Abstract 711: Antioxidant-1 as a Novel Copper Dependent Transcription Factor for Cell Proliferation: Role in Wound Healing
Copper plays a fundamental role in regulating cell growth and it is also involved in wound healing, a physiological repair process associated with cell proliferation. However, underlying molecular mechanisms of copper-induced cell proliferation, particularly in wound healing process, is largely unknown. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper dependent transcription factor that mediates copper-induced cell proliferation and wound repair process. Stimulation of wild-type (WT) mouse embryonic fibloblasts (MEFs) with copper, but neither iron nor zinc, markedly increased cell numbers in a dose-dependent manner by 1.6 ± 0.1 fold at 10 uM, and was significantly inhibited in Atox1−/− MEFs as well as by siRNA knockdown of Atox1 in WT MEFs. Further, both mRNA and protein levels of cyclin D1, a regulator of G1-S phase progression, was significantly increased by copper treatment in WT MEFs (2.4 ± 0.1 and 2.8 ± 0.2 fold at 10 uM, respectively). EMSA and ChIP assays revealed that Atox1 binds to the promoter region of cyclin D1 in a copper dependent manner. These findings indicate that Atox1 functions as a copper-dependent DNA binding transcription factor which mediates cyclin D1 activation in mammals. Using mice would healing model and immunocytochemical analysis, we found that Atox1 was highly expressed in BrdU-positive proliferating nuclei in healing skin wounds, suggesting that Atox1 is localized in the nuclei of highly proliferating cells in vivo. More importantly, the rate of wound closure was markedly delayed in Atox1+/− mice compared to WT mice (7.1 ± 0.2 day vs. 11.2 ± 0.3 day for complete wound closure in WT vs. Atox1+/− mice, p < 0.001), indicating that Atox1 plays an important role in the wound healing process. In summary, we provide the first evidence that Atox1 functions as a novel copper dependent transcription factor which activates the cyclin D1 promoter, thereby mediating copper-induced cell proliferation, which may contribute to promoting wound healing process in vivo.