RESUMEN
The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica , Expresión Génica , Trastornos Mieloproliferativos/genética , Subunidad p45 del Factor de Transcripción NF-E2/genética , Biomarcadores , Homólogo de la Proteína Chromobox 5 , Citocinas/metabolismo , Metilación de ADN , Decitabina/farmacología , Histonas/metabolismo , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Modelos Biológicos , Mutación , Trastornos Mieloproliferativos/metabolismo , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Fosforilación , Policitemia Vera/genética , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes and has been recognized as a hot spot for cardiomyopathy. We have previously identified Myozap as a novel cardiac-enriched ID protein, which interacts with several other ID proteins and is involved in RhoA/SRF signaling in vitro. To now study its potential role in vivo we generated a mouse model with cardiac overexpression of Myozap. Transgenic (Tg) mice developed cardiomyopathy with hypertrophy and LV dilation. Consistently, these mice displayed upregulation of the hypertrophy-associated and SRF-dependent gene expression. Pressure overload (transverse aortic constriction, TAC) caused exaggerated cardiac hypertrophy, further loss of contractility and LV dilation. Similarly, a physiological stimulus (voluntary running) also led to significant LV dysfunction. On the ultrastructural level, Myozap-Tg mouse hearts exhibited massive protein aggregates composed of Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the alterations observed in desmin-related cardiomyopathy. Interestingly, desmin was not detectable in the aggregates, yet was largely displaced from the ID. Molecular analyses revealed induction of autophagy and dysregulation of the unfolded protein response (UPR), associated with apoptosis. Taken together, cardiac overexpression of Myozap leads to cardiomyopathy, mediated, at least in part by induction of Rho-dependent SRF signaling in vivo. Surprisingly, this phenotype was also accompanied by protein aggregates in cardiomyocytes, UPR alteration, accelerated autophagy and apoptosis. Thus, this mouse model may also offer additional insight into the pathogenesis of protein-aggregate-associated cardiomyopathies and represents a new candidate gene itself.
Asunto(s)
Cardiomiopatías/genética , Proteínas Musculares/genética , Miocardio/metabolismo , Agregación Patológica de Proteínas/genética , Animales , Apoptosis , Autofagia , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Desmina/genética , Desmina/metabolismo , Expresión Génica , Ratones , Ratones Transgénicos , Proteínas Musculares/metabolismo , Miocardio/patología , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Estrés Mecánico , Respuesta de Proteína Desplegada/genética , Remodelación Ventricular , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The transcription factor nuclear factor erythroid-2 is over-expressed in patients with myeloproliferative neoplasms irrespective of the presence of the JAK2(V617F) mutation. Our transgenic mouse model over-expressing nuclear factor erythroid-2, which recapitulates many features of myeloproliferative neoplasms including transformation to acute myeloid leukemia, clearly implicates this transcription factor in the pathophysiology of myeloproliferative neoplasms. Because the targets mediating nuclear factor erythroid-2 effects are not well characterized, we conducted microarray analysis of CD34(+) cells lentivirally transduced to over-express nuclear factor erythroid-2 or to silence this transcription factor via shRNA, in order to identify novel target genes. Here, we report that the cytokine interleukin 8 is a novel target gene. Nuclear factor erythroid-2 directly binds the interleukin 8 promoter in vivo, and these binding sites are required for promoter activity. Serum levels of interleukin 8 are known to be elevated in both polycythemia vera and primary myelofibrosis patients. Recently, increased interleukin 8 levels have been shown to be predictive of inferior survival in primary myelofibrosis patients in multivariate analysis. Therefore, one of the mechanisms by which nuclear factor erythroid-2 contributes to myeloproliferative neoplasm pathology may be increased interleukin 8 expression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Interleucina-8/biosíntesis , Enfermedades Mielodisplásicas-Mieloproliferativas/metabolismo , Subunidad p45 del Factor de Transcripción NF-E2/fisiología , Animales , Antígenos CD34/genética , Marcación de Gen/métodos , Vectores Genéticos/administración & dosificación , Humanos , Interleucina-8/genética , Lentivirus/genética , Ratones , Enfermedades Mielodisplásicas-Mieloproliferativas/diagnóstico , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Valor Predictivo de las Pruebas , Unión Proteica/genética , Resultado del Tratamiento , Células Tumorales Cultivadas , Células U937RESUMEN
RATIONALE AND OBJECTIVE: The M-band represents a transverse structure in the center of the sarcomeric A-band and provides an anchor for the myosin-containing thick filaments. In contrast to other sarcomeric structures, eg, the Z-disc, only few M-band-specific proteins have been identified to date, and its exact molecular composition remains unclear. METHODS AND RESULTS: Using a bioinformatic approach to identify novel heart- and muscle-specific genes, we found a leucine rich protein, myomasp (Myosin-interacting, M-band-associated stress-responsive protein)/LRRC39. RT-PCR and Northern and Western blot analyses confirmed a cardiac-enriched expression pattern, and immunolocalization of myomasp revealed a strong and specific signal at the sarcomeric M-band. Yeast 2-hybrid screens, as well as coimmunoprecipitation experiments, identified the C terminus of myosin heavy chain (MYH)7 as an interaction partner for myomasp. Knockdown of myomasp in neonatal rat ventricular myocytes (NRVCMs) led to a significant upregulation of the stretch-sensitive genes GDF-15 and BNP. Conversely, the expression of MYH7 and the M-band proteins myomesin-1 and -2 was found to be markedly reduced. Mechanistically, knockdown of myomasp in NRVCM led to a dose-dependent suppression of serum response factor-dependent gene expression, consistent with earlier observations linking the M-band to serum response factor-mediated signaling. Finally, downregulation of myomasp/LRRC39 in spontaneously beating engineered heart tissue constructs resulted in significantly lower force generation and reduced fractional shortening. Likewise, knockdown of the myomasp/LRRC39 ortholog in zebrafish resulted in severely impaired heart function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal myomasp as a previously unrecognized component of an M-band-associated signaling pathway that regulates cardiomyocyte gene expression in response to biomechanical stress.
Asunto(s)
Proteínas Portadoras/metabolismo , Mecanotransducción Celular , Proteínas Musculares/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Proteínas/metabolismo , Sarcómeros/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Northern Blotting , Western Blotting , Miosinas Cardíacas/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Proteínas Portadoras/genética , Células Cultivadas , Clonación Molecular , Conectina , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Proteínas Repetidas Ricas en Leucina , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Péptido Natriurético Encefálico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas/genética , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Respuesta Sérica/metabolismo , Estrés Mecánico , Transfección , Técnicas del Sistema de Dos Híbridos , Pez CebraRESUMEN
RATIONALE: The intercalated disc (ID) is a highly specialized cell-cell contact structure that ensures mechanical and electric coupling of contracting cardiomyocytes. Recently, the ID has been recognized to be a hot spot of cardiac disease, in particular inherited cardiomyopathy. OBJECTIVE: Given its complex structure and function we hypothesized that important molecular constituents of the ID still remain unknown. METHODS AND RESULTS: Using a bioinformatics screen, we discovered and cloned a previously uncharacterized 54 kDa cardiac protein which we termed Myozap (Myocardium-enriched zonula occludens-1-associated protein). Myozap is strongly expressed in the heart and lung. In cardiac tissue it localized to the ID and directly binds to desmoplakin and zonula occludens-1. In a yeast 2-hybrid screen for additional binding partners of Myozap we identified myosin phosphatase-RhoA interacting protein (MRIP), a negative regulator of Rho activity. Myozap, in turn, strongly activates SRF-dependent transcription through its ERM (Ezrin/radixin/moesin)-like domain in a Rho-dependent fashion. Finally, in vivo knockdown of the Myozap ortholog in zebrafish led to severe contractile dysfunction and cardiomyopathy. CONCLUSIONS: Taken together, these findings reveal Myozap as a previously unrecognized component of a Rho-dependent signaling pathway that links the intercalated disc to cardiac gene regulation. Moreover, its subcellular localization and the observation of a severe cardiac phenotype in zebrafish, implicate Myozap in the pathogenesis of cardiomyopathy.
Asunto(s)
Cardiomiopatías/metabolismo , Proteínas Musculares/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Bovinos , Chlorocebus aethiops , Clonación Molecular , Biología Computacional , Desmoplaquinas/metabolismo , Perros , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/genética , Fosfoproteínas/metabolismo , Unión Proteica , Transfección , Técnicas del Sistema de Dos Híbridos , Pez Cebra , Proteína de la Zonula Occludens-1 , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.
Asunto(s)
Neoplasias de la Médula Ósea/genética , Mutación/genética , Trastornos Mieloproliferativos/genética , Subunidad p45 del Factor de Transcripción NF-E2/genética , Animales , Neoplasias de la Médula Ósea/patología , Trasplante de Médula Ósea , Linaje de la Célula/genética , Proliferación Celular , Células Clonales , ADN/metabolismo , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Janus Quinasa 2/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Trastornos Mieloproliferativos/patología , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Unión Proteica/genética , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/genéticaRESUMEN
The molecular pathophysiology of myeloproliferative neoplasms (MPNs) remains poorly understood. Based on the observation that the transcription factor NF-E2 is often overexpressed in MPN patients, independent of the presence of other molecular aberrations, we generated mice expressing an NF-E2 transgene in hematopoietic cells. These mice exhibit many features of MPNs, including thrombocytosis, leukocytosis, Epo-independent colony formation, characteristic bone marrow histology, expansion of stem and progenitor compartments, and spontaneous transformation to acute myeloid leukemia. The MPN phenotype is transplantable to secondary recipient mice. NF-E2 can alter histone modifications, and NF-E2 transgenic mice show hypoacetylation of histone H3. Treatment of mice with the histone deacetylase inhibitor (HDAC-I) vorinostat restored physiological levels of histone H3 acetylation, decreased NF-E2 expression, and normalized platelet numbers. Similarly, MPN patients treated with an HDAC-I exhibited a decrease in NF-E2 expression. These data establish a role for NF-E2 in the pathophysiology of MPNs and provide a molecular rationale for investigating epigenetic alterations as novel targets for rationally designed MPN therapies.