Abstract 1469: Segregation of Copper-dependent Transcription Factor and Copper Chaperone Function of Antioxidant-1 in Wound Healing
Copper plays an essential role in cell growth involved in wound repair and angiogenesis; however, underlying molecular mechanism remains unclear. We recently have found that the copper binding protein antioxidant-1 (Atox1), previously appreciated as a copper chaperone for secretory copper enzymes at the transGolgi network (TGN), mediates copper-induced cell proliferation in cultured fibroblasts. The role of Atox1 in wound healing in vivo is unknown. Using mouse wound healing model, here we show that Atox1 is highly induced at BrdU-positive nuclei in hair follicles and epidermis in wounding tissues where copper is accumulated, as assessed by X-ray fluorescence microscopy and by ICP-mass spectrometry (2.1-fold increase) as compared to non-wounding skin. Confocal microscopy and cell fractionation analysis demonstrate that Atox1 is predominantly found in cytosol in basal state. Copper or PKC activator PMA promotes translocation of Atox1 to nucleus as well as Atox1 threonine phosphorylation in a time-dependent manner. These copper-induced effects on Atox1 are prevented by a specific PKC inhibitor GF109203X. Luciferase reporter gene assays show that copper increases cyclin D1 promoter by 2.8±0.2 fold, which is blocked by the mutation of Atox1 responsive elements in the promoter and by GF109203X. Gel shift and ChIP assays demonstrate that Atox1 directly binds to the Atox1 responsive element including DNA segment in the cyclin D1 promoter. Atox1 knockout (KO) mice show a marked delay in the rate of wound closure (7.1 day) as compared to wild-type (WT) mice (11.2 day), which is associated with decrease in a number of BrdU-positive proliferating cells and cyclin D1 protein expression. All these defective responses in Atox1 KO mice are rescued by lentivirus-mediated re-expression of nuclear targeted Atox1 and Atox1-WT, but not TGN-targeted Atox1. In summary, copper-dependent transcription factor function of Atox1, but not copper chaperone function, plays a major role in wound healing in vivo through PKC-dependent phosphorylation and binding to the cyclin D1 promoter involved in cell proliferation. These findings provide novel insight into Atox1 as a potential therapeutic target for wound repair process or angiogenesis in response to injury.