Novel mechanism for regulation of extracellular SOD transcription and activity by copper: role of antioxidant-1.
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 elements (REs) 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 reexpression in Atox1(-/-) cells of nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, reexpression 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 the transcription factor function of Atox1, whereas the full activity of SOD3 requires both the copper chaperone and the transcription factor functions of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.
Itoh
Shinichi
S
Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
Ozumi
Kiyoshi
K
Kim
Ha Won
HW
Nakagawa
Osamu
O
McKinney
Ronald D
RD
Folz
Rodney J
RJ
Zelko
Igor N
IN
Ushio-Fukai
Masuko
M
Fukai
Tohru
T
D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D003300:Copper / Q000494:pharmacology*; D000079922:Copper Transport Proteins; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D055786:Gene Knockout Techniques; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D015533:Transcriptional Activation / Q000187:drug effects*; D014161:Transduction, Genetic; D021601:trans-Golgi Network
C499247:Atox1 protein, mouse; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D003300:Copper; C000606291:Sod1 protein, mouse; C499090:Sod3 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C029892:cupric chloride