Positive Role of MKP-1 in Angiogenesis.

描述（由申请方提供）：丝裂原活化蛋白激酶磷酸酶-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剂。MKP-1通常作为MAPK的关闭开关;然而，在血管生成中，通过内皮细胞（EC）发芽形成新血管，发现MKP-1作为打开开关，使EC迁移。由于MAPK介导促血管生成过程，如迁移和增殖，这些观察结果表明，MKP-1可能有助于血管生成独立于其典型的MAPK磷酸酶活性。来自申请人实验室的先前报告鉴定了组蛋白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的转录控制来促进血管生成。因此，本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，并评估其在临床相关的血管损伤、生长和疾病动物模型中的作用。本提案的第一个目的是通过验证MKP-1通过修饰组蛋白积极介导血管生成并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。将通过MKP-1耗竭以及人细胞中的外源性重建来评价MKP-1在人和小鼠EC迁移、增殖和管形成中的作用。初步数据表明，MKP-1与血管生成基因fractalkine的外显子DNA免疫沉淀，并且是VEGF诱导所需的。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。这些将使用后肢缺血、VEGF诱导的皮下基质胶栓塞血管生成和B16黑色素瘤肿瘤生长和血管生成的MKP-1敲除小鼠模型进行评价。将通过免疫组织化学和实时定量PCR评价体内fractalkine诱导的MKP-1依赖性。这些目标的成功完成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在血管生成基因调控中的新作用，这可能导致刺激或抑制血管生成的治疗策略的改进。

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.