MOLECULAR REGULATION OF IGFBP-3

IGFBP-3 的分子调控

• 批准号: 3081122
• 负责人: BETTY C VILLAFUERTE
• 金额: $ 8.96万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3081122
• 关键词: DNA footprinting; antireceptor antibody; binding proteins; dexamethasone; gel mobility shift assay; gene mutation; genetic regulation; hormone regulation /control mechanism; insulin; insulinlike growth factor; laboratory rabbit; laboratory rat; molecular cloning; nucleic acid sequence; receptor coupling; tissue /cell culture

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.

糖尿病的生长受损涉及代谢率减少和 胰岛素样生长因子(IGFS)的作用。IGF是有丝分裂原 与胰岛素样生长因子结合循环的多肽 结合蛋白，IGFBP-3。IGFBP-3延长循环半衰期 IGFS有助于将IGFS靶向于特定组织和细胞类型， 并可增强和抑制IGF的作用。IGFBP-3的丰度 MRNA支持肝脏作为循环合成的主要部位 IGFBP-3，但调节的特定代谢和激素因素 肝脏中的IGFBP-3尚不清楚。IGFBP-3未在中发布 传统的肝细胞系统，将目前的研究主要局限于 活体模型，在那里很难分离监管事件。 P.I.的初步研究表明，大鼠的共培养 具有完整或热灭活的非实质细胞的肝细胞显示 激素调节IGFBP-3的表达。IGFBP之间的强相关性- 3条件培养液中的mRNA量和蛋白质水平表明 翻译前监管。因此，这项建议强调机制。 分子调控，有以下具体目的：1. 评估IGFBP-3细胞调控研究的模型，我们将 不同浓度胰岛素样生长因子结合蛋白-3在肝细胞共培养中的表达 非实质细胞亚群，并解剖允许的 非实质细胞产生的可溶性和不溶性因子的贡献 细胞。2.明确与受体相关的调节机制 胰岛素或IGF-1刺激IGFBP-3，我们将评估IGFBP-3 在添加多克隆抗体的共培养体系中的表达 胰岛素样生长因子-1和/或胰岛素受体，以及添加胰岛素样生长因子-1突变体 与IGFBPs或I型IGF受体亲和力改变。3.至 勾勒出转录的关键作用，我们将量化 正常和糖尿病动物的转录速率和mRNA稳定性，以及 在添加或不添加胰岛素和/或 地塞米松--导致IGFBP-3重复性变化的条件 表达，代表在活体中看到的主要异常。4.至 鉴定介导IGFBP-3调控的DNA区域 转录，DNase I超敏分析将用于鉴定 可能涉及转录调控、瞬时转染区 将用于定义激素敏感启动子区域，以及 将使用足迹和凝胶迁移率移动分析来表征 可能受激素调节的转录因子所接触的区域。 如果时间允许，5.尝试分离代谢调节的 转录因子，我们将通过以下途径筛选lambdaZap表达文库 连锁式探针反映关键DNA--“西南印迹” 结合位点，并克隆和测序已鉴定的cDNA。这些 研究应该提供对分子调控的理解 关键的IGF结合蛋白，并提供对 糖尿病生长发育受损的病理生理学研究。