Molecular Mechanisms of MEKK3-Signaling in Angiogenesis

MEKK3 信号在血管生成中的分子机制

• 批准号: 6875604
• 负责人: BING SU
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6875604
• 关键词: JUN kinase; angiogenesis; angiogenesis factor; apoptosis; cell growth regulation; cell line; chromatography; embryo /fetus tissue /cell culture; enzyme activity; enzyme induction /repression; gene targeting; genetically modified animals; laboratory mouse; mitogen activated protein kinase; molecular cloning; tissue /cell culture; tumor antigens; vascular endothelium; yeast two hybrid system

DESCRIPTION (provided by applicant): Angiogenesis is essential for normal physiological processes such as organ development and wound healing. It is also critical to pathological processes such as tumor growth, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. Therefore, blocking angiogenesis could be a powerful therapeutic intervention for treating diseases requiring formation of new blood vessels. However, the underlying molecular mechanism of intracellular signal transduction in this process remains largely unexplored. We recently generated Mekk3-knockout mice through homologous recombination and demonstrated that MEKK3, a Ser/Thr protein kinase belonging to the mitogen-activated protein kinase (MAPK) kinase gene family, is essential for angiogenesis. The long-term goals of this study are to elucidate the molecular mechanisms of angiogenesis regulated by MEKK3 signaling. Our SPECIFIC AIM 1 is to determine the function of MEKK3 in endothelial cells (ECs) during embryonic development by examining the morphology, proliferation and apoptosis of ECs in the E8.5 to E10 wild-type, Mekk3+/- and Mekk3-/- fetuses. We will also isolate embryonic ECs from the E9-E9.5 fetuses and establish EC lines by using polyoma middle T antigen to transform the primary cultures. In SPECIFIC AIM 2, we will determine whether MEKK3 is a specific upstream activator of JNK1/2, ERK1/2, p38 and ERK5 MAPKs in ECs by using in vitro kinase assays. We will determine whether the induction of these MAPKs is defective in Mekk3-/- embryos, purified ECs and EC lines. In addition, we will determine whether MEKK3 is specifically activated by angiogenic stimulation by using in vitro kinase, in-gel kinase and MEKK3 phosphorylation assays. Furthermore, we will isolate and clone MEKK3-associated proteins by coprecipitation, chromatography, and yeast two-hybrid screening. In Specific Aim 4, we plan to generate Mekk3 floxed ES cells and mice, and use these mice to create EC-specific Mekk3 knock out (Mekk3 (EC-null)) mice and Mekk3-deficient ECs with Cre recombinase. Function of MEKK3-signaling will be studied specifically in ECs using these mice. In addition to revealing the MEKK3-signaling in angiogenesis, the outcome from this study will provide conceptual and material resources for studying MEKK3 and its homologues in many other physiological and pathological processes. Most importantly, this study may discover novel molecular targets for therapeutic intervention for treating diseases requiring formation of new blood vessels like cancer.

描述(由申请人提供)：血管生成对于器官发育和伤口愈合等正常生理过程是必不可少的。它也是肿瘤生长、动脉粥样硬化、类风湿性关节炎和糖尿病视网膜病变等病理过程的关键。因此，阻断血管生成可能是治疗需要新血管形成的疾病的强有力的治疗干预措施。然而，在这一过程中细胞内信号转导的潜在分子机制仍然很大程度上尚不清楚。我们最近通过同源重组获得了MEKK3基因敲除小鼠，并证明了MEKK3是丝裂原活化蛋白激酶(MAPK)基因家族中的一种丝氨酸/苏氨酸蛋白激酶，对血管生成是必不可少的。本研究的长期目标是阐明MEKK3信号调控血管生成的分子机制。我们的特异目的1是通过检测E8.5到E10野生型、MEKK3+/-和MEKK3-/-胚胎内皮细胞的形态、增殖和凋亡来确定MEKK3在胚胎发育过程中内皮细胞的功能。我们还将从E9-E9.5胎儿中分离胚胎内皮细胞，并用多瘤中T抗原转化原代培养的方法建立EC系。在特定的AIM 2中，我们将通过体外激酶试验来确定MEKK3是否是ECs中JNK1/2、ERK1/2、p38和ERK5 MAPKs的特异性上游激活剂。我们将确定这些MAPK在MEKK3-/-胚胎、纯化的ECs和EC系中的诱导是否存在缺陷。此外，我们还将通过体外激活法、凝胶内激活法和MEKK3磷酸化试验来确定MEKK3是否被血管生成刺激特异性激活。此外，我们将通过共沉淀、层析和酵母双杂交筛选来分离和克隆MEKK3相关蛋白。在特定的目标4中，我们计划产生MEKK3的ES细胞和小鼠，并利用这些小鼠来创造EC特异性的MEKK3基因敲除(MEKK3(EC-空))小鼠和带有Cre重组酶的MEKK3缺陷的ECs。MEKK3信号的功能将利用这些小鼠在内皮细胞中进行专门的研究。除了揭示MEKK3信号在血管生成中的作用外，本研究的结果还将为研究MEKK3及其同系物在许多其他生理和病理过程中的作用提供概念和物质资源。最重要的是，这项研究可能会发现新的分子靶点，用于治疗癌症等需要形成新血管的疾病的治疗干预。