Abstract 1380: Role of Copper Transport System for Extracellular Superoxide Dismutase in Angiotensin II-Induced Hypertension
Extracellular superoxide dismutase (ecSOD) is a secretory copper enzyme highly expressed in the vasculature, and plays an important role in protecting angiotensin II (Ang II)-induced hypertension by reducing the extracellular levels of vascular superoxide. Copper-containing enzymes obtain cofactor copper through specific copper transport system including the copper uptake transporter Ctr1 and the copper chaperone/transcription factor, antioxidant-1 (Atox1). We previously demonstrated that Ang II upregulates ecSOD expression and activity; however, underlying molecular mechanisms remain unclear. Here we show that ecSOD expression and activity are markedly decreased in fibroblasts lacking Atox1 or Ctr1. Western and immunohistochemical analysis reveal that chronic Ang II infusion (500 ng/kg/min) for 7 days increases nuclear staining of Atox1 and ecSOD protein expression in mouse aorta, which are abolished in Atox1−/− mice. In cultured vascular smooth muscle cells, Ang II stimulates translocation of Atox1 from cytosol to nucleus with peak at 30 min, which is prevented in Ctr1−/− cells. Luciferase reporter gene assays show that Ang II increases ecSOD promoter by 4.1±0.2 fold, which is blocked by the mutation of Atox1 responsive elements (Atox1-RE) and by siRNA knockdown of Ctr1. DNA pull down assays show that Ang II markedly increases binding of Atox1 to the Atox1-RE including DNA segment in the ecSOD promoter. Of note, Atox1 co-immunoprecipitates with Ctr1 in a copper dependent manner. These findings suggest that Atox1 functions as a transcription factor for ecSOD in a Ctr1-dependent manner. Finally, Ang II infusion-induced increase in systolic blood pressure and vascular superoxide production, as measured by lucigenin assay, are further enhanced in Atox1−/− mice compared to those in WT mice (149±2.3 (KO) vs. 137±1.2 (WT) mmHg; 4.0±0.2 (KO) vs. 2.54±0.2 (WT) fold), both of which are reduced by treatment of the SOD mimetic tempol. In summary, copper-dependent transcription factor function of Atox1 plays an important role in Ang II-induced upregulation of ecSOD expression and activity through Ctr1-Atox1 pathway, which may contribute to preventing oxidant-stress-dependent Ang II-induced hypertension.