MAPK-activated protein kinase 2 regulation of endothelial cell migration

MAPK 激活的蛋白激酶 2 对内皮细胞迁移的调节

• 批准号: 7858464
• 负责人: SHUANG HUANG
• 金额: $ 36.75万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858464
• 关键词: ATP phosphohydrolase; Adenoviruses; Affect; Angiogenic Factor; Arthritis; Binding; Blood Vessels; Cells; Dependence; Development; Diabetic Retinopathy; Disease; Dominant-Negative Mutation; Effectiveness; Endothelial Cells; Environment; Enzymes; Experimental Models; Family; Goals; Half-Life; Hypoxia; In Vitro; Indium; Intercept; Knowledge; Link; Mediating; Messenger RNA; Pathologic Neovascularization; Phosphorylation; Physiological; Protein Kinase; Proteins; Psoriasis; Publishing; RNA; RNA Helicase; RNA-Binding Proteins; Recruitment Activity; Regulation; Role; Screening procedure; Small Interfering RNA; System; Therapeutic; Untranslated Regions; Up-Regulation; Urokinase; Vascular Endothelial Growth Factors; Work; Wound Healing; activating transcription factor; angiogenesis; antiangiogenesis therapy; cell motility; experience; helicase; in vivo; inhibitor/antagonist; interest; knock-down; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; member; migration; mutant; novel; novel therapeutic intervention; overexpression; prevent; public health relevance; tumor growth; yeast two hybrid system

DESCRIPTION (provided by applicant): New blood vessel formation, angiogenesis, is required for normal development and wound healing. Aberrant angiogenesis contributes to many diseases including tumor growth, diabetic retinopathy, arthritis and psoriasis. Endothelial cell migration is one of the critical steps in angiogenesis and is promoted by angiogenic stimulating factors such as VEGF. Our early studies demonstrate that, under normaxic conditions, the activity of MAPK-activated protein kinase 2 (MK2) is required for VEGF- stimulated endothelial cell migration and that MK2 participates in endothelial cell migration by regulating urokinase plasminogen activator (uPA) expression. As angiogenesis occurs in the hypoxic environment, we investigated the involvement of MK2 and uPA in endothelial cell migration under the hypoxia. We show that, similar to what we have observed in normaxia, inhibiting MK2 activity also abrogates uPA expression and VEGF-stimulated endothelial cell migration while restoring uPA expression prevents MK2 inhibitor-caused inhibition in endothelial cell migration under hypoxia. These findings demonstrate a general role of the MK2-uPA axis in endothelial cell migration under both normaxia and hypoxia. In an effort to define the mechanism by which MK2 regulates uPA expression, we found that the activity of MK2 is important for relatively stable uPA mRNA in endothelial cells. Through a two-hybrid screening, we identified an RNA binding protein DDX5 that not only specifically interacts with MK2 but also serves as a direct substrate of MK2. Overexpression of DDX5 destabilizes uPA mRNA and silencing DDX5 expression prolongs the half-life of uPA mRNA in MK2-inhibited cells. DDX5 directly interacts with uPA mRNA and the degree of DDX5-uPA mRNA interaction is negatively regulated by MK2 activity. These results suggest that the MK2 may stabilize uPA mRNA by preventing DDX5 to interact with uPA mRNA and thus impeding DDX5's ability to mediate uPA mRNA decay. In our latest studies, we further investigated the potential role of the exosome in DDX5-mdiated uPA mRNA degradation. DDX5 interacts with the exosome in MK2-inhibited cells and knocking down the expression of the exosome subunits prolongs uPA mRNA stability in MK2-inhibited or DDX5- overexpressed cells. These results firmly link the exosome to MK2-DDX5 regulation of uPA mRNA stability. This proposal is to capitalize on our previous work and contains three aims: 1) determine how MK2 prevents DDX5 from facilitating uPA mRNA turnover; 2) determine the mechanism associated with DDX5-exosome interaction and its role of the exosome in uPA mRNA degradation; and 3) determine the effectiveness of intercepting the MK2-DDX5-uPA axis for suppressing angiogenesis. The proposed studies should increase our understanding of how endothelial cell migration is regulated, and may also help to develop a novel therapeutic approach to suppress pathological angiogenesis. PUBLIC HEALTH RELEVANCE New blood vessel formation, or called angiogenesis, is required for normal development and wound healing. Aberrant angiogenesis contributes to many diseases including tumor growth, diabetic retinopathy, arthritis and psoriasis. This application focuses on one of the critical steps of angiogenesis, directional endothelial cell migration. In our published studies and studies presented in this application, we found that a protein called MAPK-activated protein kinase 2 (MK2) is required for directional migration of endothelial cells in patho/physiological condition (hypoxia), and that MK2 participates in endothelial cell migration by regulating urokinase plasminogen activator (uPA) expression. To understand MK2 regulation of uPA expression, our preliminary studies revealed that MK2 promotes the levels of uPA by preventing DDX5 to interact with uPA mRNA and the exosome (consisting of RNA enzymes) and thus prolonging uPA mRNA stability. These findings demonstrate a novel mechanism involving MK2-DDX5 axis to regulate uPA level and endothelial cell migration. In this application, we wish to further investigate the functional link among MK2, DDX5 and uPA mRNA turnover. We also wish to employ the knowledge obtained from these studies to develop a therapeutic approach for inhibiting pathological angiogenesis.

描述（由申请人提供）：正常发育和伤口愈合需要新血管形成（血管生成）。异常的血管生成导致许多疾病，包括肿瘤生长、糖尿病视网膜病变、关节炎和牛皮癣。内皮细胞迁移是血管生成的关键步骤之一，并由血管生成刺激因子如VEGF促进。我们的早期研究表明，在正常条件下，MAPK激活的蛋白激酶2（MK 2）的活性是VEGF刺激的内皮细胞迁移所必需的，并且MK 2通过调节尿激酶纤溶酶原激活物（uPA）表达参与内皮细胞迁移。由于缺氧环境中发生血管生成，我们研究了MK 2和uPA在缺氧条件下内皮细胞迁移中的参与情况。我们发现，类似于我们在常氧下观察到的，抑制MK2活性也废除了uPA表达和VEGF刺激的内皮细胞迁移，而恢复uPA表达防止了缺氧下MK2激酶引起的内皮细胞迁移抑制。这些发现证明了在常氧和缺氧下MK2-uPA轴在内皮细胞迁移中的一般作用。为了明确MK2调节uPA表达的机制，我们发现MK2的活性对于内皮细胞中相对稳定的uPA mRNA是重要的。通过双杂交筛选，我们鉴定了一种RNA结合蛋白DDX 5，它不仅与MK2特异性相互作用，而且还作为MK2的直接底物。DDX 5的过表达使uPA mRNA不稳定，并且沉默DDX 5表达延长了MK 2抑制细胞中uPA mRNA的半衰期。DDX 5直接与uPA mRNA相互作用，并且DDX 5-uPA mRNA相互作用的程度受MK2活性负调控。这些结果表明，MK 2可能通过阻止DDX 5与uPA mRNA相互作用来稳定uPA mRNA，从而阻碍DDX 5介导uPA mRNA衰变的能力。在我们最新的研究中，我们进一步研究了外泌体在DDX 5介导的uPA mRNA降解中的潜在作用。DDX 5与MK2抑制细胞中的外泌体相互作用，并敲低MK2抑制或DDX 5过表达细胞中外泌体亚基UPA mRNA稳定性的表达。这些结果将外泌体与UPA mRNA稳定性的MK2-DDX 5调节牢固地联系起来。本研究的目的是利用我们以前的工作，包括三个目标：1）确定MK2如何阻止DDX 5促进uPA mRNA周转; 2）确定DDX 5-外泌体相互作用的机制及其在uPA mRNA降解中的作用; 3）确定阻断MK2-DDX 5-uPA轴抑制血管生成的有效性。拟议的研究将增加我们对内皮细胞迁移是如何调节的理解，也可能有助于开发一种新的治疗方法来抑制病理性血管生成。 新血管形成，或称为血管生成，是正常发育和伤口愈合所必需的。异常的血管生成导致许多疾病，包括肿瘤生长、糖尿病视网膜病变、关节炎和牛皮癣。本申请集中于血管生成的关键步骤之一，定向内皮细胞迁移。在我们已发表的研究和本申请中提出的研究中，我们发现一种称为MAPK活化蛋白激酶2（MK2）的蛋白质是病理/生理条件（缺氧）下内皮细胞定向迁移所必需的，并且MK2通过调节尿激酶纤溶酶原激活物（uPA）表达参与内皮细胞迁移。为了了解MK2对uPA表达的调节，我们的初步研究表明，MK2通过阻止DDX 5与uPA mRNA和外泌体（由RNA酶组成）相互作用，从而延长uPA mRNA的稳定性，从而促进uPA的水平。这些结果表明，一种新的机制，涉及MK 2-DDX 5轴调节uPA水平和内皮细胞迁移。在本申请中，我们希望进一步研究MK 2，DDX 5和uPA mRNA周转之间的功能联系。我们还希望利用从这些研究中获得的知识来开发用于抑制病理性血管生成的治疗方法。