Abstract 16637: Antioxidant-1, a Novel Cu-Dependent Transcription Factor for NADPH Oxidase p47phox, Promotes Wound Healing by Regulating Inflammation and Angiogenesis

Background: Wound healing is important repair process after injury associated with inflammation and angiogenesis. Copper (Cu) plays an important role in wound healing and angiogenesis; however, underlying mechanism is unknown. Bioavailability of Cu is tightly controlled by transport proteins (Cu importer CTR1 and Cu exporter ATP7A) and chaperone (antioxidant-1, Atox1) which transfers Cu to ATP7A. We reported that Atox1 also functions as a Cu-dependent transcription factor; however, its role in wound healing is unclear. Results: Using mouse skin puncture model, here we show that, Atox 1 protein (8 fold) and Cu level (by X-ray Fluorescence Microscope; 2.5 fold) increased in skin after wounding in WT mice at day 7 when Atox1 was localised in the nucleus of dermis endothelial cell (ECs) and ATP7A was markedly decreased. ATP7A transgenic mice showed impaired wound healing. Atox1 knockout (KO) mice exhibited decrease in wound repair (40% inhibition vs. WT, day 7) which was rescued by Atox1 gene transfer by lentivirus. Macrophage which secrets angiogenic cytokines/chemokines was significantly reduced which resulted in decreased VEGF and SDF-1α protein and angiogenesis at day 7. In cultured human ECs, inflammatory cytokine TNF-stimulation significantly decreased ATP7A protein (80%) and increased intracellular Cu and Atox1 in nucleus. ATP7A knockdown with siRNA in ECs increased intracellular Cu and promoted nuclear Atox1, which were rescued by Cu importer CTR1 siRNA or Cu chelator BCS, indicating ATP7A downregulation-induced Cu accumulation is required for Atox1 nuclear translocation. By screening Atox1 target genes, we found that NADPH oxidase p47phox promoter contains Atox1 binding responsible element. p47phox protein expression was significantly increased by wounding or overexpression of nuclear-targeted Atox1, which was markedly decreased in Atox1 KO mice with its downstream VCAM1/ICAM1. Conclusions: Cu chaperone Atox1 functions as a transcription factor by sensing ATP7A downregulation-induced intracellular Cu to upregulate NADPH oxidase p47phox and its downstream redox-sensitive VCAM1/ICAM1 expression in response to injury. This in turn promotes inflammatory cell recruitment and angiogenesis, and thus stimulating wound repair.