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. 2008 Apr 4;283(14):9157-67.
doi: 10.1074/jbc.M709463200. Epub 2008 Feb 2.

Novel role of antioxidant-1 (Atox1) as a copper-dependent transcription factor involved in cell proliferation

Shinichi Itoh  1 , Ha Won Kim, Osamu Nakagawa, Kiyoshi Ozumi, Susan M Lessner, Hiroki Aoki, Kamran Akram, Ronald D McKinney, Masuko Ushio-Fukai, Tohru Fukai
Affiliations

Novel role of antioxidant-1 (Atox1) as a copper-dependent transcription factor involved in cell proliferation

Shinichi Itoh et al. J Biol Chem. .

V体育安卓版 - Abstract

Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. 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. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1(-/-) MEFs. Promoter analysis and EMSA revealed that copper stimulates the Atox1 binding to a previously undescribed cis element in the cyclin D1 promoter. The ChIP assay confirms that copper stimulates Atox1 binding to the DNA in vivo VSports手机版. Transfection of Atox1 fused to the DNA-binding domain of Gal4 demonstrated a copper-dependent transactivation in various cell types, including endothelial and cancer cells. Furthermore, Atox1 translocated to the nucleus in response to copper through its highly conserved C-terminal KKTGK motif and N-terminal copper-binding sites. Finally, the functional role of nuclear Atox1 is demonstrated by the observation that re-expression of nuclear-targeted Atox1 in Atox1(-/-) MEFs rescued the defective copper-induced cell proliferation. Thus, Atox1 functions as a novel transcription factor that, when activated by copper, undergoes nuclear translocation, DNA binding, and transactivation, thereby contributing to cell proliferation. .

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Figures

FIGURE 1.
FIGURE 1.
Copper-induced cell proliferation requires Atox1. A, copper-induced cell proliferation in wild-type and Atox1-/- MEFs. Cells (1.5 × 103 cells/cm2) were serum-starved for 24 h and treated with either the copper chelator BCS (200 μm) or CuCl2 at the dose indicated for 72 h at 37 °C. The time of addition was considered as the t = 0 h of the experiments. Cell numbers were counted at 1-day intervals using a hemocytometer and expressed as % of initial cell number at 0 h. The data are shown as mean ± S.E. for three separate experiments. *, p < 0.01; #, p < 0.001 versus Atox1-/- cells. B, effect of copper on cell proliferation in Atox1-/- cells re-expressed with Atox1. Cell numbers were counted 72 h after transfection with either pcDNA/Atox1 or its empty vector (pcDNA) in the presence of either CuCl2 (10 μm) or BCS (200 μm). Data are the mean ± S.E. for three separate experiments. *, p < 0.01.
FIGURE 2.
FIGURE 2.
Copper stimulates cell proliferation by increasing cyclin D1 expression through Atox1. A, involvement of Atox1 in copper-dependent S-phase entry. Cells were treated either with BCS (200 μm) (-Cu) or Cu (10 μm) (+Cu) for 24 h. DNA synthesis was assessed by fluorescent-activated cell sorting (FACS) analysis. Copper-induced S-phase entry in WT and Atox1-KO cells is shown as the mean ± S.E. from four separate experiments (*, p < 0.01 versus BCS-treated cells; NS, not significant). B and C, effect of copper on cyclin D1 mRNA (B) and cyclin D1 (A), and B1 protein (C) levels in WT and Atox1-KO MEFs. Cells were exposed to either BCS (200 μm) or CuCl2 at the dose indicated for 12 h. *, p < 0.01; #, p < 0.001 versus BCS-treated cells. The data depict the mean ± S.E. for four separate experiments. D, effects of cyclin D1 or cyclin B1 siRNAs on copper-induced cell proliferation in wild-type MEFs. Cells were transfected with cyclin D1, cyclin B1, or control siRNAs in the presence of either CuCl2 (10 μm) or BCS (200 μm). 48 or 72 h after transfection, cell numbers were counted. Copper-induced increases in cell number were expressed as % increase over the increase in cell number in BCS-treated cells. The data are shown as the mean ± S.E. for three separate experiments (*, p < 0.01 versus control siRNA-treated cells). Lysates prepared 72 h after transfection were immunoblotted with anti-cyclin D1 or anti-cyclin B1 antibody. Results are representative of three separate experiments.
FIGURE 3.
FIGURE 3.
Atox1 binds to and activates the cyclin D1 promoter in a copper-dependent manner. A, effect of copper on transactivation of the cyclin D1 gene promoter in WT and Atox1-KO MEFs. Cells were transiently transfected with cyclin D1 promoter luciferase reporter constructs (pGL3-cyclin D1 (-962/+134)) or empty reporter constructs (pGL3-Basic) along with either pcDNA/Atox1 or pcDNA. Cells were treated with either BCS (200 μm) or CuCl2 at the dose indicated. Two days after transfection, the luciferase activity was assayed and normalized to the Renilla luciferase activity produced by the co-transfected control plasmid pRL-CMV. Results shown are means ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (*, p < 0.01; #, p < 0.001 versus BCS-treated WT cells, or BCS-treated Atox1-/- cells transfected with pcDNA/Atox1). B, identification of copper/Atox1-responsive elements in a proximal 90-bp cyclin D1 promoter element. WT and Atox1-KO MEFs were transiently transfected with 5′ deletion constructs of cyclin D1 in the presence of either CuCl2 (10 μm, +Cu) or BCS (200 μm, -Cu). Left and middle panels, relative luciferase activity in WT or Atox1-KO MEFs in the presence of either CuCl2 (10 μm, +Cu) or the copper chelator BCS (200 μm, -Cu). Right panel, relative luciferase activity in wild-type and Atox1-/- cells in the presence of CuCl2 (10 μm). Results shown are means ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (*, p < 0.01 versus BCS-treated WT (left panel), or CuCl2-treated Atox1-/-cells (right panel)). C and D, EMSA, showing the binding of Atox1 to the region -535 to -530 in the cyclin D1 promoter in a copper-dependent manner. C, nuclear extracts from MEFs were incubated with the biotinylated cyclin D1 promoter fragment with indicated treatments. D, left panel shows purified GST and GST-Atox1. Right panel, purified GST or GST-Atox1 was incubated with the DNA probe with indicated treatments. E, ChIP assay showing association of Atox1 with the cyclin D1 promoter in a copper-dependent manner in vivo. Cells were treated with either indicated treatments (upper panel) or CuCl2 (10 μm) (lower panel) and cross-linked with 1% formaldehyde. Nuclear lysates were immunoprecipitated (IP) with anti-Atox1 antibody or normal IgG, and the promoter region of either cyclin D1, cyclin B1, or cyclin A was amplified by PCR. A small aliquot of lysates before IP were used for PCR amplification as the input control (Input). Results are representative of three independent experiments. F, region -535 to -530 is required for copper-induced activation of the cyclin D1 promoter. MEFs were transfected with a cyclin D1 promoter luciferase reporter construct (pGL3-cyclin D1 (-962/+134)) with or without mutation of the copper/Atox1-responsive element (-535 to -530 region). *, p < 0.01 versus BCS-treated cells.
FIGURE 4.
FIGURE 4.
Copper stimulates transactivation activity of Atox1. Activation of gene expression by Atox1 in MEFs (A) and other cell types (B). Different GAL4-Atox1 hybrid constructs were cotransfected into the indicated cells along with the luciferase reporter vector containing GAL4-binding sites in the presence of either CuCl2 (10 μm, +Cu) or BCS (200 μm, -Cu). Results shown are means ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (*, p < 0.01 versus BCS-treated cells).
FIGURE 5.
FIGURE 5.
Copper stimulates nuclear translocation of Atox1. A, copper-induced nuclear translocation of Atox1 is an active process. MEFs were stimulated with copper (10 μm) at either 37 °C or 4 °C for 1–60 min. The nuclear extract (left panel) and non-nuclear fraction (right panel) were subjected to Western blotting with anti-Atox1, anti-histone H3 (a marker for nuclear fraction), or anti-tubulin (a marker for cytosolic fraction). The fold change in Atox1 protein levels is normalized to histone-H3 or tubulin as a loading control for each fraction. The bottom panel shows the mean ± S.E. for four separate experiments (*, p < 0.01 versus control cells). B, identification of the region responsible for the nuclear localization of Atox1. Atox1-/- MEFs were transiently transfected with pcDNA containing wild-type, truncated, or mutant forms of Atox1 with the Flag tag. After transfection, cells were cultured for 12 h in the presence of CuCl2 (10 μm). Immunofluorescence was performed using a Flag-M2 antibody followed by a fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Arrow shows the absence of Atox1 in the nucleus. Immunoblotting was performed using a Flag-M2 antibody. Equal amounts of protein (20 μg) were loaded in each lane.
FIGURE 6.
FIGURE 6.
Nuclear Atox1 is essential for copper-induced cell proliferation. A, right panel, generation of subcellular targeting Atox1 fusion constructs. Nuclear-targeted Atox1 (Flag-Atox1-NLS) was created by fusing a tripartite NLS sequence derived from SV40 large T antigen (NLS) to the C terminus of Flag-tagged WT-Atox1 (24, 25). Left panel, subcellular localization of Atox1-WT and Atox1-NLS fusion proteins. Atox1-/- mouse fibroblasts were transfected with Flag-Atox1-WT or Flag-Atox1 mutants, immunolabeled with antibodies for Flag tag (left panels, green) and nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI) (middle panel, blue). Upper panel, WT Atox1 was found in both the nucleus and cytoplasm. Lower panel, Atox1-NLS was mainly localized at the nucleus. B, effect of re-expression of Atox1-WT and Atox1-NLS on transactivation of the cyclin D1 promoter in Atox1-/- MEFs. Cells were transiently transfected with cyclin D1 promoter luciferase reporter constructs along with Atox1-WT or Atox1-NLS in the presence of either BCS (200 μm) or CuCl2 at the dose indicated. Bars are the mean ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (*, p < 0.01 versus BCS-treated wild-type cells or BCS-treated Atox1-/- cells transfected with pcDNA). C, effect of re-expression of Atox1-WT or Atox1-NLS on cell proliferation in Atox1-/- MEFs. Atox1-/- MEFs were transfected with Atox1-WT or Atox1-NLS in the presence of either CuCl2 (10 μm) or BCS (200 μm). 72 h after transfection, cell numbers were counted. The data are the means ± S.E. from three separate experiments. (*, p < 0.01 versus BCS-treated WT cells or Atox1-/- cells transfected with either pcDNA/Atox1-WT or pcDNA/Atox1-NLS).
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