MAPK-activated protein kinase 2 regulation of endothelial cell migration

描述(申请人提供)：新的血管形成，血管生成，是正常发育和伤口愈合所必需的。血管生成异常可导致多种疾病，包括肿瘤生长、糖尿病视网膜病变、关节炎和牛皮癣。血管内皮细胞迁移是血管生成过程中的关键步骤之一，血管生成刺激因子如血管内皮生长因子可促进内皮细胞迁移。我们早期的研究表明，在正常条件下，MAPK激活的蛋白激酶2(MK2)的活性是血管内皮生长因子(VEGF)刺激的内皮细胞迁移所必需的，MK2通过调节尿激酶型纤溶酶原激活物(UPA)的表达参与内皮细胞迁移。由于血管生成发生在低氧环境中，我们研究了MK2和uPA在低氧下内皮细胞迁移中的作用。我们发现，与我们在常氧条件下观察到的情况类似，抑制MK2活性也会抑制uPA的表达和血管内皮生长因子刺激的内皮细胞迁移，而恢复uPA的表达则可以阻止MK2抑制剂抑制低氧下内皮细胞的迁移。这些发现证明了MK2-uPA轴在常氧和低氧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，我们发现了一个RNA结合蛋白DDX5，它不仅能与MK2特异地相互作用，还能作为MK2的直接底物。在MK2抑制的细胞中，DDX5的过度表达破坏了uPA mRNA的稳定，而沉默DDX5的表达延长了uPA mRNA的半衰期。DDX5与uPA mRNA直接相互作用，其相互作用程度受MK2活性的负调节。这些结果表明，MK2可能通过阻止DDX5与uPA mRNA的相互作用，从而阻止DDX5‘S介导uPA mRNA的衰变，从而稳定uPA mRNA。在我们最新的研究中，我们进一步研究了外切体在DDX5修饰的uPA mRNA降解中的潜在作用。在MK2抑制的细胞中，DDX5与外切体相互作用，在MK2抑制或DDX5过表达的细胞中，下调外切体亚单位的表达可延长uPA mRNA的稳定性。这些结果将外切体与MK2-DDX5对uPA mRNA稳定性的调节紧密联系在一起。这项建议是利用我们以前的工作，包含三个目标：1)确定MK2如何阻止DDX5促进uPA mRNA的周转；2)确定DDX5-外切体相互作用的机制及其在uPA mRNA降解中的作用；以及3)确定拦截MK2-DDX5-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如何调控的理解，并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。血管生成异常可导致多种疾病，包括肿瘤生长、糖尿病视网膜病变、关节炎和牛皮癣。这一应用主要集中在血管生成的关键步骤之一，即内皮细胞的定向迁移。在我们已发表的研究和在本应用中介绍的研究中，我们发现在病理/生理条件下(缺氧)内皮细胞的定向迁移需要一种名为MAPK激活的蛋白激酶2(MK2)，并且MK2通过调节尿激酶型纤溶酶原激活物(UPA)的表达来参与内皮细胞的迁移。为了了解MK2对uPA表达的调控，我们的初步研究表明，MK2通过阻止DDX5与uPA mRNA和外体(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。这些发现表明了一种涉及MK2-DDX5轴调节uPA水平和内皮细胞迁移的新机制。在这一应用中，我们希望进一步研究MK2、DDX5和uPA mRNA转换之间的功能联系。我们还希望利用从这些研究中获得的知识来开发一种抑制病理性血管生成的治疗方法。

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.