Composition of TWIST1 dimers regulates fibroblast activation and tissue fibrosis.

OBJECTIVES: TWIST1 is a member of the class B of basic helix-loop-helix transcription factors that regulates cell lineage determination and differentiation and has been implicated in epithelial-to-mesenchymal transition. Here, we aimed to investigate the role of TWIST1 for the activation of resident fibroblasts in systemic sclerosis (SSc). METHODS: The expression of Twist1 in fibroblasts was modulated by forced overexpression or siRNA-mediated knockdown. Interaction of Twist1, E12 and inhibitor Of differentiation (Id) was analysed by co-immunoprecipitation. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitous or fibroblast-specific depletion of Twist1. Mice were either challenged with bleomycin or overexpressing a constitutively active transforming growth factor (TGF)ß receptor I. RESULT: The expression of TWIST1 was increased in fibroblasts in fibrotic human and murine skin in a TGFß/SMAD3-dependent manner. TWIST1 in turn enhanced TGFß-induced fibroblast activation in a p38-dependent manner. The stimulatory effects of TWIST1 on resident fibroblasts were mediated by TWIST1 homodimers. TGFß promotes the formation of TWIST1 homodimers by upregulation of TWIST1 and by induction of inhibitor of DNA-binding proteins, which have high affinity for E12/E47 and compete against TWIST1 for E12/E47 binding. Mice with selective depletion of Twist1 in fibroblasts are protected from experimental skin fibrosis in different murine models to a comparable degree as mice with ubiquitous depletion of Twist1. CONCLUSIONS: Our data identify TWIST1 as a central pro-fibrotic factor in SSc, which facilitates fibroblast activation by amplifying TGFß signalling. Targeting of TWIST1 may thus be a novel approach to normalise aberrant TGFß signalling in SSc.

Sirt1 regulates canonical TGF-ß signalling to control fibroblast activation and tissue fibrosis.

BACKGROUND: Sirt1 is a member of the sirtuin family of proteins. Sirt1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. As aberrant epigenetic modifications have been linked to the pathogenesis of systemic sclerosis (SSc), we aimed to investigate the role of Sirt1 in fibroblast activation. METHODS: Sirt1 expression was analysed by real-time PCR, western blot and immunohistochemistry. Sirt1 signalling was modulated with the Sirt1 agonist resveratrol and by fibroblast-specific knockout. The role of Sirt1 was evaluated in bleomycin-induced skin fibrosis and in mice overexpressing a constitutively active transforming growth fac-tor-ß (TGF-ß) receptor I (TBRIact). RESULTS: The expression of Sirt1 was decreased in patients with SSc and in experimental fibrosis in a TGF-ß-dependent manner. Activation of Sirt1 potentiated the profibrotic effects of TGF-ß with increased Smad reporter activity, elevated transcription of TGF-ß target genes and enhanced release of collagen. In contrast, knockdown of Sirt1 inhibited TGF-ß/SMAD signalling and reduced release of collagen in fibroblasts. Consistently, mice with fibroblast-specific knockdown of Sirt1 were less susceptible to bleomycin- or TBRIact-induced fibrosis. CONCLUSIONS: We identified Sirt1 as a crucial regulator of TGF-ß/Smad signalling in SSc. Although Sirt1 is downregulated, this decrease is not sufficient to counterbalance the excessive activation of TGF-ß signalling in SSc. However, augmentation of this endogenous regulatory mechanism, for example, by knockdown of Sirt1, can effectively inhibit TGF-ß signalling and exerts potent antifibrotic effects. Sirt1 may thus be a key regulator of fibroblast activation in SSc.

Activating transcription factor 3 regulates canonical TGFß signalling in systemic sclerosis.

BACKGROUND: Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc). METHODS: ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation. RESULTS: Transforming growth factor-ß (TGFß) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFß, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFß. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFß and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFß receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3. CONCLUSIONS: We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFß on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

Tribbles homologue 3 stimulates canonical TGF-ß signalling to regulate fibroblast activation and tissue fibrosis.

OBJECTIVES: Tribbles homologue 3 (TRB3) is a pseudokinase that modifies the activation of various intracellular signalling pathways to control fundamental processes extending from mitosis and cell activation to apoptosis and modulation of gene expression. Here, we aimed to analyse the role of TRB3 in fibroblast activation in systemic sclerosis (SSc). METHODS: The expression of TRB3 was quantified by quantitative PCR, western blot and immunohistochemistry. The role of TRB3 was analysed in cultured fibroblasts and in experimental fibrosis using small interfering RNA (siRNA)-mediated knockdown and overexpression of TRB3. RESULTS: TRB3 expression was increased in fibroblasts of patients with SSc and in murine models of SSc in a transforming growth factor-ß (TGF-ß)/Smad-dependent manner. Overexpression of TRB3 stimulated canonical TGF-ß signalling and induced an activated phenotype in resting fibroblasts. In contrast, knockdown of TRB3 reduced the profibrotic effects of TGF-ß and decreased the collagen synthesis. Moreover, siRNA-mediated knockdown of TRB3 exerted potent antifibrotic effects and ameliorated bleomycin as well as constitutively active TGF-ß receptor I-induced fibrosis with reduced dermal thickening, decreased hydroxyproline content and impaired myofibroblast differentiation. CONCLUSIONS: The present study characterises TRB3 as a novel profibrotic mediator in SSc. TGF-ß induces TRB3, which in turn activates canonical TGF-ß/Smad signalling and stimulates the release of collagen, thereby inducing a positive feedback loop that may contribute to aberrant TGF-ß signalling in SSc.

S100A4 amplifies TGF-ß-induced fibroblast activation in systemic sclerosis.

OBJECTIVES: S100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we evaluated the role of S100A4 in fibroblast activation in systemic sclerosis (SSc). METHODS: The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4(-/-)) mice. Transforming growth factor ß (TGF-ß) signalling was assessed by reporter assays, staining for phosphorylated Smad2/3 and analyses of target genes. RESULTS: The expression of S100A4 was increased in SSc skin and in experimental fibrosis in a TGF-ß/Smad-dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-ß and decreased the release of collagen. S100A4(-/-) mice were protected from bleomycin-induced skin fibrosis with reduced dermal thickening, decreased hydroxyproline content and lower myofibroblast counts. Deficiency of S100A4 also ameliorated fibrosis in the tight-skin-1 (Tsk-1) mouse model. CONCLUSIONS: We characterised S100A4 as a downstream mediator of the stimulatory effects of TGF-ß on fibroblasts in SSc. TGF-ß induces the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-ß and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies.

Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFß signalling.

OBJECTIVES: We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor ß (TGFß) signalling that mediates the antifibrotic effects of the sGC. METHODS: Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFß. sGC knockout fibroblasts were isolated from sGCI(fl/fl) mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-ß1 receptor. RESULTS: sGC stimulation inhibited TGFß-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFß-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFß target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFß-driven dermal fibrosis but did not change p-SMAD2 and 3 levels and TGFß target gene expression, confirming that non-canonical TGFß pathways mediate the antifibrotic sGC activity. CONCLUSIONS: We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFß signalling and inhibits experimental fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of fibrosis and vascular disease in systemic sclerosis.

Activation of liver X receptors inhibits experimental fibrosis by interfering with interleukin-6 release from macrophages.

OBJECTIVES: To investigate the role of liver X receptors (LXRs) in experimental skin fibrosis and evaluate their potential as novel antifibrotic targets. METHODS: We studied the role of LXRs in bleomycin-induced skin fibrosis, in the model of sclerodermatous graft-versus-host disease (sclGvHD) and in tight skin-1 (Tsk-1) mice, reflecting different subtypes of fibrotic disease. We examined both LXR isoforms using LXRα-, LXRß- and LXR-α/ß-double-knockout mice. Finally, we investigated the effects of LXRs on fibroblasts and macrophages to establish the antifibrotic mode of action of LXRs. RESULTS: LXR activation by the agonist T0901317 had antifibrotic effects in bleomycin-induced skin fibrosis, in the sclGvHD model and in Tsk-1 mice. The antifibrotic activity of LXRs was particularly prominent in the inflammation-driven bleomycin and sclGvHD models. LXRα-, LXRß- and LXRα/ß-double-knockout mice showed a similar response to bleomycin as wildtype animals. Low levels of the LXR target gene ABCA-1 in the skin of bleomycin-challenged and control mice suggested a low baseline activation of the antifibrotic LXR signalling, which, however, could be specifically activated by T0901317. Fibroblasts were not the direct target cells of LXRs agonists, but LXR activation inhibited fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic interleukin-6. CONCLUSIONS: We identified LXRs as novel targets for antifibrotic therapies, a yet unknown aspect of these nuclear receptors. Our data suggest that LXR activation might be particularly effective in patients with inflammatory disease subtypes. Activation of LXRs interfered with the release of interleukin-6 from macrophages and, thus, inhibited fibroblast activation and collagen release.

Inhibition of casein kinase II reduces TGFß induced fibroblast activation and ameliorates experimental fibrosis.

OBJECTIVES: Casein kinase II (CK2) is a constitutively active serine/threonine protein kinase that plays a key role in cellular transformation and tumorigenesis. The purpose of the study was to characterise whether CK2 contributes to the pathologic activation of fibroblasts in patients with SSc and to evaluate the antifibrotic potential of CK2 inhibition. METHODS: Activation of CK2, JAK2 and STAT3 in human skin and in experimental fibrosis was analysed by immunohistochemistry. CK2 signalling was inhibited by the selective CK2 inhibitor 4, 5, 6, 7-Tetrabromobenzotriazole (TBB). The mouse models of bleomycin-induced and TGFß receptor I (TBR)-induced dermal fibrosis were used to evaluate the antifibrotic potential of specific CK2 inhibition in vivo. RESULT: Increased expression of CK2 was detected in skin fibroblasts of SSc patients. Inhibition of CK2 by TBB abrogated the TGFß-induced activation of JAK2/STAT3 signalling and prevented the stimulatory effects of TGFß on collagen release and myofibroblasts differentiation in cultured fibroblasts. Inhibition of CK2 prevented bleomycin-induced and TBR-induced skin fibrosis with decreased dermal thickening, lower myofibroblast counts and reduced accumulation of collagen. Treatment with TBB also induced regression of pre-established fibrosis. The antifibrotic effects of TBB were accompanied by reduced activation of JAK2/STAT3 signalling in vivo. CONCLUSIONS: We provide evidence that CK2 is activated in SSc and contributes to fibroblast activation by regulating JAK2/STAT3 signalling. Inhibition of CK2 reduced the pro-fibrotic effects of TGFß and inhibited experimental fibrosis. Targeting of CK2 may thus be a novel therapeutic approach for SSc and other fibrotic diseases.

Vitamin D receptor regulates TGF-ß signalling in systemic sclerosis.

BACKGROUND: Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. Its ligand, 1,25-(OH)2D, is a metabolically active hormone derived from vitamin D3. The levels of vitamin D3 are decreased in patients with systemic sclerosis (SSc). Here, we aimed to analyse the role of VDR signalling in fibrosis. METHODS: VDR expression was analysed in SSc skin, experimental fibrosis and human fibroblasts. VDR signalling was modulated by siRNA and with the selective agonist paricalcitol. The effects of VDR on Smad signalling were analysed by reporter assays, target gene analyses and coimmunoprecipitation. The effects of paricalcitol were evaluated in the models of bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active transforming growth factor-ß (TGF-ß) receptor I (TBRI(CA)). RESULTS: VDR expression was decreased in fibroblasts of SSc patients and murine models of SSc in a TGF-ß-dependent manner. Knockdown of VDR enhanced the sensitivity of fibroblasts towards TGF-ß. In contrast, activation of VDR by paricalcitol reduced the stimulatory effects of TGF-ß on fibroblasts and inhibited collagen release and myofibroblast differentiation. Paricalcitol stimulated the formation of complexes between VDR and phosphorylated Smad3 in fibroblasts to inhibit Smad-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI(CA)-induced fibrosis. CONCLUSIONS: We characterise VDR as a negative regulator of TGF-ß/Smad signalling. Impaired VDR signalling with reduced expression of VDR and decreased levels of its ligand may thus contribute to hyperactive TGF-ß signalling and aberrant fibroblast activation in SSc.

Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease.

OBJECTIVES: IL-35 is a member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. IL-35 exerts immunomodulatory activities in experimental and human autoimmune inflammatory conditions. Our aim was to assess IL-35 expression in the skin and circulation of SSc patients and to characterize its potential association with SSc-related features. METHODS: Expression of IL-35 in skin and dermal fibroblasts was quantified by quantitative PCR, immunohistochemistry and immunofluorescence. Serum levels of IL-35 (by ELISA), CRP (by turbidimetry), ANA (by immunofluorescence) and autoantibodies of the ENA complex (by immunoblot) were measured in 40 SSc patients. Serum IL-35 was determined in 40 age- and sex-matched healthy controls. RESULTS: IL-35 expression was increased in SSc skin and dermal fibroblasts in a TGF-ß-dependent manner. IL-35 induced an activated phenotype in resting fibroblasts and enhanced the release of collagen. IL-35 serum levels were increased in patients with SSc compared with healthy controls [median 83.9 (interquartile range 45.1-146.1) vs 36.2 (interquartile range 17.2-49.4) pg/ml, P < 0.0001]. Serum IL-35 was negatively correlated with disease duration (r = -0.4339, P = 0.0052). In line with this finding, serum IL-35 was increased in patients with an early SSc pattern on capillaroscopy assessment compared with those with active and late SSc patterns. CONCLUSION: The present study demonstrates overexpression of IL-35 in SSc skin, dermal fibroblasts and serum. TGF-ß induces IL-35, which in turn activates resting fibroblasts and enhances the release of collagen, thereby contributing to aberrant TGF-ß signalling in SSc. Increased serum IL-35 is associated with early, inflammatory stages of SSc.

Heat shock protein 90 (Hsp90) inhibition targets canonical TGF-ß signalling to prevent fibrosis.

OBJECTIVES: Targeted therapies for systemic sclerosis (SSc) and other fibrotic diseases are not yet available. We evaluated the efficacy of heat shock protein 90 (Hsp90) inhibition as a novel approach to inhibition of aberrant transforming growth factor (TGF)-ß signalling and for the treatment of fibrosis in preclinical models of SSc. METHODS: Expression of Hsp90 was quantified by quantitative PCR, western blot and immunohistochemistry. The effects of Hsp90 inhibition were analysed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, in tight-skin (Tsk-1) mice and in mice overexpressing a constitutively active TGF-ß receptor I (TßRI). RESULTS: Expression of Hsp90ß was increased in SSc skin and in murine models of SSc in a TGF-ß-dependent manner. Inhibition of Hsp90 by 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) inhibited canonical TGF-ß signalling and completely prevented the stimulatory effects of TGF-ß on collagen synthesis and myofibroblast differentiation. Treatment with 17-DMAG decreased the activation of canonical TGF-ß signalling in murine models of SSc and exerted potent antifibrotic effects in bleomycin-induced dermal fibrosis, in Tsk-1 mice and in mice overexpressing a constitutively active TßRI. Dermal thickness, number of myofibroblasts and hydroxyproline content were all significantly reduced on treatment with 17-DMAG. No toxic effects were observed with 17-DMAG at antifibrotic doses. CONCLUSIONS: Hsp90 is upregulated in SSc and is critical for TGF-ß signalling. Pharmacological inhibition of Hsp90 effectively blocks the profibrotic effects of TGF-ß in cultured fibroblasts and in different preclinical models of SSc. These results have translational implications, as several Hsp90 inhibitors are in clinical trials for other indications.

The Wnt antagonists DKK1 and SFRP1 are downregulated by promoter hypermethylation in systemic sclerosis.

OBJECTIVES: Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists. METHODS: The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2'-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for ß-catenin. RESULTS: The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated experimental fibrosis. CONCLUSIONS: We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.

Inhibition of hedgehog signaling for the treatment of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a prognosis limiting complication of allogeneic stem cell transplantation. The molecular mechanisms underlying cGVHD are incompletely understood, and targeted therapies are not yet established for clinical use. Here we examined the role of the hedgehog pathway in sclerodermatous cGVHD. Hedgehog signaling was activated in human and murine cGVHD with increased expression of sonic hedgehog and accumulation of the transcription factors Gli-1 and Gli-2. Treatment with LDE223, a highly selective small-molecule antagonist of the hedgehog coreceptor Smoothened (Smo), abrogated the activation of hedgehog signaling and protected against experimental cGVHD. Preventive therapy with LDE223 almost completely impeded the development of clinical and histologic features of sclerodermatous cGVHD. Treatment with LDE223 was also effective, when initiated after the onset of clinical manifestations of cGVHD. Hedgehog signaling stimulated the release of collagen from cultured fibroblasts but did not affect leukocyte influx in murine cGVHD, suggesting direct, leukocyte-independent stimulatory effects on fibroblasts as the pathomechanism of hedgehog signaling in cGVHD. Considering the high morbidity of cGVHD, the current lack of efficient molecular therapies for clinical use, and the availability of well-tolerated inhibitors of Smo, targeting hedgehog signaling might be a novel strategy for clinical trials in cGVHD.

Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis.

OBJECTIVES: Epigenetic modifications such as DNA methylation and histone acetylation have been implicated in the pathogenesis of systemic sclerosis. However, histone methylation has not been investigated so far. We therefore aimed to evaluate the role of the trimethylation of histone H3 on lysine 27 (H3K27me3) on fibroblast activation and fibrosis. METHODS: H3K27me3 was inhibited by 3-deazaneplanocin A (DZNep) in cultured fibroblasts and in two murine models of dermal fibrosis. Fibrosis was analysed by assessment of the dermal thickening, determination of the hydroxyproline content and by quantification of the numbers of myofibroblasts. The expression of fos-related antigen 2 (fra-2) was assessed by real-time PCR, western blot and immunohistochemistry and modulated by siRNA. RESULTS: Inhibition of H3K27me3 stimulated the release of collagen in cultured fibroblasts in a time and dose-dependent manner. Treatment with DZNep exacerbated fibrosis induced by bleomycin or by overexpression of a constitutively active transforming growth factor ß receptor type I. Moreover, treatment with DZNep alone was sufficient to induce fibrosis. Inhibition of H3K27me3 induced the expression of the profibrotic transcription factor fra-2 in vitro and in vivo. Knockdown of fra-2 completely prevented the profibrotic effects of DZNep. CONCLUSIONS: These data demonstrate a novel role of H3 Lys27 histone methylation in fibrosis. In contrast to other epigenetic modifications such as DNA methylation and histone acetylation, H3 Lys27 histone methylation acts as a negative regulator of fibroblast activation in vitro and in vivo by repressing the expression of fra-2.

Combined inhibition of c-Abl and PDGF receptors for prevention and treatment of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic bone marrow transplantation, and has a major effect on the long-term prognosis. The molecular mechanisms underlying cGvHD have been only partially revealed, and molecular targeted therapies have not yet been established for clinical use. We examined the effects of the combined inhibition of the Abelson kinase (c-Abl) and platelet-derived growth factor receptors (PDGFR) in experimental sclerodermatous cGvHD. Treatment using imatinib or nilotinib abolished the aberrant activation of c-Abl and PDGFR and protected against experimental cGvHD. Preventive therapy using imatinib or nilotinib inhibited the development of sclerodermatous cGvHD. Clinical features such as weight loss, alopecia, and skin ulcers, and histologic features with dermal thickening and accumulation of collagen were significantly reduced in mice that received imatinib or nilotinib therapy, but not in mice that received prednisone therapy. Of note, imatinib and nilotinib were also effective for treatment of experimental cGvHD that had already been clinically manifested. In summary, the combined inhibition of c-Abl and PDGFR is effective for prevention and treatment of experimental sclerodermatous cGvHD. Considering the high morbidity associated with cGvHD, the lack of efficient molecular therapies for clinical use, and first positive signals from uncontrolled studies of imatinib, combined inhibition of c-Abl and PDGFR might be a promising future strategy for treatment of sclerodermatous cGvHD.

Hedgehog signaling controls fibroblast activation and tissue fibrosis in systemic sclerosis.

OBJECTIVE: Hedgehog signaling not only plays crucial roles during human development but also has been implicated in the pathogenesis of several diseases in adults. The aim of the present study was to investigate the role of the hedgehog pathway in fibroblast activation in systemic sclerosis (SSc). METHODS: Activation of the hedgehog pathway was analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR). The effects of sonic hedgehog (SHH) on collagen synthesis were analyzed by reporter assays, real-time PCR, and Sircol assays. Myofibroblast differentiation was assessed by quantification of α-smooth muscle actin and stress fiber staining. The role of hedgehog signaling in vivo was analyzed by adenoviral overexpression of SHH and using mice lacking 1 allele of the gene for inhibitory receptor Patched homolog 1 (Ptch(+/-) mice). RESULTS: SHH was overexpressed and resulted in activation of hedgehog signaling in patients with SSc, with accumulation of the transcription factors Gli-1 and Gli-2 and increased transcription of hedgehog target genes. Activation of hedgehog signaling induced an activated phenotype in cultured fibroblasts, with differentiation of resting fibroblasts into myofibroblasts and increased release of collagen. Adenoviral overexpression of SHH in the skin of mice was sufficient to induce skin fibrosis. Moreover, Ptch(+/-) mice with increased hedgehog signaling were more sensitive to bleomycin-induced dermal fibrosis. CONCLUSION: We demonstrated that the hedgehog pathway is activated in patients with SSc. Hedgehog signaling potently stimulates the release of collagen and myofibroblast differentiation in vitro and is sufficient to induce fibrosis in vivo. These findings identify the hedgehog cascade as a profibrotic pathway in SSc.

Inhibition of activator protein 1 signaling abrogates transforming growth factor ß-mediated activation of fibroblasts and prevents experimental fibrosis.

OBJECTIVE: To investigate whether c-Jun and c-Fos contribute to the pathologic activation of fibroblasts in systemic sclerosis (SSc) and to evaluate the antifibrotic potential of selective activator protein 1 (AP-1) inhibition. METHODS: Expression of c-Jun and c-Fos was determined by real-time polymerase chain reaction (PCR) and immunohistochemical analysis. Fibroblasts were stimulated with transforming growth factor ß (TGFß) and incubated with T-5224, a small-molecule inhibitor of AP-1, or were transfected with small interfering RNA (siRNA) duplexes against c-Jun and c-Fos. Collagen synthesis was quantified by real-time PCR and hydroxyproline assay. Differentiation of resting fibroblasts into myofibroblasts was assessed by staining for α-smooth muscle actin and stress fibers. The antifibrotic potential of T-5224 was evaluated in mouse models of dermal fibrosis induced by bleomycin or by adenoviral overexpression of a constitutively active TGFß receptor type I. RESULTS: Up-regulation of c-Jun and c-Fos was detected in mouse models of SSc and in the skin and dermal fibroblasts of patients with SSc. Stimulation of healthy fibroblasts with TGFß induced the expression of c-Jun and c-Fos. Treatment with T-5224 or nucleofection with siRNA directed against c-Jun and c-Fos abrogated the profibrotic effects of TGFß. T-5224 decreased the release of collagen selectively in SSc fibroblasts. T-5224 was well tolerated and prevented dermal fibrosis induced by bleomycin or by adenoviral activation of TGFß signaling. CONCLUSION: AP-1 is up-regulated in a TGFß-dependent manner in SSc. The selective AP-1 inhibitor T-5224 reduced collagen synthesis selectively in SSc fibroblasts and efficiently prevented the development of experimental dermal fibrosis. Thus, AP-1 might be a promising new molecular target for the treatment of SSc.

JAK-2 as a novel mediator of the profibrotic effects of transforming growth factor ß in systemic sclerosis.

OBJECTIVE: To investigate whether JAK-2 contributes to the pathologic activation of fibroblasts in patients with systemic sclerosis (SSc) and to evaluate the antifibrotic potential of JAK-2 inhibition for the treatment of SSc. METHODS: Activation of JAK-2 in human skin and in experimental fibrosis was determined by immunohistochemical analysis. JAK-2 signaling was inhibited by the selective JAK-2 inhibitor TG101209 or by small interfering RNA. Bleomycin-induced dermal fibrosis in mice and TSK-1 mice were used to evaluate the antifibrotic potential of specific JAK-2 inhibition in vivo. RESULTS: Increased activation of JAK-2 was detected in the skin of patients with SSc, particularly in fibroblasts. The activation of JAK-2 was dependent on transforming growth factor ß (TGFß) and persisted in cultured SSc fibroblasts. Inhibition of JAK-2 reduced basal collagen synthesis selectively in SSc fibroblasts but not in resting healthy dermal fibroblasts. Moreover, inhibition of JAK-2 prevented the stimulatory effects of TGFß on fibroblasts. Treatment with TG101209 not only prevented bleomycin-induced fibrosis but also effectively reduced skin fibrosis in TSK-1 mice. CONCLUSION: We demonstrated that JAK-2 is activated in a TGFß-dependent manner in SSc. Considering the potent antifibrotic effects of JAK-2 inhibition, our study might have direct translational implications, because inhibitors of JAK-2 are currently being evaluated in clinical trials for myeloproliferative disorders and would also be available for evaluation in patients with SSc.

Blockade of the hedgehog pathway inhibits osteophyte formation in arthritis.

BACKGROUND: Osteophyte formation is a common phenomenon in arthritis. Bone formation by endochondral ossification is considered a key pathophysiological process in the formation of osteophytes. OBJECTIVE: To examine the hypothesis that inhibition of smoothened (Smo), a key component of the hedgehog pathway inhibits osteophyte formation as the hedgehog pathway mediates endochondral ossification. METHODS: Arthritis was induced in 8-week-old C57/BL6 mice by serum transfer (K/BxN model). Mice were then treated by daily administration of either vehicle or LDE223, a specific small molecule inhibitor for Smo, over 2 weeks starting at the onset of disease. Clinical course of arthritis, histological and molecular changes of bone in the affected joints as well as systemic bone changes were assessed. RESULTS: Serum transfer-induced arthritis led to severe osteophyte formation within 2 weeks of onset. Blockade of Smo inhibited hedgehog signalling in vivo and also significantly inhibited osteophyte formation, whereas the clinical and histopathological signs of arthritis were not affected. Also, systemic bone mass did not change. Smo inhibitor particularly blocked the formation of hypertrophic chondrocytes and collagen type X expression. CONCLUSIONS: The data indicate that blockade of hedgehog signalling by targeting Smo specifically inhibits osteophyte formation in arthritis without affecting inflammation and without eliciting bone destruction at the local and systemic level. Blockade of Smo may thus be considered as a strategy to specifically influence the periosteal bone response in arthritis associated with bone apposition.

Inhibition of hedgehog signalling prevents experimental fibrosis and induces regression of established fibrosis.

OBJECTIVES: Tissue fibrosis is a leading cause of death in patients with systemic sclerosis (SSc). Effective antifibrotic treatments are not available. Here, the authors investigated inhibition of hedgehog signalling by targeting Smoothened (Smo) as a novel antifibrotic approach. METHODS: The activation status of the hedgehog pathway was assessed by immunohistochemistry for Gli transcription factors and by quantification of hedgehog target genes. Hedgehog signalling was inhibited by the selective inhibitor LDE223 and by small interfering RNA against Smo in the models of bleomycin-induced dermal fibrosis and in tight-skin-1 mice. RESULTS: Hedgehog signalling is activated in SSc and in murine models of SSc. Inhibition of Smo either by LDE223 or by small interfering RNA prevented dermal thickening, myofibroblast differentiation and accumulation of collagen upon challenge with bleomycin. Targeting Smo also exerted potent antifibrotic effects in tight-skin-1 mice and did prevent progression of fibrosis and induced regression of pre-established fibrosis. CONCLUSIONS: Inhibition of hedgehog signalling exerted potent antifibrotic effects in preclinical models of SSc in both preventive and therapeutic settings. These findings might have direct translational implications because inhibitors of Smo are already available and yielded promising results in initial clinical trials.