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Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo

View Article: PubMed Central - PubMed

ABSTRACT

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1−/− mice. Endothelial cell (EC)-specific Atox1−/− mice and gene transfer of nuclear-target Atox1 in Atox1−/− mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1−/− mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2− production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.

No MeSH data available.


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Gene transfer of nuclear-targeted Atox1 rescues impaired wound healing of Atox1−/− mice.(A) Immunofluorescence showing Atox1 staining (red) in the nucleus of dermal region and in the cytosol of epidermal region and DAPI staining (blue, nuclear marker) at day 7 after wounding. Upper panel scale bars = 50 μm, lower panel scale bars = 10 μm (B) Nuclear and cytoplasmic fractions of wound tissue lysates were immunoblotted with anti-Atox1, α-tubulin (cytoplasmic marker), and laminin B1 (nuclear marker) antibody. (C) Lentiviruses expressing either control GFP vector virus or Atox1-WT or Atox1 with nuclear-target sequence (NLS) (Atox1-NLS) were injected to the cutaneous wounds of Atox1−/− mice right after injury. Representative images (left) and a graph representing mean ± SE of wound closure rates. (n > 3 per group, #p < 0.001; *p < 0.01 vs. control virus). Ruler notches 1 mm.
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f7: Gene transfer of nuclear-targeted Atox1 rescues impaired wound healing of Atox1−/− mice.(A) Immunofluorescence showing Atox1 staining (red) in the nucleus of dermal region and in the cytosol of epidermal region and DAPI staining (blue, nuclear marker) at day 7 after wounding. Upper panel scale bars = 50 μm, lower panel scale bars = 10 μm (B) Nuclear and cytoplasmic fractions of wound tissue lysates were immunoblotted with anti-Atox1, α-tubulin (cytoplasmic marker), and laminin B1 (nuclear marker) antibody. (C) Lentiviruses expressing either control GFP vector virus or Atox1-WT or Atox1 with nuclear-target sequence (NLS) (Atox1-NLS) were injected to the cutaneous wounds of Atox1−/− mice right after injury. Representative images (left) and a graph representing mean ± SE of wound closure rates. (n > 3 per group, #p < 0.001; *p < 0.01 vs. control virus). Ruler notches 1 mm.

Mentions: We previously reported that Atox1 functions not only as Cu chaperone but also as Cu-dependent transcription factor in ECs25. Indeed, immunofluorescence analysis showed that Atox1 was in the nucleus and the cytosol in the dermis area while in the cytosol in the epidermis area at day 7 (Fig. 7A). Consistent with this, subcellular fractionation assay in wounded skin lysates demonstrated that Atox1 expression in nuclear fraction was markedly increased at day 7 after wounding (Fig. 7B). To demonstrate the functional role of nuclear Atox1 in wound healing, we performed lentivirus-mediated gene transfer of nuclear targeted Atox1 (Atox1-NLS)2434 in wound sites from Atox1−/− mice. Impaired wound healing in Atox1−/− mice was significantly rescued by Atox1-NLS and Atox1-WT (Fig. 7C). Of note, Atox1-NLS restored wound healing more rapidly than Atox1-WT. These findings indicate that nuclear Atox1 (transcription factor function of Atox1) is required for wound healing process.


Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo
Gene transfer of nuclear-targeted Atox1 rescues impaired wound healing of Atox1−/− mice.(A) Immunofluorescence showing Atox1 staining (red) in the nucleus of dermal region and in the cytosol of epidermal region and DAPI staining (blue, nuclear marker) at day 7 after wounding. Upper panel scale bars = 50 μm, lower panel scale bars = 10 μm (B) Nuclear and cytoplasmic fractions of wound tissue lysates were immunoblotted with anti-Atox1, α-tubulin (cytoplasmic marker), and laminin B1 (nuclear marker) antibody. (C) Lentiviruses expressing either control GFP vector virus or Atox1-WT or Atox1 with nuclear-target sequence (NLS) (Atox1-NLS) were injected to the cutaneous wounds of Atox1−/− mice right after injury. Representative images (left) and a graph representing mean ± SE of wound closure rates. (n > 3 per group, #p < 0.001; *p < 0.01 vs. control virus). Ruler notches 1 mm.
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Related In: Results  -  Collection

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f7: Gene transfer of nuclear-targeted Atox1 rescues impaired wound healing of Atox1−/− mice.(A) Immunofluorescence showing Atox1 staining (red) in the nucleus of dermal region and in the cytosol of epidermal region and DAPI staining (blue, nuclear marker) at day 7 after wounding. Upper panel scale bars = 50 μm, lower panel scale bars = 10 μm (B) Nuclear and cytoplasmic fractions of wound tissue lysates were immunoblotted with anti-Atox1, α-tubulin (cytoplasmic marker), and laminin B1 (nuclear marker) antibody. (C) Lentiviruses expressing either control GFP vector virus or Atox1-WT or Atox1 with nuclear-target sequence (NLS) (Atox1-NLS) were injected to the cutaneous wounds of Atox1−/− mice right after injury. Representative images (left) and a graph representing mean ± SE of wound closure rates. (n > 3 per group, #p < 0.001; *p < 0.01 vs. control virus). Ruler notches 1 mm.
Mentions: We previously reported that Atox1 functions not only as Cu chaperone but also as Cu-dependent transcription factor in ECs25. Indeed, immunofluorescence analysis showed that Atox1 was in the nucleus and the cytosol in the dermis area while in the cytosol in the epidermis area at day 7 (Fig. 7A). Consistent with this, subcellular fractionation assay in wounded skin lysates demonstrated that Atox1 expression in nuclear fraction was markedly increased at day 7 after wounding (Fig. 7B). To demonstrate the functional role of nuclear Atox1 in wound healing, we performed lentivirus-mediated gene transfer of nuclear targeted Atox1 (Atox1-NLS)2434 in wound sites from Atox1−/− mice. Impaired wound healing in Atox1−/− mice was significantly rescued by Atox1-NLS and Atox1-WT (Fig. 7C). Of note, Atox1-NLS restored wound healing more rapidly than Atox1-WT. These findings indicate that nuclear Atox1 (transcription factor function of Atox1) is required for wound healing process.

View Article: PubMed Central - PubMed

ABSTRACT

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1&minus;/&minus; mice. Endothelial cell (EC)-specific Atox1&minus;/&minus; mice and gene transfer of nuclear-target Atox1 in Atox1&minus;/&minus; mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1&minus;/&minus; mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2&minus; production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.

No MeSH data available.


Related in: MedlinePlus