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.
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