RESUMEN
Transcription factors (TFs) form homo- or hetero-dimeric DNA binding complexes along with associated co-regulators that can have transcriptional repressor or activator functions. Defining the specific composition of the complexes is therefore key to understanding their biological role. Here, we utilized bimolecular fluorescence complementation (BiFC) to visualize the formation of defined TF dimers and associated co-regulators derived from the activator protein-1 (AP-1) and myocyte enhancer factor 2 (MEF2) families. Firstly, BiFC signals were observed in cells co-expressing TFs tagged with complimentary combinations of the split fluorescent protein, demonstrating the engineered formation of defined dimer complexes. Next, we applied this approach and determined that defined AP-1 dimers localized at discrete sub-nuclear locations. Subsequently, a combination of BiFC coupled with GFP binding peptide (GBP)-nanotrap allowed observation of protein-protein interactions between a co-regulator, HDAC4, and defined BiFC-MEF2 engineered dimers. To determine transactivation properties of defined TF dimers in a cellular system, the Gal4-DNA binding domain fused to GBP was utilized to assess the transcriptional properties of the BiFC-TF dimers using a generically applicable Gal4/UAS luciferase reporter gene assay system. Here, we report efficacy of a BiFC/GBP-nanobody approach that allows engineering, visualization, and functional analysis of defined TF dimers.
Asunto(s)
Proteínas Fluorescentes Verdes , Humanos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Unión Proteica , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/genética , Factores de Transcripción MEF2/metabolismo , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/química , Histona Desacetilasas/metabolismo , Multimerización de Proteína , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/genética , Células HEK293 , Mapeo de Interacción de Proteínas/métodos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , AnimalesRESUMEN
Myocyte enhancer factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein-protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors. After bioinformatic analysis, we further characterized the interaction of MEF2A with a transcriptional repressor, FOXP1. FOXP1 coprecipitated with MEF2A in proliferating myogenic cells which diminished upon differentiation (myotube formation). Ectopic expression of FOXP1 inhibited MEF2A driven myogenic reporter genes (derived from the creatine kinase muscle and myogenin genes) and delayed induction of endogenous myogenin during differentiation. Conversely, FOXP1 depletion enhanced MEF2A transactivation properties and myogenin expression. The FoxP1:MEF2A interaction is also preserved in cardiomyocytes and FoxP1 depletion enhanced cardiomyocyte hypertrophy. FOXP1 prevented MEF2A phosphorylation and activation by the p38MAPK pathway. Overall, these data implicate FOXP1 in restricting MEF2A function in order to avoid premature differentiation in myogenic progenitors and also to possibly prevent re-activation of embryonic gene expression in cardiomyocyte hypertrophy.
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Animales , Factores de Transcripción MEF2/genética , Miogenina , Cromatografía Liquida , Músculo Esquelético/fisiología , HipertrofiaRESUMEN
Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. Whereas the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state - proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contained multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contained one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support the idea that there is an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.
Asunto(s)
Nucléolo Celular , Serina-Treonina Quinasas TOR , Nucléolo Celular/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transducción de Señal , Diferenciación Celular/genética , Desarrollo de Músculos/genéticaRESUMEN
Hippo signaling in Drosophila and mammals is prominent in regulating cell proliferation, death and differentiation. Hippo signaling effectors (YAP and TAZ; also known as YAP1 and WWTR1, respectively) exhibit crosstalk with transforming growth factor-ß (TGF-ß)-Smad and Wnt/ß-catenin pathways. Previously, we implicated Smad7 and ß-catenin in mammalian myogenesis. Therefore, we assessed a potential role of TAZ on the Smad7-ß-catenin complex in muscle cells. Here, we document functional interactions between Smad7, TAZ and ß-catenin in mouse myogenic cells. Ectopic TAZ expression resulted in repression of the muscle-specific creatine kinase muscle (Ckm) gene promoter and its corresponding protein level. Depletion of endogenous TAZ enhanced Ckm promoter activation. Ectopic TAZ, while potently active on a TEAD reporter (HIP-HOP), repressed myogenin (Myog) and Myod1 enhancer regions and myogenin protein level. Additionally, a Wnt/ß-catenin readout (TOP flash) demonstrated TAZ-mediated inhibition of ß-catenin activity. In myoblasts, TAZ was predominantly localized in nuclear speckles, while in differentiation conditions TAZ was hyperphosphorylated at Ser89, leading to enhanced cytoplasmic sequestration. Finally, live-cell imaging indicated that TAZ exhibits properties of liquid-liquid phase separation (LLPS). These observations indicate that TAZ, as an effector of Hippo signaling, suppresses the myogenic differentiation machinery.
Asunto(s)
Desarrollo de Músculos , beta Catenina , Animales , Diferenciación Celular , Ratones , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Nucleoli are well defined for their function in ribosome biogenesis, but only a small fraction of the nucleolar proteome has been characterized. Here, we report that the proto-oncogene, c-Jun, is targeted to the nucleolus. Using live cell imaging in myogenic cells, we document that the c-Jun basic domain contains a unique, evolutionarily conserved motif that determines nucleolar targeting. Fos family Jun dimer partners, such as Fra2, while nuclear, do not co-localize with c-Jun in the nucleolus. A point mutation in c-Jun that mimics Fra2 (M260E) in its Nucleolar Localization sequence (NoLS) results in loss of c-Jun nucleolar targeting while still preserving nuclear localization. Fra2 can sequester c-Jun in the nucleoplasm, indicating that the stoichiometric ratio of heterodimeric partners regulates c-Jun nucleolar targeting. Finally, nucleolar localization of c-Jun modulates nucleolar architecture and ribosomal RNA accumulation. These studies highlight a novel role for Jun family proteins in the nucleolus, having potential implications for a diverse array of AP-1-regulated cellular processes.
Asunto(s)
Nucléolo Celular/genética , Antígeno 2 Relacionado con Fos/genética , Genes jun/genética , Ribosomas/genética , Secuencia de Aminoácidos/genética , Línea Celular , Regulación de la Expresión Génica/genética , Humanos , Señales de Localización Nuclear/genética , Proteínas Nucleares/genética , Transporte de Proteínas/genética , Proteoma/genéticaRESUMEN
Transforming growth factor ß (TGFß) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFß potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFß canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFß is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFß did not reverse TGFß's inhibitory effect on myogenesis. In considering other pathways, we observed that TGFß potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFß's inhibitory effect on myogenesis, as indicated by a myogenin promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFß activation. Taken together, these observations support a model in which TGFß activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.
Asunto(s)
Diferenciación Celular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Desarrollo de Músculos/fisiología , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Núcleo Celular/metabolismo , Citocinas/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Miogenina/metabolismo , Fosforilación/fisiología , Células Madre/fisiología , Transactivadores/metabolismoRESUMEN
The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.
Asunto(s)
Corazón/fisiopatología , Proteínas de la Membrana/deficiencia , Retículo Sarcoplasmático/patología , Animales , Calcio/metabolismo , Células Cultivadas , Estrés del Retículo Endoplásmico , Técnicas de Inactivación de Genes , Silenciador del Gen , Corazón/crecimiento & desarrollo , Cardiopatías/metabolismo , Cardiopatías/patología , Cardiopatías/fisiopatología , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Pez CebraRESUMEN
Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor ß (TGF-ß) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-ß signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-ß signalling and likely represents a mechanism to limit fibrosis.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismo , Fosfatasa Alcalina/genética , Animales , Fibrosis , Ratones , Ratones Noqueados , Miocardio/patología , Proteína Smad2/genética , Proteína smad3/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Recent reports indicate that Smad7 promotes skeletal muscle differentiation and growth. We previously documented a non-canonical role of nuclear Smad7 during myogenesis, independent of its role in TGF-ß signaling. Here further characterization of the myogenic function of Smad7 revealed ß-catenin as a Smad7 interacting protein. Biochemical analysis identified a Smad7 interaction domain (SID) between aa575 and aa683 of ß-catenin. Reporter gene analysis and chromatin immunoprecipitation demonstrated that Smad7 and ß-catenin are cooperatively recruited to the extensively characterized ckm promoter proximal region to facilitate its muscle restricted transcriptional activation in myogenic cells. Depletion of endogenous Smad7 and ß-catenin in muscle cells reduced ckm promoter activity indicating their role during myogenesis. Deletion of the ß-catenin SID substantially reduced the effect of Smad7 on the ckm promoter and exogenous expression of SID abolished ß-catenin function, indicating that SID functions as a trans dominant-negative regulator of ß-catenin activity. ß-catenin interaction with the Mediator kinase complex through its Med12 subunit led us to identify MED13 as an additional Smad7-binding partner. Collectively, these studies document a novel function of a Smad7-MED12/13-ß-catenin complex at the ckm locus, indicating a key role of this complex in the program of myogenic gene expression underlying skeletal muscle development and regeneration.
Asunto(s)
Desarrollo de Músculos/genética , Proteína smad7/fisiología , beta Catenina/fisiología , Animales , Diferenciación Celular , Línea Celular , Regulación de la Expresión Génica , Ratones , Proteína smad7/genética , Proteína smad7/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Canonical Wnt/ß-catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/ß-catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context-dependent mechanisms regulating this pathway. Since protein-protein interactions underpin ß-catenin function and localization, we sought to identify novel ß-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where ß-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC ß-catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for ß-catenin, particularly in mRNA translation, and we confirm a direct interaction between ß-catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of ß-catenin to the messenger ribonucleoprotein and translational pre-initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering ß-catenin away from the pre-initiation complex. In conclusion, we present evidence that ß-catenin fulfills a previously unrecognized function in translational repression.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Iniciación de la Cadena Peptídica Traduccional , beta Catenina/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cicloheximida/farmacología , Ontología de Genes , Células HEK293 , Humanos , Ratones , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.
Asunto(s)
Retículo Endoplásmico/ultraestructura , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Mitocondrias Cardíacas/ultraestructura , Mioblastos/ultraestructura , Miocitos Cardíacos/ultraestructura , Membrana Nuclear/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Ratones , Mitocondrias Cardíacas/metabolismo , Mioblastos/metabolismo , Miocitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Proteína ORAI1/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Molécula de Interacción Estromal 1/metabolismo , TransfecciónRESUMEN
Vascular smooth muscle cells (VSMCs) do not terminally differentiate; they modulate their phenotype between proliferative and differentiated states, which is a major factor contributing to vascular diseases. TGFß signalling has been implicated in inducing VSMC differentiation, although the exact mechanism remains largely unknown. Our goal was to assess the network of transcription factors involved in the induction of VSMC differentiation, and to determine the role of TAZ in promoting the quiescent VSMC phenotype. TGFß robustly induces VSMC marker genes in 10T1/2 mouse embryonic fibroblast cells and the potent transcriptional regulator TAZ has been shown to retain Smad complexes on DNA. Thus, the role of TAZ in regulation of VSMC differentiation was studied. Using primary aortic VSMCs coupled with siRNA-mediated gene silencing, our studies reveal that TAZ is required for TGFß induction of smooth muscle genes and is also required for the differentiated VSMC phenotype; synergy between TAZ and SRF, and TAZ and Myocardin (MyoC856), in regulating smooth muscle gene activation was observed. These data provide evidence of components of a novel signalling pathway that links TGFß signalling to induction of smooth muscle genes through a mechanism involving regulation of TAZ and SRF proteins. In addition, we report a physical interaction of TAZ and MyoC856. These observations elucidate a novel level of control of VSMC induction which may have implications for vascular diseases and congenital vascular malformations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Desarrollo de Músculos/fisiología , Miocitos del Músculo Liso/citología , Proteínas Nucleares/fisiología , Factor de Respuesta Sérica/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/fisiología , Actinas/biosíntesis , Actinas/genética , Animales , Aorta , Línea Celular , Células Cultivadas , Fibroblastos , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Ratones Endogámicos C3H , Desarrollo de Músculos/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Mapeo de Interacción de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [(35)S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.
Asunto(s)
Autofagia , Proteínas de Unión al Calcio/metabolismo , Metformina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Animales , Células HEK293 , Humanos , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Proteolisis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPß (Δ184-C/EBPß) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184-C/EBPß-expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double-membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184-C/EBPß expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro-apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Embrión de Pollo , Pollos , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs) and has been shown to have potent antiapoptotic properties. Because the mechanism by which cryAB prevents apoptosis has not been fully characterized, we examined its protective effects at the cellular level by silencing cryAB in mouse neonatal CMs using lentivector-mediated transduction of short hairpin RNAs. Subcellular fractionation of whole hearts showed that cryAB is cytosolic under control conditions, and after H(2)O(2) exposure, it translocates to the mitochondria. Phosphorylated cryAB (PcryAB) is mainly associated with the mitochondria, and any residual cytosolic PcryAB translocates to the mitochondria after H(2)O(2) exposure. H(2)O(2) exposure caused increases in cryAB and PcryAB levels, and cryAB silencing resulted in increased levels of apoptosis after exposure to H(2)O(2). Coimmunoprecipitation assays revealed an apparent interaction of both cryAB and PcryAB with mitochondrial voltage-dependent anion channels (VDAC), translocase of outer mitochondrial membranes 20 kDa (TOM 20), caspase 3, and caspase 12 in mouse cardiac tissue. Our results are consistent with the conclusion that the cardioprotective effects of cryAB are mediated by its translocation from the cytosol to the mitochondria under conditions of oxidative stress and that cryAB interactions with VDAC, TOM 20, caspase 3, and caspase 12 may be part of its protective mechanism.
Asunto(s)
Apoptosis/fisiología , Peróxido de Hidrógeno/toxicidad , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/fisiología , Cadena B de alfa-Cristalina/genética , Factores de Edad , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Citosol/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Oxidantes/toxicidad , Estrés Oxidativo/efectos de los fármacos , Fosforilación/fisiología , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Cadena B de alfa-Cristalina/metabolismoRESUMEN
The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase ß isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a ß sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.
Asunto(s)
Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/genética , Homología de Secuencia de AminoácidoRESUMEN
Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Colorantes Fluorescentes/metabolismo , Mitocondrias/metabolismo , Células Musculares/citología , Células Musculares/metabolismo , Animales , Línea Celular , Supervivencia Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Desarrollo de Músculos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Proteína MioD/metabolismo , Miogenina/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Coloración y EtiquetadoRESUMEN
In the "canonical" view of transforming growth factor beta (TGF-beta) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-beta, it does not reverse the inhibitory effect of TGF-beta on myogenesis, suggesting a different function in myogenic cells. We previously reported a promyogenic role of Smad7 mediated by an interaction with MyoD. Based on this association, we hypothesized a possible nuclear function of Smad7 independent of its role at the level of the receptor. We therefore engineered a chimera of Smad7 with a nuclear localization signal (NLS), which serves to prevent and therefore bypass binding to the TGF-beta receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-beta but did retain its ability to enhance myogenic gene activation and phenotypic myogenesis, indicating that the nuclear, receptor-independent function of Smad7 is sufficient to promote myogenesis. Furthermore, Smad7 physically interacts with MyoD and antagonizes the repressive effects of active MEK on MyoD. Reporter and myogenic conversion assays indicate a pivotal regulation of MyoD transcriptional properties by the balance between Smad7 and active MEK. Thus, Smad7 has a nuclear coactivator function that is independent of TGF-beta signaling and necessary to promote myogenic differentiation.
Asunto(s)
Núcleo Celular/metabolismo , Desarrollo de Músculos/fisiología , Señales de Localización Nuclear/metabolismo , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Citocinas/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Músculos/metabolismo , Proteína MioD/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteína smad3/metabolismo , TransfecciónRESUMEN
Menin plays an established role in the differentiation of mesenchymal cells to the osteogenic lineage. Conversely, whether Menin influences the commitment of mesenschymal cells to the myogenic lineage, despite expression in the developing somite was previously unclear. We observed that Menin is down-regulated in C2C12 and C3H10T1/2 mesenchymal cells when muscle differentiation is induced. Moreover, maintenance of Menin expression by constitutive ectopic expression inhibited muscle cell differentiation. Reduction of Menin expression by siRNA technology results in precocious muscle differentiation and concomitantly attenuates BMP-2 induced osteogenesis. Reduced Menin expression antagonizes BMP-2 and TGF-beta1 mediated inhibition of myogenesis. Furthermore, Menin was found to directly interact with and potentiate the transactivation properties of Smad3 in response to TGF-beta1. Finally in concert with these observations, tissue-specific inactivation of Men1 in Pax3-expressing somite precursor cells leads to a patterning defect of rib formation and increased muscle mass in the intercostal region. These data invoke a pivotal role for Menin in the competence of mesenchymal cells to respond to TGF-beta1 and BMP-2 signals. Thus, by modulating cytokine responsiveness Menin functions to alter the balance of multipotent mesenchymal cell commitment to the osteogenic or myogenic lineages.
Asunto(s)
Linaje de la Célula , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Desarrollo de Músculos/genética , Osteogénesis/genética , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eliminación de Gen , Humanos , Músculos Intercostales/anatomía & histología , Músculos Intercostales/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteína smad3/metabolismo , Somitos/citología , Somitos/efectos de los fármacos , Somitos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Skeletal myogenesis is potently regulated by the extracellular milieu of growth factors and cytokines. We observed that cardiotrophin-1 (CT-1), a member of the interleukin-6 (IL-6) family of cytokines, is a potent regulator of skeletal muscle differentiation. The normal up-regulation of myogenic marker genes, myosin heavy chain (MyHC), myogenic regulatory factors (MRFs), and myocyte enhancer factor 2s (MEF2s) were inhibited by CT-1 treatment. CT-1 also represses myogenin (MyoG) promoter activation. CT-1 activated two signaling pathways: signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase kinase (MEK), a component of the extracellular signal-regulated MAPK (ERK) pathway. In view of the known connection between CT-1 and STAT3 activation, we surprisingly found that pharmacological blockade of STAT3 activity had no effect on the inhibition of myogenesis by CT-1 suggesting that STAT3 signaling is dispensable for myogenic repression. Conversely, MEK inhibition potently reversed the inhibition of myotube formation and attenuated the repression of MRF transcriptional activity mediated by CT-1. Taken together, these data indicate that CT-1 represses skeletal myogenesis through interference with MRF activity by activation of MEK/ERK signaling. In agreement with these in vitro observations, exogenous systemic expression of CT-1 mediated by adenoviral vector delivery increased the number of myonuclei in normal post-natal mouse skeletal muscle and also delayed skeletal muscle regeneration induced by cardiotoxin injection. The expression pattern of CT-1 in embryonic and post-natal skeletal muscle and in vivo effects of CT-1 on myogenesis implicate CT-1 in the maintenance of the undifferentiated state in muscle progenitor cells.