RESUMEN
Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.
Asunto(s)
Colágeno/genética , PPAR gamma/fisiología , Proteínas Smad/fisiología , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Ratones , Transporte de Proteínas , Activación Transcripcional , Factor de Crecimiento Transformador beta/fisiología , Factores de Transcripción p300-CBP/genéticaRESUMEN
Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Factor de Crecimiento Transformador beta/farmacología , Adolescente , Adulto , Anciano , Animales , Bleomicina , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibrosis , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Esclerodermia Sistémica/enzimología , Piel/metabolismo , Piel/patología , Proteína smad3/metabolismo , Transcripción Genética , Regulación hacia Arriba/efectos de los fármacos , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
BACKGROUND: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos. METHODS: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. RESULTS: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. CONCLUSION: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.
Asunto(s)
Fibrosis/metabolismo , Proteínas Inmediatas-Precoces/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colágeno/genética , Colágeno Tipo I , Factor de Crecimiento del Tejido Conjuntivo , Embrión de Mamíferos , Matriz Extracelular/genética , Fibroblastos , Expresión Génica , Ratones , Transducción de Señal , Proteínas Smad/metabolismo , Proteínas Smad/fisiologíaRESUMEN
Transforming growth factor- beta (TGF-beta) stimulates Type I collagen synthesis by fibroblasts and is implicated in tissue fibrosis. Here, we demonstrate that histone deacetylase inhibitor Trichostatin A (TSA) suppresses the TGF-beta-induced Type I collagen synthesis but not induced PAI-1 synthesis suggesting the influence of TSA is gene specific. Results further reveal that there is no significant alteration in Smad activation and function in presence of TSA suggesting suppression of TGF-beta-induced collagen synthesis is not due to impaired Smad signaling. TGF-beta induces the levels of Sp1, an essential transcription factor of Smad-dependent stimulation of collagen synthesis. However, in presence of TSA, TGF-beta fails to induce Sp1 levels, its interaction with Smad complex and Sp1 binding site in COL1A2 promoter. Furthermore, overexpressed Sp1 reverses the TSA-mediated inhibition of TGF-beta-induced collagen gene expression. Collectively, these results suggest that TSA-mediated suppression of Smad-dependent TGF-beta-induced collagen synthesis is due to suppression of Sp1 activity in skin fibroblasts.
Asunto(s)
Colágeno/antagonistas & inhibidores , Colágeno/genética , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Factor de Transcripción Sp1/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/fisiología , Células Cultivadas , Colágeno/biosíntesis , Inhibidores de Histona Desacetilasas , HumanosRESUMEN
Fibrosis, the hallmark of scleroderma, is characterized by excessive synthesis of collagen and extracellular matrix proteins and accumulation of myofibroblasts. Transforming growth factor-beta (TGF-beta), a potent inducer of collagen synthesis, cytokine production, and myofibroblast transdifferentiation, is implicated in fibrosis. Profibrotic TGF-beta responses are induced primarily via the type I activin-like receptor kinase 5 (ALK5) TGF-beta receptor coupled to Smad signal transducers. Here, we investigated the effect of blocking ALK5 function with SM305, a novel small-molecule kinase inhibitor, on fibrotic TGF-beta responses. In normal dermal fibroblasts, SM305 abrogated the ligand-induced phosphorylation, nuclear import, and DNA-binding activity of Smad2/3 and Smad4, and inhibited Smad2/3-dependent transcriptional responses. Furthermore, SM305 blocked TGF-beta-induced extracellular matrix gene expression, cytokine production, and myofibroblast transdifferentiation. In unstimulated scleroderma fibroblasts, SM305 caused a variable and modest reduction in type I collagen levels, and failed to abrogate constitutive nuclear accumulation of Smad2/3, or alter the proportion of smooth muscle actin stress fiber-positive fibroblasts. In vivo, SM305 prevented TGF-beta-induced Smad2/3 phosphorylation type I collagen (COL1)A2 promoter activation in dermal fibroblasts. Taken together, these results indicate that SM305 inhibits intracellular TGF-beta signaling through selective interference with ALK5-mediated Smad activation, resulting in marked suppression of profibrotic responses induced by TGF-beta in vivo and in vitro.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Quinolinas/farmacología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/metabolismo , Proteínas Smad/metabolismo , Animales , Biopsia , División Celular/efectos de los fármacos , Células Cultivadas , Dermis/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Esclerodermia Sistémica/patología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismoRESUMEN
Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.
Asunto(s)
Colágeno/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Regulación de la Expresión Génica , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Secuencia de Bases , Colágeno/metabolismo , Colágeno Tipo I , Fibroblastos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
By inhibiting collagen synthesis, interferon-gamma (IFN-gamma) plays a key role in maintaining connective tissue homeostasis, but the mechanisms are not well-understood. In addition to intracellular signaling through the canonical JAK-STAT transduction pathway, IFN-gamma was recently shown to regulate gene expression via the CCAAT/enhancer-binding protein beta (C/EBPbeta) as well. Because C/EBPbeta is a crucial mediator of immune and inflammatory responses, and has been implicated in regulation of collagen synthesis by tumor necrosis factor-alpha, we examined its role in the inhibitory effects of IFN-gamma. The results demonstrated that IFN-gamma caused increased C/EBPbeta expression in dermal fibroblasts and enhanced its binding to cognate DNA sequences in the alpha2(I) procollagen gene (COL1A2) promoter in vitro and in vivo. Disruption of C/EBP binding by deletion or site-directed mutagenesis abrogated the inhibition of collagen promoter activity in transient transfection assays, as did cotransfection with dominant negative C/EBPbeta, indicating a functional role of cellular C/EBPbeta in mediating the IFN-gamma response. Rapid phosphorylation of the ERK1/2 MAP kinases induced by IFN-gamma was accompanied by phosphorylation and nuclear translocation of cellular C/EBPbeta, and pretreatment of fibroblasts with ERK1/2 kinase inhibitor blocked C/EBPbeta phosphorylation, as well as inhibition of COL1A2 promoter activity, elicited by IFN-gamma. These results provide compelling evidence for a novel C/EBPbeta-dependent IFN-gamma signaling pathway responsible for inhibition of collagen gene transcription. Taken together with recent reports, the findings indicate that intracellular pathways mediating negative regulation of collagen synthesis in response to distinct inflammatory signals that converge on C/EBPbeta.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Colágeno/genética , Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Colágeno Tipo I , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavonoides/farmacología , Expresión Génica/genética , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Oligodesoxirribonucleótidos/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica , Elementos de Respuesta/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , TransfecciónRESUMEN
BACKGROUND: Eosinophils are frequently associated with tissue remodeling and fibrosis in allergic and other diseases and animal models. Their close physical proximity to fibroblasts at sites of tissue remodeling strongly implicates them in fibrogenesis, including subepithelial fibrosis and airway remodeling characteristic of asthma. OBJECTIVE: To identify the mediators and characterize the mechanisms underlying the fibrogenic activities of eosinophils. METHODS: A coculture system of blood eosinophils or eosinophil cell lines with normal fibroblasts was used to assess their ability to induce a fibrogenic fibroblast phenotype, including IL-6 secretion and mRNA expression, and induction of genes involved in extracellular matrix production and homeostasis. The mediators of these responses were identified by using transwell barrier cocultures, eosinophil-conditioned media, and cytokine-specific antibody neutralization. RESULTS: Eosinophil-fibroblast coculture induced potent fibroblast IL-6 secretion and mRNA expression, responses further enhanced by IL-5. The soluble nature of the eosinophil-derived mediators was demonstrated by using eosinophil-fibroblast coculture in the presence of permeable transwell barriers, and fibroblast culture in eosinophil-conditioned media, indicating that cell contact was not required. Induction of fibroblast IL-6 expression was accompanied by increased expression of fibronectin and the extracellular matrix regulatory genes plasminogen activator inhibitor 1 and tissue inhibitor of metalloproteinase 1. Antibody neutralization identified the principal eosinophil-derived mediator of fibroblast IL-6 expression as IL-1beta (>60%), with lesser contributions from IL-1alpha, IL-4, and TGF-beta (10% to 20%). CONCLUSION: Eosinophils express at least 2 potent mediators (IL-1beta and TGF-beta) that induce a fibrogenic fibroblast phenotype, strongly supporting a role for the eosinophil in the dysregulation of extracellular matrix homeostasis and consequent tissue remodeling and fibrosis in eosinophil-associated diseases.
Asunto(s)
Eosinófilos/inmunología , Fibroblastos/inmunología , Interleucina-6/biosíntesis , Comunicación Celular , Técnicas de Cocultivo , Eosinófilos/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Fibroblastos/fisiología , Fibrosis , Expresión Génica , Humanos , Interleucina-1/biosíntesis , Interleucina-6/genética , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
OBJECTIVE: Transforming growth factor beta (TGFbeta) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smad-dependent profibrotic responses elicited by TGFbeta. METHODS: Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry. RESULTS: In normal fibroblasts, TGFbeta induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGFbeta responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGFbeta-induced collagen synthesis and promoter activity. Furthermore, TGFbeta lost its ability to induce Smad-dependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGFbeta signaling was not due to alterations in the levels or the ligand-dependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versus-host disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls. CONCLUSION: These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGFbeta regulation, is an essential component of the cellular TGFbeta signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts. Since the cellular abundance of p300 appears to govern the intensity of profibrotic responses elicited by TGFbeta, elevated p300 expression in lesional tissue may contribute to the progression of skin fibrosis in scleroderma.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Fibroblastos/enzimología , Fibrosis/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Esclerodermia Sistémica/enzimología , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacología , Acetiltransferasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos/patología , Fibrosis/genética , Fibrosis/patología , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Histona Acetiltransferasas , Humanos , Recién Nacido , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , ARN Catalítico , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Piel/enzimología , Piel/patología , Proteína smad3 , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción p300-CBPRESUMEN
OBJECTIVE: Members of the transforming growth factor beta (TGFbeta) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGFbeta signals through the activin receptor-like kinase 5 (ALK-5) type I TGFbeta and triggers Smad and MAP kinase signaling pathways. Because targeting of TGFbeta signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGFbeta signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described. METHODS: Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGFbeta production was evaluated by enzyme-linked immunosorbent assay. RESULTS: SB431542 abrogated TGFbeta-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3- and Smad2-dependent gene transcription. Treatment with SB431542 prevented TGFbeta-induced stimulation of collagen, fibronectin, plasminogen activator inhibitor 1, and connective tissue growth factor gene expression, TGFbeta autoinduction, and myofibroblast transdifferentiation, and it could reverse stimulation even when added to the cultures after TGFbeta. In contrast, STAT-6-mediated stimulation of collagen gene expression induced by interleukin-13 was not prevented by SB431542, indicating the specificity of blockade for ALK-5-dependent signaling. Furthermore, in contrast to its effects on receptor-activated Smad activation, SB431542 failed to prevent TGFbeta-induced activation of MAP kinases. CONCLUSION: The results indicate that SB431542 is a potent inhibitor of intracellular TGFbeta signaling in normal fibroblasts through selective interference with ALK-5-mediated Smad activation and Smad-dependent transcriptional responses. Therefore, SB431542 is useful as a novel experimental tool for gaining a detailed understanding of normal and aberrant TGFbeta signaling in SSc. Furthermore, as an anti-TGFbeta agent, SB431542 may represent a potential new approach to the treatment of fibrosis.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Benzamidas/farmacología , Dioxoles/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/enzimología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Piel/enzimología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptores de Activinas Tipo I/metabolismo , Adulto , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis/tratamiento farmacológico , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica , Humanos , Hibridación in Situ , Recién Nacido , Masculino , Proteínas Serina-Treonina Quinasas , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Piel/efectos de los fármacos , Proteínas Smad , Transactivadores/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The tumor suppressor protein ARF (alternate reading frame) inhibits MDM2 to stabilize and activate the functions of p53. Here we provide evidence for an additional activity of ARF that attenuates cell cycle progression independently of p53 activation. We show that ARF interacts with c-Myc independently of MDM2 or p53. Consequently, ARF relocalizes c-Myc from the nucleoplasm to the nucleolus. Binding and relocalization by ARF correlate with an inhibition of the c-Myc-activated transcription in both p53-positive and -negative cells. Using inducible cell lines, we show that the wild type ARF, but not a mutant, inhibits expression of the c-Myc-induced genes before inhibiting S phase. Moreover, ARF inhibits Myc-induced progression into S phase in cells lacking p53 or expressing a defective p53, indicating that ARF inhibits the S phase stimulatory function of c-Myc independently of p53. Our results strongly suggest that cMyc is a bona fide target of ARF and that ARF attenuates c-Myc independently of the ARF-p53 axis.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína p14ARF Supresora de Tumor/fisiología , Empalme Alternativo , Animales , Northern Blotting , Western Blotting , Bromodesoxiuridina/farmacología , Línea Celular Tumoral , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Colorantes/farmacología , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Epítopos , Fibroblastos/metabolismo , Fase G1 , Genes Reporteros , Células HeLa , Humanos , Ratones , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Mutación , Proteínas Nucleares/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Sistemas de Lectura , Fase S , Tetraciclina/farmacología , Transcripción Genética , Transfección , Transgenes , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVE: In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses. METHODS: The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs. RESULTS: The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7. CONCLUSION: By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.
Asunto(s)
Dermis/citología , Fibroblastos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Actinas/metabolismo , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibrosis , Expresión Génica , Humanos , Receptores Citoplasmáticos y Nucleares/genética , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Proteínas Smad , Transactivadores/metabolismo , Factores de Transcripción/genética , TransfecciónRESUMEN
OBJECTIVE: Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor beta (TGFbeta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGFbeta signaling, in scleroderma fibroblasts. METHODS: Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGFbeta was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFbeta blockade on SMAD subcellular distribution was determined using anti-TGFbeta antibodies as well as a dominant-negative TGFbeta receptor type II (TGFbetaRII) vector to disrupt TGFbeta responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts. RESULTS: Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGFbeta, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGFbeta signaling with antibodies or by expression of dominant-negative TGFbetaRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFbeta receptors. The activity of a SMAD-responsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts. CONCLUSION: This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGFbeta/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/fisiopatología , Transactivadores/genética , Transactivadores/metabolismo , Adulto , Núcleo Celular/metabolismo , Células Cultivadas , Femenino , Fibroblastos/citología , Fibrosis , Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Esclerodermia Sistémica/patología , Piel/citología , Proteína Smad2 , Proteína smad3 , Proteína Smad4 , Proteína smad7 , Activación Transcripcional/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Anticuerpos/farmacología , Bleomicina/farmacología , Células Cultivadas , Proteínas de Unión al ADN/genética , Dermatitis/metabolismo , Dermatitis/patología , Dermatitis/fisiopatología , Modelos Animales de Enfermedad , Femenino , Fibroblastos/citología , Ratones , Ratones Endogámicos C3H , Fosforilación , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/fisiopatología , Transducción de Señal/efectos de los fármacos , Proteína Smad2 , Proteína smad3 , Proteína smad7 , Transactivadores/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Several distinct entities associated with dermal fibrosis can mimic scleroderma/systemic sclerosis. The list of scleroderma-like conditions or scleroderma variants includes eosinophilic fasciitis, localized forms of scleroderma, scleredema and scleromyxedema, keloids, and environmental exposure-associated conditions including eosinophilia-myalgia syndrome and pseudosclerodermas induced by various drugs. Although these conditions are relatively uncommon, their accurate recognition is essential to avoid misdiagnosis and inappropriate therapy. The pathogenesis of these scleroderma variants appears to share similarities with each other and with that of scleroderma. Better understanding of scleroderma-like disorders is emerging through epidemiologic investigations, and in vivo and in vitro experimental research. Activation of eosinophils and disordered regulation of fibroblast collagen synthesis, apoptosis, and proliferation are recurrent findings in these disorders. The etiologic role of infection with Borrelia species or other microorganisms remains controversial. Cytokines such as transforming growth factor-beta, interleukin-4, interleukin-13, and connective tissue growth factor contribute to fibrosis in these disorders by inducing an accentuated and persistent fibrogenic response to tissue injury. The role of genetic factors in susceptibility and clinical expression of scleroderma-like conditions remains to be systematically addressed. Because of the relative rarity of these conditions, few well-controlled clinical treatment trials have been performed. In addition, there is no consensus on optimal management. Much anecdotal information and small clinical series indicate that phototherapy may have a role in the treatment of scleroderma-like conditions.