MAPK-activated protein kinase 2 regulation of endothelial cell migration

描述（由申请人提供）：新血管的形成，血管生成，是正常发育和伤口愈合所必需的。异常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 （MK2）的活性是VEGF刺激的内皮细胞迁移所必需的，MK2通过调节尿激酶纤溶酶原激活物（uPA）的表达参与内皮细胞迁移。由于血管生成发生在缺氧环境中，我们研究了MK2和uPA在缺氧环境下内皮细胞迁移中的作用。我们发现，与我们在正常缺氧条件下观察到的结果类似，抑制MK2活性也会消除uPA表达和vegf刺激的内皮细胞迁移，而恢复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轴在正常和缺氧情况下内皮细胞迁移中的一般作用。为了确定MK2调控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非常重要。通过双杂交筛选，我们发现了一种RNA结合蛋白DDX5，它不仅可以与MK2特异性相互作用，还可以作为MK2的直接底物。在mk2抑制的细胞中，过表达DDX5会破坏uPA mRNA的稳定性，沉默DDX5的表达会延长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MK2活性的负调控。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，从而阻碍DDX5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延长了mk2抑制或DDX5-过表达细胞中uPA mRNA的稳定性。这些结果牢固地将外泌体与MK2-DDX5调控uPA mRNA稳定性联系起来。该建议是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的转换；2)确定ddx5 -外泌体相互作用的相关机制及其外泌体在uPA mRNA降解中的作用；3)确定阻断MK2-DDX5-uPA轴抑制血管生成的有效性。提出的研究应该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。

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.