Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription
Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription
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DOI：
10.1074/jbc.m510204200
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发表时间：
2006-02-10
影响因子：
4.8
通讯作者：
Rotwein, P
中科院分区：
文献类型：
--
作者：
Chia, DJ;Ono, M;Rotwein, P
生长激素（GH）的合成代谢作用中的关键剂是胰岛素样生长因子-I（IGF-I），其是一种70个氨基酸的分泌蛋白，对体细胞生长和组织维持和修复具有直接作用。GH通过独立于新蛋白质合成的机制快速有效地刺激IGF-I基因转录，最近的研究将转录因子Stat 5 b与连接细胞膜上活化的GH受体和细胞核中IGF-I基因的调控网络联系起来。在这里，我们分析了两个不同的保守的GH反应元件在大鼠IGF-I基因座，含有配对的Stat 5 b网站。每个反应元件结合Stat 5 b在体内生长激素依赖性的方式，通过染色质免疫沉淀试验进行评估，并由一个高亲和力和一个较低的亲和力Stat 5 b网站，定性和定量蛋白质-DNA结合研究确定。在生化重建实验中，当与含有主要IGF-I启动子或最小中性启动子的报告基因融合时，两种应答元件都能够介导GH刺激的和Stat 5 b依赖的转录，尽管位于第二IGF-I内含子中的成对Stat 5 b位点的有效性是与IGF-I外显子1的5'端73 kb相似的应答元件的两倍以上。两者合计，我们的研究结果定义了一个复杂的生长激素调控的转录途径的初始分子结构，并表明，显然是多余的激素反应元件提供了一种机制，放大生长激素作用在生理上重要的靶基因。
A key agent in the anabolic actions of growth hormone (GH) is insulin-like growth factor-I (IGF-I), a 70-amino acid secreted protein with direct effects on somatic growth and tissue maintenance and repair. GH rapidly and potently stimulates IGF-I gene transcription by mechanisms independent of new protein synthesis, and recent studies have linked the transcription factor Stat5b to a regulatory network connecting the activated GH receptor on the cell membrane to the IGF-I gene in the nucleus. Here we analyze two distinct conserved GH response elements in the rat IGF-I locus that contain paired Stat5b sites. Each response element binds Stat5b in vivo in a GH-dependent way, as assessed by chromatin immunoprecipitation assays, and consists of one high affinity and one lower affinity Stat5b site, as determined by both qualitative and quantitative protein-DNA binding studies. In biochemical reconstitution experiments, both response elements are able to mediate GH-stimulated and Stat5b-dependent transcription when fused to a reporter gene containing either the major IGF-I promoter or a minimal neutral promoter, although the paired Stat5b sites located in the second IGF-I intron were more than twice as effective as the response element that mapped similar to 73 kb 5' to the IGF-I exon 1. Taken together, our results define the initial molecular architecture of a complicated GH-regulated transcriptional pathway, and suggest that apparently redundant hormone response elements provide a mechanism for amplifying GH action at a physiologically important target gene.