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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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Bosentan alleviates the accelerated wound-healing phenotype of CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, the mice were orally administered 100 mg/kg/d bosentan monohydrate daily for 11 d (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E staining, and IHC staining for β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated or untreated with bosentan at 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 after bosentan treatment in CXXC5+/+ and CXXC5−/− mice are 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 = 10 mice/group). (C) Western blot analyses of wound tissue lysates from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 12 d after wounding (n = 2 mice/group) were performed to detect β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, and Erk (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wound tissues obtained from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 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 bosentan 12 d after wounding are shown (*, P < 0.05; ***, 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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fig5: Bosentan alleviates the accelerated wound-healing phenotype of CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, the mice were orally administered 100 mg/kg/d bosentan monohydrate daily for 11 d (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E staining, and IHC staining for β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated or untreated with bosentan at 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 after bosentan treatment in CXXC5+/+ and CXXC5−/− mice are 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 = 10 mice/group). (C) Western blot analyses of wound tissue lysates from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 12 d after wounding (n = 2 mice/group) were performed to detect β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, and Erk (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wound tissues obtained from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 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 bosentan 12 d after wounding are shown (*, P < 0.05; ***, 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: Endothelin-1 is a direct target of the Wnt/β-catenin pathway in NIH3T3 fibroblasts (Chen et al., 2007) and is associated with wound healing and fibrosis (Rizvi et al., 1996). To investigate whether the accelerated wound-healing phenotype in CXXC5−/− mice was dependent on endothelin-1, CXXC5+/+ and CXXC5−/− mice were fed bosentan on a daily basis for 11 d. Bosentan reversed the accelerated wound-healing phenotype observed in CXXC5−/− mice and decreased levels of β-catenin, keratin 14, collagen I, and PCNA (Fig. 5, A and B). Western blot analyses also showed that bosentan reduced β-catenin and wound-healing markers in CXXC5−/− mice through an endothelin-1–dependent mechanism (Fig. 5 C). Bosentan also decreased collagen deposition in CXXC5−/− mice, as determined by Masson’s trichrome, picrosirius red, and van Gieson staining and hydroxyproline analyses (Fig. 5, D and E). Quantitative TissueFAXS analyses also showed that induction of β-catenin and wound-healing markers (keratin 14 and collagen I) in keratinocytes and fibroblasts of CXXC5−/− mouse wounds was reversed by bosentan (Fig. 5 F).


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)

Bosentan alleviates the accelerated wound-healing phenotype of CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, the mice were orally administered 100 mg/kg/d bosentan monohydrate daily for 11 d (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E staining, and IHC staining for β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated or untreated with bosentan at 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 after bosentan treatment in CXXC5+/+ and CXXC5−/− mice are 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 = 10 mice/group). (C) Western blot analyses of wound tissue lysates from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 12 d after wounding (n = 2 mice/group) were performed to detect β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, and Erk (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wound tissues obtained from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 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 bosentan 12 d after wounding are shown (*, P < 0.05; ***, 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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fig5: Bosentan alleviates the accelerated wound-healing phenotype of CXXC5−/− mice. After generation of full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice, the mice were orally administered 100 mg/kg/d bosentan monohydrate daily for 11 d (n = 10 mice/group). (A) Representative images of macroscopic wounds, H&E staining, and IHC staining for β-catenin, CXXC5, keratin 14, collagen I, and PCNA in the wounds of CXXC5+/+ and CXXC5−/− mice treated or untreated with bosentan at 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 after bosentan treatment in CXXC5+/+ and CXXC5−/− mice are 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 = 10 mice/group). (C) Western blot analyses of wound tissue lysates from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 12 d after wounding (n = 2 mice/group) were performed to detect β-catenin, CXXC5, keratin 14, α-SMA, collagen I, PCNA, endothelin-1, and Erk (n = 2 independent experiments). Relative densitometric ratios of each protein to Erk protein are shown. (D) Representative images of Masson’s trichrome, picrosirius red, or van Gieson staining of wound tissues obtained from CXXC5+/+ and CXXC5−/− mice treated with or without bosentan 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 bosentan 12 d after wounding are shown (*, P < 0.05; ***, 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: Endothelin-1 is a direct target of the Wnt/β-catenin pathway in NIH3T3 fibroblasts (Chen et al., 2007) and is associated with wound healing and fibrosis (Rizvi et al., 1996). To investigate whether the accelerated wound-healing phenotype in CXXC5−/− mice was dependent on endothelin-1, CXXC5+/+ and CXXC5−/− mice were fed bosentan on a daily basis for 11 d. Bosentan reversed the accelerated wound-healing phenotype observed in CXXC5−/− mice and decreased levels of β-catenin, keratin 14, collagen I, and PCNA (Fig. 5, A and B). Western blot analyses also showed that bosentan reduced β-catenin and wound-healing markers in CXXC5−/− mice through an endothelin-1–dependent mechanism (Fig. 5 C). Bosentan also decreased collagen deposition in CXXC5−/− mice, as determined by Masson’s trichrome, picrosirius red, and van Gieson staining and hydroxyproline analyses (Fig. 5, D and E). Quantitative TissueFAXS analyses also showed that induction of β-catenin and wound-healing markers (keratin 14 and collagen I) in keratinocytes and fibroblasts of CXXC5−/− mouse wounds was reversed by bosentan (Fig. 5 F).

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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