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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 collagen production in human dermal fibroblasts. (A) PTD-DBM is a synthetic peptide that includes a PTD for enhanced protein delivery, a linker for flexibility, DBM, and lysine conjugated with FITC for visualization. (B) Human dermal fibroblasts (n = 2–3 cells), which were treated with 2 or 10 µM PTD-DBM for 2 d, were analyzed using Western blotting to detect β-catenin, α-SMA, collagen I, endothelin-1, c-Myc, cyclin D1, and α-tubulin. Endothelin-1 and GAPDH expression were measured by RT-PCR analysis (n = 2 independent experiments). Relative densitometry values are shown below the blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). (C) Human dermal fibroblasts, treated with 2 or 10 µM PTD-DBM for 2 d, were subjected to ICC analyses to detect β-catenin or collagen I. Representative ICC images are shown (top), and mean intensity values are presented (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with PTD-DBM for 2 d after treatment with β-catenin siRNA (β-catenin si). Western blot analyses of WCLs were performed with antibodies against β-catenin, α-SMA, collagen I, endothelin-1, or α-tubulin (n = 2 independent experiments). (E) Human dermal fibroblasts, treated with 2 µM PTD-DBM and/or 10 µM bosentan for 2 d, were analyzed by Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). (D and E) Relative densitometry values are shown below the blots. (F) Wnt reporter activity (left), Col1a2 reporter activity (middle), and the concentration (right) of collagen in the supernatants of human dermal fibroblasts were measured after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (G) A collagen gel contraction assay after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β was performed and representative images are shown (left). Gel weight quantitation is shown (right; ***, P < 0.0005; n = 3 independent experiments). (H) An in vitro wound healing assay was performed after treatment with or without 2 or 10 µM PTD-DBM. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the wounds scratched at the start of the assay are indicated with dashed lines. (I) ICC staining for phalloidin (left) and α-SMA (right) was performed in human dermal fibroblasts treated with or without 2 or 10 µM PTD-DBM. Representative ICC images are shown (n = 3 independent experiments). Bars: (C and I) 50 µm; (H) 500 µm. Means ± SD.
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fig7: PTD-DBM induces collagen production in human dermal fibroblasts. (A) PTD-DBM is a synthetic peptide that includes a PTD for enhanced protein delivery, a linker for flexibility, DBM, and lysine conjugated with FITC for visualization. (B) Human dermal fibroblasts (n = 2–3 cells), which were treated with 2 or 10 µM PTD-DBM for 2 d, were analyzed using Western blotting to detect β-catenin, α-SMA, collagen I, endothelin-1, c-Myc, cyclin D1, and α-tubulin. Endothelin-1 and GAPDH expression were measured by RT-PCR analysis (n = 2 independent experiments). Relative densitometry values are shown below the blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). (C) Human dermal fibroblasts, treated with 2 or 10 µM PTD-DBM for 2 d, were subjected to ICC analyses to detect β-catenin or collagen I. Representative ICC images are shown (top), and mean intensity values are presented (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with PTD-DBM for 2 d after treatment with β-catenin siRNA (β-catenin si). Western blot analyses of WCLs were performed with antibodies against β-catenin, α-SMA, collagen I, endothelin-1, or α-tubulin (n = 2 independent experiments). (E) Human dermal fibroblasts, treated with 2 µM PTD-DBM and/or 10 µM bosentan for 2 d, were analyzed by Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). (D and E) Relative densitometry values are shown below the blots. (F) Wnt reporter activity (left), Col1a2 reporter activity (middle), and the concentration (right) of collagen in the supernatants of human dermal fibroblasts were measured after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (G) A collagen gel contraction assay after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β was performed and representative images are shown (left). Gel weight quantitation is shown (right; ***, P < 0.0005; n = 3 independent experiments). (H) An in vitro wound healing assay was performed after treatment with or without 2 or 10 µM PTD-DBM. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the wounds scratched at the start of the assay are indicated with dashed lines. (I) ICC staining for phalloidin (left) and α-SMA (right) was performed in human dermal fibroblasts treated with or without 2 or 10 µM PTD-DBM. Representative ICC images are shown (n = 3 independent experiments). Bars: (C and I) 50 µm; (H) 500 µm. Means ± SD.

Mentions: To activate the Wnt/β-catenin pathway by disrupting the negative feedback regulation of CXXC5, we used competitor peptides that interfere with the CXXC5-Dvl interaction. These synthetic peptides were composed of a PTD for delivery (Matsushita and Matsui, 2005), a linker for flexibility, DBM, and lysine conjugated with FITC for visualization (Fig. 7 A). The PTD-DBM peptide induced the expression of β-catenin, α-SMA, and collagen I in a concentration-dependent manner in human dermal fibroblasts (Fig. 7 B). PTD-DBM specifically increased both mRNA and protein levels of endothelin-1 but did not affect c-Myc and cyclin D1 (Fig. 7 B). ICC analyses also showed that PTD-DBM increased the nuclear translocation of β-catenin and induced collagen I production (Fig. 7 C). Transfection with β-catenin siRNA caused a reduction in the levels of β-catenin and endothelin-1 in human dermal fibroblasts (Fig. 7 D). Knockdown of β-catenin abolished the PTD-DBM–induced increases in α-SMA and collagen I (Fig. 7 D). Bosentan also blocked the increases of α-SMA and collagen I induced by PTD-DBM (Fig. 7 E). PTD-DBM induced the Wnt reporter activity, Col1a2 promoter activity, and collagen concentration in the supernatants in a dose-dependent manner (Fig. 7 F). The stimulatory effects of PTD-DBM were comparable with those of Wnt3a or TGF-β, which are key mediators of dermal fibrosis (Fig. 7 F; Sato, 2006; Carthy et al., 2011). PTD-DBM also enhanced the ability of fibroblasts to contract collagen gels (Fig. 7 G). We next investigated the effect of PTD-DBM on fibroblast migration after scratching of cell monolayers. 24 h after PTD-DBM treatment, the numbers of cells that migrated in the scratched area were significantly increased (Fig. 7 H). In addition, fibroblasts treated with PTD-DBM showed increased stress fibers and a thicker cortical network compared with control fibroblasts as determined by ICC analyses with anti-phalloidin or anti–α-SMA antibody (Fig. 7 I).


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 collagen production in human dermal fibroblasts. (A) PTD-DBM is a synthetic peptide that includes a PTD for enhanced protein delivery, a linker for flexibility, DBM, and lysine conjugated with FITC for visualization. (B) Human dermal fibroblasts (n = 2–3 cells), which were treated with 2 or 10 µM PTD-DBM for 2 d, were analyzed using Western blotting to detect β-catenin, α-SMA, collagen I, endothelin-1, c-Myc, cyclin D1, and α-tubulin. Endothelin-1 and GAPDH expression were measured by RT-PCR analysis (n = 2 independent experiments). Relative densitometry values are shown below the blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). (C) Human dermal fibroblasts, treated with 2 or 10 µM PTD-DBM for 2 d, were subjected to ICC analyses to detect β-catenin or collagen I. Representative ICC images are shown (top), and mean intensity values are presented (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with PTD-DBM for 2 d after treatment with β-catenin siRNA (β-catenin si). Western blot analyses of WCLs were performed with antibodies against β-catenin, α-SMA, collagen I, endothelin-1, or α-tubulin (n = 2 independent experiments). (E) Human dermal fibroblasts, treated with 2 µM PTD-DBM and/or 10 µM bosentan for 2 d, were analyzed by Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). (D and E) Relative densitometry values are shown below the blots. (F) Wnt reporter activity (left), Col1a2 reporter activity (middle), and the concentration (right) of collagen in the supernatants of human dermal fibroblasts were measured after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (G) A collagen gel contraction assay after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β was performed and representative images are shown (left). Gel weight quantitation is shown (right; ***, P < 0.0005; n = 3 independent experiments). (H) An in vitro wound healing assay was performed after treatment with or without 2 or 10 µM PTD-DBM. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the wounds scratched at the start of the assay are indicated with dashed lines. (I) ICC staining for phalloidin (left) and α-SMA (right) was performed in human dermal fibroblasts treated with or without 2 or 10 µM PTD-DBM. Representative ICC images are shown (n = 3 independent experiments). Bars: (C and I) 50 µm; (H) 500 µm. Means ± SD.
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fig7: PTD-DBM induces collagen production in human dermal fibroblasts. (A) PTD-DBM is a synthetic peptide that includes a PTD for enhanced protein delivery, a linker for flexibility, DBM, and lysine conjugated with FITC for visualization. (B) Human dermal fibroblasts (n = 2–3 cells), which were treated with 2 or 10 µM PTD-DBM for 2 d, were analyzed using Western blotting to detect β-catenin, α-SMA, collagen I, endothelin-1, c-Myc, cyclin D1, and α-tubulin. Endothelin-1 and GAPDH expression were measured by RT-PCR analysis (n = 2 independent experiments). Relative densitometry values are shown below the blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). (C) Human dermal fibroblasts, treated with 2 or 10 µM PTD-DBM for 2 d, were subjected to ICC analyses to detect β-catenin or collagen I. Representative ICC images are shown (top), and mean intensity values are presented (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with PTD-DBM for 2 d after treatment with β-catenin siRNA (β-catenin si). Western blot analyses of WCLs were performed with antibodies against β-catenin, α-SMA, collagen I, endothelin-1, or α-tubulin (n = 2 independent experiments). (E) Human dermal fibroblasts, treated with 2 µM PTD-DBM and/or 10 µM bosentan for 2 d, were analyzed by Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). (D and E) Relative densitometry values are shown below the blots. (F) Wnt reporter activity (left), Col1a2 reporter activity (middle), and the concentration (right) of collagen in the supernatants of human dermal fibroblasts were measured after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (G) A collagen gel contraction assay after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β was performed and representative images are shown (left). Gel weight quantitation is shown (right; ***, P < 0.0005; n = 3 independent experiments). (H) An in vitro wound healing assay was performed after treatment with or without 2 or 10 µM PTD-DBM. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the wounds scratched at the start of the assay are indicated with dashed lines. (I) ICC staining for phalloidin (left) and α-SMA (right) was performed in human dermal fibroblasts treated with or without 2 or 10 µM PTD-DBM. Representative ICC images are shown (n = 3 independent experiments). Bars: (C and I) 50 µm; (H) 500 µm. Means ± SD.
Mentions: To activate the Wnt/β-catenin pathway by disrupting the negative feedback regulation of CXXC5, we used competitor peptides that interfere with the CXXC5-Dvl interaction. These synthetic peptides were composed of a PTD for delivery (Matsushita and Matsui, 2005), a linker for flexibility, DBM, and lysine conjugated with FITC for visualization (Fig. 7 A). The PTD-DBM peptide induced the expression of β-catenin, α-SMA, and collagen I in a concentration-dependent manner in human dermal fibroblasts (Fig. 7 B). PTD-DBM specifically increased both mRNA and protein levels of endothelin-1 but did not affect c-Myc and cyclin D1 (Fig. 7 B). ICC analyses also showed that PTD-DBM increased the nuclear translocation of β-catenin and induced collagen I production (Fig. 7 C). Transfection with β-catenin siRNA caused a reduction in the levels of β-catenin and endothelin-1 in human dermal fibroblasts (Fig. 7 D). Knockdown of β-catenin abolished the PTD-DBM–induced increases in α-SMA and collagen I (Fig. 7 D). Bosentan also blocked the increases of α-SMA and collagen I induced by PTD-DBM (Fig. 7 E). PTD-DBM induced the Wnt reporter activity, Col1a2 promoter activity, and collagen concentration in the supernatants in a dose-dependent manner (Fig. 7 F). The stimulatory effects of PTD-DBM were comparable with those of Wnt3a or TGF-β, which are key mediators of dermal fibrosis (Fig. 7 F; Sato, 2006; Carthy et al., 2011). PTD-DBM also enhanced the ability of fibroblasts to contract collagen gels (Fig. 7 G). We next investigated the effect of PTD-DBM on fibroblast migration after scratching of cell monolayers. 24 h after PTD-DBM treatment, the numbers of cells that migrated in the scratched area were significantly increased (Fig. 7 H). In addition, fibroblasts treated with PTD-DBM showed increased stress fibers and a thicker cortical network compared with control fibroblasts as determined by ICC analyses with anti-phalloidin or anti–α-SMA antibody (Fig. 7 I).

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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