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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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Wnt3a induces CXXC5 expression and enhances CXXC5-Dvl interaction. (A and B) Human dermal fibroblasts (n = 2–3 cells) were treated with or without Wnt3a (50 or 200 ng/ml). (A) WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, α-SMA, collagen I, or α-tubulin and to RT-PCR analyses with primers for CXXC5 or GAPDH (n = 2 independent experiments). (B) Relative densitometry values are shown underneath blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). The cells were also immunocytochemically stained for β-catenin, CXXC5, or collagen I. Representative ICC images are shown (top), and mean intensity quantitation was performed (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) Human dermal fibroblasts were incubated with 50 ng/ml Wnt3a for 1, 6, 12, 24, 48, or 72 h, and WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, or α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (D and E) Human dermal fibroblasts were treated with 50 ng/ml Wnt3a for 2 d after treatment with control siRNA (Con si) or CXXC5 siRNA (CXXC5 si). (D) Western blot analysis (n = 3 independent experiments) of WCLs was performed with antibodies against β-catenin, α-SMA, collagen I, CXXC5, or α-tubulin. Relative densitometry values are shown underneath the blots as ratios relative to the levels of α-tubulin. ICC analysis (n = 3 independent experiments) of samples in D and E was performed with β-catenin or CXXC5. (E) Representative ICC images are shown (left), and mean intensity values were calculated (right; ***, P < 0.0005). (B and E) Bars, 50 µm. Means ± SD. (F) Human dermal fibroblasts were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by immunoblotting to detect Dvl-1, Myc, β-catenin, or α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots. (G) WCLs from human dermal fibroblasts treated with (Wnt3a) or without (Con) 50 ng/ml Wnt3a for 2 d were subjected to immunoprecipitation with anti–Dvl-1 antibody, and Western blot analyses were subsequently performed to detect Dvl-1, CXXC5, β-catenin, and α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots.
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fig3: Wnt3a induces CXXC5 expression and enhances CXXC5-Dvl interaction. (A and B) Human dermal fibroblasts (n = 2–3 cells) were treated with or without Wnt3a (50 or 200 ng/ml). (A) WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, α-SMA, collagen I, or α-tubulin and to RT-PCR analyses with primers for CXXC5 or GAPDH (n = 2 independent experiments). (B) Relative densitometry values are shown underneath blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). The cells were also immunocytochemically stained for β-catenin, CXXC5, or collagen I. Representative ICC images are shown (top), and mean intensity quantitation was performed (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) Human dermal fibroblasts were incubated with 50 ng/ml Wnt3a for 1, 6, 12, 24, 48, or 72 h, and WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, or α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (D and E) Human dermal fibroblasts were treated with 50 ng/ml Wnt3a for 2 d after treatment with control siRNA (Con si) or CXXC5 siRNA (CXXC5 si). (D) Western blot analysis (n = 3 independent experiments) of WCLs was performed with antibodies against β-catenin, α-SMA, collagen I, CXXC5, or α-tubulin. Relative densitometry values are shown underneath the blots as ratios relative to the levels of α-tubulin. ICC analysis (n = 3 independent experiments) of samples in D and E was performed with β-catenin or CXXC5. (E) Representative ICC images are shown (left), and mean intensity values were calculated (right; ***, P < 0.0005). (B and E) Bars, 50 µm. Means ± SD. (F) Human dermal fibroblasts were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by immunoblotting to detect Dvl-1, Myc, β-catenin, or α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots. (G) WCLs from human dermal fibroblasts treated with (Wnt3a) or without (Con) 50 ng/ml Wnt3a for 2 d were subjected to immunoprecipitation with anti–Dvl-1 antibody, and Western blot analyses were subsequently performed to detect Dvl-1, CXXC5, β-catenin, and α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots.

Mentions: To confirm the role of the Wnt/β-catenin pathway in CXXC5 function, we examined the effect of Wnt3a in human dermal fibroblasts. Wnt3a treatment induced expressions of β-catenin, α-SMA, and collagen I (Fig. 3 A). Interestingly, both mRNA and protein levels of CXXC5 were increased by treatment with Wnt3a (Fig. 3 A). ICC analysis also showed that Wnt3a increased expression of CXXC5, in concert with that of β-catenin and collagen I, in a dose-dependent manner (Fig. 3 B). Both β-catenin and CXXC5 were concomitantly increased in a time-dependent manner by Wnt3a treatment, and maximal levels were reached at 48 h (Fig. 3 C). Considering the role of CXXC5 as a negative regulator of the Wnt/β-catenin pathway, we hypothesized that Wnt3a-dependent induction of CXXC5 may be the negative feedback mechanism. This notion was further indicated by synergistic increases in the expression of β-catenin, α-SMA, and collagen I by co-treatment with CXXC5 siRNA and Wnt3a (Fig. 3 D). Both β-catenin and CXXC5 were also increased in the nucleus and cytosol, respectively, by Wnt3a treatment, and β-catenin levels were synergistically increased by co-treatment with CXXC5 siRNA and Wnt3a, as shown by ICC analysis (Fig. 3 E). To further characterize the role of CXXC5 as a negative feedback regulator that functions via Dvl-1, we assessed the interaction of wild-type CXXC5 or its deletion mutant CXXC5 ΔDBM with Dvl-1. CXXC5, but not CXXC5 ΔDBM, interacted with Dvl-1, and this interaction was enhanced by Wnt3a treatment (Fig. 3 F). Consistently, Wnt3a-induced increase in β-catenin was significantly reduced by overexpression of CXXC5 but not of CXXC5 ΔDBM. We also observed an enhanced interaction between CXXC5 and Dvl-1 by Wnt3a in the endogenous level (Fig. 3 G). Overall, CXXC5 is a negative feedback regulator of the Wnt/β-catenin pathway in human dermal fibroblasts and functions via binding to Dvl in a manner dependent on Wnt3a.


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)

Wnt3a induces CXXC5 expression and enhances CXXC5-Dvl interaction. (A and B) Human dermal fibroblasts (n = 2–3 cells) were treated with or without Wnt3a (50 or 200 ng/ml). (A) WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, α-SMA, collagen I, or α-tubulin and to RT-PCR analyses with primers for CXXC5 or GAPDH (n = 2 independent experiments). (B) Relative densitometry values are shown underneath blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). The cells were also immunocytochemically stained for β-catenin, CXXC5, or collagen I. Representative ICC images are shown (top), and mean intensity quantitation was performed (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) Human dermal fibroblasts were incubated with 50 ng/ml Wnt3a for 1, 6, 12, 24, 48, or 72 h, and WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, or α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (D and E) Human dermal fibroblasts were treated with 50 ng/ml Wnt3a for 2 d after treatment with control siRNA (Con si) or CXXC5 siRNA (CXXC5 si). (D) Western blot analysis (n = 3 independent experiments) of WCLs was performed with antibodies against β-catenin, α-SMA, collagen I, CXXC5, or α-tubulin. Relative densitometry values are shown underneath the blots as ratios relative to the levels of α-tubulin. ICC analysis (n = 3 independent experiments) of samples in D and E was performed with β-catenin or CXXC5. (E) Representative ICC images are shown (left), and mean intensity values were calculated (right; ***, P < 0.0005). (B and E) Bars, 50 µm. Means ± SD. (F) Human dermal fibroblasts were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by immunoblotting to detect Dvl-1, Myc, β-catenin, or α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots. (G) WCLs from human dermal fibroblasts treated with (Wnt3a) or without (Con) 50 ng/ml Wnt3a for 2 d were subjected to immunoprecipitation with anti–Dvl-1 antibody, and Western blot analyses were subsequently performed to detect Dvl-1, CXXC5, β-catenin, and α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots.
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fig3: Wnt3a induces CXXC5 expression and enhances CXXC5-Dvl interaction. (A and B) Human dermal fibroblasts (n = 2–3 cells) were treated with or without Wnt3a (50 or 200 ng/ml). (A) WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, α-SMA, collagen I, or α-tubulin and to RT-PCR analyses with primers for CXXC5 or GAPDH (n = 2 independent experiments). (B) Relative densitometry values are shown underneath blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). The cells were also immunocytochemically stained for β-catenin, CXXC5, or collagen I. Representative ICC images are shown (top), and mean intensity quantitation was performed (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (C) Human dermal fibroblasts were incubated with 50 ng/ml Wnt3a for 1, 6, 12, 24, 48, or 72 h, and WCLs were subjected to Western blot analyses with antibodies against β-catenin, CXXC5, or α-tubulin (n = 2 independent experiments). Relative densitometric ratios of each protein to α-tubulin are shown. (D and E) Human dermal fibroblasts were treated with 50 ng/ml Wnt3a for 2 d after treatment with control siRNA (Con si) or CXXC5 siRNA (CXXC5 si). (D) Western blot analysis (n = 3 independent experiments) of WCLs was performed with antibodies against β-catenin, α-SMA, collagen I, CXXC5, or α-tubulin. Relative densitometry values are shown underneath the blots as ratios relative to the levels of α-tubulin. ICC analysis (n = 3 independent experiments) of samples in D and E was performed with β-catenin or CXXC5. (E) Representative ICC images are shown (left), and mean intensity values were calculated (right; ***, P < 0.0005). (B and E) Bars, 50 µm. Means ± SD. (F) Human dermal fibroblasts were transfected with pcDNA3.1, pcDNA3.1-CXXC5-Myc, or pcDNA3.1-CXXC5ΔDBM-Myc. WCLs or cell lysates immunoprecipitated with anti-Myc were analyzed by immunoblotting to detect Dvl-1, Myc, β-catenin, or α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots. (G) WCLs from human dermal fibroblasts treated with (Wnt3a) or without (Con) 50 ng/ml Wnt3a for 2 d were subjected to immunoprecipitation with anti–Dvl-1 antibody, and Western blot analyses were subsequently performed to detect Dvl-1, CXXC5, β-catenin, and α-tubulin (n = 2 independent experiments). Relative densitometry values are shown below the blots.
Mentions: To confirm the role of the Wnt/β-catenin pathway in CXXC5 function, we examined the effect of Wnt3a in human dermal fibroblasts. Wnt3a treatment induced expressions of β-catenin, α-SMA, and collagen I (Fig. 3 A). Interestingly, both mRNA and protein levels of CXXC5 were increased by treatment with Wnt3a (Fig. 3 A). ICC analysis also showed that Wnt3a increased expression of CXXC5, in concert with that of β-catenin and collagen I, in a dose-dependent manner (Fig. 3 B). Both β-catenin and CXXC5 were concomitantly increased in a time-dependent manner by Wnt3a treatment, and maximal levels were reached at 48 h (Fig. 3 C). Considering the role of CXXC5 as a negative regulator of the Wnt/β-catenin pathway, we hypothesized that Wnt3a-dependent induction of CXXC5 may be the negative feedback mechanism. This notion was further indicated by synergistic increases in the expression of β-catenin, α-SMA, and collagen I by co-treatment with CXXC5 siRNA and Wnt3a (Fig. 3 D). Both β-catenin and CXXC5 were also increased in the nucleus and cytosol, respectively, by Wnt3a treatment, and β-catenin levels were synergistically increased by co-treatment with CXXC5 siRNA and Wnt3a, as shown by ICC analysis (Fig. 3 E). To further characterize the role of CXXC5 as a negative feedback regulator that functions via Dvl-1, we assessed the interaction of wild-type CXXC5 or its deletion mutant CXXC5 ΔDBM with Dvl-1. CXXC5, but not CXXC5 ΔDBM, interacted with Dvl-1, and this interaction was enhanced by Wnt3a treatment (Fig. 3 F). Consistently, Wnt3a-induced increase in β-catenin was significantly reduced by overexpression of CXXC5 but not of CXXC5 ΔDBM. We also observed an enhanced interaction between CXXC5 and Dvl-1 by Wnt3a in the endogenous level (Fig. 3 G). Overall, CXXC5 is a negative feedback regulator of the Wnt/β-catenin pathway in human dermal fibroblasts and functions via binding to Dvl in a manner dependent on Wnt3a.

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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