MOLECULAR REGULATION OF IGF BINDING PROTEIN 1

胰岛素样生长因子（IGFs）是细胞生长的重要调节因子， 在许多组织中生长和分化。 IGFs循环于 与特定 结合蛋白（IGFBPs）。 研究的 和其他实验室表明，IGFBP-1是一个主要的短期 IGF生物利用度调节剂和IGFBP-1的肝脏产生 在基因水平上受糖皮质激素和胰岛素的快速调节 转录。 IGFBP-1表达和IGF生物利用度的变化可能 在低血糖和高血压并发症的发病机制中起重要作用， 和高胰岛素血症 此外，迄今为止的研究表明， IGFBP-1提供了一个重要的模型系统， 胰岛素和糖皮质激素相互作用调节 肝基因转录 在NIH FIRST奖期间，我们鉴定并纯化了大鼠IGFBP-1， 鉴定并利用体内和细胞培养模型系统， IGFBP-1的调控，克隆了IGFBP-1基因及其5'启动子 区域和确定的连续糖皮质激素（GRE）和胰岛素（IRS） IGFBP-1启动子近端的应答序列。 竞争性结合 超位移研究表明， 序列为肝细胞核因子-3（HNF-3），C/EBP蛋白 也与这个序列相互作用。 据我们所知，这代表了 首次证明HNF-3蛋白可能与IRS相互作用， 可能有助于胰岛素对基因表达的影响。 功能研究证实HNF-3蛋白增强IGFBP-1启动子 在该位点的活性和该HNF-3结合位点的突变也 破坏糖皮质激素的作用并降低胰岛素的作用， 糖皮质激素对启动子功能的影响。 凝胶位移研究表明， 核提取物中的C/EPB蛋白也与该位点相互作用， 胰岛素可能还需要其它因素来发挥其作用， 基础IGFBP-1启动子功能。 根据这些观察，我们现在 将利用精细定位、定点诱变和共转染 系统来检查HNF-3和/或C/EBP蛋白在介导 糖皮质激素与胰岛素在IGFBP-1启动子上的相互作用 活动，并检查之间的相互作用和/或 HNF-3、C/EBP和其他因子的修饰可能在介导 胰岛素对IGFBP-1表达的影响。 体内足迹也将 以确定胰岛素是否改变了特定的 影响该位点或IGFBP-1启动子的构象。 的 这些研究的结果应该提供深入了解具体机制 糖皮质激素和胰岛素通过相互作用调节肝脏基因 表达和调节IGFs的生物学效应， 新陈代谢.

Insulin-like growth factors (IGFs) are important regulators of cell growth and differentiation in many tissues. IGFs circulate in association with specific binding proteins (IGFBPs). Studies in this and other laboratories indicate that IGFBP-1 is a major short-term modulator of IGF bioavailability and that hepatic production of IGFBP-1 is rapidly regulated by glucocorticoids and insulin at the level of gene transcription. Changes in IGFBP-1 expression and IGF bioavailability may play an important role in the pathogenesis of complications of both hypo- and hyperinsulinemic states. Moreover, studies to date indicate that IGFBP-1 provides in important model system for understanding basic mechanisms by which insulin and glucocorticoids interact to regulate hepatic gene transcription. During an NIH FIRST Award, we identified and purified rat IGFBP-1, identified and utilized in vivo and cell culture model systems to examine the regulation of IGFBP-1, cloned the IGFBP-1 gene and its 5' promoter region and identified contiguous glucocorticoid (GRE) and insulin (IRS) response sequences in the proximal IGFBP-1 promoter. Competitive binding and supershift studies show that the major protein binding to this sequence is hepatocyte nuclear factor-3 (HNF-3) and that C/EBP proteins also interact with this sequence. To our knowledge, this represented the first demonstration that HNF-3 proteins may interact with an IRS and potentially contribute to effects of insulin on gene expression. Functional studies confirm that HNF-3 proteins enhance IGFBP-1 promoter activity at this site and mutation of this HNF-3 binding site also disrupts glucocorticoid effects and reduces the effect of insulin and glucocorticoids on promoter function. Gel shift studies indicate that C/EPB proteins in nuclear extracts also interact with this site and that still other factors may be required for insulin to exert its effects on basal IGFBP-1 promoter function. Based on these observations, we now will utilize fine mapping, site directed mutagenesis and co-transfection systems to examine the role of HNF-3 and/or C/EBP proteins in mediating interactions between glucocorticoids and insulin on IGFBP-1 promoter activity, and examine the role that interactions between and/or modifications on HNF-3, C/EBP and other factors may play in mediating effects of insulin on IGFBP-1 expression. In vivo footprinting will also be performed to determine whether insulin alters access of specific factors this site or the conformation of the IGFBP-1 promoter. The results of these studies should provide insight into specific mechanisms by which glucocorticoids and insulin interact to regulate hepatic gene expression and modulate biological effects of IGFs in disorders of metabolism.