Growth hormone regulation of IGF-I gene expression

描述（申请人提供）：生长激素（GH）是体细胞生长的主要调节剂。几十年来，人们已经知道生长激素主要通过刺激组织（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，但生长激素刺激IGF-I基因表达的分子机制尚不清楚。理解这一机制的关键是鉴定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，直接鉴定这种顺式调控区域一直很困难。最近采用敲除小鼠模型和体内基因转移技术的研究表明，信号换能器和转录激活因子5 （STAT5）是GH信号通路的一个既定组成部分，对于GH刺激的IGF-I基因在肝脏中的表达至关重要。基于这些信息并使用一种独特的方法，我们已经确定了远端IGF-I 5'侧区域，该区域可以作为STAT5响应增强子，包含多个共识的STAT5结合位点，并且是进化保守的。基于这些初步观察，我们假设这种远端增强子介导GH-STAT5激活肝脏中IGF-I基因转录。我们建议在小鼠系统中验证这一假设。在具体目标1中，我们将使用DNase I足迹、凝胶转移和共转染分析来确认远端IGFI增强子包含功能性STAT5结合位点。在特定目标2中，我们将确定GH是否导致STAT5与这种远端IGF-I增强子结合，以及STAT5的结合是否在GH诱导的IGF-I转录之前发生在GH缺乏/点燃小鼠的肝脏中。体内STAT5-DNA结合将通过染色质免疫沉淀法（CHIP）结合实时PCR来定量。在特定目标3中，我们确定了igf - 1增强子是否足以诱导GH诱导转基因小鼠的基因表达。本研究结果为进一步阐明生长激素调控igf - 1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.