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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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PTD-DBM induces β-catenin, wound-healing markers, and endothelin-1 during cutaneous wound healing. (A and B, top) Representative images of macroscopic wounds, H&E staining and IHC staining for β-catenin, keratin 14, collagen I, or PCNA in wounds (n = 3 mice group) 4 (A) and 8 d (B) after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (bottom) Mean intensity values are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) RT-PCR analysis was performed 12 d after wounding on wounds treated with or without 100 µM PTD-DBM to detect the mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath the blots. (D, top) Representative images of X-gal staining of wounded tissue from Axin2LacZ/+ mice (n = 3 mice/group) treated with 100 µM PTD-DBM for 11 d and magnified images in epidermal keratinocytes and dermal fibroblasts are shown. (bottom) Relative number of lacZ-positive cells is presented in keratinocytes and fibroblasts (***, P < 0.0005; n = 3 independent experiments). (E) Representative images of H&E staining and IHC staining for c-Myc or cyclin D1 in wounds (n = 3 mice/group) at 6 mo after wounding (top) and mean intensity values are presented for keratinocytes (left) and fibroblasts (right; bottom) are shown (n = 3 independent experiments). Bars: (A, B, and D) 100 µm; (E) 50 µm. Means ± SD.
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fig10: PTD-DBM induces β-catenin, wound-healing markers, and endothelin-1 during cutaneous wound healing. (A and B, top) Representative images of macroscopic wounds, H&E staining and IHC staining for β-catenin, keratin 14, collagen I, or PCNA in wounds (n = 3 mice group) 4 (A) and 8 d (B) after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (bottom) Mean intensity values are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) RT-PCR analysis was performed 12 d after wounding on wounds treated with or without 100 µM PTD-DBM to detect the mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath the blots. (D, top) Representative images of X-gal staining of wounded tissue from Axin2LacZ/+ mice (n = 3 mice/group) treated with 100 µM PTD-DBM for 11 d and magnified images in epidermal keratinocytes and dermal fibroblasts are shown. (bottom) Relative number of lacZ-positive cells is presented in keratinocytes and fibroblasts (***, P < 0.0005; n = 3 independent experiments). (E) Representative images of H&E staining and IHC staining for c-Myc or cyclin D1 in wounds (n = 3 mice/group) at 6 mo after wounding (top) and mean intensity values are presented for keratinocytes (left) and fibroblasts (right; bottom) are shown (n = 3 independent experiments). Bars: (A, B, and D) 100 µm; (E) 50 µm. 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)

PTD-DBM induces β-catenin, wound-healing markers, and endothelin-1 during cutaneous wound healing. (A and B, top) Representative images of macroscopic wounds, H&E staining and IHC staining for β-catenin, keratin 14, collagen I, or PCNA in wounds (n = 3 mice group) 4 (A) and 8 d (B) after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (bottom) Mean intensity values are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) RT-PCR analysis was performed 12 d after wounding on wounds treated with or without 100 µM PTD-DBM to detect the mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath the blots. (D, top) Representative images of X-gal staining of wounded tissue from Axin2LacZ/+ mice (n = 3 mice/group) treated with 100 µM PTD-DBM for 11 d and magnified images in epidermal keratinocytes and dermal fibroblasts are shown. (bottom) Relative number of lacZ-positive cells is presented in keratinocytes and fibroblasts (***, P < 0.0005; n = 3 independent experiments). (E) Representative images of H&E staining and IHC staining for c-Myc or cyclin D1 in wounds (n = 3 mice/group) at 6 mo after wounding (top) and mean intensity values are presented for keratinocytes (left) and fibroblasts (right; bottom) are shown (n = 3 independent experiments). Bars: (A, B, and D) 100 µm; (E) 50 µm. Means ± SD.
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fig10: PTD-DBM induces β-catenin, wound-healing markers, and endothelin-1 during cutaneous wound healing. (A and B, top) Representative images of macroscopic wounds, H&E staining and IHC staining for β-catenin, keratin 14, collagen I, or PCNA in wounds (n = 3 mice group) 4 (A) and 8 d (B) after wounding are shown. Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (bottom) Mean intensity values are presented for keratinocytes (left) and fibroblasts (right; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) RT-PCR analysis was performed 12 d after wounding on wounds treated with or without 100 µM PTD-DBM to detect the mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath the blots. (D, top) Representative images of X-gal staining of wounded tissue from Axin2LacZ/+ mice (n = 3 mice/group) treated with 100 µM PTD-DBM for 11 d and magnified images in epidermal keratinocytes and dermal fibroblasts are shown. (bottom) Relative number of lacZ-positive cells is presented in keratinocytes and fibroblasts (***, P < 0.0005; n = 3 independent experiments). (E) Representative images of H&E staining and IHC staining for c-Myc or cyclin D1 in wounds (n = 3 mice/group) at 6 mo after wounding (top) and mean intensity values are presented for keratinocytes (left) and fibroblasts (right; bottom) are shown (n = 3 independent experiments). Bars: (A, B, and D) 100 µm; (E) 50 µm. 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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