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1.
Basic Res Cardiol ; 118(1): 4, 2023 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-36670288

RESUMEN

During embryonic development, cardiomyocytes undergo differentiation and maturation, processes that are tightly regulated by tissue-specific signaling cascades. Although redox signaling pathways involved in cardiomyogenesis are established, the exact sources responsible for reactive oxygen species (ROS) formation remain elusive. The present study investigates whether ROS produced by the mitochondrial flavoenzyme monoamine oxidase A (MAO-A) play a role in cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs). Wild type (WT) and MAO-A knock out (KO) hiPSCs were generated by CRISPR/Cas9 genome editing and subjected to cardiomyocyte differentiation. Mitochondrial ROS levels were lower in MAO-A KO compared to the WT cells throughout the differentiation process. MAO-A KO hiPSC-derived cardiomyocytes (hiPSC-CMs) displayed sarcomere disarray, reduced α- to ß-myosin heavy chain ratio, GATA4 upregulation and lower macroautophagy levels. Functionally, genetic ablation of MAO-A negatively affected intracellular Ca2+ homeostasis in hiPSC-CMs. Mechanistically, MAO-A generated ROS contributed to the activation of AKT signaling that was considerably attenuated in KO cells. In addition, MAO-A ablation caused a reduction in WNT pathway gene expression consistent with its reported stimulation by ROS. As a result of WNT downregulation, expression of MESP1 and NKX2.5 was significantly decreased in MAO-A KO cells. Finally, MAO-A re-expression during differentiation rescued expression levels of cardiac transcription factors, contractile structure, and intracellular Ca2+ homeostasis. Taken together, these results suggest that MAO-A mediated ROS generation is necessary for the activation of AKT and WNT signaling pathways during cardiac lineage commitment and for the differentiation of fully functional human cardiomyocytes.


Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Humanos , Miocitos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Diferenciación Celular/fisiología , Vía de Señalización Wnt
2.
Cells ; 11(17)2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-36078109

RESUMEN

Diabetes leads to cardiomyopathy and heart failure, the leading cause of death for diabetic patients. Monoamine oxidase (MAO) inhibition in diabetic cardiomyopathy prevents oxidative stress, mitochondrial and endoplasmic reticulum stress and the development of diastolic dysfunction. However, it is unclear whether, in addition to the direct effects exerted on the mitochondria, MAO activity is able to post-transcriptionally regulate cardiomyocyte function and survival in diabetes. To this aim, we performed gene and miRNA expression profiling in cardiac tissue from streptozotocin-treated mice (model of type 1 diabetes (T1D)), administered with either vehicle or MAOs inhibitor pargyline for 12 weeks. We found that inhibition of MAO activity in T1D hearts leads to profound transcriptomic changes, affecting autophagy and pro-survival pathways activation. MAO activity in T1D hearts increased miR-133a-3p, -193a-3p and -27a-3p expression. These miRNAs target insulin-like growth factor receptor 1 (Igf1r), growth factor receptor bound protein 10 and inositol polyphosphate 4 phosphatase type 1A, respectively, all components of the IGF1R/PI3K/AKT signaling pathway. Indeed, AKT activation was significantly downregulated in T1D hearts, whereas MAO inhibition restored the activation of this pro-survival pathway. The present study provides an important link between MAO activity, transcriptomic changes and activation of pro-survival signaling and autophagy in diabetic cardiomyopathy.


Asunto(s)
Diabetes Mellitus Tipo 1 , Cardiomiopatías Diabéticas , MicroARNs , Animales , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/metabolismo , Ratones , MicroARNs/genética , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal
3.
Antioxid Redox Signal ; 34(7): 531-550, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-32524823

RESUMEN

Aims: Doxorubicin cardiomyopathy is a lethal pathology characterized by oxidative stress, mitochondrial dysfunction, and contractile impairment, leading to cell death. Although extensive research has been done to understand the pathophysiology of doxorubicin cardiomyopathy, no effective treatments are available. We investigated whether monoamine oxidases (MAOs) could be involved in doxorubicin-derived oxidative stress, and in the consequent mitochondrial, cardiomyocyte, and cardiac dysfunction. Results: We used neonatal rat ventricular myocytes (NRVMs) and adult mouse ventricular myocytes (AMVMs). Doxorubicin alone (i.e., 0.5 µM doxorubicin) or in combination with H2O2 induced an increase in mitochondrial formation of reactive oxygen species (ROS), which was prevented by the pharmacological inhibition of MAOs in both NRVMs and AMVMs. The pharmacological approach was supported by the genetic ablation of MAO-A in NRVMs. In addition, doxorubicin-derived ROS caused lipid peroxidation and alterations in mitochondrial function (i.e., mitochondrial membrane potential, permeability transition, redox potential), mitochondrial morphology (i.e., mitochondrial distribution and perimeter), sarcomere organization, intracellular [Ca2+] homeostasis, and eventually cell death. All these dysfunctions were abolished by MAO inhibition. Of note, in vivo MAO inhibition prevented chamber dilation and cardiac dysfunction in doxorubicin-treated mice. Innovation and Conclusion: This study demonstrates that the severe oxidative stress induced by doxorubicin requires the involvement of MAOs, which modulate mitochondrial ROS generation. MAO inhibition provides evidence that mitochondrial ROS formation is causally linked to all disorders caused by doxorubicin in vitro and in vivo. Based upon these results, MAO inhibition represents a novel therapeutic approach for doxorubicin cardiomyopathy.


Asunto(s)
Doxorrubicina/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Monoaminooxidasa/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Ventrículos Cardíacos/metabolismo , Ratones , Mitocondrias , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/análisis
4.
Pharmacol Rev ; 72(4): 801-828, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32859763

RESUMEN

Reactive oxygen species (ROS) have been correlated with almost every human disease. Yet clinical exploitation of these hypotheses by pharmacological modulation of ROS has been scarce to nonexistent. Are ROS, thus, irrelevant for disease? No. One key misconception in the ROS field has been its consideration as a rather detrimental metabolic by-product of cell metabolism, and thus, any approach eliminating ROS to a certain tolerable level would be beneficial. We now know, instead, that ROS at every concentration, low or high, can serve many essential signaling and metabolic functions. This likely explains why systemic, nonspecific antioxidants have failed in the clinic, often with neutral and sometimes even detrimental outcomes. Recently, drug development has focused, instead, on identifying and selectively modulating ROS enzymatic sources that in a given constellation cause disease while leaving ROS physiologic signaling and metabolic functions intact. As sources, the family of NADPH oxidases stands out as the only enzyme family solely dedicated to ROS formation. Selectively targeting disease-relevant ROS-related proteins is already quite advanced, as evidenced by several phase II/III clinical trials and the first drugs having passed registration. The ROS field is expanding by including target enzymes and maturing to resemble more and more modern, big data-enhanced drug discovery and development, including network pharmacology. By defining a disease based on a distinct mechanism, in this case ROS dysregulation, and not by a symptom or phenotype anymore, ROS pharmacology is leaping forward from a clinical underperformer to a proof of concept within the new era of mechanism-based precision medicine. SIGNIFICANCE STATEMENT: Despite being correlated to almost every human disease, nearly no ROS modulator has been translated to the clinics yet. Here, we move far beyond the old-fashioned misconception of ROS as detrimental metabolic by-products and suggest 1) novel pharmacological targeting focused on selective modulation of ROS enzymatic sources, 2) mechanism-based redefinition of diseases, and 3) network pharmacology within the ROS field, altogether toward the new era of ROS pharmacology in precision medicine.


Asunto(s)
Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Oxidación-Reducción/efectos de los fármacos , Ensayos Clínicos Controlados Aleatorios como Asunto
5.
Pharmacol Res ; 151: 104548, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31759087

RESUMEN

Ischemia/reperfusion (I/R) injury is mediated in large part by opening of the mitochondrial permeability transition pore (PTP). Consequently, inhibitors of the PTP hold great promise for the treatment of a variety of cardiovascular disorders. At present, PTP inhibition is obtained only through the use of drugs (e.g. cyclosporine A, CsA) targeting cyclophilin D (CyPD) which is a key modulator, but not a structural component of the PTP. This limitation might explain controversial findings in clinical studies. Therefore, we investigated the protective effects against I/R injury of small-molecule inhibitors of the PTP (63 and TR002) that do not target CyPD. Both compounds exhibited a dose-dependent inhibition of PTP opening in isolated mitochondria and were more potent than CsA. Notably, PTP inhibition was observed also in mitochondria devoid of CyPD. Compounds 63 and TR002 prevented PTP opening and mitochondrial depolarization induced by Ca2+ overload and by reactive oxygen species in neonatal rat ventricular myocytes (NRVMs). Remarkably, both compounds prevented cell death, contractile dysfunction and sarcomeric derangement induced by anoxia/reoxygenation injury in NRVMs at sub-micromolar concentrations, and were more potent than CsA. Cardioprotection was observed also in adult mouse ventricular myocytes and human iPSc-derived cardiomyocytes, as well as ex vivo in perfused hearts. Thus, this study demonstrates that 63 and TR002 represent novel cardioprotective agents that inhibit PTP opening independent of CyPD targeting.


Asunto(s)
Cardiotónicos/uso terapéutico , Poro de Transición de la Permeabilidad Mitocondrial/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Cardiotónicos/farmacología , Línea Celular , Células Cultivadas , Humanos , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Ratas Sprague-Dawley , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/farmacología
7.
iScience ; 16: 340-355, 2019 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-31203189

RESUMEN

Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described.

8.
Free Radic Biol Med ; 134: 678-687, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30731114

RESUMEN

Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.


Asunto(s)
Modelos Animales de Enfermedad , Hormesis , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/citología , Paraquat/farmacología , Superóxidos/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Herbicidas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Wistar
9.
Basic Res Cardiol ; 113(5): 39, 2018 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-30120595
10.
Cell Death Differ ; 25(9): 1671-1685, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29459772

RESUMEN

Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.


Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Estrés del Retículo Endoplásmico , Mitocondrias/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Remodelación Ventricular/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glucosa/farmacología , Interleucina-1beta/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Monoaminooxidasa/química , Monoaminooxidasa/genética , Células Musculares/citología , Células Musculares/metabolismo , Células Musculares/fisiología , Miocardio/metabolismo , Miocardio/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo
11.
Curr Opin Pharmacol ; 33: 64-69, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28528298

RESUMEN

In the past decade, accumulating evidence highlighted the role of monoamine oxidases (MAOs) in cardiovascular disease (CVD). MAOs are flavoenzymes located in the outer mitochondrial membrane, responsible for the degradation of neurotransmitters and biogenic amines. During this process they generate hydrogen peroxide, aldehydes and ammonia, species that can target mitochondria and induce mitochondrial dysfunction and cardiomyocyte death. Indeed, MAO inhibition affords cardioprotection in several models of CVD, such as ischemia/reperfusion, heart failure and diabetes. Importantly, a few studies provided encouraging results suggesting that MAO inhibition might be beneficial also in patients with CVD. Thus, selective and reversible MAO inhibitors, currently used as therapy for depression and neurodegenerative disorders, might be considered as candidate drugs for the treatment of CVD.


Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Inhibidores de la Monoaminooxidasa/uso terapéutico , Monoaminooxidasa/metabolismo , Animales , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo
12.
Nat Commun ; 6: 8825, 2015 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-26593099

RESUMEN

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family. We previously showed that disruption of the Speg gene locus in mice leads to a dilated cardiomyopathy with immature-appearing cardiomyocytes. Here we show that cardiomyopathy of Speg(-/-) mice arises as a consequence of defects in cardiac progenitor cell (CPC) function, and that neonatal cardiac dysfunction can be rescued by in utero injections of wild-type CPCs into Speg(-/-) foetal hearts. CPCs harvested from Speg(-/-) mice display defects in clone formation, growth and differentiation into cardiomyocytes in vitro, which are associated with cardiac dysfunction in vivo. In utero administration of wild-type CPCs into the hearts of Speg(-/-) mice results in CPC engraftment, differentiation and myocardial maturation, which rescues Speg(-/-) mice from neonatal heart failure and increases the number of live births by fivefold. We propose that in utero administration of CPCs may have future implications for treatment of neonatal heart diseases.


Asunto(s)
Cardiomiopatías/terapia , Tratamiento Basado en Trasplante de Células y Tejidos , Enfermedades Fetales/terapia , Terapias Fetales , Miocitos Cardíacos/trasplante , Animales , Cardiomiopatías/embriología , Cardiomiopatías/enzimología , Cardiomiopatías/fisiopatología , Diferenciación Celular , Femenino , Enfermedades Fetales/enzimología , Enfermedades Fetales/genética , Enfermedades Fetales/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/enzimología , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Embarazo , Trasplante de Células Madre , Células Madre/citología , Células Madre/enzimología , Útero
13.
Eur J Pharm Sci ; 63: 22-8, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24993177

RESUMEN

No genes are yet directly implicated in etiology of male infertility. Identification of genes critical at various stages of spermatogenesis is pivotal for the timely diagnostic and treatment of infertility. We previously found that L-GILZ deficiency in a mouse KO model leads to hyperactivation of Ras signaling and increased proliferation in spermatogonia, resulting in male sterility. The possibility to establish culture cell system that maintains spermatogonial cells in vitro allowed us to delivery a recombinant protein TAT-L-GILZ able to restore normal proliferation rate in gilz KO spermatogonia. We also found that N-terminal part of L-GILZ protein is responsible for Ras/L-GILZ protein-to-protein interaction, important for the control of proliferation rate of spermatogonia. Therefore, treatments increasing L-GILZ expression, such as delivering small molecules or peptides that mimic L-GILZ functions, are approaches with great potential of applicability for new therapeutic strategies based on gene/protein delivery to the affected testes.


Asunto(s)
Glucocorticoides/farmacología , Espermatogonias/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Glucocorticoides/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Proteínas Recombinantes/metabolismo , Espermatogonias/citología
14.
Cell Rep ; 7(2): 464-475, 2014 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-24703841

RESUMEN

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) control immune responses and prevent autoimmunity. Treatment with glucocorticoids (GCs) has been shown to increase Treg cell frequency, but the mechanisms of their action on Treg cell induction are largely unknown. Here, we report that glucocorticoid-induced leucine zipper (GILZ), a protein induced by GCs, promotes Treg cell production. In mice, GILZ overexpression causes an increase in Treg cell number, whereas GILZ deficiency results in impaired generation of peripheral Treg cells (pTreg), associated with increased spontaneous and experimental intestinal inflammation. Mechanistically, we found that GILZ is required for GCs to cooperate with TGF-ß in FoxP3 induction, while it enhances TGF-ß signaling by binding to and promoting Smad2 phosphorylation and activation of FoxP3 expression. Thus, our results establish an essential GILZ-mediated link between the anti-inflammatory action of GCs and the regulation of TGF-ß-dependent pTreg production.


Asunto(s)
Glucocorticoides/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Colitis Ulcerosa/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Factores de Transcripción/genética
15.
J Biol Chem ; 287(2): 1242-51, 2012 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-22110132

RESUMEN

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.


Asunto(s)
Diferenciación Celular/fisiología , Transducción de Señal/fisiología , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Meiosis/fisiología , Ratones , Ratones Noqueados , Fosforilación/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Espermatogonias/citología , Factores de Transcripción/genética , Proteínas ras/genética
16.
J Biol Chem ; 285(14): 10385-96, 2010 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-20124407

RESUMEN

Myogenesis is a process whereby myoblasts differentiate and fuse into multinucleated myotubes, the precursors of myofibers. Various signals and factors modulate this process, and glucocorticoids (GCs) are important regulators of skeletal muscle metabolism. We show that glucocorticoid-induced leucine zipper (GILZ), a GC-induced gene, and the newly identified isoform long GILZ (L-GILZ) are expressed in skeletal muscle tissue and in C2C12 myoblasts where GILZ/L-GILZ maximum expression occurs during the first few days in differentiation medium. Moreover, we observed that GC treatment of myoblasts, which increased GILZ/L-GILZ expression, resulted in reduced myotube formation, whereas GILZ and L-GILZ silencing dampened GC effects. Inhibition of differentiation caused by GILZ/L-GILZ overexpression correlated with inhibition of MyoD function and reduced expression of myogenin. Notably, results indicate that GILZ and L-GILZ bind and regulate MyoD/HDAC1 transcriptional activity, thus mediating the anti-myogenic effect of GCs.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Glucocorticoides/farmacología , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/citología , Mioblastos/citología , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Animales Recién Nacidos , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Técnicas para Inmunoenzimas , Inmunoprecipitación , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Miogenina/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcripción Genética , Transfección
17.
Pharmacol Res ; 61(5): 405-9, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20056147

RESUMEN

Silymarin, a mixture of bioactive flavonolignans isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. In this study, the in vitro immunomodulatory activity of silymarin was investigated using CD4+ splenocytes from C57/Bl6 mice. Proliferation assay revealed that silymarin, at 50 microM concentration, significantly inhibited CD4+ cells proliferation. ELISA analyses indicated that silymarin significantly inhibited IL-2 and IFN-gamma production. Immunofluorescence staining performed on the mouse hybridoma T cell line (3DO) revealed a block of nuclear translocation of transcription factor kappaB (NF-kappaB), which is known to be responsible for IL-2 transcriptional activation. Moreover, silymarin inhibited p65/NF-kappaB phosphorylation in CD4+ T cell. These results suggest that silymarin is able to inhibit T cell activation and proliferation, notably acting on pathways of NF-kappaB activation/translocation.


Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Factores Inmunológicos , Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Silimarina/farmacología , Animales , Anexina A5 , Biotransformación/efectos de los fármacos , Western Blotting , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas/efectos de los fármacos , Bazo/citología , Factor de Transcripción ReIA/metabolismo
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