Positive Role of MKP-1 in Angiogenesis.

描述（由申请人提供）：丝裂原激活蛋白激酶磷酸酶-1 (MKP-1) 是一种使 MAPK 去磷酸化的酶，MAPK 是细胞行为的主要调节因子。 MKP-1 通常充当 MAPK 的关闭开关；然而，在血管生成（内皮细胞（EC）萌芽形成新血管）中，MKP-1被发现充当开关，使内皮细胞能够迁移。由于 MAPK 介导促血管生成过程，例如迁移和增殖，这些观察结果表明 MKP-1 可能独立于其典型的 MAPK 磷酸酶活性而促进血管生成。申请人实验室之前的报告鉴定出组蛋白 H3（一种有助于 DNA 包装的蛋白质）作为 MKP-1 的新型底物。总之，这些数据表明 MKP-1 可能通过组蛋白修饰对血管生成基因进行转录控制来促进血管生成。因此，该项目的总体目标是通过体外鉴定 MKP-1 介导的血管生成机制来阐明 MKP-1 在血管生成中的作用，并评估其在血管损伤、生长和疾病的临床相关动物模型中的作用。该提案的首要目的是通过测试 MKP-1 正介导血管生成并通过修饰组蛋白调节促血管生成基因（如 fractalkine）表达的假设，来表征内皮 MKP-1 在血管内皮生长因子（VEGF）诱导的体外血管生成中的作用和机制。 MKP-1 在人和小鼠 EC 迁移、增殖和管形成中的作用将通过 MKP-1 耗竭以及人细胞中的外源重建来评估。初步数据表明，MKP-1 与血管生成基因 fractalkine 的外显子 DNA 发生免疫沉淀，并且是 VEGF 诱导其所必需的。该提案将通过染色质免疫沉淀评估 MKP-1 与 fractalkine DNA 相互作用的机制，并将通过蛋白质阵列筛选鉴定其他潜在的 MKP-1 调节的促血管生成基因。该提案的第二个目的是通过测试 MKP-1 积极介导缺血性损伤的恢复、通过血管生成的新血管生长和病理性血管生成的假设来评估 MKP-1 在体内血管生成中的作用。这些将使用 MKP-1 敲除小鼠后肢缺血模型、VEGF 诱导的皮下基质胶塞血管生成以及 B16 黑色素瘤肿瘤生长和血管生成进行评估。体内 fractalkine 诱导的 MKP-1 依赖性将通过免疫组织化学和实时定量 PCR 进行评估。成功完成这些目标将增强我们对治疗性和病理性血管生成机制的基础知识，并将确定 MKP-1 在血管生成基因调节中的新作用，这可能会导致改善刺激或抑制血管生成的治疗策略。

DESCRIPTION (provided by applicant): Mitogen activated protein kinase phosphatase-1 (MKP-1) is an enzyme that dephosphorylates MAPK, which act as master regulators of cellular behavior. MKP-1 canonically acts as an off-switch for the MAPK; however, in angiogenesis, the formation of new blood vessels by endothelial cell (EC) sprouting, MKP-1 was found to act as an on-switch, enabling EC migration. Since the MAPK mediate pro-angiogenic processes such as migration and proliferation, these observations suggest that MKP-1 may contribute to angiogenesis independently of its canonical MAPK phosphatase activity. Previous reports from the applicant's laboratory identified histone H3, a protein that aids in packaging of DNA, as a novel substrate of MKP-1. Together, these data suggest that MKP-1 may facilitate angiogenesis by transcriptional control of angiogenic genes through histone modification. The overall objective of this project is therefore to elucidate the role of MKP-1 in angiogenesis by identifyin the mechanism of MKP-1-mediated angiogenesis in vitro and evaluate its effects in clinically relevant animal models of vascular injury, growth, and disease. The first aim of this proposal is to characterize the role and mechanism of endothelial MKP-1 in vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro by testing the hypothesis that MKP-1 positively mediates angiogenesis and regulates expression of pro-angiogenic genes such as fractalkine by modifying histones. The role of MKP-1 in both human and mouse EC migration, proliferation, and tube formation will be evaluated by MKP-1 depletion as well as exogenous reconstitution in human cells. Preliminary data demonstrated that MKP-1 immunoprecipitates with exonic DNA of the angiogenic gene fractalkine and is required for its induction by VEGF. This proposal will evaluate the mechanism by which MKP-1 interacts with fractalkine DNA by chromatin immunoprecipitation, and will identify other potential MKP-1-regulated pro-angiogenic genes by protein array screening. The second aim of this proposal is to evaluate the role of MKP-1 in angiogenesis in vivo by testing the hypotheses that MKP-1 positively mediates recovery from ischemic injury, neovascular growth through angiogenesis, and pathological angiogenesis. These will be evaluated using MKP-1 knockout mouse models of hind limb ischemia, VEGF-induced subcutaneous Matrigel plug angiogenesis, and B16 melanoma tumor growth and angiogenesis. The MKP-1-dependence of fractalkine induction in vivo will be evaluated by immunohistochemistry and real-time quantitative PCR. Successful completion of these aims will enhance our fundamental knowledge of the mechanisms governing therapeutic and pathological angiogenesis, and will identify a novel role for MKP-1 in angiogenic gene regulation, which may lead to improved therapeutic strategies for stimulating or inhibiting angiogenesis.