Abstract 992: Novel Role of Copper-dependent Transcription Factor Antioxidant-1 in Cell Proliferation and Wound Healing

Abstract

Copper plays a fundamental role in regulating cell proliferation involved in wound healing and angiogenesis. Recently, we found that antioxidant-1 (Atox1), previously appreciated as a copper chaperone at the transGolgi network (TGN), stimulates cyclin D1 transcription and cell proliferation in a copper dependent manner. However, functional role of Atox1 in wound healing in vivo remains unknown. Here we show that Atox1 was highly expressed at the BrdU positive nuclei in the wounding tissue, which was associated with an increase in copper content (2.1-fold), as assessed by ICP-mass spectrometry, as compared to non-wounded skin. In Atox1 knockout (KO) mice, the rate of wound closure was markedly delayed as compared to wild-type (WT) mice (7.1 day vs. 11.2 day for complete wound closure in WT vs. Atox1 KO mice, p < 0.001), and BrdU-positive proliferating cells and cyclin D1 protein expression were significantly decreased. Capillary density in healing wound tissue was also reduced in Atox1 KO mice (27.8% decrease). To segregate a copper-dependent transcription factor and a copper chaperone function of Atox1, we have generated lentivirus expressing nuclear targeted Atox1 (Atox1-NLS), TGN-targeted Atox1 (Atox1-TGN) or Atox1-WT. and examine their effect on cell proliferation and wound healing in vitro and in vivo. In cultured Atox1 KO mouse fibroblasts, copper-induced cell proliferation and cyclin D1 expression were abolished, which was rescued by re-expression of both Atox1-NLS and Atox1-WT, but not Atox1-TGN. Promoter deletion analysis identified Atox1 responsive element (Atox1-RE) at cyclin D1 promoter and EMSA and ChIP assays demonstrated that Atox1 binds to Atox1-RE at the cyclin D1 promoter in a copper dependent manner. The impairment of wound healing in Atox1 KO mice was rescued by re-expression with lenti-Atox1-NLS and Atox1-WT, but not Atox1-TGN, which was associated with the increase in cyclin D1 expression as well as number of BrdU positive proliferating nuclei. In summary, transcription factor function of Atox1, but not copper-chaperone function of Atox1, plays a critical role in copper-induced cell proliferation, which may contribute to wound repair process in response to injury in vivo.