Modulation of Vascular Extracellular Superoxide Dismutase

描述（由申请人提供）：细胞外超氧化物歧化酶（ecSOD）是一种在血管中高表达的含铜分泌酶，通过降低血管壁细胞外O2-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起重要作用。我们之前发现，铜伴侣抗氧化1 （Atox1）和铜转运蛋白Menkes蛋白（MNK）是将铜传递到ecSOD的辅助因子，从而提高ecSOD的比活性所必需的。我们还发现Ang II上调血管平滑肌细胞（VSMC）和小鼠主动脉ecSOD mRNA和蛋白水平。在angii诱导的高血压中，ecSOD的特异性活性和转录调控的分子机制尚不清楚。我们的初步数据显示，Atox1-/-小鼠成纤维细胞和主动脉中ecSOD的表达明显降低，并且Atox1不仅存在于细胞质中，也存在于细胞核中。因此，我们假设核Atox1作为铜依赖的转录因子和细胞质Atox1作为ecSOD的铜伴侣，从而在Ang ii诱导的高血压期间调节ecSOD的活性、O2-产生和内皮功能。Aim1将研究Atox1是否通过在反式高尔基网络上与MNK结合而作为ecSOD的铜伴侣，从而通过缺乏MNK功能的小鼠VSMC （MASM）和通过Atox1-MNK结合抑制肽递送的MASM，促进Ang ii诱导的ecSOD特异性活性的增加。目的2将研究Atox1的核定位信号（NLS）和铜结合域（CBD）是否对angii诱导的ecSOD mRNA转录增加至关重要，使用转染NLS或CBD基序突变Atox1的Atox1-/- masm。我们还将研究核靶向Atox1的过表达对ecSOD启动子中Atox1与其顺式元件的结合活性的影响，以及Atox1的反激活。目的3将利用Atox1-/-小鼠和Atox1-/-小鼠与转录不受Atox1控制而受β -肌动蛋白启动子控制的ecSOD过表达转基因小鼠杂交，研究Atox1在angii诱导的高血压、血管O27-水平和内皮功能中的功能意义。这些研究将为ecSOD及其调节因子Atox1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。

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.