Positive Role of MKP-1 in Angiogenesis.

描述(申请人提供)：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因子。MKP-1典型地充当MAPK的关断开关；然而，在血管生成中，通过内皮细胞(EC)萌芽形成新血管，MKP-1被发现作为开关，使EC迁移。由于MAPK介导了迁移和增殖等促血管生成过程，这些观察表明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确认组蛋白H3，一种帮助包装DNA的蛋白质，是MKP-1的一种新底物。综上所述，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，从而促进血管生成。因此，本项目的总体目标是通过确定MKP-1介导的体外血管生成的机制来阐明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。本研究的第一个目的是通过验证MKP-1正向调节血管生成和通过修饰组蛋白调节促血管生成基因表达的假说，来研究内皮MKP-1在血管内皮细胞生长因子(VEGF)诱导的血管生成中的作用和机制。MKP-1在人和小鼠内皮细胞迁移、增殖和管状形成中的作用将通过MKP-1在人类细胞中的耗竭和外源性重建来评估。初步数据表明，MKP-1与血管生成基因Fractalkine的外显子DNA一起免疫沉淀，并被血管内皮生长因子诱导。这项建议将评估MKP-1通过染色质免疫沉淀与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。这项建议的第二个目的是通过验证MKP-1积极地介导缺血损伤的恢复、通过血管生成的新生血管生长和病理性血管生成的假说来评估MKP-1在体内血管生成中的作用。这些将使用MKP-1基因敲除小鼠的后肢缺血模型、血管内皮生长因子诱导的皮下Matrigel栓血管生成以及B16黑色素瘤的生长和血管生成来进行评估。免疫组织化学和实时定量聚合酶链式反应将评估体内诱导的MKP-1的依赖性。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角色，这可能导致改进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.