Novel regulatory mechanism of vascular endothelial function mediated by BMK1/ERK5 and its application for anti-atherosclerosis therapy

利用表达Gal-DNA结合区与BMK1/ERK5融合蛋白的哺乳动物表达载体，通过双荧光素酶报告基因检测，建立了BMK1/ERK5活性的定量检测体系。Pitavastatin以剂量依赖方式显著增强培养的血管内皮细胞BMK1/ERK5活性。Pitavastatin诱导的BMK1/ERK5激活被认为是他汀类效应，因为其他他汀类药物阿托伐他汀和辛伐他汀也激活BMK1/ERK5。Pitavastatin的作用可通过给予香叶基香叶基焦磷酸(GGPP)来消除，GGPP是胆固醇生物合成中的一种类异戊二烯中间体。以往的报道表明，抑制GGPP的合成通过抑制Rho激酶而引起多效性效应。然而，由于Rho激酶抑制剂Y27632不能激活BMK1/ERK5，因此BMK1/ERK5的激活被认为是一种新的血管内皮细胞功能Rho激酶的独立调节系统。Pitavastatin可降低肿瘤坏死因子α诱导的血管细胞黏附分子-1的表达，但显性负性MEK5β对BMK1/ERK5表达的抑制可消除其抗炎作用。另外，转染BMK1/ERK5 siRNA后，Pitavastatin诱导的eNOS启动子活性增加几乎完全消失。我们报道了匹伐他汀能增强PPARγ1的转录活性，并且BMK1/ERK5与PPARγ1结合，从而激活其活性。最近的报道表明，剪切力通过增加KLF2的表达来增强eNOS启动子的活性。这些结果提示，他汀类药物诱导的BMK1/ERK5的激活对血管内皮细胞功能的调节起着重要作用，其机制可能是通过激活PPARγ1抑制黏附分子的表达，并通过KLF2的表达增加eNOS的表达。

We established the quantitative measurement system of BMK1/ERK5 activity by dual-luciferase reporter gene assay using mammalian expression vector which expressed the fusion protein of Gal-DNA binding domain and BMK1/ERK5. Pitavastatin markedly enhanced BMK1/ERK5 activity in dose-dependent manner in cultured vascular endothelial cells. Pitavastatin-induced BMK1/ERK5 activation is thought to be the class effect of statin because other statins, atorvastatin and simvastatin, also activate BMK1/ERK5. The effect of pitavastatin is abolished by the administration of geranylgeranylpyrophosphate (GGPP), one of isoprenoid intermediates in cholesterol biosynthesis. Previous reports showed inhibition of GGPP synthesis causes pleiotropic effect via the inhibition of Rho kinase. However, activation of BMK1/ERK5 is thought to be a novel regulatory system of vascular endothelial function Rho kinase-independently because Y27632, Rho kinase inhibitor, can not activate BMK1/ERK5. Pitavastatin decreased TNFα-induced VCAM-1 expression, but the inhibition of BMK1/ERK5 by the transfection of dominant-negative MEK5β abolished the anti-inflammatory effect. In addition, pitavastatin-induced increase of eNOS promoter activity was almost abolished by transfection of BMK1/ERK5 siRNA. We reported that pitavastatin enhanced PPARγ1 transcriptional activity and that BMK1/ERK5 associated with PPARγ1, leading to activate its activity. Recent report showed that shear stress enhanced eNOS promoter activity via increased expression of KLF2. These finding suggested that statin-induced BMK1/ERK5 activation plays an important role for regulation of vascular endothelial function through the suppression of adhesion molecule expression via PPARγ1 activation and the increase of eNOS expression via KLF2 expression.