Objective Glycogen synthase kinase 3β (GSK-3) regulates the phosphorylation and subsequent degradation of β-catenin, thereby preventing aberrant activation of the canonical Wnt pathway. A study was undertaken to define the role of GSK-3 in fibroblast activation and in experimental models of systemic sclerosis (SSc).
Methods siRNA and specific inhibitors were used to inhibit GSK-3 in cultured fibroblasts and in mice. Activation of the canonical Wnt signalling was analysed by determining the levels of nuclear β-catenin and by measuring the mRNA levels of the Wnt target gene Axin2. The effects of GSK-3 on the release of collagen were evaluated in human dermal fibroblasts and in the mouse model of bleomycin-induced skin fibrosis in tight-skin-1 (tsk-1) mice.
Results Targeting GSK-3 potently activated the canonical Wnt pathway in fibroblasts in vitro and in vivo. Inactivation of GSK-3 dose-dependently stimulated the release of collagen from cultured fibroblasts in a β-catenin-dependent manner and further resulted in progressive accumulation of collagen and dermal thickening in mice. Inhibition of GSK-3 aggravated experimental fibrosis in bleomycin-challenged mice and in tsk-1 mice.
Conclusion Inhibition of GSK-3 activates the canonical Wnt pathway in fibroblasts, stimulates the release of collagen from fibroblasts, exacerbates experimental fibrosis and is sufficient to induce fibrosis. GSK-3 is therefore a key regulator of the canonical Wnt signalling in fibroblasts and inhibition of GSK-3 results in fibroblast activation and increased release of collagen.
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Systemic sclerosis (SSc) is a chronic fibrosing disease of unknown aetiology. The most obvious histopathological finding of involved tissues is the excessive accumulation of extracellular matrix components.1 The resulting tissue fibrosis often disrupts the physiological tissue structure and results in dysfunction of affected organs. Activated fibroblasts are key players in SSc that produce and release the extracellular matrix components. The molecular mechanisms for the uncontrolled activation of SSc fibroblasts are only partially known.1
Wnt signalling profoundly affects developmental processes during embryogenesis and tissue homeostasis in adults. 2 3 The canonical Wnt signalling pathway is best characterised among the different Wnt cascades. Aberrant Wnt signalling has been implicated in a variety of different diseases and might also play a role in fibrotic disorders such as SSc. First evidence suggested that several Wnt proteins might be overexpressed in tight-skin-1 mice, a common animal model for studies in SSc.4 Moreover, overexpression of Wnt 10b in the skin resulted in dermal fibrosis (J Varga, unpublished results). Thus, the canonical Wnt signalling might be a key player in the pathogenesis of SSc.
GSK-3 is a ubiquitously expressed serine/threonine kinase with a pivotal role in the regulation of the canonical Wnt pathway.2 5 In the absence of Wnt proteins, GSK-3 forms a complex with axin, adenomatous polypsis coli (APC) protein and casein kinase Iα. This catalytically active complex phosphorylates cytosolic β-catenin which, in turn, induces ubiquitinylation and subsequent proteasomal degradation of β-catenin.5 Mutations that inhibit the phosphorylation of β-catenin by GSK-3 highlight the importance of GSK-3 for the regulation of the canonical Wnt pathway; in various tumours such as hepatocellular carcinoma and carcinomas of the ovaries and endometrium, these mutations result in ligand-independent accumulation of β-catenin with uncontrolled transcription of Wnt target genes.6 7 This study examines the consequences of GSK-3 inhibition for fibroblast activation and the development of tissue fibrosis.
Material and methods
Patients and fibroblast cultures
All patients fulfilled the criteria for systemic sclerosis (SSc) as suggested by LeRoy et al.8 Biopsies from patients with SSc (n=9) were taken from involved skin (see table 1 in online supplement). Fibroblasts from healthy controls (n=6) were obtained from skin biopsies of age- and sex-matched volunteers and prepared as described previously.9
Incubation with small molecule inhibitors of GSK-3
Stimulation experiments were performed in Dulbecco's Modified Eagle Medium of 0.1% fetal calf serum. Dermal fibroblasts were incubated with two small molecule inhibitors of GSK-3: N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418; Calbiochem, Darmstadt, Germany) and 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5dione (SB216763; Tocris, Bristol, UK) in concentrations from 10 nM to 10 μM for 24 h. Fibroblasts incubated with the same volumes of the solvent dimethyl sulfoxide (DMSO) were used as controls. Both inhibitors are highly specific for GSK-3. AR-A014418 inhibits GSK-3 with a half maximal inhibitory concentration (IC)50 value of 104 nM and does not inhibit related kinases such as cyclin-dependent kinase 2 (CDK2) or cyclin-dependent kinase 5 (CDK5) (IC50 for CDK2 and CDK5 >100 μmol/l).10 Similarly, SB216763 inhibits GSK-3 with an IC50 value of 34 nM and an IC50 for related protein kinases of >10 μM.11
Knockdown of GSK-3 in dermal fibroblasts by siRNA
In addition to pharmacological inhibition, GSK-3 was targeted by siRNA-mediated knockdown using a predesigned siRNA and the nucleofection technique as described previously.12
Quantitative real-time PCR
Gene expression was quantified by TaqMan or by SYBR Green real-time PCR using the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, California, USA) as described elsewhere.13
The collagen content was quantified by the hydroxyproline assay.14
Western blot analysis
Western blot analyses were performed using polyclonal antibodies against human β-catenin (R&D Systems, Minneapolis, Minnesota, USA) at a dilution of 1:1000 and antihuman α-tubulin antibodies (Sigma; dilution 1:1000) as primary antibodies.
Microtitre tetrazolium assay
The colorimetric microtitre tetrazolium (MTT) assay is an established method of analysing cell viability in cytotoxicity investigations.15 Briefly, the number of viable cells directly correlates with the release of formazan following exposure to MTT (3(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide).15 16 Formazan is detected colorimetrically. Cultured SSc fibroblasts were incubated with SB216763 and AR-A014418 at concentrations of 1 μM and 10 μM, respectively. The MTT assay was performed as described previously.17 Untreated fibroblasts were used as negative controls and fibroblasts incubated with 50% DMSO served as positive controls.
Immunhistochemistry for β-catenin and α-smooth muscle actin
β-Catenin was detected by incubating skin sections with polyclonal goat-anti-mouse β-catenin antibodies (R&D Systems) at a dilution of 1:300 at 4°C overnight. To identify nuclear accumulation of β-catenin, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz Biotechnology, Santa Cruz, California, USA) at a dilution of 1:1000 for 10 min. In each section, cells positive for nuclear β-catenin were counted in 20 randomly chosen high-power fields. To confirm that Wnt signalling is active in fibroblasts, skin sections were triple-stained for β-catenin, DAPI and the selective fibroblast marker prolyl-4-hydroxylase-β (Acris Antibodies, Herfold, Germany) at a dilution of 1:50 at 4°C overnight. Myofibroblasts were identified by staining for α-smooth muscle actin (SMA) as described previously.18 In each section, α-SMA-positive cells were counted in six randomly chosen high-power fields.
Dermal thickness at the injection sites was analysed as described previously.19
Effects of GSK-3 inhibition on the release of extracellular matrix in vivo
Six-week-old female dilute brown non-Agouti mouse (DBA)/2 mice (Janvier, Le Genest St Isle, France) were treated with intraperitoneal injections of the GSK-3 inhibitor SB216763 at concentrations of 0.6 mg/kg every other day. To analyse potential changes caused by the activation of GSK-3 over time, one group of mice was treated for 4 weeks and another group of mice was treated for 8 weeks. DBA/2 mice given intraperitoneal injections of 0.9% NaCl containing 20% DMSO were used as controls.
Bleomycin-induced dermal fibrosis
Skin fibrosis was induced in 6-week-old female DBA/2 mice by local injections of bleomycin for 21 days.17 20 AR-A014418 was applied in final concentrations of 4 mg/kg twice daily and SB21676 at concentrations of 0.6 mg/kg twice daily by intraperitoneal injections every other day. Mice from the control group and the bleomycin group were injected with the solvent of the GSK-3 inhibitors (0.9% NaCl dissolved in 20% DMSO) to control for the intraperitoneal injections in the treatment groups. After 21 days the mice were killed by cervical dislocation. A total of 32 mice were analysed.
Inhibition of GSK-3 in the tight-skin-1 mouse model
Three groups of mice were analysed. The first group was a control group consisting of pa/pa mice not bearing the tsk-1 mutation, the second group consisted of mock-treated tsk-1 mice and the third group consisted of tsk-1 mice receiving intraperitoneal injections of SB216763 at a concentration of 0.6 mg/kg every other day. The treatment was started at the age of 5 weeks. All mice were killed at 10 weeks of age. A total of 21 mice were analysed.
Data are expressed as mean±SEM. The Wilcoxon signed rank test for related samples and the Mann–Whitney U test for non-related samples were used for statistical analyses. A p value of <0.05 was considered statistically significant.
Inhibition of GSK-3 stimulates the release of collagen from SSc fibroblasts
Incubation of SSc fibroblasts with SB216763, a highly specific inhibitor of GSK-3, increased the mRNA levels of col1a1 by 178±28% and 210±28% at concentrations of 0.1 μM and 1 μM, respectively. The stimulatory effects of SB216763 on the mRNA levels of col1a1 peaked at a concentration of 10 μM with a mean increase of 260±49% (p<0.05, figure 1A). Consistent with the induction of mRNA for col1a1, incubation with SB216763 increased the release of collagen with a maximal induction of 161±2% (p<0.05, figure 1B). Similar results were obtained with AR-A014418, another structurally unrelated inhibitor of GSK-3 (data not shown). Comparable increases in col1a1 mRNA and collagen protein upon incubation with inhibitors of GSK-3 were observed in healthy dermal fibroblasts, suggesting an ubiquitous response pattern rather than an SSc-specific phenomenon (data not shown).
We then targeted the expression of GSK-3 with siRNA to confirm the effects of the pharmacological inhibition by an independent approach. Transfection with siRNA against GSK-3 reduced the mRNA levels of GSK-3 by 91±2%. Consistent with pharmacological inhibition, siRNA against GSK-3 increased the mRNA levels of col1a1 by 50±11% compared with fibroblasts transfected with scrambled siRNA (figure 1C).
To exclude the possibility that the increased release of collagen with pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) resulted from toxic effects, we determined the effects of SB216763 and AR-A014418 on the cell viability using the MTT assay. Treatment with SB216763 and AR-A014418 in concentrations up to 10 μM did not alter the cell viability of SSc fibroblasts (see figure 1 in online supplement). In contrast, we observed a reduction of viable cells to 11±24% in control fibroblasts treated with DMSO.
Inhibition of GSK-3 activates the canonical Wnt signalling cascade in dermal fibroblasts
We next investigated whether pharmacological inhibition of GSK-3 activates canonical Wnt signalling. In this context, we assessed nuclear β-catenin and the mRNA levels of Axin2, an established target gene of the canonical Wnt cascade.2 Incubation with SB216763 significantly increased the nuclear accumulation of β-catenin in cultured dermal fibroblasts of patients with SSc by 296±41% compared with controls (see figure 2A in online supplement). Moreover, incubation with SB216763 increased the mRNA levels of Axin2 by 930±360% (p<0.05; see figure 2B in online supplement). Similar results were also observed with fibroblasts isolated from healthy volunteers (data not shown). These data demonstrate that GSK-3 is a crucial regulator of the canonical Wnt pathway in dermal fibroblasts.
SB216763 stimulates the collagen synthesis in a β-catenin-dependent manner
To investigate whether the canonical Wnt pathway mediates the profibrotic effects observed upon inactivation of GSK-3, we inhibited the canonical Wnt pathway by knockdown of β-catenin in healthy dermal fibroblasts. siRNA against β-catenin decreased the mRNA levels of β-catenin by 87±5%, confirming an effective inactivation of this central mediator of canonical Wnt signalling. In cells treated with non-targeting siRNA, SB216763 increased the expression of col1a1 mRNA by 187±30% (p<0.05) compared with controls. By contrast, incubation with SB216763 did not alter the levels of col1a1 mRNA in cells transfected with siRNA against β-catenin compared with mock-treated controls (figure 2A). Consistent with this finding, siRNA against β-catenin also abolished the stimulatory effects of SB216763 on the release of collagen protein (figure 2B). Thus, inhibition of GSK-3 induces collagen synthesis by activating canonical Wnt signalling.
Inhibition of GSK-3 is sufficient to induce fibrosis
To study the effects of GSK-3 in vivo, DBA/2 mice were treated with SB216763. No signs of toxicity related to treatment with SB216761 were observed. Body weight, texture of the fur and the activity did not differ between mice treated with SB216761 and sham-treated controls. Gross examination of the colon also did not reveal tumour formation. However, no in-depth histological or molecular examination was performed. In addition, the treatment period with inhibitors of GSK-3 may have been too short to induce tumour formation.
SB216763 potently activated the canonical Wnt pathway in murine skin. The number of fibroblasts with nuclear staining for β-catenin increased from 11±4% in mock-treated mice to 50±5% upon treatment with SB216763 for 8 weeks (p=0.02). Consistent with this, the mRNA levels of the Wnt target gene Axin2 were induced by 598±139% (p=0.003) in mice treated with SB216763 (figure 3A,B and supplementary figure 3).
Activation of the canonical Wnt cascade by SB216763 resulted in progressive dermal fibrosis (figure 3C and supplementary figure 4A). The dermal thickness increased by 35±6% compared with mock-treated controls after 4 weeks of treatment with SB216763 (p=0.04). This effect increased further to 65±9% after 8 weeks of treatment (p=0.001; figure 3D). Consistent with this, mice treated with SB216763 had an increased hydroxyproline content in the skin (129±22% compared with controls, p=0.04; figure 3E). Treatment with SB216763 also enhanced the differentiation of resting fibroblasts into myofibroblasts with increases of 264±12% after 8 weeks (p=0.002) (figure 3F and supplementary figure 4B).
Inhibition of GSK-3 enhances the activation of the canonical Wnt cascade and exacerbates bleomycin-induced dermal fibrosis
We next investigated whether inhibition of GSK-3 also exacerbates fibrosis in different experimental models of SSc.
We first examined the effects of GSK-3 in the mouse model of bleomycin-induced dermal fibrosis, an established model for early inflammatory stages of SSc. Challenge with bleomycin or SB216763 activated the canonical Wnt pathway. Inhibition of GSK-3 in bleomycin-treated mice resulted in further activation of the canonical Wnt pathway. The number of fibroblasts positive for nuclear β-catenin increased from 57±6% in bleomycin-challenged mock-treated mice and 45±4% in SB216763-treated mice to 79±4% in bleomycin-challenged SB216763-treated mice (p=0.05 vs bleomycin alone, figure 4A). Moreover, the mRNA levels of Axin2 were upregulated by 277±168% in bleomycin-challenged SB216763-treated mice compared with bleomycin-challenged mock-treated mice (p=0.01, figure 4B).
We further analysed whether inhibition of GSK-3 and subsequent activation of the Wnt pathway affects the outcome of bleomycin-induced dermal fibrosis. Bleomycin and SB216763 were both sufficient to induce fibrosis alone (figures 3 and 4). However, inhibition of GSK-3 exacerbated bleomycin-induced dermal fibrosis. Treatment of bleomycin-challenged mice with SB216763 increased dermal thickening by a further 84±15% compared with bleomycin alone (p<0.001, figure 4C,D). SB216763 also enhanced the hydroxyproline content (65±11%, p=0.04, figure 4E) and myofibroblast counts (84±23%, p=0.005, figure 4F) compared with bleomycin or SB216763 alone.
Inhibition of GSK-3 exacerbates the tight skin phenotype
We next investigated the effects of GSK-3 inhibition in tsk-1 mice, which serve as a model of later less inflammatory stages of SSc.21 Inhibition of GSK-3 further enhanced the activation of the canonical Wnt pathway in this model and increased the number of fibroblasts positive for nuclear β-catenin from 57±4% in mock-treated tsk-1 mice to 84±5% (p=0.03). The number of fibroblasts positive for nuclear β-catenin in pa/pa control mice treated with SB216763 was 37±5% (p=0.04, figure 5A). Treatment of pa/pa mice with SB216763 modestly increased the hypodermal thickness and the numbers of myofibroblasts compared with sham-treated pa/pa mice. Inhibition of GSK-3 by SB216763 exacerbated the tsk-1 phenotype and increased hypodermal thickness by 59±5% compared with mock-treated tsk-1 mice (p=0.05, figure 5B,C). The differentiation of resting fibroblasts into myofibroblasts also increased with GSK-3 inhibition by 63±15% (p=0.05, figure 5D).
This study shows that GSK-3 is a central regulator of fibroblast activation and collagen synthesis. Inhibition of GSK-3 by specific inhibitors or siRNA prevented the degradation of β-catenin, resulted in nuclear accumulation of β-catenin and in the transcription of Wnt target genes in dermal fibroblasts in vitro as well as in vivo. Inhibition of GSK-3 activated the canonical Wnt pathway in resting fibroblasts and healthy mice. In addition, inhibition of GSK-3 further stimulated the Wnt pathway in different models of experimental fibrosis in which the canonical Wnt pathway was already activated. We found nuclear accumulation of β-catenin and raised levels of Axin2 mRNA, both common markers of activated canonical Wnt signalling,2 in dermal fibroblasts of mice challenged with bleomycin and in tsk-1 mice. Of note, both markers further increased upon inhibition of GSK-3 in both mouse models. Thus, GSK-3 controls the canonical Wnt signalling pathway under physiological conditions and in the context of fibrosis.
Our study highlights the importance of a proper function of GSK-3 and a tight regulation of the canonical Wnt pathway in fibroblasts. Inactivation of GSK-3 resulted in increased release of collagen from cultured fibroblasts in vitro. Moreover, GSK-3 inhibition induced dermal fibrosis in vivo with accumulation of collagen, dermal thickening and differentiation of resting fibroblasts into myofibroblasts. Treatment with the small molecule inhibitor SB216763 also enhanced dermal fibrosis in the mouse model of bleomycin-induced fibrosis and in tsk-1 mice. However, one limitation of the study is that the effect of the inhibition of GSK-3 on organs other than the skin has not been analysed. Inhibition of the canonical Wnt pathway by siRNA-mediated knockdown of β-catenin completely prevented the profibrotic effects of the inactivation of GSK-3 and abrogated the induction of collagen by SB216763. Our data indicate that inhibition or loss of GSK-3 potently stimulates the release of collagen in fibroblasts and that this stimulation is mediated by the canonical Wnt pathway. However, GSK-3 does regulate the canonical Wnt pathway and integrates signals from other cascades such as the Notch and the Hedgehog pathway.22 23 Interestingly, Notch and Hedgehog signalling both have recently been suggested to contribute to tissue fibrosis in SSc.24,–,27 Although inhibition of canonical Wnt signalling by knockdown of β-catenin alone was sufficient to fully abrogate the profibrotic effects of the inhibition of GSK-3 in cultured fibroblasts, we cannot exclude the possibility that dysregulation of the Notch and the Hedgehog pathways might have contributed to the profibrotic effects of SB216763 in vivo.
Inhibition of GSK-3 by SB216763 has also been described to reduce the inflammatory response upon the activation of Toll-like-receptor 2 (TLR2) signalling in human monocytes by shifting the balance between proinflammatory and anti-inflammatory cytokines.28 Interestingly, activation of TLR2 signalling also contributes to bleomycin-induced dermal fibrosis.29 Thus, the effects of SB216763 on TLR2 might have influenced the results obtained with SB216763 in vitro and in vivo. However, considering the profibrotic effects of TLR2 and the inhibitory effects of SB216763 on TLR2 signalling, the effects of SB216763 on TLR2 would have ameliorated rather than exacerbated the profibrotic effects of SB216763. Moreover, AR-A014418, another inhibitor of GSK-3 not closely related structurally to SB216763 that has not been reported to alter TLR2 signalling and specific siRNA-mediated knockdown of GSK-3, stimulated the release of collagen in cultured fibroblasts to a similar degree to SB216763.
In addition to fibrosis, GSK-3 might also play a crucial role in wound healing. Mice harbouring a fibroblast-specific knockout of GSK-3 exhibited accelerated wound closure and excessive scarring in a dermal punch wound model.30 Consistent with our results, fibroblasts deficient in GSK-3 were activated and showed enhanced adhesion, migration and gel contraction.30 Furthermore, GSK-3 might regulate the release of extracellular matrix in osteoblasts as well as in fibroblasts. A recent study showed that locally increased levels of vascular endothelial growth factor (VEGF) increased bone mass.31 Overexpression of VEGF resulted in aberrant activation of osteoblasts with uncontrolled release of bone matrix. VEGF induced osteoblast activation by inhibited GSK-3 activity and stimulated the canonical Wnt signalling pathway.31 In turn, Wnt 3a-mediated inhibition of GSK-3 also enhances the release of VEGF in osteoblasts.32 This effect is also mimicked by inhibition of GSK-3 with SB216763.32 Thus, GSK-3-mediated inhibition of the canonical Wnt pathway is important for proper regulation of fibroblast function in fibrotic diseases and also plays a central role in tissue homeostasis in other cell types.
In summary, we have shown that inhibition of GSK-3 stimulates the canonical Wnt pathway in fibroblasts and induces fibrosis in a Wnt-dependent manner. Moreover, inhibition of GSK-3 exacerbates fibrosis in different experimental models of SSc. Our data show that GSK-3 is a crucial regulator of the canonical Wnt pathway that prevents the uncontrolled activation of fibroblasts and development of tissue fibrosis.
The authors thank Maria Halter for excellent technical support.
Funding This study was supported by grant A40 of the Interdisciplinary Center of Clinical Research (IZKF) in Erlangen, Deutsche Forschungsgesellschaft and the Career Support Award of Medicine of the Ernst Jung Foundation.
Competing interests None.
Ethics approval All patients and controls signed an informed consent form approved by the local institutional review boards.
Provenance and peer review Not commissioned; externally peer reviewed.