RESUMO
Coordinated changes in signaling pathways and gene expression in hearts subjected to prolonged stress maintain cardiac function. Loss of steroid receptor coactivator-2 (SRC-2) results in a reversal to the fetal gene program and disrupts the response to pressure overload, accompanied by prominent effects on metabolism and growth signaling, including increased AMPK activation. We proposed that early metabolic stress driven by AMPK activation induces contractile dysfunction in mice lacking SRC-2. We used 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK transiently before transverse aortic constriction (TAC) in wild-type and cardiomyocyte-specific SRC-2 knockout (CKO) animals. In contrast to AMPK activities during stress, in unstressed hearts, AICAR induced a mild activation of Akt signaling, and, in SRC-2-CKO mice, partially relieved an NAD+ deficiency and increased antioxidant signaling. These molecular changes translated to a mild hypertrophic response to TAC with decreased maladaptive remodeling, including markedly decreased fibrosis. Additionally, preactivation of AMPK in SRC-2-CKO mice was accompanied by a dramatic improvement in cardiac function compared with saline-treated SRC-2-CKO mice. Our results show that altered molecular signaling before stress onset has extended effects on sustained cardiac stress responses, and prestress modulation of transient growth and metabolism pathways may control those effects.-Nam, D. H., Kim, E., Benham, A., Park, H.-K., Soibam, B., Taffet, G. E., Kaelber, J. T., Suh, J. H., Taegtmeyer, H., Entman, M. L., Reineke, E. L. Transient activation of AMPK preceding left ventricular pressure overload reduces adverse remodeling and preserves left ventricular function.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Cardiomegalia/prevenção & controle , Coativador 2 de Receptor Nuclear/fisiologia , Ribonucleotídeos/farmacologia , Função Ventricular Esquerda/fisiologia , Pressão Ventricular , Remodelação Ventricular/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/farmacologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1α and -2α in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.
Assuntos
Coativador 2 de Receptor Nuclear/metabolismo , Indutores da Angiogênese/metabolismo , Angiotensina II/metabolismo , Animais , Ventrículos do Coração/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neovascularização Patológica/metabolismo , Comunicação Parácrina/fisiologia , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/metabolismo , Remodelação VentricularRESUMO
We have previously demonstrated the potential role of steroid receptor coactivator-2 (SRC-2) as a co-regulator in the transcription of critical molecules modulating cardiac function and metabolism in normal and stressed hearts. The present study seeks to extend the previous information by demonstrating SRC-2 fulfills this role by serving as a critical coactivator for the transcription and activity of critical transcription factors known to control cardiac growth and metabolism as well as in their downstream signaling. This knowledge broadens our understanding of the mechanism by which SRC-2 acts in normal and stressed hearts and allows further investigation of the transcriptional modifications mediating different types and degrees of cardiac stress. Moreover, the genetic manipulation of SRC-2 in this study is specific for the heart and thereby eliminating potential indirect effects of SRC-2 deletion in other organs. We have shown that SRC-2 is critical to transcriptional control modulated by MEF2, GATA-4, and Tbx5, thereby enhancing gene expression associated with cardiac growth. Additionally, we describe SRC-2 as a novel regulator of PPARα expression, thus controlling critical steps in metabolic gene expression. We conclude that through regulation of cardiac transcription factor expression and activity, SRC-2 is a critical transcriptional regulator of genes important for cardiac growth, structure, and metabolism, three of the main pathways altered during the cardiac stress response.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Fatores de Transcrição/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/citologia , Coativador 2 de Receptor Nuclear/genética , Fatores de Transcrição/genéticaRESUMO
The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.
Assuntos
Carcinoma Hepatocelular/genética , Análise Mutacional de DNA/métodos , Genes Supressores de Tumor , Neoplasias Hepáticas/genética , Coativador 2 de Receptor Nuclear/genética , Transposases/genética , Alquilantes/toxicidade , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Feminino , Genes myc/genética , Células HEK293 , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Transplante HeterólogoRESUMO
Promyelocytic leukemia protein (PML) was originally identified as part of a chromosomal translocation that contributes to the development of acute promyelocytic leukemia (APL). Since its discovery, PML has been found to play diverse roles in different cellular processes. Notably, PML has anti-proliferative and pro-apoptotic activity that supports its role as a tumor suppressor. We have previously shown that the peptidyl-prolyl isomerase Pin1 is able to affect cell proliferation and hydrogen peroxide (H(2)O(2))-mediated cell death through modulation of the steady-state levels of PML. We have extended these studies to show that the interaction between PML and Pin1 is targeted by multiple extracellular signals in the cell. We show that H(2)O(2) up-regulates and IGF-1 down-regulates PML expression in a Pin1-dependent manner. Interestingly, we found that H(2)O(2)- and IGF-1-mediated alteration in PML accumulation regulate MDA-MB-231 cell migration. Furthermore, we show that the control of cell migration by PML, and thus H(2)O(2) and IGF-1, results from PML-dependent decreased expression of integrin beta1 (ITGB1). Knockdown of Pin1 leads to decreased cell migration, lower levels of ITGB1 expression and resistance to IGF-1- and H(2)O(2)-induced changes in cell migration and ITGB1 expression. Taken together, our work identifies PML as a common target for H(2)O(2) and IGF-1 and supports a novel tumor suppressive role for PML in controlling cell migration through the expression of ITGB1.
Assuntos
Regulação Leucêmica da Expressão Gênica , Peróxido de Hidrogênio/química , Fator de Crescimento Insulin-Like I/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Humanos , Integrina beta1/metabolismo , Microscopia de Fluorescência/métodos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , CicatrizaçãoRESUMO
Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here, we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multifaceted role in vasculature. Of the three ERR subtypes, ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration, and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation but increased migration and tube formation. ERRα depletion increased basal as well as vascular endothelial growth factor A (VEGFA)- and ANG1/2-stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to that in wild-type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets, such as metabolism, mitochondrial biogenesis, and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory, and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, proangiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis and potentially fine-tunes growth factor-mediated angiogenesis.
Assuntos
Células Endoteliais/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Indutores da Angiogênese/metabolismo , Animais , Adesão Celular/genética , Movimento Celular/genética , Metabolismo Energético/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
In most clinical cases, left ventricular hypertrophy (LVH) occurs over time from persistent cardiac stress. At the molecular level, this results in both transient and long-term changes to metabolic, sarcomeric, ion handling, and stress signaling pathways. Although this is initially an adaptive change, the mechanisms underlying LVH eventually lead to maladaptive changes including fibrosis, decreased cardiac function, and failure. Understanding the regulators of long-term changes, which are largely driven by transcriptional remodeling, is a crucial step in identifying novel therapeutic targets for preventing the downstream negative effects of LVH and treatments that could reverse or prevent it. The development of effective therapeutics, however, will require a critical understanding of what to target, how to modify important pathways, and how to identify the stage of pathology in which a specific treatment should be used.
Assuntos
Fármacos Cardiovasculares/administração & dosagem , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Tempo para o Tratamento , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Progressão da Doença , Esquema de Medicação , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica , Resultado do TratamentoRESUMO
Synchrony of the mammalian circadian clock is achieved by complex transcriptional and translational feedback loops centered on the BMAL1:CLOCK heterodimer. Modulation of circadian feedback loops is essential for maintaining rhythmicity, yet the role of transcriptional coactivators in driving BMAL1:CLOCK transcriptional networks is largely unexplored. Here, we show diurnal hepatic steroid receptor coactivator 2 (SRC-2) recruitment to the genome that extensively overlaps with the BMAL1 cistrome during the light phase, targeting genes that enrich for circadian and metabolic processes. Notably, SRC-2 ablation impairs wheel-running behavior, alters circadian gene expression in several peripheral tissues, alters the rhythmicity of the hepatic metabolome, and deregulates the synchronization of cell-autonomous metabolites. We identify SRC-2 as a potent coregulator of BMAL1:CLOCK and find that SRC-2 targets itself with BMAL1:CLOCK in a feedforward loop. Collectively, our data suggest that SRC-2 is a transcriptional coactivator of the BMAL1:CLOCK oscillators and establish SRC-2 as a critical positive regulator of the mammalian circadian clock.
Assuntos
Ritmo Circadiano , Metaboloma , Coativador 2 de Receptor Nuclear/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Coativador 2 de Receptor Nuclear/genética , Especificidade de Órgãos , TranscriptomaRESUMO
The rapidly growing family of transcriptional coregulators includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have emerged as important regulators of metabolic homeostasis, including the p160 steroid receptor coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis, fat absorption and storage in the liver, and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of the SRC family members, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockouts (KOs) of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 KO mice and their wild-type littermates under fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC. Specifically, SRC-1 ablation revealed the most significant impact on hepatic metabolism, whereas SRC-2 appeared to impact cardiac metabolism. Conversely, ablation of SRC-3 primarily affected brain and skeletal muscle metabolism. Surprisingly, we identified very few metabolites that changed universally across the three SRC KO models. The findings of this Research Resource demonstrate that coactivator function has very limited metabolic redundancy even within the homologous SRC family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.
Assuntos
Metaboloma , Coativadores de Receptor Nuclear/metabolismo , Aminoácidos/metabolismo , Animais , Encéfalo/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Coativador 1 de Receptor Nuclear/deficiência , Coativador 1 de Receptor Nuclear/genética , Coativador 2 de Receptor Nuclear/deficiência , Coativador 2 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/deficiência , Coativador 3 de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/sangue , Coativadores de Receptor Nuclear/genética , Plasma/metabolismoRESUMO
A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks. Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the "fetal gene program" in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction. In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.
Assuntos
Hipertrofia Ventricular Esquerda/metabolismo , Coativador 2 de Receptor Nuclear/genética , Disfunção Ventricular Esquerda/metabolismo , Animais , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação Ventricular/genéticaRESUMO
Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of ß-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.
Assuntos
Carnitina Aciltransferases/genética , Carnitina Aciltransferases/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Coativador 3 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/metabolismo , Animais , Carnitina Aciltransferases/deficiência , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hipoglicemia/genética , Hipoglicemia/metabolismo , Cetose/genética , Cetose/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Doenças Musculares/enzimologia , Coativador 3 de Receptor Nuclear/deficiência , OxirreduçãoRESUMO
Though originally discovered as a tumor suppressor in Acute Promyelocytic Leukemia (APL), the importance of promyelocytic leukemia protein (PML) in cancers of other origins has not been widely studied. Recent studies have shown that multiple types of cancers show decreased expression of PML protein, though the mechanisms leading to this down-regulation are unknown. Decreased expression of PML can result in loss of cell cycle control and prevention of apoptosis and is likely a key event in the promotion of oncogenesis. Many of these effects are due to changes in the transcriptional profile of the cell as a result of decreased size and number of PML nuclear bodies. Several mouse studies confirm the contribution of PML to oncogenesis and cancer progression. It is important to not only further define a role for PML as a tumor suppressor, but also to begin to develop strategies to target PML therapeutically.
RESUMO
The promyelocytic leukemia protein (PML) is involved in many cellular processes including cell cycle progression, DNA damage response, transcriptional regulation, viral infection, and apoptosis. These cellular activities often rely on the localization of PML to unique subnuclear structures known as PML nuclear bodies (NBs). More than 50 cellular proteins are known to traffic in and out of PML NBs, either transiently or constitutively. In order to understand the dynamics of these NBs, it is important to delineate the regulation of PML itself. PML is subject to extensive regulation at transcriptional, post-transcriptional, and post-translational levels. Many of these modes of regulation depend on the cellular context and the presence of extracellular signals. This review focuses on the current knowledge of regulation of PML under normal cellular conditions as well as the role for regulation of PML in viral infection and cancer.
Assuntos
Núcleo Celular/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Humanos , Corpos de Inclusão Intranuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteína da Leucemia Promielocítica , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Peptidilprolil Isomerase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Neoplasias da Mama/etiologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Fenótipo , Proteína da Leucemia Promielocítica , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
The aberrant association of promyelocytic leukemia protein-retinoic acid receptor-alpha (PML-RARalpha) with corepressor complexes is generally thought to contribute to the ability of PML-RARalpha to regulate transcription. We report here that PML-RARalpha acquires aberrant association with coactivators. We show that endogenous PML-RARalpha interacts with the histone acetyltransferases CBP, p300, and SRC-1 in a hormoneindependent manner, an association not seen for RARalpha. This hormone-independent coactivator binding activity requires an intact ligand-binding domain and the NR box of the coactivators. Confocal microscopy studies demonstrate that exogenous PML-RARalpha sequesters and colocalizes with coactivators. These observations correlate with the ability of PML-RARalpha to attenuate the transcription activation of the Notch signaling downstream effector, CBF1, and of the glucocorticoid receptor. This includes attenuation of the glucocorticoid-induced leucine zipper (GILZ) and FLJ25390 target genes of the endogenous glucocorticoid receptor. Furthermore, treatment of NB4 cells with all-trans-retinoic acid, which promotes PML-RARalpha degradation, resulted in increased activation of GILZ. On the basis of these findings, we propose a model in which the hormone-independent association between PML-RARalpha and coactivators contributes to its ability to regulate gene expression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , Glutationa Transferase/metabolismo , Células HL-60 , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Plasmídeos/metabolismo , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Notch/metabolismo , Receptor alfa de Ácido Retinoico , Transdução de SinaisRESUMO
Histone deacetylase 7 (HDAC7) is a member of class IIa HDACs that regulate myocyte enhancer factor-2 (MEF2)-mediated transcription and participate in multiple cellular processes such as T cell apoptosis. We have identified alpha-actinin 1 and 4 as class IIa HDAC-interacting proteins. The interaction domains are mapped to C terminus of alpha-actinin 4 and amino acids 72-172 of HDAC7. A point mutation in HDAC7 that disrupts its association with MEF2A also disrupts its association with alpha-actinin 4, indicating that MEF2A and alpha-actinin 4 binding sites largely overlap. We have also isolated a novel splice variant of alpha-actinin 4 that is predominantly localized in the nucleus, a pattern distinct from the full-length alpha-actinin 4, which is primarily distributed in the cytoplasm and plasma membrane. Using small interfering RNA, chromatin immunoprecipitation, and transient transfection assays, we show that alpha-actinin 4 potentiates expression of TAF55, a putative MEF2 target gene. Loss of MEF2A interaction correlates with loss of the ability of alpha-actinin 4 to potentiate TAF55 promoter activity. Ectopic expression of alpha-actinin 4, but not the mutant defective in MEF2A association, leads to disruption of HDAC7.MEF2A association and enhancement of MEF2-mediated transcription. Taken together, we have identified a novel mechanism by which HDAC7 activity is negatively regulated and uncovered a previously unknown function of alpha-actinin 4.
Assuntos
Actinina/metabolismo , Histona Desacetilases/metabolismo , Proteínas de Domínio MADS/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fatores de Regulação Miogênica/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , Proteínas de Domínio MADS/genética , Fatores de Transcrição MEF2 , Fatores de Regulação Miogênica/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers control gene expression through recruitment of co-repressors or co-activators, depending on their hormone binding status. We show that the helix 12 of RXRalpha and RARalpha is critical for recruitment of the co-regulators and transcriptional regulation by RXRalpha, RARalpha, and RXRalpha/RARalpha. LG268, an RXR-specific agonist, was able to promote co-activator association with the heterodimers, but was unable to dissociate co-repressors. Reconstitution experiments in yeast demonstrated that LG268 was capable of activating transcription by RXRalpha/RARalpha through recruitment of the co-activator. We hypothesize that the inability to release co-repressors from RXRalpha/RARalpha is responsible for the inability of LG268 to activate RXRalpha/RARalpha heterodimers in mammalian cells. Deletion of RARalpha helix 12 (RXRalpha/RARalpha Delta403) abolished both hormone-dependent dissociation from co-repressors and hormone-dependent association with co-activators. Deletion of RXRalpha helix 12 (RXRalpha Delta443/RARalpha) resulted in a higher binding affinity for co-repressors. Unexpectedly, RXRalpha Delta443/RARalpha also gained hormone-independent co-activator binding activity. Moreover, LG268 became an antagonist to RXRalpha Delta443/RARalpha heterodimers. These data suggest that the helix 12 of RXRalpha plays an inhibitory role in the recruitment of co-activators by unliganded RXRalpha/RARalpha.