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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 attenuates myofibroblast differentiation and collagen production in human dermal fibroblasts. (A–D) Human dermal fibroblasts (n = 2–3 cells) were transfected with pcDNA3.0-CXXC5-Flag (A) at the indicated concentration, 100 nM CXXC5 siRNA (B and C), and pcDNA3.1-CXXC5-Myc and pcDNA3.1-CXXC5ΔDBM-Myc (D), together with corresponding control plasmid (Con) or control siRNA (Con si). The cells transfected with CXXC5 siRNA (C) or control siRNA were treated with or without 10 µM bosentan. The cells were further cultured for 2 d and harvested, and whole cell lysates (WCLs) were subjected to Western blot analyses to detect the protein levels of β-catenin, endothelin-1, α-SMA, and collagen I (n = 2 independent experiments). Relative densitometric ratios of each protein to loading control (α-tubulin or lamin A/C) are shown. (E) Human dermal fibroblasts were transfected with pEGFP-c3 or GFP-CXXC5 and further cultured for 2 d. The cells were immunocytochemically stained for collagen I. Representative confocal (ICC) images are shown (left), and mean intensity quantitation in GFP-positive cells was performed (right; ***, P < 0.0005; n = 3 independent experiments). Red arrows indicate the expression of collagen I, and green arrows indicate the expression of GFP. (F) Wnt luciferase reporter activity (left), Col1a2 luciferase reporter activity (middle), and collagen concentration (right) in the supernatant of human dermal fibroblasts were measured after transfection with pcDNA3.0 or pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Values are presented relative to control (**, P < 0.005; ***, P < 0.0005). (G) Contraction of collagen gels was monitored by measuring gel weight after transfection with pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Representative images (left) and quantitation (right) are shown (***, P < 0.0005). (H) The cells transfected with pcDNA3.0 or pcDNA3.0-CXXC5-Flag were scratched with sterile pipette tips and incubated in medium containing 5% serum for 36 h. Cells were then fixed with 4% PFA and stained with crystal violet. Representative microscopy images are shown (left), and cell migration was measured by counting migrating cells (right; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Dashed lines represent the widths of the initially scratched wounds. (I) ICC staining of human dermal fibroblasts transfected with GFP or GFP-CXXC5 was performed to detect phalloidin (left) and α-SMA (right). Representative ICC images are shown (n = 3 independent experiments). Means ± SD. Bars: (E and I) 50 µm; (H) 500 µm.
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fig2: CXXC5 attenuates myofibroblast differentiation and collagen production in human dermal fibroblasts. (A–D) Human dermal fibroblasts (n = 2–3 cells) were transfected with pcDNA3.0-CXXC5-Flag (A) at the indicated concentration, 100 nM CXXC5 siRNA (B and C), and pcDNA3.1-CXXC5-Myc and pcDNA3.1-CXXC5ΔDBM-Myc (D), together with corresponding control plasmid (Con) or control siRNA (Con si). The cells transfected with CXXC5 siRNA (C) or control siRNA were treated with or without 10 µM bosentan. The cells were further cultured for 2 d and harvested, and whole cell lysates (WCLs) were subjected to Western blot analyses to detect the protein levels of β-catenin, endothelin-1, α-SMA, and collagen I (n = 2 independent experiments). Relative densitometric ratios of each protein to loading control (α-tubulin or lamin A/C) are shown. (E) Human dermal fibroblasts were transfected with pEGFP-c3 or GFP-CXXC5 and further cultured for 2 d. The cells were immunocytochemically stained for collagen I. Representative confocal (ICC) images are shown (left), and mean intensity quantitation in GFP-positive cells was performed (right; ***, P < 0.0005; n = 3 independent experiments). Red arrows indicate the expression of collagen I, and green arrows indicate the expression of GFP. (F) Wnt luciferase reporter activity (left), Col1a2 luciferase reporter activity (middle), and collagen concentration (right) in the supernatant of human dermal fibroblasts were measured after transfection with pcDNA3.0 or pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Values are presented relative to control (**, P < 0.005; ***, P < 0.0005). (G) Contraction of collagen gels was monitored by measuring gel weight after transfection with pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Representative images (left) and quantitation (right) are shown (***, P < 0.0005). (H) The cells transfected with pcDNA3.0 or pcDNA3.0-CXXC5-Flag were scratched with sterile pipette tips and incubated in medium containing 5% serum for 36 h. Cells were then fixed with 4% PFA and stained with crystal violet. Representative microscopy images are shown (left), and cell migration was measured by counting migrating cells (right; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Dashed lines represent the widths of the initially scratched wounds. (I) ICC staining of human dermal fibroblasts transfected with GFP or GFP-CXXC5 was performed to detect phalloidin (left) and α-SMA (right). Representative ICC images are shown (n = 3 independent experiments). Means ± SD. Bars: (E and I) 50 µm; (H) 500 µm.

Mentions: CXXC5 is known as a negative regulator of the Wnt/β-catenin pathway (Kim et al., 2010a). Considering the relationship between the Wnt/β-catenin pathway and fibrosis (Wei et al., 2011; Akhmetshina et al., 2012), we examined the effect of CXXC5 transfection on myofibroblast differentiation and collagen production in human dermal fibroblasts. Transfection with CXXC5 markedly inhibited β-catenin expression in a dose-dependent manner (Fig. 2 A). The levels of α-smooth muscle actin (α-SMA) and collagen I were diminished by CXXC5 transfection (Fig. 2 A). Expression of endothelin-1, a target of the Wnt/β-catenin pathway (Kim et al., 2005; Chen et al., 2007) and a marker for collagen production (Rizvi et al., 1996), was also significantly decreased after CXXC5 transfection (Fig. 2 A). To further characterize the role of CXXC5 in myofibroblast differentiation and collagen production, we examined the effect of CXXC5 knockdown on myofibroblast differentiation and collagen production. Expressions of β-catenin, α-SMA, collagen I, and endothelin-1 were increased by siRNA-mediated CXXC5 knockdown (Fig. 2 B). Moreover, CXXC5 knockdown induced β-catenin nuclear translocation as shown by cell fractionation and Western blotting (Fig. 2 B). Collagen production induced by CXXC5 knockdown was abolished by bosentan, a dual endothelin receptor antagonist, indicating that CXXC5 knockdown induced collagen production through an endothelin-1–dependent mechanism (Fig. 2 C). CXXC5 contains a C-terminal Dvl-binding motif (DBM) that is essential for CXXC5 function as a negative regulator of the Wnt/β-catenin pathway (London et al., 2004). Inhibition of β-catenin expression, as well as reduced expressions of α-SMA, collagen I, and endothelin-1, was abolished when CXXC5ΔDBM, a CXXC5 mutant with a DBM deletion (London et al., 2004), was expressed (Fig. 2 D). Immunocytochemical (ICC) analysis showed that collagen I expression was specifically abolished in GFP-CXXC5–transfected cells (Fig. 2 E). The Wnt/β-catenin reporter activity, Col1a2 promoter activity, and the level of collagen in the supernatant were significantly lowered by CXXC5 overexpression (Fig. 2 F). Moreover, overexpression of CXXC5 inhibited the ability of fibroblasts to contract collagen gels (Fig. 2 G), cell motility (Fig. 2 H), and the formation of stress fibers (Fig. 2 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)

CXXC5 attenuates myofibroblast differentiation and collagen production in human dermal fibroblasts. (A–D) Human dermal fibroblasts (n = 2–3 cells) were transfected with pcDNA3.0-CXXC5-Flag (A) at the indicated concentration, 100 nM CXXC5 siRNA (B and C), and pcDNA3.1-CXXC5-Myc and pcDNA3.1-CXXC5ΔDBM-Myc (D), together with corresponding control plasmid (Con) or control siRNA (Con si). The cells transfected with CXXC5 siRNA (C) or control siRNA were treated with or without 10 µM bosentan. The cells were further cultured for 2 d and harvested, and whole cell lysates (WCLs) were subjected to Western blot analyses to detect the protein levels of β-catenin, endothelin-1, α-SMA, and collagen I (n = 2 independent experiments). Relative densitometric ratios of each protein to loading control (α-tubulin or lamin A/C) are shown. (E) Human dermal fibroblasts were transfected with pEGFP-c3 or GFP-CXXC5 and further cultured for 2 d. The cells were immunocytochemically stained for collagen I. Representative confocal (ICC) images are shown (left), and mean intensity quantitation in GFP-positive cells was performed (right; ***, P < 0.0005; n = 3 independent experiments). Red arrows indicate the expression of collagen I, and green arrows indicate the expression of GFP. (F) Wnt luciferase reporter activity (left), Col1a2 luciferase reporter activity (middle), and collagen concentration (right) in the supernatant of human dermal fibroblasts were measured after transfection with pcDNA3.0 or pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Values are presented relative to control (**, P < 0.005; ***, P < 0.0005). (G) Contraction of collagen gels was monitored by measuring gel weight after transfection with pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Representative images (left) and quantitation (right) are shown (***, P < 0.0005). (H) The cells transfected with pcDNA3.0 or pcDNA3.0-CXXC5-Flag were scratched with sterile pipette tips and incubated in medium containing 5% serum for 36 h. Cells were then fixed with 4% PFA and stained with crystal violet. Representative microscopy images are shown (left), and cell migration was measured by counting migrating cells (right; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Dashed lines represent the widths of the initially scratched wounds. (I) ICC staining of human dermal fibroblasts transfected with GFP or GFP-CXXC5 was performed to detect phalloidin (left) and α-SMA (right). Representative ICC images are shown (n = 3 independent experiments). Means ± SD. Bars: (E and I) 50 µm; (H) 500 µm.
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fig2: CXXC5 attenuates myofibroblast differentiation and collagen production in human dermal fibroblasts. (A–D) Human dermal fibroblasts (n = 2–3 cells) were transfected with pcDNA3.0-CXXC5-Flag (A) at the indicated concentration, 100 nM CXXC5 siRNA (B and C), and pcDNA3.1-CXXC5-Myc and pcDNA3.1-CXXC5ΔDBM-Myc (D), together with corresponding control plasmid (Con) or control siRNA (Con si). The cells transfected with CXXC5 siRNA (C) or control siRNA were treated with or without 10 µM bosentan. The cells were further cultured for 2 d and harvested, and whole cell lysates (WCLs) were subjected to Western blot analyses to detect the protein levels of β-catenin, endothelin-1, α-SMA, and collagen I (n = 2 independent experiments). Relative densitometric ratios of each protein to loading control (α-tubulin or lamin A/C) are shown. (E) Human dermal fibroblasts were transfected with pEGFP-c3 or GFP-CXXC5 and further cultured for 2 d. The cells were immunocytochemically stained for collagen I. Representative confocal (ICC) images are shown (left), and mean intensity quantitation in GFP-positive cells was performed (right; ***, P < 0.0005; n = 3 independent experiments). Red arrows indicate the expression of collagen I, and green arrows indicate the expression of GFP. (F) Wnt luciferase reporter activity (left), Col1a2 luciferase reporter activity (middle), and collagen concentration (right) in the supernatant of human dermal fibroblasts were measured after transfection with pcDNA3.0 or pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Values are presented relative to control (**, P < 0.005; ***, P < 0.0005). (G) Contraction of collagen gels was monitored by measuring gel weight after transfection with pcDNA3.0-CXXC5-Flag (n = 3 independent experiments). Representative images (left) and quantitation (right) are shown (***, P < 0.0005). (H) The cells transfected with pcDNA3.0 or pcDNA3.0-CXXC5-Flag were scratched with sterile pipette tips and incubated in medium containing 5% serum for 36 h. Cells were then fixed with 4% PFA and stained with crystal violet. Representative microscopy images are shown (left), and cell migration was measured by counting migrating cells (right; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). Dashed lines represent the widths of the initially scratched wounds. (I) ICC staining of human dermal fibroblasts transfected with GFP or GFP-CXXC5 was performed to detect phalloidin (left) and α-SMA (right). Representative ICC images are shown (n = 3 independent experiments). Means ± SD. Bars: (E and I) 50 µm; (H) 500 µm.
Mentions: CXXC5 is known as a negative regulator of the Wnt/β-catenin pathway (Kim et al., 2010a). Considering the relationship between the Wnt/β-catenin pathway and fibrosis (Wei et al., 2011; Akhmetshina et al., 2012), we examined the effect of CXXC5 transfection on myofibroblast differentiation and collagen production in human dermal fibroblasts. Transfection with CXXC5 markedly inhibited β-catenin expression in a dose-dependent manner (Fig. 2 A). The levels of α-smooth muscle actin (α-SMA) and collagen I were diminished by CXXC5 transfection (Fig. 2 A). Expression of endothelin-1, a target of the Wnt/β-catenin pathway (Kim et al., 2005; Chen et al., 2007) and a marker for collagen production (Rizvi et al., 1996), was also significantly decreased after CXXC5 transfection (Fig. 2 A). To further characterize the role of CXXC5 in myofibroblast differentiation and collagen production, we examined the effect of CXXC5 knockdown on myofibroblast differentiation and collagen production. Expressions of β-catenin, α-SMA, collagen I, and endothelin-1 were increased by siRNA-mediated CXXC5 knockdown (Fig. 2 B). Moreover, CXXC5 knockdown induced β-catenin nuclear translocation as shown by cell fractionation and Western blotting (Fig. 2 B). Collagen production induced by CXXC5 knockdown was abolished by bosentan, a dual endothelin receptor antagonist, indicating that CXXC5 knockdown induced collagen production through an endothelin-1–dependent mechanism (Fig. 2 C). CXXC5 contains a C-terminal Dvl-binding motif (DBM) that is essential for CXXC5 function as a negative regulator of the Wnt/β-catenin pathway (London et al., 2004). Inhibition of β-catenin expression, as well as reduced expressions of α-SMA, collagen I, and endothelin-1, was abolished when CXXC5ΔDBM, a CXXC5 mutant with a DBM deletion (London et al., 2004), was expressed (Fig. 2 D). Immunocytochemical (ICC) analysis showed that collagen I expression was specifically abolished in GFP-CXXC5–transfected cells (Fig. 2 E). The Wnt/β-catenin reporter activity, Col1a2 promoter activity, and the level of collagen in the supernatant were significantly lowered by CXXC5 overexpression (Fig. 2 F). Moreover, overexpression of CXXC5 inhibited the ability of fibroblasts to contract collagen gels (Fig. 2 G), cell motility (Fig. 2 H), and the formation of stress fibers (Fig. 2 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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