Role of Copper Transporters in Vascular Remodeling

铜转运蛋白在血管重塑中的作用

• 批准号: 8143009
• 负责人: TOHRU FUKAI
• 金额: --
• 依托单位: JESSE BROWN VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8143009
• 关键词: Actins; Antioxidants; Atherosclerosis; Binding; Binding Sites; Biological Assay; Biological Availability; Blood Vessels; Cardiovascular Diseases; Carrier Proteins; Caveolae; Caveolins; Cell Fractionation; Cell membrane; Cells; Chelating Agents; Cholesterol; Complex; Copper; Coupled; Cytoplasmic Vesicles; Cytoskeleton; Data; Enzyme Precursors; Enzymes; Event; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Growth Factor; Homeostasis; Image Analysis; Immunofluorescence Immunologic; In Vitro; Injury; Label; Laboratories; Life; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane Microdomains; Metabolic; Modeling; Molecular; Molecular Chaperones; Movement; Mus; Mutant Strains Mice; Nutrient; Pathology; Pathway interactions; Peptides; Plasma; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor beta Receptor; Play; Population; Process; Protein-Lysine 6-Oxidase; Proteins; Recruitment Activity; Resistance; Roentgen Rays; Role; Scaffolding Protein; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Synchrotrons; Testing; Transfection; Vascular Diseases; Vascular remodeling; Veterans; base; caveolin 1; cell motility; cellular imaging; fluorescence microscope; in vivo; insight; migration; mortality; mutant; new therapeutic target; novel; public health relevance; response; restenosis; therapeutic target; trans-Golgi Network; treatment strategy; tyrosine receptor; vascular smooth muscle cell migration

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.

描述（由申请人提供）： 血管移位是血管损伤后动脉粥样硬化和再狭窄的标志性血管病理学，是退伍军人人群死亡的主要原因。铜是一种必需的营养素，与血管重塑和动脉粥样硬化有关。关于这种反应所涉及的机制知之甚少。细胞内铜的生物利用度不仅受铜输入者CTR 1的调节，还受铜输出者ATP 7A的调节，ATP 7A的功能是通过铜依赖性的从trans-Golgi网络（TGN）的转运以及铜伴侣抗氧化剂1（Atox 1）介导的，铜伴侣抗氧化剂1从CTR 1获得铜并将其转移到ATP 7A。血小板源性生长因子（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刺激铜和CTR 1依赖性的ATP 7A从TGN转移到位于前沿的脂筏，在那里它招募Rac 1，并降低铜含量和分泌铜酶，赖氨酰氧化酶（pro-LOX）。这反过来又刺激板状伪足形成和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一个支架蛋白IQGAP 1作为一种新的结合伴侣的ATP 7A。IQGAP 1直接与Rac 1的活性形式结合，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CTR 1-Atox 1通路和ATP 7A与IQGAP 1的结合在PDGF诱导的铜依赖性ATP 7A和Rac 1易位至前缘、板状伪足形成和VSMC迁移中起重要作用。此外，小窝/脂筏是PDGF刺激ATP 7A依赖性LOX活化的重要信号传导结构域，其参与铜依赖性VSMC迁移。为了验证这一点，将提出三个具体目标。目的1探讨PDGF刺激ATP 7A和Rac 1向细胞前缘移位的分子机制，从而参与片状伪足的形成和VSMC的迁移。我们将使用体外下拉或体内共转染试验鉴定ATP 7A-IQGAP 1结合位点，并确定其在PDGF诱导的反应中结合的功能意义。FRET分析将用于在活细胞图像分析中检查铜转运蛋白在调节Rac 1活性和易位中的作用。目的2将确定ATP 7A运动到富含小窝蛋白的脂筏在PDGF诱导的pro-LOX分泌和铜稳态中的功能意义，这是LOX活化和VSMC迁移所必需的。将使用亚细胞分级分离、来自小窝蛋白-1缺陷小鼠的VSMC、64 Cu代谢标记分析、电感耦合等离子体质谱法（ICP-MS）和X射线荧光显微镜。目的3将评估ATP 7A及其调节因子在血管损伤后新生内膜形成中的功能作用。将使用ATP 7A突变小鼠和IQGAP 1缺陷小鼠和线损伤模型。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。