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The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing.

Lee SH, Kim MY, Kim HY, Lee YM, Kim H, Nam KA, Roh MR, Min do S, Chung KY, Choi KY - J. Exp. Med. (2015)

Bottom Line: We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein.A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production.Protein transduction domain (PTD)-Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro.

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Affiliation: Translational Research Center for Protein Function Control; Department of Biotechnology, College of Life Science and Biotechnology; and Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, College of Medicine; Yonsei University, Seoul 120-749, South Korea Translational Research Center for Protein Function Control; Department of Biotechnology, College of Life Science and Biotechnology; and Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, College of Medicine; Yonsei University, Seoul 120-749, South Korea.

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Co-treatment with PTD-DBM and VPA accelerates cutaneous wound healing and synergistically induces collagen production in mice. The wounded skin of 7-wk-old male C3H mice was treated daily with 100 µM PTD-DBM and/or 500 mM VPA or with 100 µM EGF for 11 d (n = 16 mice/group). (A) IHC staining of wounds treated with PTD-DBM was performed to detect β-catenin. Representative images are shown (n = 3 independent experiments). (B) Representative H&E-stained images of wounds at 12 d after wounding with the different treatments are shown (n = 3 independent experiments). The arrowheads represent the edges of the wounds. (C) Representative images of macroscopic wounds and H&E and IHC staining (n = 5 mice group) for β-catenin, keratin 14, collagen I, PCNA, or p-Erk in wounds 12 d after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary (n = 3 independent experiments). F, fibroblasts; K, keratinocytes. (D) Relative wound closure rates for wounds from mice treated with PTD-DBM and/or VPA or EGF were quantified as percent wound closure as shown. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (E) Western blot analyses of β-catenin, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, p-Erk, and Erk in wounds (n = 2 mice/group) 12 d after wounding were performed (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (F) Representative images of Masson’s trichrome, picrosirius red, and van Gieson staining of wounds (n = 5 mice/group) 12 d after wounding are shown (n = 3 independent experiments). Bars: (A, C, and F) 100 µm; (B) 500 µm. (G) Hydroxyproline levels were measured in wounds at 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 5 mice/group). (H) Quantitative TissueFAXS analyses of immunohistochemical staining shown in C are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
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fig9: Co-treatment with PTD-DBM and VPA accelerates cutaneous wound healing and synergistically induces collagen production in mice. The wounded skin of 7-wk-old male C3H mice was treated daily with 100 µM PTD-DBM and/or 500 mM VPA or with 100 µM EGF for 11 d (n = 16 mice/group). (A) IHC staining of wounds treated with PTD-DBM was performed to detect β-catenin. Representative images are shown (n = 3 independent experiments). (B) Representative H&E-stained images of wounds at 12 d after wounding with the different treatments are shown (n = 3 independent experiments). The arrowheads represent the edges of the wounds. (C) Representative images of macroscopic wounds and H&E and IHC staining (n = 5 mice group) for β-catenin, keratin 14, collagen I, PCNA, or p-Erk in wounds 12 d after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary (n = 3 independent experiments). F, fibroblasts; K, keratinocytes. (D) Relative wound closure rates for wounds from mice treated with PTD-DBM and/or VPA or EGF were quantified as percent wound closure as shown. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (E) Western blot analyses of β-catenin, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, p-Erk, and Erk in wounds (n = 2 mice/group) 12 d after wounding were performed (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (F) Representative images of Masson’s trichrome, picrosirius red, and van Gieson staining of wounds (n = 5 mice/group) 12 d after wounding are shown (n = 3 independent experiments). Bars: (A, C, and F) 100 µm; (B) 500 µm. (G) Hydroxyproline levels were measured in wounds at 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 5 mice/group). (H) Quantitative TissueFAXS analyses of immunohistochemical staining shown in C are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.

Mentions: To investigate the effect of co-treatment with PTD-DBM and VPA on cutaneous wound healing in vivo, we created cutaneous wounds (diameter = 1.5 cm) on the dorsal skin of C3H mice and applied PTD-DBM and/or VPA topically to the wounds on a daily basis. As a positive control, one group of mice was treated with epidermal growth factor (EGF), a currently prescribed wound-healing agent (Kim et al., 2010b, 2014b). When the wounds were treated with PTD-DBM, we observed a strong increase in β-catenin expression (Fig. 9 A). Treatment with PTD-DBM or VPA accelerated cutaneous wound healing as efficiently as EGF (Fig. 9, B–D); however, combination treatment with PTD-DBM and VPA accelerated cutaneous wound healing much more efficiently than treatment with EGF alone (Fig. 9, B–D). The wounds were completely reepithelialized by combination treatment with PTD-DBM and VPA (Fig. 9, B and C) with reduction in inflammatory cells (Fig. 9 C). Notably, the combination treatment group exhibited 42.8% reepithelialization, whereas the control group only exhibited 4.9% reepithelialization 3 d after wounding (Fig. 9 D). Treatment with a combination of PTD-DBM and VPA induced expression of β-catenin, keratin 14, collagen I, endothelin-1, and PCNA much more effectively than treatment with EGF only (Fig. 9, C and E). The levels of phosphorylated ERK induced by the different treatment agents also correlated with the levels of β-catenin and wound-healing markers (Fig. 9, C and E). The combination treatment groups showed higher levels of collagen than other groups, including the EGF treatment group, as determined by collagen staining and hydroxyproline assay (Fig. 9, F and G). Quantitative TissueFAXS analyses also showed that β-catenin and the wound-healing markers were significantly induced in keratinocytes and fibroblasts by treatment with both PTD-DBM and VPA (Fig. 9 H). The more efficient improvement of cutaneous wound healing after combination treatment with PTD-DBM and VPA compared with after EGF treatment was convincingly shown by time course analyses during the wound-healing process (Fig. 10, A and B). However, the levels of c-Myc and cyclin D1 were not obviously changed in all treatment groups (Figs. 9 E and 10 C). Finally, we treated PTD-DBM on the wounds of Axin2LacZ/+ mice (Gay et al., 2013; Whyte et al., 2013) to monitor the effect of PTD-DBM on Wnt activation in vivo. Axin2-LacZ expression was significantly increased in dermal fibroblasts of wounds treated with PTD-DBM (Fig. 10 D).


The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing.

Lee SH, Kim MY, Kim HY, Lee YM, Kim H, Nam KA, Roh MR, Min do S, Chung KY, Choi KY - J. Exp. Med. (2015)

Co-treatment with PTD-DBM and VPA accelerates cutaneous wound healing and synergistically induces collagen production in mice. The wounded skin of 7-wk-old male C3H mice was treated daily with 100 µM PTD-DBM and/or 500 mM VPA or with 100 µM EGF for 11 d (n = 16 mice/group). (A) IHC staining of wounds treated with PTD-DBM was performed to detect β-catenin. Representative images are shown (n = 3 independent experiments). (B) Representative H&E-stained images of wounds at 12 d after wounding with the different treatments are shown (n = 3 independent experiments). The arrowheads represent the edges of the wounds. (C) Representative images of macroscopic wounds and H&E and IHC staining (n = 5 mice group) for β-catenin, keratin 14, collagen I, PCNA, or p-Erk in wounds 12 d after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary (n = 3 independent experiments). F, fibroblasts; K, keratinocytes. (D) Relative wound closure rates for wounds from mice treated with PTD-DBM and/or VPA or EGF were quantified as percent wound closure as shown. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (E) Western blot analyses of β-catenin, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, p-Erk, and Erk in wounds (n = 2 mice/group) 12 d after wounding were performed (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (F) Representative images of Masson’s trichrome, picrosirius red, and van Gieson staining of wounds (n = 5 mice/group) 12 d after wounding are shown (n = 3 independent experiments). Bars: (A, C, and F) 100 µm; (B) 500 µm. (G) Hydroxyproline levels were measured in wounds at 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 5 mice/group). (H) Quantitative TissueFAXS analyses of immunohistochemical staining shown in C are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
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fig9: Co-treatment with PTD-DBM and VPA accelerates cutaneous wound healing and synergistically induces collagen production in mice. The wounded skin of 7-wk-old male C3H mice was treated daily with 100 µM PTD-DBM and/or 500 mM VPA or with 100 µM EGF for 11 d (n = 16 mice/group). (A) IHC staining of wounds treated with PTD-DBM was performed to detect β-catenin. Representative images are shown (n = 3 independent experiments). (B) Representative H&E-stained images of wounds at 12 d after wounding with the different treatments are shown (n = 3 independent experiments). The arrowheads represent the edges of the wounds. (C) Representative images of macroscopic wounds and H&E and IHC staining (n = 5 mice group) for β-catenin, keratin 14, collagen I, PCNA, or p-Erk in wounds 12 d after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary (n = 3 independent experiments). F, fibroblasts; K, keratinocytes. (D) Relative wound closure rates for wounds from mice treated with PTD-DBM and/or VPA or EGF were quantified as percent wound closure as shown. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (E) Western blot analyses of β-catenin, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, p-Erk, and Erk in wounds (n = 2 mice/group) 12 d after wounding were performed (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (F) Representative images of Masson’s trichrome, picrosirius red, and van Gieson staining of wounds (n = 5 mice/group) 12 d after wounding are shown (n = 3 independent experiments). Bars: (A, C, and F) 100 µm; (B) 500 µm. (G) Hydroxyproline levels were measured in wounds at 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 5 mice/group). (H) Quantitative TissueFAXS analyses of immunohistochemical staining shown in C are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
Mentions: To investigate the effect of co-treatment with PTD-DBM and VPA on cutaneous wound healing in vivo, we created cutaneous wounds (diameter = 1.5 cm) on the dorsal skin of C3H mice and applied PTD-DBM and/or VPA topically to the wounds on a daily basis. As a positive control, one group of mice was treated with epidermal growth factor (EGF), a currently prescribed wound-healing agent (Kim et al., 2010b, 2014b). When the wounds were treated with PTD-DBM, we observed a strong increase in β-catenin expression (Fig. 9 A). Treatment with PTD-DBM or VPA accelerated cutaneous wound healing as efficiently as EGF (Fig. 9, B–D); however, combination treatment with PTD-DBM and VPA accelerated cutaneous wound healing much more efficiently than treatment with EGF alone (Fig. 9, B–D). The wounds were completely reepithelialized by combination treatment with PTD-DBM and VPA (Fig. 9, B and C) with reduction in inflammatory cells (Fig. 9 C). Notably, the combination treatment group exhibited 42.8% reepithelialization, whereas the control group only exhibited 4.9% reepithelialization 3 d after wounding (Fig. 9 D). Treatment with a combination of PTD-DBM and VPA induced expression of β-catenin, keratin 14, collagen I, endothelin-1, and PCNA much more effectively than treatment with EGF only (Fig. 9, C and E). The levels of phosphorylated ERK induced by the different treatment agents also correlated with the levels of β-catenin and wound-healing markers (Fig. 9, C and E). The combination treatment groups showed higher levels of collagen than other groups, including the EGF treatment group, as determined by collagen staining and hydroxyproline assay (Fig. 9, F and G). Quantitative TissueFAXS analyses also showed that β-catenin and the wound-healing markers were significantly induced in keratinocytes and fibroblasts by treatment with both PTD-DBM and VPA (Fig. 9 H). The more efficient improvement of cutaneous wound healing after combination treatment with PTD-DBM and VPA compared with after EGF treatment was convincingly shown by time course analyses during the wound-healing process (Fig. 10, A and B). However, the levels of c-Myc and cyclin D1 were not obviously changed in all treatment groups (Figs. 9 E and 10 C). Finally, we treated PTD-DBM on the wounds of Axin2LacZ/+ mice (Gay et al., 2013; Whyte et al., 2013) to monitor the effect of PTD-DBM on Wnt activation in vivo. Axin2-LacZ expression was significantly increased in dermal fibroblasts of wounds treated with PTD-DBM (Fig. 10 D).

Bottom Line: We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein.A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production.Protein transduction domain (PTD)-Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Translational Research Center for Protein Function Control; Department of Biotechnology, College of Life Science and Biotechnology; and Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, College of Medicine; Yonsei University, Seoul 120-749, South Korea Translational Research Center for Protein Function Control; Department of Biotechnology, College of Life Science and Biotechnology; and Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, College of Medicine; Yonsei University, Seoul 120-749, South Korea.

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