Role of Antioxidant-1 in Extracellular Superoxide Dismutase Function and Expression

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Abstract
The extracellular superoxide dismutase (ecSOD or SOD3) is a copper-containing enzyme which is highly expressed in the vasculature. Copper-containing enzymes require copper chaperones for their activity however the chaperone which delivers copper to SOD3 has not previously been defined. Atox1 is a copper chaperone proposed to deliver copper to the trans-Golgi network. Because SOD3 is secreted via the trans-Golgi network, we sought to determine whether Atox1 acts as a copper chaperone for SOD3. Using recombinant human SOD3, we found that the specific activity of SOD3 directly correlates with its copper content (R2=0.99). SOD3 specific activity in the conditioned medium from cultured Atox1−/− fibroblasts was markedly decreased, but could be recovered to that of wild-type cells by copper addition. These results indicated that Atox1 is required for delivering copper to SOD3 for its full activity. Unexpectedly, the protein and mRNA levels of SOD3 were dramatically decreased in cultured Atox1−/− fibroblasts. This was associated with a marked decrease in SOD3 transcription rate but no change in SOD3 mRNA stability. Overexpression of Atox1 markedly increased SOD3 mRNA in both Atox1−/− and Atox1+/+ cells. These findings indicate that Atox1 positively regulates SOD3 transcription. Because SOD3 protein is upregulated in atherosclerotic vessels, we examined expression of Atox1 in vessels from ApoE−/− mice. Western and immunohistochemical analysis in ApoE−/− mice revealed that both Atox1 and SOD3 protein levels are markedly increased in atherosclerotic intimal lesions. In summary, Atox1 functions not only as a copper chaperone for SOD3 but also as a positive regulator for SOD3 transcription and may have an important role in modulating oxidative stress in the cardiovascular system.