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, 118 (10), 3279-90

GSK-3beta in Mouse Fibroblasts Controls Wound Healing and Fibrosis Through an endothelin-1-dependent Mechanism

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GSK-3beta in Mouse Fibroblasts Controls Wound Healing and Fibrosis Through an endothelin-1-dependent Mechanism

Mohit Kapoor et al. J Clin Invest.

Retraction in

Abstract

Glycogen synthase kinase-3 (GSK-3) is a widely expressed and highly conserved serine/threonine protein kinase encoded by 2 genes, GSK3A and GSK3B. GSK-3 is thought to be involved in tissue repair and fibrogenesis, but its role in these processes is currently unknown. To investigate the function of GSK-3beta in fibroblasts, we generated mice harboring a fibroblast-specific deletion of Gsk3b and evaluated their wound-healing and fibrogenic responses. We have shown that Gsk3b-conditional-KO mice (Gsk3b-CKO mice) exhibited accelerated wound closure, increased fibrogenesis, and excessive scarring compared with control mice. In addition, Gsk3b-CKO mice showed elevated collagen production, decreased cell apoptosis, elevated levels of profibrotic alpha-SMA, and increased myofibroblast formation during wound healing. In cultured Gsk3b-CKO fibroblasts, adhesion, spreading, migration, and contraction were enhanced. Both Gsk3b-CKO mice and fibroblasts showed elevated expression and production of endothelin-1 (ET-1) compared with control mice and cells. Antagonizing ET-1 reversed the phenotype of Gsk3b-CKO fibroblasts and mice. Thus, GSK-3beta appears to control the progression of wound healing and fibrosis by modulating ET-1 levels. These results suggest that targeting the GSK-3beta pathway or ET-1 may be of benefit in controlling tissue repair and fibrogenic responses in vivo.

Figures

Figure 1
Figure 1. Conditional deletion of Gsk3b in fibroblasts.
(A) PCR analysis of genomic DNA isolated from tails of Col1a2-creER(T)/0; Gsk3bF/F mice treated with corn oil (Gsk3b-C) or tamoxifen (Gsk3b-CKO) for 1 week. Col1a2-creER(T)/0; Gsk3bF/F mice treated with tamoxifen generated fibroblast-specific CKO mice. All genomic DNA samples were run on the same gel. Genotype of mice: lanes 1–3, Col1a2-creER(T)/0; Gsk3bF/F mice; lanes 4 and 5, Gsk3bF/F mice (B). GSK-3β immunofluorescence (in vivo). Immunofluorescence using GSK-3β antibody in day 0 unwounded skin of Gsk3b-C and Gsk3b-CKO mice. Scale bars: top row, 200 μm; bottom row, 100 μm. White arrows indicate GSK-3β–positive staining in the epidermis; yellow arrow, GSK-3β–positive staining in the dermis; blue arrows, GSK-3β–positive staining in hair follicles. Representative data from n = 4 animals per group are shown. (C) Percentage of GSK-3β–positive fibroblasts in Gsk3b-C versus Gsk3b-CKO mice. (D) GSK-3β immunofluorescence (in vitro). Immunofluorescence using GSK-3β antibody in dermal fibroblasts isolated from Gsk3b-C and -CKO mice. Representative data from n = 8 cell lines from 8 mice. Scale bar: 50 μm. (E) Loss of GSK-3β protein expression in fibroblasts isolated from Gsk3b-CKO mice. n = 8 cell lines from 8 mice is shown. *P < 0.05.
Figure 2
Figure 2. Loss of GSK-3β expression in fibroblasts results in enhanced wound closure.
(A and B) Kinetics of wound closure. Microphotographs of wounds were captured on days 0, 3, 7, and 10 after wounding to determine the degree of wound closure in Gsk3b-CKO compared with Gsk3b-C mice. Total area of wounds at days 0, 3, and 7 after wounding was measured using Northern Eclipse software as described in Methods. Gsk3b-CKO mice showed a significant (P < 0.05) acceleration in wound closure compared with Gsk3b-C mice on days 7 and 10 after wounding. Representative data from n = 8 animals per group are shown. Scale bar: 1 mm. (C and D) Histomorphometric analysis of wound closure. Histomorphometric evaluation of wound closure was performed by measuring the diameter of day 0 and 7 wounds of Gsk3b-CKO versus Gsk3b-C mice. Gsk3b-CKO mice showed a significant (P < 0.05) decrease in wound diameter compared with Gsk3b-C mice on day 7 after wounding. Representative data from n = 6 animals per group are shown. Arrows indicate the leading edges of wounded epidermis. Scale bar: 200 μm. *P < 0.05.
Figure 3
Figure 3. Conditional deletion of Gsk3b in fibroblasts results in elevated collagen, α-SMA production, and fibroblast proliferation and decreased apoptosis in vivo.
(A) Wound collagen synthesis. Hydroxyproline levels (a marker of collagen synthesis) were assessed in day 7 wounds. Significantly (P < 0.05) higher levels of hydroxyproline were detected in day 7 wounds of Gsk3b-CKO mice compared with Gsk3b-C mice (n = 6 animals per group). (B) α-SMA protein expression. Western blot analysis showed a significant (P < 0.05) increase in the protein expression of α-SMA in day 7 wounds of Gsk3b-CKO compared with Gsk3b-C mice. Representative data from n = 4 animals per group are shown. (C) α-SMA staining. Immunofluorescence for α-SMA in day 7 wound sections showed a significant (P < 0.05) increase in the number of α-SMA–positive cells in Gsk3b-CKO compared with Gsk3b-C mice (n = 6 animals per group). Arrows indicate α-SMA–positive staining. Scale bar: 100 μm. (D) PCNA staining. Immunohistochemistry for PCNA in day 7 wound sections showed a significant (P < 0.05) increase in the number of PCNA-positive cells in Gsk3b-CKO compared with Gsk3b-C mice (n = 4 animals per group). Arrows indicate PCNA-positive staining. Scale bar: 100 μm. (E) Apoptosis assay in day 7 wound sections showed a significant (P < 0.05) decrease in the number of apoptotic cells in Gsk3b-CKO compared with Gsk3b-C mice (n = 4 animals per group). Arrows indicate apoptotic cells. Scale bar: 100 μm. *P < 0.05.
Figure 4
Figure 4. Conditional deletion of Gsk3b in fibroblasts results in excessive scarring during wound healing in vivo.
(A and B) Quality of scarring. (A) Quality of scarring and collagen deposition was assessed by van Gieson collagen staining in day 21 and 28 wounds. A greater amount of collagen deposition in the dermis was observed in Gsk3b-CKO compared with Gsk3b-C mice on days 21 and 28 after wounding. By day 28 after wounding, Gsk3b-C mice exhibited a morphologically normal-looking scar, whereas Gsk3b-CKO mice exhibited excessive collagen deposition, with excessive scarring. Representative data from n = 6 animals per group are shown. Scale bars: top row, 200 μm; middle row, 200 μm; bottom row, 100 μm. (B) The van Gieson staining intensity was evaluated (see Methods) on the photomicrographs in a blinded manner and analysis performed on day 28. Van Gieson–stained wound sections showed more mature collagen fibers (deep red color of collagen fibers) in Gsk3b-CKO compared with Gsk3b-C mice (pink color of collagen fibers) (n = 6 animals per group). (C) Wound collagen synthesis. Significantly (P < 0.05) higher levels of hydroxyproline were detected in day 21 wounds of Gsk3b-CKO compared with Gsk3b-C mice (n = 6 animals per group). *P < 0.05. S, scar.
Figure 5
Figure 5. Loss of GSK-3β results in an enhanced ability of fibroblasts to migrate, spread, adhere, and contract on ECM in vitro.
(A and B) Migration. Scratch assay was performed to assess the rate of migration of Gsk3b-C and Gsk3b-CKO dermal fibroblasts at 0, 24, and 36 hours after scratch injury. A significant (P < 0.05) increase in the migration rate was observed in Gsk3b-CKO compared with Gsk3b-C fibroblasts at 36 hours after scratch injury (n = 4 cell lines from 4 mice). Scale bar: 200 μm. (C) Cell spreading over fibronectin. Dermal fibroblasts were plated on fibronectin-coated plates for 30, 60, 90, and 120 minutes and stained with F-actin and vinculin. Gsk3b-CKO fibroblasts showed greater spreading over fibronectin compared with Gsk3b-C fibroblasts (n = 4 cell lines from 4 mice). Scale bar: 50 μm. (D) Percent adhesion to fibronectin. Dermal fibroblasts were plated on a 96-well plate coated with fibronectin and assessed for percent adhesion to fibronectin at 30, 60, 90, and 120 minutes by MTT assay. Gsk3b-CKO fibroblasts showed significantly greater adhesion to fibronectin compared with Gsk3b-C fibroblasts (n = 4 cell lines from 4 mice). (E) Contraction of collagen gel matrices. Loss of GSK-3β results in an enhanced ability of fibroblasts to contract a collagen gel matrix (n = 4 cell lines from 4 mice; mean ± SEM is indicated). *P < 0.05.
Figure 6
Figure 6. Loss of GSK-3β in fibroblasts results in increased α-SMA and type I collagen expression.
(AC) α-SMA and Col1a2 (type I collagen) expression. (A) α-SMA protein expression was assessed in Gsk3b-CKO versus Gsk3b-C dermal fibroblasts by Western blotting. Gsk3b-CKO fibroblasts showed significantly (P < 0.05) higher expression of α-SMA protein versus Gsk3b-C fibroblasts. Representative data from n = 4 cell lines from 4 mice are shown. (B) Immunofluorescence for α-SMA showed greater stress fiber formation and higher expression of α-SMA in Gsk3b-CKO versus Gsk3b-C fibroblasts (n = 4 cell lines from 4 mice). Arrows indicate α-SMA–positive stress fibers. Scale bar: 50 μm. (C) α-SMA and type I collagen mRNA expression was assessed in Gsk3b-CKO versus Gsk3b-C fibroblasts by real-time PCR. Gsk3b-CKO fibroblasts showed significantly (P < 0.05) higher expression of both α-SMA and type I collagen (Type I Col) protein versus Gsk3b-C fibroblasts (n = 5 cell lines from 5 mice). *P < 0.05.
Figure 7
Figure 7. Loss of GSK-3β results in increased ET-1 expression.
(A) ET-1 protein expression. Western blot analysis showed a significant (P < 0.05) increase in the protein expression of ET-1 in day 7 wounds of Gsk3b-CKO compared with Gsk3b-C mice. Representative data for n = 8 animals per group are shown. (B) ET-1 immunohistochemistry. Higher expression levels of ET-1 were detected in day 7 wounds of Gsk3b-CKO compared with Gsk3b-C mice. Representative data for n = 4 animals per group are shown. Arrows indicate ET-1–positive staining. Scale bar: 100 μm. (C) ET-1 mRNA expression was assessed in Gsk3b-CKO versus Gsk3b-C fibroblasts by real-time PCR. Gsk3b-CKO fibroblasts showed significantly (P < 0.05) higher expression of ET-1 mRNA versus Gsk3b-C fibroblasts (n = 4 cell lines from 4 mice). (D) ET-1 ELISA. ET-1 levels were assessed in the cell culture supernatants of Gsk3b-CKO versus Gsk3b-C fibroblasts by ELISA. Gsk3b-CKO fibroblasts showed significantly (P < 0.05) higher levels of ET-1 versus Gsk3b-C fibroblasts (n = 6 cell lines from 6 mice). *P < 0.05.
Figure 9
Figure 9. The dual ETA/B receptor antagonist bosentan alleviates the phenotype of GSK-3β–deficient mice (in vivo).
(A and B) Kinetics of wound closure was determined in Gsk3b-C and Gsk3b-CKO mice treated with bosentan (100 mg/kg) or gum arabic (vehicle) on days 0, 7, and 10 after wounding. Bosentan treatment significantly (P < 0.05) reversed the enhanced rate of wound closure in Gsk3b-CKO mice without affecting the wound closure of Gsk3b-C mice (n = 4 wounds from 4 mice). Scale bar: 1 mm. (C) Effect of bosentan treatment on collagen synthesis was assessed using van Gieson staining in day 21 wounds. Note the reduction in the intensity of pink/red staining in Gsk3b-CKO animals in the presence of bosentan. Representative data from n = 4 wounds from 4 mice are shown. Scale bar: 100 μm. (D) Hydroxyproline levels. Bosentan (P < 0.05) reversed elevated wound collagen (hydroxyproline) synthesis in Gsk3b-CKO mice on day 21 after wounding (n = 4 wounds from 4 mice). (E) Bosentan (P < 0.05) reduced and reversed elevated α-SMA expression in Gsk3b-CKO mice on day 21 after wounding (n = 4 wounds from 4 mice). Scale bar: 100 μm. (F) Bosentan significantly (P < 0.05) reduced and reversed elevated α-SMA protein expression in Gsk3b-CKO mice on day 21 after wounding (n = 3). *P < 0.05. (G and H) Bosentan (P < 0.05) rescued loss of apoptosis in Gsk3b-CKO mice on day 7 after wounding (n = 4 wounds from 4 mice). Arrows indicate apoptotic cells. Scale bar: 100 μm. *P < 0.05.
Figure 10
Figure 10. Loss of GSK-3β results in increased β-catenin, which is responsible for ET-1 overexpression.
(A and B) β-Catenin protein expression. Western blot analysis showed a significant (P < 0.05) increase in the expression of β-catenin in day 7 wounds and dermal fibroblasts of Gsk3b-CKO compared with GSK-3β C mice. Representative data for n = 4 animals per group are shown. (C) siRNA recognizing β-catenin reduces β-catenin protein expression in Gsk3b-CKO fibroblasts. Transfection of Gsk3b-CKO fibroblasts with siRNA recognizing β-catenin, compared with control siRNA, reduced the expression of β-catenin as revealed by Western blot analysis with anti–β-catenin antibody. Hours after transfection are indicated. (D and E) siRNA recognizing β-catenin reduces ET-1 protein and mRNA expression in Gsk3b-CKO fibroblasts. Forty-eight hours after transfection with control or β-catenin siRNA, Gsk3b-CKO fibroblasts were examined for ET-1 protein production by Western blot analysis with anti–ET-1 antibody or by real-time PCR to detect ET-1 mRNA. Fibroblasts from 6 mice were used. *P < 0.05. (F) Model showing effect of loss of GSK-3β expression on tissue repair in vivo.
Figure 8
Figure 8. The dual ETA/B receptor antagonist bosentan alleviates the phenotype of GSK-3β–deficient fibroblasts (in vitro).
(A) α-SMA immunofluorescence. Gsk3b-C and Gsk3b-CKO fibroblasts were treated with bosentan for 48 hours, and α-SMA immunofluorescence was performed. Representative data from n = 4 cell lines from 4 mice are shown. Arrows indicate α-SMA–positive stress fibers. Scale bar: 50 μm. (B) α-SMA protein expression. Bosentan treatment significantly (P < 0.05) reversed the enhanced protein expression of α-SMA in Gsk3b-CKO fibroblasts. Representative data from n = 4 cell lines from 4 mice are shown. (C and D) α-SMA and Col1a2 mRNA expression. Bosentan treatment significantly (P < 0.05) reduced the expression of both α-SMA and type I collagen in Gsk3b-CKO fibroblasts (n = 5 cell lines from 5 mice). (E) Migration scratch assay. Bosentan treatment significantly (P < 0.05) decreased migration of Gsk3b-CKO fibroblasts (n = 4 cell lines from 4 mice). Scale bar: 200 μm. (F) Percent adhesion to fibronectin. Bosentan treatment significantly (P < 0.05) reversed the enhanced adhesion of Gsk3b-CKO dermal fibroblasts to fibronectin (n = 4 cell lines from 4 mice). (G) Gel contraction assay. Bosentan treatment significantly (P < 0.05) reversed the enhanced ability of Gsk3b-CKO fibroblasts to contract the collagen gel matrix (n = 3; mean ± SEM is indicated). *P < 0.05.

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