TRANSCRIPTION FACTOR NF-E2 IN ALPHA IIB BETA 3 SIGNALING

ALPHA IIB BETA 3 信号转导中的转录因子 NF-E2

• 批准号: 6152975
• 负责人: SANFORD J SHATTIL
• 金额: $ 41.59万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6152975
• 关键词: biological signal transduction; fibrinogen; gene complementation; gene expression; genetic manipulation; human tissue; integrins; laboratory mouse; megakaryocytes; microarray technology; platelets; protein binding; regulatory gene; subtraction hybridization; transcription factor; transfection /expression vector

Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering inward signals that regulate the cytoskeleton. Since current understanding of alphaIIbbeta3 signaling is limited, we will study alphaIIbbeta3 signaling in primary murine megakaryocytes, cells that naturally express alphaIIbbeta3 and, unlike platelets, are amenable to genetic manipulation. We have discovered that agonist-induced fibrinogen binding to alphaIIbbeta3 occurs only in the largest, most mature megakaryocytes from normal mice. In contrast, large megakaryocytes from mice genetically deficient in the transcription factor NF-E2 p45 subunit (NF-E2-/-) fail to bind soluble fibrinogen and they adhere poorly to immobilized fibrinogen, despite normal expression of alphaIIbbeta3. This suggests that one or more NF-E2-regulated genes are required for inside-out alphaIIbbeta3 signaling. The goal of this project is to identify and characterize NF-E2-regulated genes that control alphaIIbbeta3 function. First, the functional defect in NF-E2-/- megakaryocytes will be characterized in detail by examining cytoskeletal reorganization in fibrinogen-adherent cells, by extending the analysis to other integrins, and by determining whether recombinant expression of p45 in NF-E2-/- megakaryocytes reconstitutes normal alphaIIbbeta3 signaling. Recombinant p45 will be expressed using retroviral and Sindbis virus systems. Second, NF-E2-regulated genes, one or more of which may re regulate alphaIIbbeta3 function, will be identified by examining differential gene expression in wild-type and NF-E2-/- megakaryocytes using a series of complementary approaches. Third, candidate genes identified by differential expression will be evaluated for their role(s) in alphaIIbbeta3 signaling by using viral vectors to express them, or relevant mutants, in megakaryocytes and determining their effects on alphaIIbbeta3 function. The unique window on alphaIIbbeta3 signaling provided by NF-E2-/- megakaryocytes promises to fill major gaps in our understanding of bidirectional integrin signaling. Taken together with the complementary work on thrombin and alphaIIbbeta3 signaling proposed by our collaborators, this mechanistic information may facilitate the development of a new class of antithrombotic drugs that inhibit alphaIIbbeta3 function from within the platelet.

血小板整联蛋白alphaiibbeta3通过结合纤维蛋白原并触发调节细胞骨架的内向信号来响应细胞内信号。 由于目前对Alphaiibbeta3信号传导的理解有限，因此我们将研究原代鼠巨核细胞中的alphaiibbeta3信号传导，自然表达alphaiibbeta3的细胞和与血小板不同的是基因操纵。 我们发现，激动剂诱导的纤维蛋白原与αibbeta3结合仅发生在正常小鼠的最大，最成熟的巨核细胞中。 相反，来自转录因子NF-E2 p45亚基（NF-E2 - / - ）在遗传上缺乏的大巨核细胞未能结合可溶性纤维蛋白原，并且尽管α33的正常表达，它们仍无法与固定化的纤维蛋白原相结合。这表明内而外的Alphaiibbeta3信号需要一个或多个NF-E2调节的基因。 该项目的目的是识别和表征控制alphaiibbeta3功能的NF-E2调节基因。 First, the functional defect in NF-E2-/- megakaryocytes will be characterized in detail by examining cytoskeletal reorganization in fibrinogen-adherent cells, by extending the analysis to other integrins, and by determining whether recombinant expression of p45 in NF-E2-/- megakaryocytes reconstitutes normal alphaIIbbeta3 signaling. 重组P45将使用逆转录病毒和信德氏病毒系统表达。其次，通过使用一系列互补方法检查野生型和NF-E2 - / - 巨核细胞中的差异基因表达，将通过检查野生型和NF-E2 - / - 巨核细胞中的差异基因表达来鉴定NF-E2调节的基因，其中一个或多个可以重新调节alphaiibbeta3功能。第三，通过使用病毒载体表达它们或相关突变体在巨核细胞中，通过差异表达鉴定的候选基因将在alphaiibbeta3信号中评估其作用，并确定其对alphaiibbeta3函数的影响。 NF-E2 - / - 巨核细胞提供的Alphaiibbeta3信号传导上的独特窗口有望填补我们对双向整合素信号传导的理解。 与我们的合作者提出的有关凝血酶和alphaiibbeta3信号的互补工作一起，这些机械信息可能有助于开发一种新的抗血栓形成药物，从而抑制血小板内抑制alphaiibbeta3的功能。