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）： Project摘要血管迁移是一种标志性的血管病理学，其血管硬化和再狭窄后血管损伤后，这是退伍军人人口死亡的主要原因。铜是一种必不可少的营养素，与血管重塑和动脉粥样硬化有关。关于此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的进口铜CTR1的调节，而且还由铜出口商ATP7A调节，其功能是通过Trans-Golgi Network（TGN）的铜依赖性转移以及铜伴侣的铜转移以及抗氧化剂-1（ATOX1）的介导的。血小板来源的生长因子（PDGF）促进血管平滑肌细胞（VSMC）迁移和新内膜形成。最近，我们的实验室表明，PDGF刺激ATP7A从TGN到脂质筏的铜和Ctr1依赖性易位，在该脂质筏位于募集RAC1的领先边缘，并减少铜含量和分泌的铜酶，并降低铜酶，Pro-LySyl氧化酶（Pro-Lox）。这反过来刺激了层状脂蛋白的形成和LOX活性，从而促进了VSMC迁移。潜在的分子事件仍然未知。我们的初步研究将脚手架蛋白IQGAP1确定为ATP7A的新型结合伴侣。 IQGAP1证明可以直接与Rac1的活动形式结合，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CTR1-ATOX1途径和ATP7A与IQGAP1结合起着PDGF诱导的铜依赖性ATP7A的重要作用，而Rac1转移到前缘，lamellipodia组的前沿和VSMC迁移。此外，小窝/脂质筏是重要的信号域，其中PDGF刺激ATP7A依赖性LOX激活，这与铜依赖性VSMC迁移有关。为了测试这一点，将提出三个具体目标。 AIM 1将确定PDGF刺激ATP7A和RAC1转移到前缘的分子机制，该机制参与了层状形成和VSMC迁移。我们将使用体外下拉或体内共染色测定法确定ATP7A-IQGAP1结合位点，并在PDGF诱导的响应中定义其结合的功能意义。 FRET分析将用于检查铜转运蛋白在调节RAC1活性和易位中的作用。 AIM 2将确定pDGF诱导的pro-lox和铜稳态分泌中ATP7A运动对小窝蛋白增强的脂质筏的功能显着性，这是LOX激活和VSMC迁移所必需的。亚细胞分馏，源自可窝蛋白-1缺陷小鼠，64CU代谢标记分析，电感耦合等离子体质谱法（ICP-MS）和X射线荧光显微镜的VSMC。 AIM 3将评估ATP7A及其调节剂在响应血管损伤的新内膜形成中的功能作用。将使用ATP7A突变小鼠，IQGAP1缺乏小鼠和电线损伤模型。这些研究将为铜转运蛋白提供新的洞察力，以此作为心血管硬化等心血管疾病的潜在治疗靶点。