MOLECULAR REGULATION OF IGFBP-3

糖尿病的生长受损涉及生成减少， 胰岛素样生长因子（IGFs）。 胰岛素样生长因子是促有丝分裂的 多肽，其主要与IGF结合循环 结合蛋白IGFBP-3。 IGFBP-3延长了循环中的半衰期， IGFs，促进IGFs靶向特定的组织和细胞类型， 并且可以增强和抑制IGF作用。 IGFBP-3的缺失 mRNA支持肝脏作为合成循环免疫球蛋白的主要场所。 IGFBP-3，但特定的代谢和激素因素，调节 肝脏中的IGFBP-3未知。 IGFBP-3未在 传统的肝细胞系统，限制了目前的研究，主要是在 体内模型，其中难以分离调节事件。 私家侦探的初步研究。已经证实， 具有完整或热灭活非实质细胞的肝细胞表现出 胰岛素样生长因子结合蛋白-3的表达受胰岛素样生长因子结合蛋白-3调节。 IGFBP- 3条件培养基中的mRNA丰度和蛋白质水平表明 翻译前调控 因此，这项建议强调机制 分子调控，具有以下具体目标：1。到 评估IGFBP-3细胞调节研究模型，我们将 定量与不同浓度胰岛素共培养的肝细胞中IGFBP-3 mRNA的丰度 非实质细胞的亚群，并解剖允许的 非实质细胞产生的可溶性和不溶性因子 细胞 2.明确受体相关的介导机制 通过胰岛素或IGF-1刺激IGFBP-3，我们将评估IGFBP-3 在与添加的多克隆抗体孵育的共培养物中表达， IGF-1和/或胰岛素受体，以及添加IGF-1突变体 对IGFBPs或1型IGF受体的亲和力改变。 3.到 描述转录的重要作用，我们将量化 正常和糖尿病动物中的转录速率和mRNA稳定性，以及 在添加或不添加胰岛素的共培养肝细胞中，和/或 地塞米松-导致IGFBP-3发生可重现变化的条件 表达，并代表体内观察到的主要异常。 4.到 表征介导IGFBP-3调节的DNA区域 转录，DNA酶I超敏反应分析将用于鉴定 可能涉及转录控制、瞬时转染 将被用于定义启动子敏感区，和 足迹法和凝胶迁移率变化分析将用于表征 区域接触假定的转录调控因子。 如果时间允许，5。试图分离出代谢调节的 转录因子，我们将通过筛选EscheridaZap表达文库， "西南印迹法"用连锁探针反映关键DNA- 结合位点，并克隆和测序鉴定的cDNA。 这些 研究应该提供对一个细胞的分子调控的理解， 关键的IGF结合蛋白，并提供深入了解 糖尿病中生长受损的病理生理学。

Impaired growth in diabetes mellitus involves reduced generation and action of insulin-like growth factors (IGFs). The IGFs are mitogenic polypeptides, which circulate in predominant association with an IGF binding protein, IGFBP-3. IGFBP-3 prolongs the halflife of circulating IGFs, facilitates targeting of IGFs to specific tissue and cell types, and can both potentiate and inhibit IGF actions. Abundance of IGFBP-3 mRNA supports the liver as a major site of synthesis of circulating IGFBP-3, but the specific metabolic and hormonal factors that regulate IGFBP-3 in the liver are not known. IGFBP-3 is not released in conventional liver cell systems, limiting present studies largely to in vivo models, where separation of regulatory events is difficult. Preliminary studies by the P.I. have established that co-cultures of rat hepatocytes with intact or heat-killed nonparenchymal cells exhibit hormone-regulated IGFBP-3 expression. Strong correlations between IGFBP- 3 mRNA abundance and levels of protein in conditioned medium indicate pre-translational regulation. Thus, this proposal emphasizes mechanisms of molecular regulation, with the following specific aims: 1. To evaluate models for study of cellular regulation of IGFBP-3, we will quantify IGFBP-3 mRNA abundance in hepatocytes co-cultured with different subpopulations of nonparenchymal cells, and dissect the permissive contributions of soluble and insoluble factors produced by nonparenchymal cells. 2. To define the receptor-related mechanisms mediating stimulation of IGFBP-3 by insulin or IGF-1, we will assess IGFBP-3 expression in co-cultures incubated with added polyclonal antibodies to IGF-1 and/or to insulin receptors, and with addition of IGF-1 mutants with altered affinity for IGFBPs or the Type 1 IGF receptor. 3. To delineate the critical role of transcription, we will quantify transcription rate and mRNA stability in normal and diabetic animals, and in co-cultured hepatocytes with or without added insulin and/or dexamethasone -- conditions which lead to reproducible changes in IGFBP-3 expression, and represent major abnormalities seen in vivo. 4. to characterize the DNA regions which mediate regulation of IGFBP-3 transcription, DNase I hypersensitivity analysis will be used to identify areas likely involved in transcriptional control, transient transfection will be used to define hormone-sensitive promoter regions, and footprinting and gel mobility shift analysis will be used to characterize regions contacted by putative hormone-regulated transcription factors. If time permits, 5. to attempt isolation of metabolically regulated transcription factors, we will screen lambdaZap expression libraries via "Southwestern blotting" with catenated probes reflecting critical DNA- binding sites, and clone and sequence the identified cDNAs. These studies should provide understanding of the molecular regulation of a critical IGF binding protein, and provide insight into the pathophysiology of impaired growth in diabetes mellitus.