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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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CXXC5 levels are inversely associated with the levels of β-catenin and wound-healing markers in human acute wounds. Wounded tissues (n = 5 samples) were excised from wound margins at 0, 7, 28, and 84 d after surgery in melanoma patients and subjected to H&E staining and immunohistochemical analyses. 0 d represents normal healthy biopsy (control). (A) Representative H&E and confocal (IHC) images (n = 5 independent experiments) for β-catenin, CXXC5, keratin 14, collagen I, or PCNA in the wounds are shown. Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B and C, top) High magnification of representative IHC images for β-catenin, CXXC5, and the indicated wound-healing markers in keratinocytes (B) and fibroblasts (C) are shown. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 5 independent experiments) were performed in five random, representative fields per each patient sample. Horizontal lines represent the mean fluorescence intensities. Student’s t test was used (***, P < 0.0005). (D and E, top) Representative images of IHC staining performed with antibodies against β-catenin (D) or CXXC5 (E) and markers for fibroblasts (vimentin), endothelial cells (CD34), or macrophages (CD68). Yellow arrowheads indicate the co-expression of β-catenin or CXXC5 and markers for fibroblasts, endothelial cells, or macrophages, and the green arrowhead indicates the cells expressing only CD68. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 3 independent experiments) were performed in three random, representative fields (**, P < 0.005; ***, P < 0.0005). Means ± SD. Bars: (A–C) 100 µm; (D and E) 50 µm.
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fig1: CXXC5 levels are inversely associated with the levels of β-catenin and wound-healing markers in human acute wounds. Wounded tissues (n = 5 samples) were excised from wound margins at 0, 7, 28, and 84 d after surgery in melanoma patients and subjected to H&E staining and immunohistochemical analyses. 0 d represents normal healthy biopsy (control). (A) Representative H&E and confocal (IHC) images (n = 5 independent experiments) for β-catenin, CXXC5, keratin 14, collagen I, or PCNA in the wounds are shown. Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B and C, top) High magnification of representative IHC images for β-catenin, CXXC5, and the indicated wound-healing markers in keratinocytes (B) and fibroblasts (C) are shown. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 5 independent experiments) were performed in five random, representative fields per each patient sample. Horizontal lines represent the mean fluorescence intensities. Student’s t test was used (***, P < 0.0005). (D and E, top) Representative images of IHC staining performed with antibodies against β-catenin (D) or CXXC5 (E) and markers for fibroblasts (vimentin), endothelial cells (CD34), or macrophages (CD68). Yellow arrowheads indicate the co-expression of β-catenin or CXXC5 and markers for fibroblasts, endothelial cells, or macrophages, and the green arrowhead indicates the cells expressing only CD68. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 3 independent experiments) were performed in three random, representative fields (**, P < 0.005; ***, P < 0.0005). Means ± SD. Bars: (A–C) 100 µm; (D and E) 50 µm.

Mentions: To identify the involvement of CXXC5 in cutaneous wound healing in a human model, we investigated the status of CXXC5 in surgical wounds after the removal of melanomas. We monitored levels of β-catenin, CXXC5, and wound-healing markers during the healing process for up to 3 mo in five acute wound tissue specimens from patients using immunohistochemical analyses. Protein levels of β-catenin gradually increased and peaked 28 d after the operation (Fig. 1 A). The expression of keratin 14, collagen I, and proliferating cell nuclear antigen (PCNA) correlated directly with that of β-catenin during wound healing (Fig. 1 A). CXXC5 expression was reduced in surgical wounds; its expression patterns were opposite of those of β-catenin in the same wounded tissue area (Fig. 1 A). We also confirmed the expression of β-catenin and wound-healing markers in epidermal keratinocytes and dermal fibroblasts, respectively. The levels of β-catenin, keratin 14, and PCNA in keratinocytes were increased in wounds compared with normal skin (Fig. 1 B). Conversely, CXXC5 expression was decreased in the keratinocytes from wounded tissues (Fig. 1 B). The concurrent increases in β-catenin, collagen I, and PCNA in fibroblasts of wound tissue were also observed in the magnified images (Fig. 1 C). In contrast, CXXC5 expression was reduced in fibroblasts of wound tissues (Fig. 1 C). Quantitative TissueFAXS analysis of immunohistochemical staining also confirmed the inverse expression patterns of β-catenin and CXXC5 in wound tissues (Fig. 1, B and C). For comprehensive analysis of β-catenin and CXXC5 expression in human skin tissues, we performed co-immunostaining with those proteins and markers for fibroblasts (vimentin), endothelial cells (CD34), and macrophages (CD68). β-Catenin was primarily expressed in endothelial cells of normal skin, and its level was significantly increased in fibroblasts, endothelial cells, or macrophages in the dermis of wounds (Fig. 1 D). CXXC5 was mainly expressed in fibroblasts or endothelial cells in the dermis of human normal skin, and its level was significantly decreased in fibroblasts in the dermis of human wounds (Fig. 1 E). Overall, these data show an inverse relationship between CXXC5 protein expression and active Wnt/β-catenin signaling in wound tissues.


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)

CXXC5 levels are inversely associated with the levels of β-catenin and wound-healing markers in human acute wounds. Wounded tissues (n = 5 samples) were excised from wound margins at 0, 7, 28, and 84 d after surgery in melanoma patients and subjected to H&E staining and immunohistochemical analyses. 0 d represents normal healthy biopsy (control). (A) Representative H&E and confocal (IHC) images (n = 5 independent experiments) for β-catenin, CXXC5, keratin 14, collagen I, or PCNA in the wounds are shown. Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B and C, top) High magnification of representative IHC images for β-catenin, CXXC5, and the indicated wound-healing markers in keratinocytes (B) and fibroblasts (C) are shown. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 5 independent experiments) were performed in five random, representative fields per each patient sample. Horizontal lines represent the mean fluorescence intensities. Student’s t test was used (***, P < 0.0005). (D and E, top) Representative images of IHC staining performed with antibodies against β-catenin (D) or CXXC5 (E) and markers for fibroblasts (vimentin), endothelial cells (CD34), or macrophages (CD68). Yellow arrowheads indicate the co-expression of β-catenin or CXXC5 and markers for fibroblasts, endothelial cells, or macrophages, and the green arrowhead indicates the cells expressing only CD68. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 3 independent experiments) were performed in three random, representative fields (**, P < 0.005; ***, P < 0.0005). Means ± SD. Bars: (A–C) 100 µm; (D and E) 50 µm.
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fig1: CXXC5 levels are inversely associated with the levels of β-catenin and wound-healing markers in human acute wounds. Wounded tissues (n = 5 samples) were excised from wound margins at 0, 7, 28, and 84 d after surgery in melanoma patients and subjected to H&E staining and immunohistochemical analyses. 0 d represents normal healthy biopsy (control). (A) Representative H&E and confocal (IHC) images (n = 5 independent experiments) for β-catenin, CXXC5, keratin 14, collagen I, or PCNA in the wounds are shown. Dashed lines indicate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. (B and C, top) High magnification of representative IHC images for β-catenin, CXXC5, and the indicated wound-healing markers in keratinocytes (B) and fibroblasts (C) are shown. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 5 independent experiments) were performed in five random, representative fields per each patient sample. Horizontal lines represent the mean fluorescence intensities. Student’s t test was used (***, P < 0.0005). (D and E, top) Representative images of IHC staining performed with antibodies against β-catenin (D) or CXXC5 (E) and markers for fibroblasts (vimentin), endothelial cells (CD34), or macrophages (CD68). Yellow arrowheads indicate the co-expression of β-catenin or CXXC5 and markers for fibroblasts, endothelial cells, or macrophages, and the green arrowhead indicates the cells expressing only CD68. (bottom) Quantitative TissueFAXS analyses of IHC staining for β-catenin and CXXC5 (n = 3 independent experiments) were performed in three random, representative fields (**, P < 0.005; ***, P < 0.0005). Means ± SD. Bars: (A–C) 100 µm; (D and E) 50 µm.
Mentions: To identify the involvement of CXXC5 in cutaneous wound healing in a human model, we investigated the status of CXXC5 in surgical wounds after the removal of melanomas. We monitored levels of β-catenin, CXXC5, and wound-healing markers during the healing process for up to 3 mo in five acute wound tissue specimens from patients using immunohistochemical analyses. Protein levels of β-catenin gradually increased and peaked 28 d after the operation (Fig. 1 A). The expression of keratin 14, collagen I, and proliferating cell nuclear antigen (PCNA) correlated directly with that of β-catenin during wound healing (Fig. 1 A). CXXC5 expression was reduced in surgical wounds; its expression patterns were opposite of those of β-catenin in the same wounded tissue area (Fig. 1 A). We also confirmed the expression of β-catenin and wound-healing markers in epidermal keratinocytes and dermal fibroblasts, respectively. The levels of β-catenin, keratin 14, and PCNA in keratinocytes were increased in wounds compared with normal skin (Fig. 1 B). Conversely, CXXC5 expression was decreased in the keratinocytes from wounded tissues (Fig. 1 B). The concurrent increases in β-catenin, collagen I, and PCNA in fibroblasts of wound tissue were also observed in the magnified images (Fig. 1 C). In contrast, CXXC5 expression was reduced in fibroblasts of wound tissues (Fig. 1 C). Quantitative TissueFAXS analysis of immunohistochemical staining also confirmed the inverse expression patterns of β-catenin and CXXC5 in wound tissues (Fig. 1, B and C). For comprehensive analysis of β-catenin and CXXC5 expression in human skin tissues, we performed co-immunostaining with those proteins and markers for fibroblasts (vimentin), endothelial cells (CD34), and macrophages (CD68). β-Catenin was primarily expressed in endothelial cells of normal skin, and its level was significantly increased in fibroblasts, endothelial cells, or macrophages in the dermis of wounds (Fig. 1 D). CXXC5 was mainly expressed in fibroblasts or endothelial cells in the dermis of human normal skin, and its level was significantly decreased in fibroblasts in the dermis of human wounds (Fig. 1 E). Overall, these data show an inverse relationship between CXXC5 protein expression and active Wnt/β-catenin signaling in wound tissues.

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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Related in: MedlinePlus