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: 7620355
• 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/7620355
• 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）：新血管形成、血管生成是正常发育和伤口愈合所必需的。异常的血管生成会导致许多疾病，包括肿瘤生长、糖尿病性视网膜病变、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，并受到血管生成刺激因子（例如 VEGF）的促进。我们的早期研究表明，在正常条件下，MAPK 激活蛋白激酶 2 (MK2) 的活性是 VEGF 刺激的内皮细胞迁移所必需的，并且 MK2 通过调节尿激酶纤溶酶原激活剂 (uPA) 表达参与内皮细胞迁移。由于血管生成发生在缺氧环境中，我们研究了MK2和uPA在缺氧条件下内皮细胞迁移的参与。我们发现，与我们在常氧条件下观察到的情况类似，抑制 MK2 活性也会消除 uPA 表达和 VEGF 刺激的内皮细胞迁移，同时恢复 uPA 表达可防止缺氧条件下 MK2 抑制剂引起的内皮细胞迁移抑制。这些发现证明了 MK2-uPA 轴在常氧和缺氧条件下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 很重要。通过双杂交筛选，我们鉴定了一种RNA结合蛋白DDX5，它不仅与MK2特异性相互作用，而且还可以作为MK2的直接底物。 DDX5 的过度表达会破坏 uPA mRNA 的稳定性，而沉默 DDX5 表达会延长 MK2 抑制细胞中 uPA mRNA 的半衰期。 DDX5 直接与 uPA mRNA 相互作用，并且 DDX5-uPA mRNA 相互作用的程度受到 MK2 活性的负调节。这些结果表明，MK2 可能通过阻止 DDX5 与 uPA mRNA 相互作用来稳定 uPA mRNA，从而阻止 DDX5 介导 uPA mRNA 衰减的能力。在我们最新的研究中，我们进一步研究了外泌体在 DDX5 介导的 uPA mRNA 降解中的潜在作用。 DDX5 与 MK2 抑制细胞中的外泌体相互作用，敲低外泌体亚基的表达可延长 MK2 抑制或 DDX5 过表达细胞中 uPA mRNA 的稳定性。这些结果将外泌体与 MK2-DDX5 对 uPA mRNA 稳定性的调节紧密联系起来。该提案是为了利用我们之前的工作，包含三个目标：1）确定 MK2 如何阻止 DDX5 促进 uPA mRNA 周转； 2)确定DDX5-外泌体相互作用的相关机制以及外泌体在uPA mRNA降解中的作用； 3)确定拦截MK2-DDX5-uPA轴以抑制血管生成的有效性。拟议的研究应该增加我们对内皮细胞迁移如何调节的理解，并且还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。 公共卫生相关性 新血管的形成，或称为血管生成，是正常发育和伤口愈合所必需的。异常的血管生成会导致许多疾病，包括肿瘤生长、糖尿病性视网膜病变、关节炎和牛皮癣。该应用重点关注血管生成的关键步骤之一，即定向内皮细胞迁移。在我们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 激活蛋白激酶 2 (MK2) 的蛋白质是内皮细胞在病理/生理条件（缺氧）下定向迁移所必需的，并且 MK2 通过调节尿激酶纤溶酶原激活剂 (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 DDX5 与 uPA mRNA 和外泌体（由 RNA 酶组成）相互作用来促进 uPA 水平，从而延长 uPA mRNA 的稳定性。这些发现证明了涉及 MK2-DDX5 轴调节 uPA 水平和内皮细胞迁移的新机制。在此应用中，我们希望进一步研究 MK2、DDX5 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抑制病理性血管生成的治疗方法。