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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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Combination treatment with PTD-DBM and Wnt3a synergistically induces collagen production in human dermal fibroblasts. (A) Human dermal fibroblasts (n = 2–3 cells) were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a for 2 d. (B) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 1 mM VPA for 2 d. (A and B) WCLs were subjected to Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (C) Representative images of ICC staining of β-catenin (top) in human dermal fibroblasts, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a, and mean intensity value (bottom) are shown (*, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a after transfection with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. The cell lysates were used for immunoprecipitation with anti-Myc antibody (n = 2 independent experiments). Relative densitometry values are shown below the blots. (E) Wnt reporter assay, Col1a2 reporter assay, and Sircol collagen assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a were measured (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (F) A collagen gel contraction assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed, and representative images are shown (left). Gel weight quantitation is also shown (right; ***, P < 0.0005; n = 3 independent experiments). (G) An in vitro wound healing assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the initially scratched wounds are indicated with dashed lines. (H) ICC staining of human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed to detect phalloidin (left) and α-SMA (right; n = 3 independent experiments). (I) Representative images of ICC staining of β-catenin (top) are shown in HaCaT keratinocytes, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a. Mean intensity values are shown below (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Bars: (C, H, and I) 50 µm; (G) 500 µm. Means ± SD.
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fig8: Combination treatment with PTD-DBM and Wnt3a synergistically induces collagen production in human dermal fibroblasts. (A) Human dermal fibroblasts (n = 2–3 cells) were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a for 2 d. (B) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 1 mM VPA for 2 d. (A and B) WCLs were subjected to Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (C) Representative images of ICC staining of β-catenin (top) in human dermal fibroblasts, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a, and mean intensity value (bottom) are shown (*, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a after transfection with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. The cell lysates were used for immunoprecipitation with anti-Myc antibody (n = 2 independent experiments). Relative densitometry values are shown below the blots. (E) Wnt reporter assay, Col1a2 reporter assay, and Sircol collagen assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a were measured (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (F) A collagen gel contraction assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed, and representative images are shown (left). Gel weight quantitation is also shown (right; ***, P < 0.0005; n = 3 independent experiments). (G) An in vitro wound healing assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the initially scratched wounds are indicated with dashed lines. (H) ICC staining of human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed to detect phalloidin (left) and α-SMA (right; n = 3 independent experiments). (I) Representative images of ICC staining of β-catenin (top) are shown in HaCaT keratinocytes, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a. Mean intensity values are shown below (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Bars: (C, H, and I) 50 µm; (G) 500 µm. Means ± SD.

Mentions: The role of CXXC5 as a negative feedback regulator of the Wnt/β-catenin pathway was confirmed by the synergistic increase in β-catenin levels after co-treatment with PTD-DBM and either Wnt3a or VPA (Fig. 8, A and B). Wnt3a or PTD-DBM treatment alone induced α-SMA and collagen I, but combination treatment induced the greatest fibrotic effect in human dermal fibroblasts (Fig. 8 A). Although VPA marginally increased the levels of α-SMA and collagen I, co-treatment with PTD-DBM and VPA also synergistically increased the expression of these markers (Fig. 8 B). We also observed synergistic increases of β-catenin in the nucleus and cytosol via ICC analysis after co-treatment with PTD-DBM and Wnt3a (Fig. 8 C). Moreover, PTD-DBM significantly disrupted the Wnt3a-induced interaction between CXXC5 and Dvl-1 (Fig. 8 D). The synergistic increases in Wnt reporter activity, Col1a2 reporter activity, and collagen concentration were also observed in fibroblasts treated with both PTD-DBM and Wnt3a (Fig. 8 E). Furthermore, co-treatment with PTD-DBM and Wnt3a significantly induced contraction of collagen gels (Fig. 8 F). Cell migration and stress fiber formation were also synergistically induced by treatment with a combination of PTD-DBM and Wnt3a (Fig. 8, G and H). Co-treatment with PTD-DBM and Wnt3a also significantly increased β-catenin in HaCaT keratinocytes (Fig. 8 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)

Combination treatment with PTD-DBM and Wnt3a synergistically induces collagen production in human dermal fibroblasts. (A) Human dermal fibroblasts (n = 2–3 cells) were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a for 2 d. (B) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 1 mM VPA for 2 d. (A and B) WCLs were subjected to Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (C) Representative images of ICC staining of β-catenin (top) in human dermal fibroblasts, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a, and mean intensity value (bottom) are shown (*, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a after transfection with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. The cell lysates were used for immunoprecipitation with anti-Myc antibody (n = 2 independent experiments). Relative densitometry values are shown below the blots. (E) Wnt reporter assay, Col1a2 reporter assay, and Sircol collagen assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a were measured (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (F) A collagen gel contraction assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed, and representative images are shown (left). Gel weight quantitation is also shown (right; ***, P < 0.0005; n = 3 independent experiments). (G) An in vitro wound healing assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the initially scratched wounds are indicated with dashed lines. (H) ICC staining of human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed to detect phalloidin (left) and α-SMA (right; n = 3 independent experiments). (I) Representative images of ICC staining of β-catenin (top) are shown in HaCaT keratinocytes, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a. Mean intensity values are shown below (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Bars: (C, H, and I) 50 µm; (G) 500 µm. Means ± SD.
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fig8: Combination treatment with PTD-DBM and Wnt3a synergistically induces collagen production in human dermal fibroblasts. (A) Human dermal fibroblasts (n = 2–3 cells) were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a for 2 d. (B) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 1 mM VPA for 2 d. (A and B) WCLs were subjected to Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (C) Representative images of ICC staining of β-catenin (top) in human dermal fibroblasts, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a, and mean intensity value (bottom) are shown (*, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a after transfection with pcDNA3.1 or pcDNA3.1-CXXC5-Myc. The cell lysates were used for immunoprecipitation with anti-Myc antibody (n = 2 independent experiments). Relative densitometry values are shown below the blots. (E) Wnt reporter assay, Col1a2 reporter assay, and Sircol collagen assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a were measured (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (F) A collagen gel contraction assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed, and representative images are shown (left). Gel weight quantitation is also shown (right; ***, P < 0.0005; n = 3 independent experiments). (G) An in vitro wound healing assay in human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the initially scratched wounds are indicated with dashed lines. (H) ICC staining of human dermal fibroblasts treated with PTD-DBM and/or Wnt3a was performed to detect phalloidin (left) and α-SMA (right; n = 3 independent experiments). (I) Representative images of ICC staining of β-catenin (top) are shown in HaCaT keratinocytes, which were treated with 2 µM PTD-DBM and/or 50 ng/ml Wnt3a. Mean intensity values are shown below (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Bars: (C, H, and I) 50 µm; (G) 500 µm. Means ± SD.
Mentions: The role of CXXC5 as a negative feedback regulator of the Wnt/β-catenin pathway was confirmed by the synergistic increase in β-catenin levels after co-treatment with PTD-DBM and either Wnt3a or VPA (Fig. 8, A and B). Wnt3a or PTD-DBM treatment alone induced α-SMA and collagen I, but combination treatment induced the greatest fibrotic effect in human dermal fibroblasts (Fig. 8 A). Although VPA marginally increased the levels of α-SMA and collagen I, co-treatment with PTD-DBM and VPA also synergistically increased the expression of these markers (Fig. 8 B). We also observed synergistic increases of β-catenin in the nucleus and cytosol via ICC analysis after co-treatment with PTD-DBM and Wnt3a (Fig. 8 C). Moreover, PTD-DBM significantly disrupted the Wnt3a-induced interaction between CXXC5 and Dvl-1 (Fig. 8 D). The synergistic increases in Wnt reporter activity, Col1a2 reporter activity, and collagen concentration were also observed in fibroblasts treated with both PTD-DBM and Wnt3a (Fig. 8 E). Furthermore, co-treatment with PTD-DBM and Wnt3a significantly induced contraction of collagen gels (Fig. 8 F). Cell migration and stress fiber formation were also synergistically induced by treatment with a combination of PTD-DBM and Wnt3a (Fig. 8, G and H). Co-treatment with PTD-DBM and Wnt3a also significantly increased β-catenin in HaCaT keratinocytes (Fig. 8 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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