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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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Loss of CXXC5 accelerates cutaneous wound healing and enhances collagen production in mice. A full-thickness wound (diameter = 1.5 cm) was made on the backs of 7-wk-old CXXC5+/+ and CXXC5−/− mice (n = 16 mice/group). (A and B) Representative macroscopic images (A) and H&E staining (B) of wounded skin of CXXC5+/+ and CXXC5−/− mice at 12 d after wounding are shown (n = 3 independent experiments). Four continuously captured images were connected for analysis of the entire wound of CXXC5+/+ and CXXC5−/− mice. Arrowheads indicate the wound margins. (C) Relative healing of wounds of CXXC5+/+ and CXXC5−/− mice were measured at 1, 3, 5, 7, 9, and 12 d after wounding and shown as percent wound closure (**, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (D) Representative images of H&E, Masson’s trichrome, picrosirius red, and van Gieson staining and IHC showing β-catenin, CXXC5, keratin 14, collagen I, and PCNA of wound sections from CXXC5+/+ and CXXC5−/− mice (n = 5 mice/group) are presented (n = 3 independent experiments). Insets represent the high magnification images of a region of interest. (E) Hydroxyproline levels in the wounds of CXXC5+/+ and CXXC5−/− mice were measured at 12 d after wounding (**, P < 0.005; n = 5 mice/group). (F) Quantitative TissueFAXS analyses of immunohistochemical staining for β-catenin, keratin 14, and collagen I were performed in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (**, P < 0.005; n = 5 mice/group) at 12 d after wounding (n = 3 independent experiments). (G) RT-PCR analysis was performed with wound tissue obtained from CXXC5+/+ and CXXC5−/− mice at 12 d after wounding to detect mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath blot as ratios relative to the levels of GAPDH. (H) IHC staining for endothelin-1 in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (n = 3 mice/group; left) and quantitative TissueFAXS analyses of IHC staining for endothelin-1 (right) were performed (***, P < 0.0005; n = 3 independent experiments). (D and H) Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. Bars: (B) 500 µm; (D) 100 µm; (H) 50 µm. Means ± SD.
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fig4: Loss of CXXC5 accelerates cutaneous wound healing and enhances collagen production in mice. A full-thickness wound (diameter = 1.5 cm) was made on the backs of 7-wk-old CXXC5+/+ and CXXC5−/− mice (n = 16 mice/group). (A and B) Representative macroscopic images (A) and H&E staining (B) of wounded skin of CXXC5+/+ and CXXC5−/− mice at 12 d after wounding are shown (n = 3 independent experiments). Four continuously captured images were connected for analysis of the entire wound of CXXC5+/+ and CXXC5−/− mice. Arrowheads indicate the wound margins. (C) Relative healing of wounds of CXXC5+/+ and CXXC5−/− mice were measured at 1, 3, 5, 7, 9, and 12 d after wounding and shown as percent wound closure (**, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (D) Representative images of H&E, Masson’s trichrome, picrosirius red, and van Gieson staining and IHC showing β-catenin, CXXC5, keratin 14, collagen I, and PCNA of wound sections from CXXC5+/+ and CXXC5−/− mice (n = 5 mice/group) are presented (n = 3 independent experiments). Insets represent the high magnification images of a region of interest. (E) Hydroxyproline levels in the wounds of CXXC5+/+ and CXXC5−/− mice were measured at 12 d after wounding (**, P < 0.005; n = 5 mice/group). (F) Quantitative TissueFAXS analyses of immunohistochemical staining for β-catenin, keratin 14, and collagen I were performed in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (**, P < 0.005; n = 5 mice/group) at 12 d after wounding (n = 3 independent experiments). (G) RT-PCR analysis was performed with wound tissue obtained from CXXC5+/+ and CXXC5−/− mice at 12 d after wounding to detect mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath blot as ratios relative to the levels of GAPDH. (H) IHC staining for endothelin-1 in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (n = 3 mice/group; left) and quantitative TissueFAXS analyses of IHC staining for endothelin-1 (right) were performed (***, P < 0.0005; n = 3 independent experiments). (D and H) Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. Bars: (B) 500 µm; (D) 100 µm; (H) 50 µm. Means ± SD.

Mentions: To identify a role for CXXC5 in cutaneous wound healing and collagen production in vivo, we used CXXC5−/− mice, which were generated by inactivating exon 2 of CXXC5 (Kim et al., 2014a). We created full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice and then monitored expression of wound-healing markers. The sizes of wounds in CXXC5−/− mice were markedly reduced compared with those of wounds in CXXC5+/+ mice (Fig. 4 A). Histological analysis also showed that the distance between the wound edges was decreased in CXXC5−/− mice compared with that in CXXC5+/+ mice (Fig. 4 B). We also found that CXXC5−/− mice showed a marked acceleration in wound closure compared with CXXC5+/+ mice, as determined by measurement of wound diameters (Fig. 4 C). The rate of wound closure in CXXC5−/− mice was accelerated by 16 to approximately 32% compared with that in CXXC5+/+ mice during the proliferative phase of wound healing. The levels of collagen deposition were increased in CXXC5−/− mice, as detected by stainings with Masson’s trichrome, picrosirius red, and van Gieson (Fig. 4 D). Expression of keratin 14, collagen I, and PCNA was increased in conjunction with β-catenin expression in CXXC5−/− mice (Fig. 4 D). Quantitative analysis of collagen synthesis using a hydroxyproline assay showed enhanced collagen production after 12 d in wound tissues of CXXC5−/− mice compared with those of CXXC5+/+ mice (Fig. 4 E). In wounded skin sections, quantitative immunofluorescence TissueFAXS analyses revealed a more than fourfold increase in β-catenin protein levels in both keratinocytes and fibroblasts of CXXC5−/− mice compared with those in wild-type mice (Fig. 4 F). Keratin 14 levels in keratinocytes were increased 2.4-fold, and collagen I levels in fibroblasts were increased 6.3-fold in CXXC5−/− mice compared with wild-type mice (Fig. 4 F). To analyze the expression of Wnt/β-catenin pathway target genes, mRNA levels of c-Myc, cyclin D1, and endothelin-1 were monitored by RT-PCR analysis of wound tissues in CXXC5+/+ and CXXC5−/− mice. Expression levels of c-Myc and cyclin D1, markers of oncogenesis (Liao et al., 2007), were not significantly changed in wounds of CXXC5−/− mice as compared with wild-type mice, but expression of endothelin-1, a marker of collagen production (Rizvi et al., 1996), was significantly increased in the CXXC5−/− mice (Fig. 4 G). Moreover, endothelin-1 was specifically increased in fibroblasts of CXXC5−/− mice, as revealed by immunohistochemical analysis (Fig. 4 H). Therefore, CXXC5 specifically regulates Wnt/β-catenin signaling target genes involved in stimulation of wound healing without changing expression of genes involved in oncogenic transformation of cells.


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)

Loss of CXXC5 accelerates cutaneous wound healing and enhances collagen production in mice. A full-thickness wound (diameter = 1.5 cm) was made on the backs of 7-wk-old CXXC5+/+ and CXXC5−/− mice (n = 16 mice/group). (A and B) Representative macroscopic images (A) and H&E staining (B) of wounded skin of CXXC5+/+ and CXXC5−/− mice at 12 d after wounding are shown (n = 3 independent experiments). Four continuously captured images were connected for analysis of the entire wound of CXXC5+/+ and CXXC5−/− mice. Arrowheads indicate the wound margins. (C) Relative healing of wounds of CXXC5+/+ and CXXC5−/− mice were measured at 1, 3, 5, 7, 9, and 12 d after wounding and shown as percent wound closure (**, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (D) Representative images of H&E, Masson’s trichrome, picrosirius red, and van Gieson staining and IHC showing β-catenin, CXXC5, keratin 14, collagen I, and PCNA of wound sections from CXXC5+/+ and CXXC5−/− mice (n = 5 mice/group) are presented (n = 3 independent experiments). Insets represent the high magnification images of a region of interest. (E) Hydroxyproline levels in the wounds of CXXC5+/+ and CXXC5−/− mice were measured at 12 d after wounding (**, P < 0.005; n = 5 mice/group). (F) Quantitative TissueFAXS analyses of immunohistochemical staining for β-catenin, keratin 14, and collagen I were performed in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (**, P < 0.005; n = 5 mice/group) at 12 d after wounding (n = 3 independent experiments). (G) RT-PCR analysis was performed with wound tissue obtained from CXXC5+/+ and CXXC5−/− mice at 12 d after wounding to detect mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath blot as ratios relative to the levels of GAPDH. (H) IHC staining for endothelin-1 in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (n = 3 mice/group; left) and quantitative TissueFAXS analyses of IHC staining for endothelin-1 (right) were performed (***, P < 0.0005; n = 3 independent experiments). (D and H) Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. Bars: (B) 500 µm; (D) 100 µm; (H) 50 µm. Means ± SD.
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fig4: Loss of CXXC5 accelerates cutaneous wound healing and enhances collagen production in mice. A full-thickness wound (diameter = 1.5 cm) was made on the backs of 7-wk-old CXXC5+/+ and CXXC5−/− mice (n = 16 mice/group). (A and B) Representative macroscopic images (A) and H&E staining (B) of wounded skin of CXXC5+/+ and CXXC5−/− mice at 12 d after wounding are shown (n = 3 independent experiments). Four continuously captured images were connected for analysis of the entire wound of CXXC5+/+ and CXXC5−/− mice. Arrowheads indicate the wound margins. (C) Relative healing of wounds of CXXC5+/+ and CXXC5−/− mice were measured at 1, 3, 5, 7, 9, and 12 d after wounding and shown as percent wound closure (**, P < 0.005; ***, P < 0.0005; n = 16 mice/group). (D) Representative images of H&E, Masson’s trichrome, picrosirius red, and van Gieson staining and IHC showing β-catenin, CXXC5, keratin 14, collagen I, and PCNA of wound sections from CXXC5+/+ and CXXC5−/− mice (n = 5 mice/group) are presented (n = 3 independent experiments). Insets represent the high magnification images of a region of interest. (E) Hydroxyproline levels in the wounds of CXXC5+/+ and CXXC5−/− mice were measured at 12 d after wounding (**, P < 0.005; n = 5 mice/group). (F) Quantitative TissueFAXS analyses of immunohistochemical staining for β-catenin, keratin 14, and collagen I were performed in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (**, P < 0.005; n = 5 mice/group) at 12 d after wounding (n = 3 independent experiments). (G) RT-PCR analysis was performed with wound tissue obtained from CXXC5+/+ and CXXC5−/− mice at 12 d after wounding to detect mRNA levels of CXXC5, endothelin-1, c-Myc, cyclin D1, and GAPDH (n = 2 mice/group). Relative densitometry values are shown underneath blot as ratios relative to the levels of GAPDH. (H) IHC staining for endothelin-1 in keratinocytes (left) and fibroblasts (right) of wounds of CXXC5+/+ and CXXC5−/− mice (n = 3 mice/group; left) and quantitative TissueFAXS analyses of IHC staining for endothelin-1 (right) were performed (***, P < 0.0005; n = 3 independent experiments). (D and H) Dashed lines demarcate the epidermal–dermal boundary. F, fibroblasts; K, keratinocytes. Bars: (B) 500 µm; (D) 100 µm; (H) 50 µm. Means ± SD.
Mentions: To identify a role for CXXC5 in cutaneous wound healing and collagen production in vivo, we used CXXC5−/− mice, which were generated by inactivating exon 2 of CXXC5 (Kim et al., 2014a). We created full-thickness wounds (diameter = 1.5 cm) on the backs of CXXC5+/+ and CXXC5−/− mice and then monitored expression of wound-healing markers. The sizes of wounds in CXXC5−/− mice were markedly reduced compared with those of wounds in CXXC5+/+ mice (Fig. 4 A). Histological analysis also showed that the distance between the wound edges was decreased in CXXC5−/− mice compared with that in CXXC5+/+ mice (Fig. 4 B). We also found that CXXC5−/− mice showed a marked acceleration in wound closure compared with CXXC5+/+ mice, as determined by measurement of wound diameters (Fig. 4 C). The rate of wound closure in CXXC5−/− mice was accelerated by 16 to approximately 32% compared with that in CXXC5+/+ mice during the proliferative phase of wound healing. The levels of collagen deposition were increased in CXXC5−/− mice, as detected by stainings with Masson’s trichrome, picrosirius red, and van Gieson (Fig. 4 D). Expression of keratin 14, collagen I, and PCNA was increased in conjunction with β-catenin expression in CXXC5−/− mice (Fig. 4 D). Quantitative analysis of collagen synthesis using a hydroxyproline assay showed enhanced collagen production after 12 d in wound tissues of CXXC5−/− mice compared with those of CXXC5+/+ mice (Fig. 4 E). In wounded skin sections, quantitative immunofluorescence TissueFAXS analyses revealed a more than fourfold increase in β-catenin protein levels in both keratinocytes and fibroblasts of CXXC5−/− mice compared with those in wild-type mice (Fig. 4 F). Keratin 14 levels in keratinocytes were increased 2.4-fold, and collagen I levels in fibroblasts were increased 6.3-fold in CXXC5−/− mice compared with wild-type mice (Fig. 4 F). To analyze the expression of Wnt/β-catenin pathway target genes, mRNA levels of c-Myc, cyclin D1, and endothelin-1 were monitored by RT-PCR analysis of wound tissues in CXXC5+/+ and CXXC5−/− mice. Expression levels of c-Myc and cyclin D1, markers of oncogenesis (Liao et al., 2007), were not significantly changed in wounds of CXXC5−/− mice as compared with wild-type mice, but expression of endothelin-1, a marker of collagen production (Rizvi et al., 1996), was significantly increased in the CXXC5−/− mice (Fig. 4 G). Moreover, endothelin-1 was specifically increased in fibroblasts of CXXC5−/− mice, as revealed by immunohistochemical analysis (Fig. 4 H). Therefore, CXXC5 specifically regulates Wnt/β-catenin signaling target genes involved in stimulation of wound healing without changing expression of genes involved in oncogenic transformation of cells.

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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