Abstract 150: Novel Mechanism for Regulation of Extracellular SOD Expression and Activity by Antioxidant-1

Abstract

Extracellular superoxide dismutase (ecSOD), a copper-containing secretory antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Little is known regarding the mechanisms by which ecSOD expression and activity are regulated. We previously demonstrated that antioxidant-1 (Atox1) functions as a copper chaperone for ecSOD at transGolgi network (TGN) to regulate its activity. Unexpectedly, we also found that ecSOD mRNA is markedly decreased in Atox1-knockout (KO) fibroblasts. We thus hypothesize that Atox1 functions not only as copper chaperone but also as transcription factor to regulate both expression and activity of ecSOD. Here we show that in ApoE-KO mouse aorta, Atox1 was intensely stained in the nucleus at the intimal lesion of atherosclerosis where ecSOD was highly expressed. In cultured mouse fibroblast, copper treatment stimulated translocation of Atox1 from the cytosol to the nucleus. The ecSOD mRNA and its promoter activity were markedly decreased in Atox1 KO cells (82.5% and 85.7% decrease, respectively). Promoter deletion analysis identified Atox1-response element (Atox1-RE) at −314/−304 segment of ecSOD promoter. Gel shift and ChIP assays showed that Atox1 directly bound to the Atox1-RE including DNA segment in a copper dependent manner. To segregate a transcription factor and a copper chaperone function of Atox1, we generated adenovirus expressing wild-type, nucleus-targeted, and TGN-targeted Atox1 (Atox1-WT, Atox1-NLS, and Atox1-TGN). Re-expression of either Atox1-NLS or Atox1-WT, but not Atox1-TGN, in Atox1-KO cells increased ecSOD mRNA by 3.2-and 2.5-fold, respectively. Importantly, re-expression of either Atox1-WT only or both Atox1-NLS and Atox1-TGN in Atox1 KO cells increased ecSOD activity (2.1- and 2.7-fold, respectively), while either Atox1-NLS or Atox1-TGN alone had no effect. In summary, Atox1 functions as a novel copper dependent transcription factor to increase ecSOD expression, and modulates ecSOD activity through both copper chaperone and transcription factor function. Thus, not only ecSOD but also Atox1 are potential therapeutic targets for oxidant stress-dependent cardiovascular disease such as atherosclerosis.