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）： 项目摘要 血管迁移是动脉粥样硬化和血管损伤后再狭窄的标志性血管病理学，是退伍军人群体死亡的主要原因。铜是一种必需营养素，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到铜输入蛋白 CTR1 的调节，还受到铜输出蛋白 ATP7A 的调节，ATP7A 的功能是通过来自反高尔基体网络 (TGN) 的铜依赖性易位以及铜伴侣、抗氧化剂 1 (Atox1) 介导的，后者从 CTR1 获取铜并将其转移到 ATP7A。血小板源性生长因子 (PDGF) 促进血管平滑肌细胞 (VSMC) 迁移和新内膜形成。最近，我们的实验室证明，PDGF 刺激铜和 CTR1 依赖性的 ATP7A 从 TGN 易位到位于前缘的脂筏，在那里招募 Rac1，并降低铜含量和分泌性铜酶、赖氨酰氧化酶原 (pro-LOX)。这反过来又刺激片状伪足的形成和 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 作为 ATP7A 的新型结合伴侣。 IQGAP1 显示直接与 Rac1 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 CTR1-Atox1 途径和 ATP7A 与 IQGAP1 的结合在 PDGF 诱导的铜依赖性 ATP7A 和 Rac1 易位到前缘、板状伪足形成和 VSMC 迁移中发挥重要作用。此外，小窝/脂筏是重要的信号传导域，PDGF 在其中刺激 ATP7A 依赖性 LOX 激活，从而参与铜依赖性 VSMC 迁移。为了测试这一点，将提出三个具体目标。目标 1 将确定 PDGF 刺激 ATP7A 和 Rac1 易位至前缘的分子机制，该机制参与板状伪足的形成和 VSMC 迁移。我们将使用体外 Pull-down 或体内共转染测定来鉴定 ATP7A-IQGAP1 结合位点，并定义它们的结合在 PDGF 诱导的反应中的功能意义。 FRET 分析将用于检查铜转运蛋白在活细胞图像分析中调节 Rac1 活性和易位的作用。目标 2 将确定 ATP7A 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 激活和 VSMC 迁移所必需的。将使用亚细胞分级分离、caveolin-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 (ICP-MS) 和 X 射线荧光显微镜。目标 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。