RESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Proteínas Nucleares/fisiología , Esclerodermia Sistémica/metabolismo , Proteína 1 Relacionada con Twist/fisiología , Animales , Estudios de Casos y Controles , Femenino , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones Noqueados , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Multimerización de Proteína/fisiología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Esclerodermia Sistémica/patología , Transducción de Señal/fisiología , Piel/patología , Factor de Crecimiento Transformador beta/farmacología , Proteína 1 Relacionada con Twist/biosíntesis , Proteína 1 Relacionada con Twist/deficiencia , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
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.
Asunto(s)
Factor de Transcripción Activador 3/genética , Fibroblastos/metabolismo , Esclerodermia Sistémica/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Western Blotting , Estudios de Casos y Controles , Dermis/citología , Femenino , Fibrosis/genética , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/metabolismo , Transducción de Señal/genética , Factor de Transcripción AP-1/metabolismo , Adulto JovenRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/genética , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Represoras/genética , Esclerodermia Sistémica/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Estudios de Casos y Controles , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , Dermis/citología , Modelos Animales de Enfermedad , Femenino , Fibrosis/inducido químicamente , Fibrosis/genética , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta , Proteínas Represoras/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/genética , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/genética , Adulto JovenRESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Sirtuina 1/fisiología , Piel/patología , Factor de Crecimiento Transformador beta/fisiología , Adulto , Anciano , Animales , Bleomicina , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Femenino , Fibrosis , Humanos , Masculino , Ratones Noqueados , Persona de Mediana Edad , Esclerodermia Sistémica/patología , Transducción de Señal/fisiología , Piel/metabolismoRESUMEN
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.
Asunto(s)
Interleucinas/biosíntesis , Esclerodermia Sistémica/inmunología , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Colágeno/biosíntesis , Femenino , Fibroblastos/inmunología , Humanos , Interleucinas/sangre , Interleucinas/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Piel/inmunología , Factor de Crecimiento Transformador beta/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-ß (TGF-ß) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-ß target genes, thereby limiting pro-fibrotic TGF-ß effects. Even though temporary upregulation of TGF-ß in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-ß signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-ß signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.
Asunto(s)
Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Cirrosis Hepática Alcohólica/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , Células Cultivadas , Proteínas Co-Represoras/metabolismo , Femenino , Fibrosis , Histona Desacetilasa 1/metabolismo , Histona Demetilasas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/patología , Hígado/patología , Cirrosis Hepática Alcohólica/patología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteínas Represoras/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal , Complejo Correpresor Histona Desacetilasa y Sin3 , Piel/citología , Piel/patología , Factor de Transcripción Sp1/metabolismo , Cicatrización de Heridas , Adulto JovenRESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Proteínas S100/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteína de Unión al Calcio S100A4 , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Cardiomyopathy has emerged as a leading cause of death in patients with systemic sclerosis (SSc). However, the pathogenesis of SSc-related cardiomyopathy is poorly understood, and new therapies as well as platforms for testing are needed. The aim of this study was to characterize the histopathologic features of cardiomyopathy in patients with SSc and in common mouse models of SSc. METHODS: The histopathologic features of myocardial tissue specimens obtained at autopsy from 5 subjects with SSc and 5 control subjects matched for sex, age, and cardiovascular risk factors were evaluated and compared with those of myocardial tissue specimens obtained from 3 common mouse models of SSc with systemic manifestations: Fra-2-transgenic mice, mice with sclerodermatous chronic graft-versus-host disease (GVHD), and TSK-1 mice. RESULTS: Myocardial tissue from autopsy subjects with SSc and no clinically manifest cardiac involvement showed endothelial cell apoptosis with reduced capillary density, perivascular inflammation, myofibroblast differentiation, and accumulation of collagen. Only selected features of SSc-related cardiomyopathy were observed in the mice with chronic GVHD and TSK-1 mice. However, the myocardial tissue of Fra-2-transgenic mice mimicked all features of SSc-related cardiomyopathy and also demonstrated comparable vascular, inflammatory, and fibrotic manifestations. Of note, the expression of Fra-2 was also increased in the myocardium of autopsy subjects with SSc. CONCLUSION: We demonstrate that all typical manifestations of SSc-related cardiomyopathy are mimicked in Fra-2-transgenic mice. Moreover, overexpression of Fra-2 in the myocardium of autopsy subjects with SSc may suggest similar underlying pathogenic mechanisms. Thus, Fra-2-transgenic mice might be a suitable preclinical model with which to study the mechanisms of and therapeutic approaches to myocardial involvement in SSc.
Asunto(s)
Cardiomiopatías/etiología , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Miocardio/patología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/patología , Adulto , Animales , Apoptosis , Autopsia , Cardiomiopatías/epidemiología , Enfermedades Cardiovasculares/epidemiología , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Antígeno 2 Relacionado con Fos/genética , Antígeno 2 Relacionado con Fos/metabolismo , Enfermedad Injerto contra Huésped/complicaciones , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Persona de Mediana Edad , Miocardio/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de RiesgoRESUMEN
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.
Asunto(s)
Fibroblastos/patología , Guanilato Ciclasa/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/fisiopatología , Transducción de Señal/fisiología , Piel/patología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Estudios de Casos y Controles , Células Cultivadas , Colágeno/metabolismo , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis/metabolismo , Fibrosis/prevención & control , Guanilato Ciclasa/deficiencia , Humanos , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Pirazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/efectos de los fármacos , Piel/metabolismo , Proteínas Smad/metabolismo , Guanilil Ciclasa Soluble , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/genética , Esclerodermia Difusa/metabolismo , Enfermedades de la Piel/metabolismo , Piel/patología , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibrosis , Humanos , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/metabolismo , Piel/efectos de los fármacos , Enfermedades de la Piel/inducido químicamente , Sulfonamidas/farmacologíaRESUMEN
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.
Asunto(s)
Fibroblastos/metabolismo , Receptores de Calcitriol/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/fisiología , Piel/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Ergocalciferoles/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , ARN Interferente Pequeño/metabolismo , Receptores de Calcitriol/agonistas , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Proteínas Smad/efectos de los fármacos , Factor de Crecimiento Transformador beta/efectos de los fármacos , Adulto JovenRESUMEN
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.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Fibroblastos/metabolismo , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis , Humanos , Janus Quinasa 2/efectos de los fármacos , Masculino , Ratones , Persona de Mediana Edad , Factor de Transcripción STAT3/efectos de los fármacos , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Factor de Crecimiento Transformador beta/efectos de los fármacos , Triazoles/farmacología , Adulto JovenRESUMEN
OBJECTIVE: Nuclear receptors regulate cell growth, differentiation, and homeostasis. Selective nuclear receptors promote fibroblast activation, which leads to tissue fibrosis, the hallmark of systemic sclerosis (SSc). This study was undertaken to investigate the effects of constitutive androstane receptor (CAR)/NR1I3, an orphan nuclear receptor, on fibroblast activation and experimental dermal fibrosis. METHODS: CAR expression was quantified by quantitative polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. CAR expression was modulated by small molecules, small interfering RNA, forced overexpression, and site-directed mutagenesis. The effects of CAR activation were analyzed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, and in mice overexpressing a constitutively active transforming growth factor ß (TGFß) receptor type I (TßRI-CA). RESULTS: Up-regulation of CAR was detected in the skin and in dermal fibroblasts in SSc patients. Stimulation of healthy fibroblasts with TGFß induced the expression of CAR messenger RNA and protein in a Smad-dependent manner. Pharmacologic activation or overexpression of CAR in healthy fibroblasts significantly increased the stimulatory effects of TGFß on collagen synthesis and myofibroblast differentiation, and amplified the stimulatory effects of TGFß on COL1A2 transcription activity. Treatment with CAR agonist increased the activation of canonical TGFß signaling in murine models of SSc and exacerbated bleomycin-induced and TßRI-CA-induced fibrosis with increased dermal thickening, myofibroblast counts, and collagen accumulation. CONCLUSION: Our findings indicate that CAR is up-regulated in SSc and regulates TGFß signaling. Activation of CAR increases the profibrotic effects of TGFß in cultured fibroblasts and in different preclinical models of SSc. Thus, inactivation of CAR might be a novel approach to target aberrant TGFß signaling in SSc and in other fibrotic diseases.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo , Esclerodermia Sistémica/metabolismo , Piel/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Bleomicina/efectos adversos , Células Cultivadas , Colágeno Tipo I/metabolismo , Receptor de Androstano Constitutivo , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
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.
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Metilación de ADN/genética , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Leucocitos Mononucleares/metabolismo , Proteínas de la Membrana/genética , Esclerodermia Sistémica/genética , Vía de Señalización Wnt/genética , Adulto , Anciano , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Estudios de Casos y Controles , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Metilación de ADN/efectos de los fármacos , Decitabina , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Vía de Señalización Wnt/efectos de los fármacos , Adulto JovenRESUMEN
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.
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Fibroblastos/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Esclerodermia Sistémica/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Benzoquinonas/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Esclerodermia Sistémica/prevención & control , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Piel/metabolismo , Factor de Crecimiento Transformador beta/efectos de los fármacosRESUMEN
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.
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Fibroblastos/enzimología , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Esclerodermia Difusa/metabolismo , Esclerodermia Difusa/patología , Adulto , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Células Cultivadas , Colágeno/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Dermis/patología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/fisiología , Fibroblastos/patología , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Fibrosis/patología , Antígeno 2 Relacionado con Fos/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Esclerodermia Difusa/inducido químicamente , Adulto JovenRESUMEN
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.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/prevención & control , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/prevención & control , Animales , Benzamidas , Enfermedad Crónica , Dermis/efectos de los fármacos , Dermis/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Fibrosis , Enfermedad Injerto contra Huésped/patología , Humanos , Mesilato de Imatinib , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Piperazinas/farmacología , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVES: Tissue fibrosis is a major hallmark and a leading cause of death in systemic sclerosis (SSc). Here, we investigated the antifibrotic effects of pomalidomide, an analogue of thalidomide with potent immunomodulatory effects, in preclinical models of skin fibrosis. METHODS: We evaluated the antifibrotic effects of pomalidomide in preventive as well as therapeutic treatment regimes using bleomycin-induced dermal fibrosis as a model of early, inflammatory stages of fibrosis and the tight-skin mouse model as a model of later stages of fibrosis with endogenous activation of fibroblasts. RESULTS: Treatment with pomalidomide in doses from 0.3 to 30 mg/kd/day prevented skin fibrosis in Tsk-1 mice and in bleomycin-induced dermal fibrosis in a dose-dependent manner and reduced the expression of transforming growth factor (TGF) ß-target genes such as PAI-1, CTGF and col 1a1. Pomalidomide was also effective in the setting of pre-established fibrosis and reduced dermal thickness, myofibroblast counts and hydroxyproline content below pretreatment levels. CONCLUSIONS: We demonstrate for the first time that pomalidomide exerts potent antifibrotic effects in different preclinical models of skin fibrosis. These findings lend preclinical support for the clinical studies of pomalidomide in SSc.
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Fibrosis/prevención & control , Inmunosupresores/farmacología , Enfermedades de la Piel/tratamiento farmacológico , Talidomida/análogos & derivados , Animales , Modelos Animales de Enfermedad , Fibrosis/inducido químicamente , Fibrosis/patología , Hidroxiprolina/metabolismo , Ratones , Ratones Endogámicos DBA , Ratones Mutantes , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Piel/efectos de los fármacos , Piel/metabolismo , Enfermedades de la Piel/inducido químicamente , Enfermedades de la Piel/patología , Talidomida/farmacologíaRESUMEN
BACKGROUND: Selective targeting of the cannabinoid receptors CB1 and CB2 by synthetic compounds has revealed opposing roles of both receptors in fibrosis. OBJECTIVES: To characterise the role of endogenous cannabinoids (endocannabinoids) and their predominant receptor in fibrosis. METHODS: The levels of endocannabinoids in mice were modulated by pharmacological or genetic inactivation of the enzyme fatty acid amide hydrolase (FAAH). The predominant receptor for endocannabinoids was determined by selective inhibition of either CB1 or CB2. The extent of fibrosis upon challenge with bleomycin was determined by quantification of dermal thickness, hydroxyproline content and myofibroblast counts. RESULTS: The expression of FAAH is decreased in systemic sclerosis fibroblasts. FAAH-deficient mice with strongly increased levels of endocannabinoids were more sensitive to bleomycin. Consistently, pharmacological inhibition of FAAH significantly exacerbated bleomycin-induced fibrosis. Inhibition of CB1 completely abrogated the profibrotic effects of FAAH inactivation. In contrast, inhibition of CB2 only modestly enhanced fibrosis, indicating that CB1 is the predominant receptor for endocannabinoids in experimental fibrosis. CONCLUSIONS: Increased levels of endocannabinoids induced by inactivation of FAAH worsen experimental fibrosis via activation of CB1. These findings highlight the profibrotic effects of endocannabinoids and suggest that CB1 maybe a more promising candidate for targeted treatments in fibrotic diseases than CB2.
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Amidohidrolasas/genética , Endocannabinoides/metabolismo , Receptor Cannabinoide CB1/metabolismo , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/patología , Amidohidrolasas/metabolismo , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Fibrosis/patología , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Cannabinoide CB2/metabolismo , Enfermedades de la Piel/inducido químicamenteRESUMEN
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.