Novel mechanism for regulation of extracellular SOD transcription and activity by copper: Role of antioxidant-1

Abstract

Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone Antioxidant-1 (Atox1) involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response element (RE) at the SOD3 promoter. Gel shift and ChIP assays reveal that Atox1 directly binds to the Atox1-RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated re-expression in Atox1-/- cells with nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, re-expression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1-/- cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through transcription factor function of Atox1, while full activity of SOD3 requires both copper chaperone and transcription factor function of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.

Regulation of Oxidative Stress in Pulmonary Artery Endothelium. Modulation of Extracellular Superoxide Dismutase and NOX4 Expression Using Histone Deacetylase Class I Inhibitors

“…For example, several other antioxidant genes were strongly up-regulated, such as glutathione peroxidase 3, sulfiredoxin 1, and epoxide hydrolase 2. Interestingly, the ATOX 1 gene involved in the delivery of copper to the active site of EC-SOD was also upregulated after exposure to scriptaid (21). Thus, attenuation of oxidative stress by scriptaid might not be attributed solely to EC-SOD and NOX4 modulation.…”

Hydrogen Peroxide Regulates Extracellular Superoxide Dismutase Activity and Expression in Neonatal Pulmonary Hypertension

“…However, the mechanisms that regulate ecSOD expression are poorly understood. Antioxidant-1 (Atox-1) increased ecSOD expression in mouse fibroblasts by binding to the ecSOD promoter in a copperdependent fashion (17). Induction of heme oxygenase-1 (HO-1) was accompanied by an increase in ecSOD expression in bovine pulmonary arteries (1), whereas exogenous H 2 O 2 increased the expression of HO-1 in human aortic SMCs (4).…”

An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

“…In the Atox1 -/ -nuclei, the accumulating copper inhibits transcription of cyclin D1 and decreases cell proliferation rate (40). Thus, it could be that the role of Atox1 in transcription is to prevent the entry and inhibitory effects of copper in the nucleus, although direct interaction of recombinant Atox1 with DNA has been observed in several independent assays, suggesting a more complex mechanism (41,62).…”