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
OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-ß (TGF-ß) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-ß receptor I. RESULTS: TGF-ß upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-ß signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.
Asunto(s)
Proteínas Hedgehog/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Anilidas/farmacología , Animales , Células Cultivadas , Colágeno Tipo I/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Técnicas de Inactivación de Genes , Humanos , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pteridinas/farmacología , Fibrosis Pulmonar/inducido químicamente , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Proteína smad3/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Adulto Joven , Proteína Gli2 con Dedos de ZincRESUMEN
OBJECTIVES: Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-ß. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts. METHODS: The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance. RESULTS: The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. CONCLUSION: Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.
Asunto(s)
Fibroblastos/efectos de los fármacos , Janus Quinasa 1/efectos de los fármacos , Janus Quinasa 2/efectos de los fármacos , Pulmón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Fibrosis Pulmonar/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Esclerodermia Sistémica , Sulfonamidas/farmacología , Adulto , Animales , Antibióticos Antineoplásicos/toxicidad , Benzoquinonas/farmacología , Bleomicina/toxicidad , Western Blotting , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Inmunohistoquímica , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Lactamas Macrocíclicas/farmacología , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Nitrilos , Fosforilación/efectos de los fármacos , Fibrosis Pulmonar/inducido químicamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
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
BACKGROUND AND OBJECTIVES: Fibrosis is a major socioeconomic burden, but effective antifibrotic therapies are not available in the clinical routine. There is growing evidence for a central role of Wnt signalling in fibrotic diseases such as systemic sclerosis, and we therefore evaluated the translational potential of pharmacological Wnt inhibition in experimental dermal fibrosis. METHODS: We examined the antifibrotic effects of PKF118-310 and ICG-001, two novel inhibitors of downstream canonical Wnt signalling, in the models of prevention and treatment of bleomycin-induced dermal fibrosis as well as in experimental dermal fibrosis induced by adenoviral overexpression of a constitutively active transforming growth factor (TGF)-ß receptor I. RESULTS: PKF118-310 and ICG-001 were well tolerated throughout all experiments. Both therapeutic approaches showed antifibrotic effects in preventing and reversing bleomycin-induced dermal fibrosis as measured by skin thickness, hydroxyproline content and myofibroblast counts. PKF118-310 and ICG-001 were effective in inhibiting TGF-ß receptor I-driven fibrosis as assessed by the same outcome measures. CONCLUSIONS: Blockade of canonical Wnt signalling by PKF118-310 and ICG-001 showed antifibrotic effects in different models of skin fibrosis. Both therapies were well tolerated. Although further experimental evidence for efficacy and tolerability is necessary, inhibition of canonical Wnt signalling is a promising treatment approach for fibrosis.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Pirimidinonas/uso terapéutico , Enfermedades de la Piel/prevención & control , Piel/patología , Triazinas/uso terapéutico , Vía de Señalización Wnt/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Modelos Animales de Enfermedad , Fibrosis , Ratones , Ratones Endogámicos DBA , Pirimidinonas/farmacología , Esclerodermia Sistémica , Transducción de Señal/efectos de los fármacos , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/patología , Resultado del Tratamiento , Triazinas/farmacologíaRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFß-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFß. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc.