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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.

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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VPA treatment further accelerates cutaneous wound healing in CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, 500 mM VPA was applied topically to the wounds daily (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E, and IHC staining showing expression of β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA treatment 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B) Relative wound closure rates describe the effects of VPA on wound healing in CXXC5+/+ and CXXC5−/− mice. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 10 mice/group). (C) Western blot analyses of β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, and Erk were performed in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA (n = 2 mice/group) 12 d after wounding (n = 2 independent experiments). Relative densitometry values are shown below blots. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). (A and D) Bars, 100 µm. (E) Hydroxyproline levels in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 4 mice/group). (F) Quantitative TissueFAXS analyses of IHC staining shown in A were performed in keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
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fig6: VPA treatment further accelerates cutaneous wound healing in CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, 500 mM VPA was applied topically to the wounds daily (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E, and IHC staining showing expression of β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA treatment 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B) Relative wound closure rates describe the effects of VPA on wound healing in CXXC5+/+ and CXXC5−/− mice. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 10 mice/group). (C) Western blot analyses of β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, and Erk were performed in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA (n = 2 mice/group) 12 d after wounding (n = 2 independent experiments). Relative densitometry values are shown below blots. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). (A and D) Bars, 100 µm. (E) Hydroxyproline levels in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 4 mice/group). (F) Quantitative TissueFAXS analyses of IHC staining shown in A were performed in keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.

Mentions: To confirm that CXXC5 functioned as a negative feedback regulator of the Wnt/β-catenin pathway in vivo, we treated the wounds of CXXC5+/+ and CXXC5−/− mice with VPA, a Wnt/β-catenin pathway activator (Gould and Manji, 2002; Gould et al., 2004). Interestingly, CXXC5−/− mice treated with VPA displayed markedly accelerated wound healing (Fig. 6, A and B). Moreover, expression levels of β-catenin, keratin 14, collagen I, α-SMA, and PCNA were synergistically increased in VPA-treated CXXC5−/− mice, as determined by immunohistochemical and Western blot analyses (Fig. 6, A and C); however, VPA-treated CXXC5−/− mice did not show significant changes in expression of c-Myc and cyclin D1 (Fig. 6 C). Stainings with Masson’s trichrome, picrosirius red, and van Gieson in combination with results of the hydroxyproline assay also confirmed that collagen expression was synergistically increased in VPA-treated CXXC5−/− mice (Fig. 6, D and E). Quantitative TissueFAXS analyses also showed that β-catenin and wound-healing markers were significantly increased in keratinocytes and fibroblasts of VPA-treated CXXC5−/− mouse wounds (Fig. 6 F). Overall, blockade of CXXC5 function in concert with activation of the Wnt/β-catenin pathway is a potential approach for the development of drugs to enhance wound healing.


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)

VPA treatment further accelerates cutaneous wound healing in CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, 500 mM VPA was applied topically to the wounds daily (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E, and IHC staining showing expression of β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA treatment 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B) Relative wound closure rates describe the effects of VPA on wound healing in CXXC5+/+ and CXXC5−/− mice. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 10 mice/group). (C) Western blot analyses of β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, and Erk were performed in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA (n = 2 mice/group) 12 d after wounding (n = 2 independent experiments). Relative densitometry values are shown below blots. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). (A and D) Bars, 100 µm. (E) Hydroxyproline levels in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 4 mice/group). (F) Quantitative TissueFAXS analyses of IHC staining shown in A were performed in keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
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fig6: VPA treatment further accelerates cutaneous wound healing in CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, 500 mM VPA was applied topically to the wounds daily (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E, and IHC staining showing expression of β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA treatment 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B) Relative wound closure rates describe the effects of VPA on wound healing in CXXC5+/+ and CXXC5−/− mice. Wound sizes were measured at 1, 3, 5, 7, 9, and 12 d after wounding (**, P < 0.005; ***, P < 0.0005; n = 10 mice/group). (C) Western blot analyses of β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, c-Myc, cyclin D1, and Erk were performed in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA (n = 2 mice/group) 12 d after wounding (n = 2 independent experiments). Relative densitometry values are shown below blots. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding (n = 4 mice/group) are shown (n = 3 independent experiments). (A and D) Bars, 100 µm. (E) Hydroxyproline levels in wounds of CXXC5+/+ and CXXC5−/− mice treated with or without VPA 12 d after wounding are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 4 mice/group). (F) Quantitative TissueFAXS analyses of IHC staining shown in A were performed in keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Means ± SD.
Mentions: To confirm that CXXC5 functioned as a negative feedback regulator of the Wnt/β-catenin pathway in vivo, we treated the wounds of CXXC5+/+ and CXXC5−/− mice with VPA, a Wnt/β-catenin pathway activator (Gould and Manji, 2002; Gould et al., 2004). Interestingly, CXXC5−/− mice treated with VPA displayed markedly accelerated wound healing (Fig. 6, A and B). Moreover, expression levels of β-catenin, keratin 14, collagen I, α-SMA, and PCNA were synergistically increased in VPA-treated CXXC5−/− mice, as determined by immunohistochemical and Western blot analyses (Fig. 6, A and C); however, VPA-treated CXXC5−/− mice did not show significant changes in expression of c-Myc and cyclin D1 (Fig. 6 C). Stainings with Masson’s trichrome, picrosirius red, and van Gieson in combination with results of the hydroxyproline assay also confirmed that collagen expression was synergistically increased in VPA-treated CXXC5−/− mice (Fig. 6, D and E). Quantitative TissueFAXS analyses also showed that β-catenin and wound-healing markers were significantly increased in keratinocytes and fibroblasts of VPA-treated CXXC5−/− mouse wounds (Fig. 6 F). Overall, blockade of CXXC5 function in concert with activation of the Wnt/β-catenin pathway is a potential approach for the development of drugs to enhance wound healing.

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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