RESUMEN
Objective- Cardiac progenitor cells reside in the heart in adulthood, although their physiological relevance remains unknown. Here, we demonstrate that after myocardial infarction, adult Bmi1+ (B lymphoma Mo-MLV insertion region 1 homolog [PCGF4]) cardiac cells are a key progenitor-like population in cardiac neovascularization during ventricular remodeling. Approach and Results- These cells, which have a strong in vivo differentiation bias, are a mixture of endothelial- and mesenchymal-related cells with in vitro spontaneous endothelial cell differentiation capacity. Genetic lineage tracing analysis showed that heart-resident Bmi1+ progenitor cells proliferate after acute myocardial infarction and differentiate to generate de novo cardiac vasculature. In a mouse model of induced myocardial infarction, genetic ablation of these cells substantially deteriorated both heart angiogenesis and the ejection fraction, resulting in an ischemic-dilated cardiac phenotype. Conclusions- These findings imply that endothelial-related Bmi1+ progenitor cells are necessary for injury-induced neovascularization in adult mouse heart and highlight these cells as a suitable therapeutic target for preventing dysfunctional left ventricular remodeling after injury.
Asunto(s)
Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Neovascularización Patológica , Complejo Represivo Polycomb 1/fisiología , Células Madre/patología , Células Madre/fisiología , Remodelación Ventricular , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factores de Transcripción/metabolismoRESUMEN
MicroRNAs (miR) have considerable potential as therapeutic tools in cardiac diseases. Alterations in atrial miR are involved in the development of atrial fibrillation (AF), but the molecular mechanism underlying their contribution to atrial remodeling in chronic atrial fibrillation (CAF) is only partially understood. Here we used miR array to analyze the miR profile of atrial biopsies from sinus rhythm (SR) and CAF patients. qRT-PCR identified a distinctive CAF-miR signature and described conserved miR-208b upregulation in human and ovine AF atrial tissue. We used bioinformatics analysis to predict genes and signaling pathways as putative miR-208b targets, which highlighted genes from the cardiac muscle gene program and from canonical WNT, gap-junction and Ca2+ signaling networks. Results from analysis of miR-208b-overexpressing HL-1 atrial myocytes and from myocytes isolated from CAF patients showed that aberrant miR-208b levels reduced the expression and function of L-type Ca2+ channel subunits (CACNA1C and CACNB2) as well as the sarcoplasmic reticulum-Ca2+ pump SERCA2. These findings clearly pointed to CAF-specific upregulated miR-208b as an important mediator in Ca2+ handling impairment during atrial remodeling.
Asunto(s)
Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Calcio/metabolismo , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Regiones no Traducidas 3' , Animales , Fibrilación Atrial/fisiopatología , Remodelación Atrial , Secuencia de Bases , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Línea Celular , Enfermedad Crónica , Conexina 43/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Atrios Cardíacos/fisiopatología , Humanos , Miosinas/genética , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Ovinos , Proteínas Wnt/metabolismoRESUMEN
Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.
Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Evolución Molecular , Redes Reguladoras de Genes , Mamíferos/embriología , Mamíferos/genética , Células Madre Pluripotentes/metabolismo , Animales , Secuencia de Bases , Embrión de Pollo , Secuencia Conservada/genética , Elementos de Facilitación Genéticos/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Especificidad de la EspecieRESUMEN
In the mouse blastocyst, Eomes and Cdx2 are critical for establishing the trophoectoderm, the precursor of the placenta. To better understand how the trophoectoderm lineage arose in mammals during evolution, we examined the expression of their orthologues in the pregastrulation chick embryo and found that, while both genes are expressed in extraembryonic tissues, their temporal pattern of expression differs from what occurs in mouse. Moreover, we failed to detect expression of other genes specific from the mouse trophoectoderm in extraembryonic regions of the chick. Also unlike the mouse, chick Eomes is expressed in primordial germ cells. Finally, conserved noncoding elements in the Eomes genomic region are unable to drive trophoectoderm restricted expression in the mouse blastocyst, but do so in conserved sites of expression such as the forebrain. These results suggest that critical changes in the gene regulatory networks controlling extraembryonic development accompanied the appearance of the trophoectoderm in mammals.
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Evolución Biológica , Linaje de la Célula , Embrión de Pollo/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Animales , Blastocisto/metabolismo , Factor de Transcripción CDX2 , Secuencia Conservada , Elementos de Facilitación Genéticos , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Placenta/embriología , EmbarazoRESUMEN
Adult progenitor cells reside in specialized microenvironments which maintain their undifferentiated cell state and trigger regenerative responses following injury. Although these environments are well described in several tissues, the cellular components that comprise the cardiac environment where progenitor cells are located remain unknown. Here we use Bmi1CreERT and Bmi1GFP mice as genetic tools to trace cardiac damage-responsive cells throughout the mouse lifespan. In adolescent mice, Bmi1+ damage-responsive cells are broadly distributed throughout the myocardium. In adult mice, however, Bmi1+ cells are confined predominately in perivascular areas with low levels of reactive oxygen species (ROS) and their number decline in an age-dependent manner. In vitro co-culture experiments with endothelial cells supported a regulatory role of the endothelium in damage-responsive cell behavior. Accordingly, in vivo genetic decrease of ROS levels in adult heart disengaged Bmi1+ cells from the cardiovascular network, recapitulating an adolescent-like Bmi1 expression profile. Thus, we identify cardiac perivascular regions as low-stress microenvironments that favor the maintenance of adult damage-responsive cells.
Asunto(s)
Miocardio/metabolismo , Estrés Oxidativo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Edad , Animales , Permeabilidad Capilar , Proliferación Celular , Células Endoteliales/metabolismo , Inmunohistoquímica , Ratones , Ratones Transgénicos , Estrés Oxidativo/genética , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Accumulation of reactive oxygen species (ROS) is associated with several cardiovascular pathologies and with cell cycle exit by neonanatal cardiomyocytes, a key limiting factor in the regenerative capacity of the adult mammalian heart. The polycomb complex component BMI1 is linked to adult progenitors and is an important partner in DNA repair and redox regulation. We found that high BMI1 expression is associated with an adult Sca1+ cardiac progenitor sub-population with low ROS levels. In homeostasis, BMI1 repressed cell fate genes, including a cardiogenic differentiation program. Oxidative damage nonetheless modified BMI1 activity in vivo by derepressing canonical target genes in favor of their antioxidant and anticlastogenic functions. This redox-mediated mechanism is not restricted to damage situations, however, and we report ROS-associated differentiation of cardiac progenitors in steady state. These findings demonstrate how redox status influences the cardiac progenitor response, and identify redox-mediated BMI1 regulation with implications in maintenance of cellular identity in vivo.
Asunto(s)
Células Madre Adultas/metabolismo , Diferenciación Celular , Miocardio/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Madre Adultas/citología , Animales , Ratones , Ratones Transgénicos , Miocardio/citología , Oxidación-Reducción , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
BACKGROUND: Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. AIM AND SCOPE: A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. METHODS: We compared hADSC cultures at short (PS) and prolonged (PL) passages, both in standard and low [O2] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. RESULTS: Comparison of hADSCs cultured at PL or PS surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in PL cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O2], was also significantly higher in PL than in PS passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. CONCLUSION: A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.
Asunto(s)
Impresión Genómica , Péptidos y Proteínas de Señalización Intercelular/genética , Yoduro Peroxidasa/genética , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al Calcio , Proliferación Celular/genética , Células Cultivadas , Senescencia Celular/genética , Cromosomas Humanos Par 14/genética , Epigénesis Genética , Regulación de la Expresión Génica , Humanos , Ratones , MicroARNs/genética , Regulación hacia ArribaRESUMEN
miRNA-1 (miR-1) and miRNA-133a (miR-133a) are muscle-specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR-1 and miR-133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR-1 and miR-133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)-mediated mesodermal and cardiac differentiation by gain- and loss-of-function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR-1 and miR-133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR-1 enhanced the cardiac differentiation of P19.CL6, while miR-133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR-1 and miR-133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR-1 and miR-133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , MicroARNs/metabolismo , Miocardio/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mesodermo/citología , Ratones SCID , MicroARNs/genética , Modelos Biológicos , Neuronas/citología , Neuronas/metabolismoRESUMEN
Non-homologous end joining (NHEJ) is the main mechanism for double strand break (DSB) DNA repair. The error-prone DNA polymerase mu (Polµ) is involved in immunoglobulin variable region rearrangement and in general, NHEJ in non-lymphoid cells. Deletion of NHEJ factors in P53-/- mice, which are highly prone to development of T cell lymphoma, generally increases cancer incidence and shifts the tumor spectrum towards aggressive pro-B lymphoma. In contrast, Polµ deletion increased sarcoma incidence, proportionally reducing pro-B lymphoma development on the P53-deficient background. Array comparative genomic hybridization (aCGH) analyses showed DNA copy number alterations in both P53-/- and Polµ-/-P53-/- lymphomas. Our results also indicate that the increase in sarcoma incidence in Polµ-/-P53-/- mice could be associated with Cdk4 and Kub3 amplification and overexpression. These results identify a role for Polµ in the prevention of sarcomagenesis on a murine P53-deficient background, in contrast to most other NHEJ factors.
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Carcinogénesis , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/genética , Sarcoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Proteínas Portadoras/genética , Quinasa 4 Dependiente de la Ciclina/genética , ADN/metabolismo , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Eliminación de Gen , Inestabilidad Genómica , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Sarcoma/genética , Sarcoma/patología , Regulación hacia ArribaRESUMEN
Insulin-like growth factors (IGFs) have a pivotal role during nervous system development and in its functional maintenance. IGF-I and its high affinity receptor (IGF1R) are expressed in the developing inner ear and in the postnatal cochlear and vestibular ganglia. We recently showed that trophic support by IGF-I is essential for the early neurogenesis of the chick cochleovestibular ganglion (CVG). In the chicken embryo otic vesicle, IGF-I regulates developmental death dynamics by regulating the activity and/or levels of key intracellular molecules, including lipid and protein kinases such as ceramide kinase, Akt and Jun N-terminal kinase (JNK). Mice lacking IGF-I lose many auditory neurons and present increased auditory thresholds at early postnatal ages. Neuronal loss associated to IGF-I deficiency is caused by apoptosis of the auditory neurons, which presented abnormally increased levels of activated caspase-3. It is worth noting that in man, homozygous deletion of the IGF-1 gene causes sensory-neural deafness. IGF-I is thus necessary for normal development and maintenance of the inner ear. The trophic actions of IGF-I in the inner ear suggest that this factor may have therapeutic potential for the treatment of hearing loss.
Asunto(s)
Oído Interno/embriología , Factor I del Crecimiento Similar a la Insulina/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Senescencia Celular/fisiología , Cóclea/citología , Cóclea/crecimiento & desarrollo , Desarrollo Embrionario/fisiologíaRESUMEN
The first lineage choice in mammalian embryogenesis is that between the trophectoderm, which gives rise to the trophoblast of the placenta, and the inner cell mass, from which is derived the embryo proper and the yolk sac. The establishment of these lineages is preceded by the inside-versus-outside positioning of cells in the early embryo and stochastic expression of key transcription factors, which is then resolved into lineage-restricted expression. The regulatory inputs that drive this restriction and how they relate to cell position are largely unknown. Here, we show an unsuspected role of Notch signaling in regulating trophectoderm-specific expression of Cdx2 in cooperation with TEAD4. Notch activity is restricted to outer cells and is able to influence positional allocation of blastomeres, mediating preferential localization to the trophectoderm. Our results show that multiple signaling inputs at preimplantation stages specify the first embryonic lineages.
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Blastocisto/metabolismo , Linaje de la Célula , Ectodermo/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Notch1/metabolismo , Factores de Transcripción/metabolismo , Animales , Blastocisto/citología , Factor de Transcripción CDX2 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ectodermo/citología , Ectodermo/embriología , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Vía de Señalización Hippo , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Receptor Notch1/genética , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
miR-133a and miR-1 are known as muscle-specific microRNAs that are involved in cardiac development and pathophysiology. We have shown that both miR-1 and miR-133a are early and progressively upregulated during in vitro cardiac differentiation of adult cardiac progenitor cells (CPCs), but only miR-133a expression was enhanced under in vitro oxidative stress. miR-1 was demonstrated to favor differentiation of CPCs, whereas miR-133a overexpression protected CPCs against cell death, targeting, among others, the proapoptotic genes Bim and Bmf. miR-133a-CPCs clearly improved cardiac function in a rat myocardial infarction model by reducing fibrosis and hypertrophy and increasing vascularization and cardiomyocyte proliferation. The beneficial effects of miR-133a-CPCs seem to correlate with the upregulated expression of several relevant paracrine factors and the plausible cooperative secretion of miR-133a via exosomal transport. Finally, an in vitro heart muscle model confirmed the antiapoptotic effects of miR-133a-CPCs, favoring the structuration and contractile functionality of the artificial tissue.
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MicroARNs/genética , Mioblastos Cardíacos/metabolismo , Infarto del Miocardio/genética , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Biología Computacional , Expresión Génica , Perfilación de la Expresión Génica , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Interferencia de ARN , ARN Mensajero/genética , RatasRESUMEN
Early mammalian development is characterized by a highly specific stage, the blastocyst, by which embryonic and extraembryonic lineages have been determined, but pattern formation has not yet begun. The blastocyst is also of interest because cell precursors of the embryo proper retain for a certain time the capability to generate all the cell types of the adult animal. This embryonic pluripotency is established and maintained by a regulatory network under the control of a small set of transcription factors, comprising Oct4, Sox2 and Nanog. This network is largely conserved in eutherian mammals, but there is scarce information about how it arose in vertebrates. We have analysed the conservation of gene regulatory networks controlling blastocyst lineages and pluripotency in the mouse by comparison with the chick. We found that few of elements of the network are novel to mammals; rather, most of them were present before the separation of the mammalian lineage from other amniotes, but acquired novel expression domains during early mammalian development. Our results strongly support the hypothesis that mammalian blastocyst regulatory networks evolved through rewiring of pre-existing components, involving the co-option and duplication of existing genes and the establishment of new regulatory interactions among them.
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Evolución Biológica , Blastocisto/citología , Linaje de la Célula/genética , Células Madre Pluripotentes/citología , Animales , Redes Reguladoras de Genes/genética , Humanos , Mamíferos , Factores de Transcripción/genéticaRESUMEN
Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor-encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.
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ADN/metabolismo , Genoma , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular , Línea Celular , Pollos , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa , Humanos , Ratones , Esclerosis Múltiple/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Nanog is a pluripotency-associated factor expressed in embryonic stem cells and in the epiblast and primordial germ cells of the mouse embryo. We have identified the chick orthologue of Nanog and found that its expression is limited to primordial germ cells in the early embryo and is not found throughout the epiblast. Genomic analysis has shown that Nanog is an amniote-specific gene and is absent from anamniotes and invertebrates. Furthermore, other pluripotency associated genes that are located in close proximity to Nanog in human and mouse are absent from the chick genome. Such observations lead to a scenario of sequential addition of novel genes to a genomic region associated to pluripotency. These results have profound implications for the study of the evolution of pluripotent lineages in the embryo and of vertebrate stem cells.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Células Germinativas/metabolismo , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Proteínas de Unión al ADN/genética , Etiquetas de Secuencia Expresada , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Zarigüeyas/embriología , Zarigüeyas/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Nerve growth factor induces cell death in organotypic cultures of otic vesicle explants. This cell death has a restricted pattern that reproduces the in vivo pattern of apoptosis occurring during inner ear development. In this study, we show that binding of nerve growth factor to its low affinity p75 neurotrophin receptor is essential to achieve the apoptotic response. Blockage of binding to p75 receptor neutralized nerve-growth-factor-induced cell death, as measured by immunoassays detecting the presence of cytosolic oligonucleosomes and by TUNEL assay to visualize DNA fragmentation. Nerve growth factor also induced a number of cell-death-related intracellular events including ceramide generation, caspase activation and poly-(ADP ribose) polymerase cleavage. Again, p75 receptor blockade completely abolished all of these effects. Concerning the intracellular pathway, ceramide increase depended on initiator caspases, whereas its actions depended on both initiator and effector caspases, as shown by using site-specific caspase inhibitors. Conversely, insulin-like growth factor I, which promotes cell growth and survival in the inner ear, abolished apoptosis induced by nerve growth factor. Insulin-like growth factor cytoprotective actions were accomplished, at least in part, by decreasing endogenous ceramide levels and activating Akt. Taken together, these results strongly suggest that regulation of nerve-growth-factor-induced apoptosis in the otocysts occurs via p75 receptor binding and is strictly controlled by the interaction with survival signalling pathways.