AMPK调节MKP1在尼古丁致动脉粥样硬化的机制研究

Smoking is a classic risk factor of atherosclerosis(AS). Nicotine, a major constituent of cigarette smoking, has been found to induces the aberrant activation of MAPK (including p38 and JNK) in macrophages and promotes AS. The degration of MKP1(a key supressor of MAPK) after ubiquitination may be the key step in nicotine-induced AS. Our recent data suggested that MKP1 may be a new substrate of AMPK. There is a AMPK-recognized phosphorylate motif in the protein sequence of MKP1. Kinase assay found that AMPK phosphorylates MKP1 drectly,,which may the key in nicotine-induced AS. However,the mechanism is still unestablished. According to lately papers，we propose the hypothesis that nicotine phosphorylates MKP1 through ROS/RNS, initiating an ubiquitination-mediated proteasome-dependent degradation of MKP1. P38 and JNK are then aberrantly activated, inducing macrophage inflammation and make them be liable to internalize cholesterol，forming foam cell, releasing inflammatory factors and increasing unstability of AS. In the present proposal, we will elucidate the mechanism of AMPK-MKP1 signaling pathway to regulate nicotine induced atherosclerosis at multiple levels(molecular, cellular，tissue and animal), which may provide the molecular biology evidence for prevention and treatment of atherosclerosis.

吸烟是动脉粥样硬化（AS）的经典危险因素，但其确切机制仍不完全明了。尼古丁（香烟主要成分）致巨噬细胞p38和JNK异常激活，p38/JNK关键抑制子MKP1泛素化后降解及丢失可能是其中的核心环节。我们发现MKP1可能是AMPK一个新的底物，MKP1蛋白序列上有AMPK识别的磷酸化基团，激酶分析示AMPK可直接磷酸化MKP1，推测其在尼古丁致AS中发挥关键调节作用。基于已有的研究，我们提出假说：尼古丁通过ROS/RNS磷酸化MKP1，使其易于发生泛素化而降解，p38和JNK失去抑制子而异常活化，介导巨噬细胞向促炎表型转化，形成泡沫细胞，释放炎症因子，致AS发展和斑块不稳定。本研究拟在已有的工作基础上，从分子（磷酸化机制）、细胞（原代及细胞系）、组织（AS病变）、动物（Knockout小鼠）多层次验证AMPK-MKP1信号通路在尼古丁致AS的具体机制，为探讨AS的防治提供分子细胞学依据。

吸烟是冠心病的重要危险因素，但其引起动脉粥样硬化（AS）确切机制仍不完全清楚。我们发现尼古丁致巨噬细胞MKP1容易发生泛素化后降解，下游p38和JNK被异常激活，引起炎症反应及细胞焦亡，通过质谱发现MKP1容易发生磷酸化，并且细胞实验证实MKP1磷酸化后导致泛素化降解，生物信息学揭示MKP1蛋白序列上有AMPK识别的磷酸化基团，双分子作用显示AMPK可直接磷酸化MKP1，我们使用AMPK与MKP1多个肽段作用、S334A质粒突变找出了MKP1具体磷酸化位点Ser334，我们制备出p-S334-MKP1特异性抗体，并且发现其受AMPK调节，给与AMPK抑制剂或SiRNA均抑制MKP1此位点的磷酸化及降解。动物实验我们发现尼古丁（1.5mg/kg/day）干预使ApoE-/-小鼠斑块形成及炎症反应，其中巨噬细胞MKP1表达很低，导致下游p38和JNK被异常激活，引起炎症因子释放，但尼古丁的此效应在ApoE-/-AMPKα2-/-小鼠却不能促进粥样硬化及血管壁炎症，在体证实尼古丁通过AMPK磷酸化介导巨噬细胞向促炎表型转化，形成泡沫细胞，释放炎症因子，致AS发展和斑块不稳定。我们的研究为AS的防治提供分子细胞学依据。