荧光定量聚合酶链式反应(RT-PCR)相结合来定量。在特定目标3中，我们确定IGF-I增强子是否足以在转基因小鼠中诱导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提供了基础。

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.