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.

血小板整合素α IIb β 3通过结合纤维蛋白原并触发调节细胞骨架的向内信号来响应细胞内信号。 由于目前对α IIb β 3信号的理解是有限的，我们将研究α IIb β 3信号在原代小鼠巨核细胞，细胞自然表达α IIb β 3，不像血小板，是服从遗传操作。 我们发现激动剂诱导的纤维蛋白原与α IIb β 3的结合仅发生在正常小鼠最大、最成熟的巨核细胞中。 相反，来自转录因子NF-E2 p45亚基（NF-E2-/-）遗传缺陷小鼠的大巨核细胞不能结合可溶性纤维蛋白原，尽管α IIb β 3表达正常，但它们与固定化纤维蛋白原的粘附性很差。这表明一个或多个NF-E2调节基因是由内而外的α IIb β 3信号传导所必需的。 该项目的目标是识别和表征控制alphaIIbbeta 3功能的NF-E2调节基因。 首先，通过检查纤维蛋白原粘附细胞中的细胞骨架重组，通过将分析扩展到其他整合素，以及通过确定NF-E2-/-巨核细胞中p45的重组表达是否重建正常的α IIb β 3信号传导，来详细表征NF-E2-/-巨核细胞中的功能缺陷。 重组p45将使用逆转录病毒和辛德毕斯病毒系统表达。第二，NF-E2调节基因，其中一个或多个可以重新调节α IIb β 3功能，将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来鉴定。第三，通过使用病毒载体在巨核细胞中表达通过差异表达鉴定的候选基因或相关突变体，并确定其对α IIb β 3功能的影响，评价其在α IIb β 3信号传导中的作用。 NF-E2-/-巨核细胞提供的alphaIIbbeta 3信号传导的独特窗口有望填补我们对双向整合素信号传导理解的主要空白。 与我们的合作者提出的凝血酶和alphaIIbbeta 3信号传导的互补工作一起，这种机制信息可能有助于开发一类新的抗血栓药物，抑制血小板内的alphaIIbbeta 3功能。