Role of Copper Transporters in Vascular Remodeling

描述(由申请人提供)： 项目概述血管移行是导致动脉粥样硬化和血管损伤后再狭窄的标志性血管病变，是导致退伍军人死亡的主要原因。铜是一种重要的营养物质，与血管重塑和动脉粥样硬化有关。关于参与这种反应的机制，人们知之甚少。细胞内铜的生物利用率不仅受铜进口体CTR1的调节，还受铜输出体ATP7A和铜伴侣抗氧化剂-1(Atox1)的调节，ATP7A的功能是通过依赖于铜从反式高尔基体网络(TGN)的转运来实现的，后者从CTR1获得铜并将其转移到ATP7A。血小板衍生生长因子(PDGF)促进血管平滑肌细胞(VSMC)迁移和新生内膜形成。最近，我们的实验室证明，PDGF刺激依赖铜和CTR1的ATP7A从TGN转移到定位在前沿的脂筏上，在那里它招募rac1，并降低铜含量和分泌铜酶赖氨酰氧化酶原(Pro-LOX)。这反过来刺激片状脂膜的形成和LOX活性，从而促进VSMC迁移。潜在的分子事件仍然未知。我们的初步研究确定了支架蛋白IQGAP1是一种新的ATP7A结合伙伴。研究表明，IQGAP1直接与活性形式的rac1结合，以保持其活性状态，并参与细胞运动。我们的初步数据与新的假设一致，即CTR1-Atox1途径和ATP7A与IQGAP1结合在PDGF诱导的铜依赖的ATP7A和rac1易位到前沿、片状脂膜形成和VSMC迁移中起重要作用。此外，小窝/脂筏是PDGF刺激ATP7A依赖的LOX激活的重要信号域，该激活参与了铜依赖的VSMC迁移。为了检验这一点，将提出三个具体目标。目的1确定PDGF刺激ATP7A和rac1易位到前缘的分子机制，该机制参与片状脂膜的形成和VSMC的迁移。我们将通过体外下拉或体内共转染的方法确定ATP7A-IQGAP1的结合部位，并确定其结合在PDGF诱导的反应中的功能意义。FRET分析将用于研究活细胞图像分析中铜转运蛋白在调节rac1活性和转位中的作用。目的2研究ATP7A在PDGF诱导LOX活化和VSMC迁移所需的前LOX和铜稳态分泌中的功能意义。将使用亚细胞分离、小窝蛋白-1缺陷小鼠来源的VSMC、64Cu代谢标记分析、电感耦合等离子体质谱(ICPMS)和X射线荧光显微镜。目的3将评估ATP7A及其调节因子在血管损伤后新生内膜形成中的功能作用。将采用ATP7A突变小鼠、IQGAP1缺陷小鼠和电线损伤模型。这些研究将为铜转运蛋白作为动脉粥样硬化等心血管疾病的潜在治疗靶点提供新的见解。

DESCRIPTION (provided by applicant): Project Summary Vascular migration is a hallmark vascular pathology underlying atherosclerosis and restenosis following vascular injury, which are the major causes of mortality in Veteran population. Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis. Little is known regarding mechanisms involved in this response. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1, but also by the copper exporter ATP7A whose function is mediated through copper-dependent translocation from trans-Golgi network (TGN) as well as copper chaperon, antioxidant-1 (Atox1) which obtains copper from CTR1 and transfer it to ATP7A. Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration and neointimal formation. Most recently, our laboratory demonstrated that PDGF stimulates copper- and CTR1-dependent translocation of ATP7A from TGN to the lipid rafts localized at the leading edge where it recruits Rac1 as well as decreases copper content and secretory copper enzyme, pro-lysyl oxidase (pro-LOX). This in turn stimulates lamellipodia formation and LOX activity, thereby promoting VSMC migration. Underlying molecular events remain unknown. Our preliminary studies identified a scaffold protein IQGAP1 as a novel binding partner for ATP7A. IQGAP1 is shown to bind directly to active form of Rac1 to keep it active state and involved in cell motility. Our preliminary data are consistent with the novel hypothesis that CTR1-Atox1 pathway and ATP7A binding to IQGAP1 plays an important role PDGF-induced copper-dependent ATP7A and Rac1 translocation to the leading edge, lamellipodia formation and VSMC migration. Moreover, caveolae/lipid rafts are important signaling domains where PDGF stimulates ATP7A- dependent LOX activation, which is involved in copper-dependent VSMC migration. To test this, three specific aims will be proposed. Aim 1 will determine the molecular mechanism by which PDGF stimulates ATP7A and Rac1 translocation to the leading edge, which is involved in lamellipodia formation and VSMC migration. We will identify ATP7A-IQGAP1 binding sites using in vitro pull-down or in vivo co-transfection assays and define the functional significance of their binding in PDGF-induced responses. FRET analysis will be used to examine role of copper transporters in regulating Rac1 activity and translocation in live cell image analysis. Aim 2 will determine the functional significance of ATP7A movement to caveolin-enriched lipid rafts in PDGF-induced secretion of pro-LOX and copper homeostasis, which are required for LOX activation and VSMC migration. Subcellular fractionation, VSMC derived from caveolin-1 deficient mice, 64Cu metabolic labeling analysis, inductively coupled plasma mass spectrometry (ICP-MS), and X-ray fluorescence microscope will be used. Aim 3 will assess the functional role of ATP7A and its regulators in neointimal formation in response to vascular injury. ATP7A mutant mice, and IQGAP1 deficient mice and wire injury model will be used. These studies will provide new insight into copper transporters as potential therapeutic targets for cardiovascular diseases such as atherosclerosis.