GENE REGULATION OF THROMBOPOIETIN EXPRESSION

血小板生成素表达的基因调控

• 批准号: 6523733
• 负责人: JOHN M MC CARTY
• 金额: $ 13.01万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6523733
• 关键词: DNA footprinting; RNase protection assay; actinomycin; cell growth regulation; clinical research; disease /disorder model; enzyme linked immunosorbent assay; gene expression; genetic promoter element; genetic regulation; genetic transcription; hematopoiesis; human tissue; in situ hybridization; laboratory mouse; megakaryocytes; messenger RNA; nuclear runoff assay; nucleic acid quantitation /detection; platelets; polymerase chain reaction; posttranscriptional RNA processing; site directed mutagenesis; thrombocytopenia; thrombocytosis; thrombopoietic factor; tissue /cell culture

One of the least understood aspects of hematopoiesis is the process of megakaryocyte development. The recent cloning of thrombopoietin (TPO), an essential cytokine regulator of this process, allows investigation of the molecular basis for maintenance of physiologically appropriate platelet levels to proceed. Northern blots have revealed several disparate tissues to express TPO, but the cells which are responsible for TPO expression in vivo have not been identified. Two main models of TPO serum level regulation have been proposed. One asserts that TPO expression is constitutive in liver and kidney, and that serum levels are mediated via protein metabolism by an expanding or contracting platelet mass. A second suggests that in states of significant platelet variability, TPO mRNA levels may vary inversely to platelet mass. We provide experimental evidence which supports constitutive TPO expression in the liver and kidney, and mRNA-based regulation in the marrow and spleen. We plan to study the molecular basis of TPO gene regulation with an eye to understanding how the basal and inducible tissue-specific expression of the TPO gene translates into physiologically appropriate serum protein levels. To achieve these ends, we propose a research plan of three specific aims: 1. To identify the cellular and histologic sites of basal and inducible TPO production in mouse models of thrombocytopenia by in situ hybridization, RNA analysis of primary cell fractions and lines and RT-PCR; 2. To refine in vitro models of constitutive and inducible tissue-specific TPO gene expression and identify the relative contribution of transcriptional enhancement and mRNA accumulation in cells which increase TPO mRNA levels in response to thrombocytopenic sera; 3. To compare the functional organization of the TPO gene by DNAseI hypersensitive site mapping, by RNAse protection assays and 5' RACE analysis to characterize hTPO 5' mRNA isoforms during perturbations in platelet and megakarcyocyte mass, by identification of functionally relevant cis-acting elements of the TPO promoter by reporter gene analysis, refining these sequences by DNAse I footprint and mobility shift assays, and confirming the functional contribution of these sequences to constitutive and inducible tissue-specific TPO expression by site directed mutagenesis and determining their functional role by gain of function/loss of function analysis in reporter gene assays. We provide data to suggest these aims are feasible and will result in useful data as a basis for future studies. Understanding the mechanisms by which this regulator of megakaryocyte maturation is controlled will provide insight into normal and dysregulated megakaryocytopoiesis.

造血的一个最鲜为人知的方面是 巨核细胞发育。血小板生成素(TPO)的最新克隆， 这一过程的一个重要的细胞因子调节因子，允许研究 维持生理上适宜的分子基础 血小板水平才能继续。北方斑点揭示了几个 不同的组织表达TPO，但负责的细胞 对于TPO在体内的表达还没有确定。两种主要模式 对TPO血清水平的调节作用已被提出。有人断言TPO 肝脏和肾脏的表达是结构性的，血清水平 是通过蛋白质新陈代谢调节的，由扩张或收缩 血小板块。第二种情况表明，在显著的血小板状态下 变异性，TPO mRNA水平可能与血小板质量成反比。我们 提供支持结构性TPO表达的实验证据 在肝和肾中，以及在骨髓和 脾。我们计划研究TPO基因调控的分子基础。 着眼于了解基础和可诱导组织特异性 TPO基因的表达转化为生理上合适的 血清蛋白质水平。为了实现这些目标，我们提出了一项研究计划 三个具体目标：1.确定细胞和组织学部位 基础和可诱导的TPO在小鼠模型中的产生 原位杂交检测血小板减少症原代细胞RNA分析 片段和线系及RT-PCR；2.体外模型的提纯 结构性和可诱导性组织特异性TPO基因表达和 确定转录增强的相对贡献和 细胞中的mRNA积累增加了TPO的mRNA水平作为响应 对血小板减少性血清的影响；3.比较其功能组织 TPO基因DNAseI超敏位点定位，核糖核酸酶保护 测定和5‘RACE分析hTPO 5’m RNA亚型 血小板和巨核细胞群的扰动，通过鉴定 TPO启动子的功能相关顺式作用元件 报告基因分析，通过DNase I足迹提炼这些序列 和迁移率变化分析，并确认功能贡献 这些序列中的每一个都是构成和可诱导的组织特异性TPO 定点突变表达及其功能鉴定 报告基因中功能获得/功能丧失的作用分析 化验。我们提供的数据表明这些目标是可行的，并将 产生了有用的数据，作为未来研究的基础。了解 这种巨核细胞成熟调节因子的机制是 受控将提供对正常和不受监管的洞察 巨核细胞生成。

项目成果

A preliminary investigation into the action of anagrelide: thrombopoietin-c-Mpl receptor interactions.

阿那格雷作用的初步研究：血小板生成素-c-Mpl 受体相互作用。

• DOI: 10.1016/j.exphem.2005.09.009
• 发表时间: 2006
• 期刊: Experimental hematology
• 影响因子: 2.6
• 作者: McCarty,JohnM;Melone,PamelaD;Simanis,JurisP;Kanamori,David;Dessypris,EmmanuelN;Warshamana-Greene,GSakuntala
• 通讯作者: Warshamana-Greene,GSakuntala