Modulation of Vascular Extracellular Superoxide Dismutase

血管细胞外超氧化物歧化酶的调节

• 批准号: 8044786
• 负责人: TOHRU FUKAI
• 金额: $ 35.33万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2013-12-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044786
• 关键词: ATP7A protein; Actins; Angiotensin II; Antioxidants; Aorta; Binding; Binding Sites; Biochemical; Biological Assay; Blood Pressure; Blood Vessels; Cardiovascular Diseases; Cell Fractionation; Cell Nucleus; Chronic; Copper; Cytosol; DNA; DNA Binding Domain; Data; EMSA; Enzymes; Extracellular Space; Fibroblasts; Fractionation; Functional disorder; Funding; Genetic Transcription; Goals; Hypertension; Immunofluorescence Immunologic; In Vitro; Indium; Infusion procedures; Measurement; Messenger RNA; Modeling; Molecular; Molecular Chaperones; Mus; Mutate; Nuclear; Nuclear Localization Signal; Oxidative Stress; Pathogenesis; Peptides; Play; Production; Promoter Regions; Protein Binding; Protein Binding Domain; Proteins; Recombinants; Role; Smooth Muscle Myocytes; Staining method; Stains; Superoxide Dismutase; Superoxides; System; Testing; Transactivation; Transfection; Transgenic Mice; beta Actin; blood pressure regulation; cofactor; copper transporter 1; extracellular; in vivo; insight; mutant; new therapeutic target; overexpression; oxidant stress; promoter; protein function; superoxide dismutase 1; trans-Golgi Network; transcription factor

DESCRIPTION (provided by applicant): Extracellular superoxide dismutase (ecSOD) is a secretory copper-containing enzyme highly expressed in the vasculature, and plays an important role in protecting angiotensin II (Ang II)-induced hypertension and endothelial dysfunction by reducing the extracellular levels of O2- in the vessel wall. We previously found that the copper chaperone Antioxidant 1 (Atox1) and the copper transporter Menkes protein (MNK) are required for delivering the cofactor copper to ecSOD, thereby increasing the specific activity of ecSOD. We have also shown that Ang II upregulates ecSOD mRNA and protein levels in vascular smooth muscle cells (VSMC) and mice aorta. Little is known about molecular mechanisms of how specific activity and transcription of ecSOD are regulated in Ang II-induced hypertension. Our preliminary data show that ecSOD expression is markedly decreased in Atox1-/- mouse fibroblast and aorta, and that Atox1 is found not only in cytosol but also in nucleus. We thus hypothesize that nuclear Atox1 functions as a copper dependent transcription factor and cytosolic Atox1 functions as a copper chaperone for ecSOD, thereby regulating full ecSOD activity, O2- production and endothelial function during Ang II-induced hypertension. Aim1 will examine whether Atox1 functions as a copper chaperone for ecSOD via binding to MNK at trans-Golgi network, thereby contributing to Ang II-induced increase in specific activity of ecSOD using mouse VSMC (MASM) lacking MNK function and in MASM delivered with Atox1-MNK binding inhibitory peptides. Aim 2 will examine whether nuclear localization signal (NLS) and copper binding domain (CBD) of Atox1 are critical for Ang II-induced increase in ecSOD mRNA transcription using Atox1-/-MASM transfected with Atox1 mutated in NLS or CBD motifs. We will also examine the overexpression of nuclear-targeted Atox1 on binding activity of Atox1 to its cis elements in the ecSOD promoter, and transactivation of Atox1. Aim 3 will examine the functional significance of Atox1 in Ang II-induced hypertension, vascular O27- levels and endothelial function using Atox1-/- mice and Atox1-/- mice crossed with ecSOD overexpressing transgenic mice whose transcription is not controlled by Atox1 but by beta-actin promoters. These studies will provide new insight into ecSOD and its regulator Atox1 as novel therapeutic targets for oxidative stress-dependent various cardiovascular diseases such as hypertension.

描述（由申请人提供）：细胞外超氧化物歧化酶（ecSOD）是一种分泌型含铜酶，在血管系统中高度表达，通过降低血管壁中O2-的细胞外水平，在保护血管紧张素II（Ang II）诱导的高血压和内皮功能障碍中发挥重要作用。我们以前发现，铜分子伴侣抗氧化剂1（Atox 1）和铜转运蛋白Menkes蛋白（MNK）是将辅因子铜传递给ecSOD所必需的，从而增加了ecSOD的比活性。我们还发现，血管紧张素II上调ecSOD的mRNA和蛋白水平在血管平滑肌细胞（VSMC）和小鼠主动脉。关于血管紧张素II诱导的高血压中ecSOD的特异性活性和转录如何调节的分子机制知之甚少。我们的初步数据表明，ecSOD的表达显着降低Atox 1-/-小鼠成纤维细胞和主动脉，Atox 1被发现不仅在胞浆中，但也在核中。因此，我们假设，核Atox 1功能作为铜依赖性转录因子和胞质Atox 1功能作为铜伴侣ecSOD，从而调节完整的ecSOD活性，O2-生产和血管内皮功能在血管紧张素II诱导的高血压。Aim 1将检查Atox 1是否通过在trans-Golgi网络处与MNK结合而作为ecSOD的铜分子伴侣发挥作用，从而有助于使用缺乏MNK功能的小鼠VSMC（MASM）和使用Atox 1-MNK结合抑制肽递送的MASM中Ang II诱导的ecSOD比活性的增加。目的2：用Atox 1-/-MASM转染Atox 1的细胞，研究Atox 1的核定位信号（NLS）和铜结合结构域（CBD）在Ang II诱导的ecSOD mRNA转录增加中的作用。我们还将研究过表达的核靶向Atox 1对Atox 1的结合活性，其顺式元件在ecSOD启动子，和反式激活Atox 1。目的3将使用Atox 1-/-小鼠和Atox 1-/-小鼠与过表达ecSOD的转基因小鼠杂交，检测Atox 1在Ang II诱导的高血压、血管O27-水平和内皮功能中的功能意义，所述转基因小鼠的转录不受Atox 1控制，而是受β-肌动蛋白启动子控制。这些研究将为ecSOD及其调节剂Atox 1作为氧化应激依赖性各种心血管疾病（如高血压）的新治疗靶点提供新的见解。