RESUMO
Friedreich ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here, we describe that frataxin-deficient dorsal root ganglia neurons display low levels of ferredoxin 1 (FDX1), a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both FDX1 and NCLX levels and restores mitochondrial membrane potential indicating an overall mitochondrial function improvement. Accordingly, reduction in apoptotic markers and neurite degeneration was observed and, as a result, cell survival was also recovered. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients.
Assuntos
Calcitriol/farmacologia , Ferredoxinas/metabolismo , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Apoptose/efeitos dos fármacos , Calcitriol/biossíntese , Calcitriol/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Vitamina D/metabolismo , FrataxinaRESUMO
The Myocyte Enhancer Factor-2 (MEF2) family of transcription factors regulates gene expression during cardiomyocyte differentiation and adaptation of the myocardium to stress. MEF2 activity is enhanced by increasing its transcription and by MAPK-dependent phosphorylation, and is reduced by binding to class-II Histone Deacetylases and by miR-1-mediated degradation of its transcript. Here we show that MEF2 protein abundance is regulated at the translational level, determining myocyte size, during hypertrophy. In order to reduce MEF2 protein expression, its silencing through RNA interference required serum deprivation and, even in this condition, MEF2 protein abundance recovered to basal levels in presence of phenylephrine. Hypertrophic agonist stimulation of neonatal ventricular cardiomyocytes increased Mef2 expression by enhancing its translation, without changing its transcription or blocking degradation of the protein. MEF2 abundance was increased by Calcineurin overexpression in vivo and was reduced by Calcineurin inhibition in vitro, without affecting Mef2 mRNA levels. Calcineurin activity influenced expression of Polypyrimidine Tract Protein (PTB), contributing to MEF2 translation. Thus, our results show a previously unrecognized but relevant level of MEF2 activity regulation through the control of its translation that involves Calcineurin and PTB.
Assuntos
Calcineurina/metabolismo , Proteínas de Domínio MADS/biossíntese , Proteínas de Domínio MADS/genética , Miócitos Cardíacos/metabolismo , Fatores de Regulação Miogênica/biossíntese , Fatores de Regulação Miogênica/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/biossíntese , Animais , Células Cultivadas , Células HEK293 , Humanos , Fatores de Transcrição MEF2 , Masculino , Camundongos , Camundongos Transgênicos , Pirimidinas/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Cardiovascular diseases are the leading cause of death globally and more than four out of five cases are due to ischemic events. Cardiac fibroblasts (CF) contribute to normal heart development and function, and produce the post-ischemic scar. Here, we characterize the biochemical and functional aspects related to CF endurance to ischemia-like conditions. Expression data mining showed that cultured human CF (HCF) express more BCL2 than pulmonary and dermal fibroblasts. In addition, gene set enrichment analysis showed overrepresentation of genes involved in the response to hypoxia and oxidative stress, respiration and Janus kinase (JAK)/Signal transducer and Activator of Transcription (STAT) signaling pathways in HCF. BCL2 sustained survival and proliferation of cultured rat CF, which also had higher respiration capacity and reactive oxygen species (ROS) production than pulmonary and dermal fibroblasts. This was associated with higher expression of the electron transport chain (ETC) and antioxidant enzymes. CF had high phosphorylation of JAK2 and its effectors STAT3 and STAT5, and their inhibition reduced viability and respiration, impaired ROS control and reduced the expression of BCL2, ETC complexes and antioxidant enzymes. Together, our results identify molecular and biochemical mechanisms conferring survival advantage to experimental ischemia in CF and show their control by the JAK2/STAT signaling pathway. The presented data point to potential targets for the regulation of cardiac fibrosis and also open the possibility of a general mechanism by which somatic cells required to acutely respond to ischemia are constitutively adapted to survive it.
Assuntos
Antioxidantes , Janus Quinase 2 , Animais , Fibroblastos/metabolismo , Isquemia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Respiração , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.
Assuntos
Encéfalo/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Neurogênese/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Neurônios Motores/fisiologia , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso , Neuroglia/metabolismo , Células PC12 , Ratos , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/metabolismo , Gânglio Cervical Superior/embriologia , Gânglio Cervical Superior/crescimento & desenvolvimento , Gânglio Cervical Superior/fisiologiaRESUMO
The nerve growth factor receptor TrkA (tropomyosin-related kinase receptor) participates in the survival and differentiation of several neuronal populations. The C-terminal tail of TrkA contains a PPXY motif, the binding site of the E3 ubiquitin-ligase Nedd4-2 (neural precursor cell expressed, developmentally down-regulated 4-2). In order to analyze the role of Nedd4-2 ubiquitination on TrkA function, we generated three TrkA mutants, by introducing point mutations on conserved hydrophobic amino acids - Leu784 and Val790 switched to Ala. TrkA mutants co-localized and co-immunoprecipitated more efficiently with Nedd4-2 and consequently a strong increase in the basal multimonoubiquitination of the mutant receptors was observed. In addition, we found a decrease in TrkA abundance because of the preferential sorting of mutant receptors towards the late endosome/lysosome pathway instead of recycling back to the plasma membrane. Despite the reduction in the amount of membrane receptor caused by the C-terminal changes, TrkA mutants were able to activate signaling cascades and were even more efficient in promoting neurite outgrowth than the wild-type receptor. Our results demonstrate that the C-terminal tail hydrophobicity of TrkA regulates Nedd4-2 binding and activity and therefore controls receptor turnover. In addition, TrkA multimonoubiquitination does not interfere with the activation of signaling cascades, but rather potentiates receptor signaling leading to differentiation.
Assuntos
Cisteína Endopeptidases/metabolismo , Lisossomos/metabolismo , Receptor trkA/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Animais , Biotina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cisteína Endopeptidases/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas de Fluorescência Verde/genética , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Mutação/genética , Fator de Crescimento Neural/farmacologia , Células PC12 , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Ratos , Receptor trkA/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção/métodos , Ubiquitinação/efeitos dos fármacos , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
EndoG influences mitochondrial DNA replication and is involved in somatic cell proliferation. Here, we investigated the effect of ENDOG/Endog expression on proliferation in different tumor models. Noteworthy, ENDOG deficiency reduced proliferation of endometrial tumor cells expressing low PTEN/high p-AKT levels, and Endog deletion blunted the growth of PTEN-deficient 3D endometrial cultures. Furthermore, ENDOG silencing reduced proliferation of follicular thyroid carcinoma and glioblastoma cell lines with high p-AKT expression. High ENDOG expression was associated with a short time to treatment in a cohort of patients with chronic lymphocytic leukemia (CLL), a B-cell lymphoid neoplasm with activation of PI3K/AKT. This clinical impact was observed in the less aggressive CLL subtype with mutated IGHV in which high ENDOG and low PTEN levels were associated with worse outcome. In summary, our results show that reducing ENDOG expression hinders growth of some tumors characterized by low PTEN activity and high p-AKT expression and that ENDOG has prognostic value for some cancer types.
RESUMO
The apoptotic nuclease EndoG is involved in mitochondrial DNA replication. Previous results suggested that, in addition to regulate cardiomyocyte hypertrophy, EndoG could be involved in cell proliferation. Here, by using in vivo and cell culture models, we investigated the role of EndoG in cell proliferation. Genetic deletion of Endog both in vivo and in cultured cells or Endog silencing in vitro induced a defect in rodent and human cell proliferation with a tendency of cells to accumulate in the G1 phase of cell cycle and increased reactive oxygen species (ROS) production. The defect in cell proliferation occurred with a decrease in the activity of the AKT/PKB-GSK-3ß-Cyclin D axis and was reversed by addition of ROS scavengers. EndoG deficiency did not affect the expression of ROS detoxifying enzymes, nor the expression of the electron transport chain complexes and oxygen consumption rate. Addition of the micropeptide Humanin to EndoG-deficient cells restored AKT phosphorylation and proliferation without lowering ROS levels. Thus, our results show that EndoG is important for cell proliferation through the control of ROS and that Humanin can restore cell division in EndoG-deficient cells and counteracts the effects of ROS on AKT phosphorylation.
Assuntos
Apoptose , Proliferação de Células , Endodesoxirribonucleases , Mitocôndrias , Espécies Reativas de Oxigênio , Animais , Ciclo Celular , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Camundongos , RatosRESUMO
Toward understanding topographically specific branching of retinal axons in their target area, we have studied the interaction between neurotrophin receptors and members of the Eph family. TrkB and its ligand BDNF are uniformly expressed in the retina and tectum, respectively, and exert a branch-promoting activity, whereas EphAs and ephrinAs are expressed in gradients in retina and tectum and can mediate a suppression of axonal branching. We have identified a novel cis interaction between ephrinA5 and TrkB on retinal ganglion cell axons. TrkB interacts with ephrinA5 via its second cysteine-rich domain (CC2), which is necessary and sufficient for binding to ephrinA5. Their functional interaction is twofold: ephrinA5 augments BDNF-promoted retinal axon branching in the absence of its activator EphA7-Fc, whereas EphA7-Fc application abolishes branching in a local and concentration-dependent manner. The importance of TrkB in this process is shown by the fact that overexpression of an isolated TrkB-CC2 domain interfering with the ephrinA/TrkB interaction abolishes this regulatory interplay, whereas knockdown of TrkB via RNA interference diminishes the ephrinA5-evoked increase in branching. The ephrinA/Trk interaction is neurotrophin induced and specifically augments the PI-3 kinase/Akt pathway generally known to be involved in the promotion of branching. In addition, ephrinAs/TrkB modulate axon branching and also synapse formation of hippocampal neurons. Our findings uncover molecular mechanisms of how spatially restricted axon branching can be achieved by linking globally expressed branch-promoting with differentially expressed branch-suppressing activities. In addition, our data suggest that growth factors and the EphA-ephrinA system interact in a way that affects axon branching and synapse development.
Assuntos
Axônios/metabolismo , Neurogênese/fisiologia , Receptor trkB/metabolismo , Receptores da Família Eph/metabolismo , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Animais , Axônios/ultraestrutura , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Embrião de Galinha , Efrina-A5/química , Efrina-A5/genética , Efrina-A5/metabolismo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Terciária de Proteína/fisiologia , RNA/metabolismo , Ratos , Receptor trkB/genética , Receptores da Família Eph/química , Receptores da Família Eph/genética , Retina/citologia , Células Ganglionares da Retina/citologia , Transdução de Sinais/fisiologia , Colículos Superiores/citologia , Colículos Superiores/embriologia , Colículos Superiores/metabolismo , Sinapses/metabolismoRESUMO
Tropomyosin-related kinase A (TrkA) receptor mediates the effects exerted by nerve growth factor on several subpopulations of neuronal cells. Ligand binding to TrkA induces receptor autophosphorylation on several tyrosine residues and the activation of signaling cascades. In this study, we describe a new site relevant for TrkA regulation, the tyrosine 701 (Y701), which is important for receptor trafficking and activation. Y701 replacement by aspartate or phenylalanine reduces receptor internalization rate and decreases the colocalization and association of TrkA with clathrin heavy chain, demonstrating that Y701 constitutes a YxxPhi (YRKF701-704) trafficking motif relevant for the regulation of receptor endocytosis. In accordance with this hypothesis, the colocalization of Y701 mutant receptors with a lysosomal marker is also reduced giving support to the involvement of the YRKF701-704 motif in the lysosomal targeting of TrkA receptors. Contrary to what was expected, substitution of Y701 for an Asp in order to mimic phosphorylation, impairs TrkA ability to mediate nerve growth factor-induced differentiation, although the mutant receptor retains its in vitro kinase activity. This is the first evidence that a Tyr residue can simultaneously regulate TrkA receptor trafficking and activity.
Assuntos
Receptor trkA/metabolismo , Tirosina/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Cadeias Pesadas de Clatrina/metabolismo , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/metabolismo , Mutação/fisiologia , Células PC12 , Transporte Proteico/fisiologia , Ratos , Transfecção/métodosRESUMO
The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3ß and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency.
Assuntos
Endodesoxirribonucleases/genética , Hipertrofia/genética , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Mitocôndrias/genética , Animais , Apoptose/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/metabolismo , Humanos , Hipertrofia/tratamento farmacológico , Hipertrofia/enzimologia , Hipertrofia/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The involvement of caspases in postmitotic cell death is controversial. Here we report that adult brain and heart are devoid of many key pro-apoptotic proteins due to a progressive postnatal silencing event involving a reduction of their transcript levels. E2F has been shown to control cell cycle progression and to be transcriptional activator of apoptotic genes. However, our data demonstrate that apoptotic gene expression in heart, brain and liver, as well as cardiac and neuronal apoptotic gene silencing during development, are E2F-independent events. Therefore, the genes regulating caspase-dependent cell death are expressed in embryonic organs in an E2F-independent manner and a developmental-related silencing event represses these genes in postmitotic adult tissues.
Assuntos
Apoptose/genética , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Mitose/genética , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/embriologia , Caspases/metabolismo , Células Cultivadas , Fatores de Transcrição E2F/metabolismo , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult null myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase-null myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.
Assuntos
Caspase 3/deficiência , Caspase 7/deficiência , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , Animais , Animais Recém-Nascidos , Caspase 3/metabolismo , Caspase 7/metabolismo , Contagem de Células , Ciclo Celular/genética , Proliferação de Células , Replicação do DNA/genética , Metabolismo Energético , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Proteômica , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-x(L). These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells.
Assuntos
Fragmentação do DNA , Endodesoxirribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/patologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Caspases/metabolismo , Marcação In Situ das Extremidades Cortadas , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Isquemia Miocárdica/enzimologia , Miocárdio/enzimologia , Transporte Proteico , Ratos , Proteína bcl-X/metabolismoRESUMO
Neurotrophic factors promote motoneuron (MN) survival through increased intracellular calcium (Ca(2+)) and regulation of the phosphatidylinositol (PI) 3-kinase/protein kinase B (PKB) pathway by calmodulin (CaM). Activation of the PI 3-kinase/PKB pathway is one of the well established mechanisms involved in MN survival. The Ca(2+)/CaM complex interacts with and modulates the functionality of a large number of proteins, including serine/threonine protein kinases such as Ca(2+)/CaM-dependent protein kinases (CaMKs). Using a primary culture of embryonic chicken spinal cord MNs, we investigated the role of CaMKIV in mediating this process. We cloned chicken CaMKIV and demonstrated its expression in purified MNs by means of reverse transcription-PCR, Western blot, and immunofluorescence. Using RNA interference, we show that endogenous CaMKIV mediates cell survival induced by neurotrophic factors or membrane depolarization. The survival effect is independent of CaMKIV kinase activity; however, CaMKIV functionality depends on the presence of Ca(2+)/CaM. Finally, CaMKIV associates to the p85 subunit of PI 3-kinase in a Ca(2+)-dependent manner, suggesting a role in regulating PI 3-kinase/PKB activation.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Potenciais da Membrana , Fatores de Crescimento Neural/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Galinhas , Primers do DNA , Embrião não Mamífero , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Tyrosine phosphorylation of beta-catenin, a component of adhesion complexes and of the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing beta-catenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that beta-catenin is required for axon growth downstream of brain-derived neurotrophic factor (BDNF) signalling and hepatocyte growth factor (HGF) signalling. We demonstrate that the receptor tyrosine kinases (RTKs) Trk and Met interact with and phosphorylate beta-catenin. Stimulation of Trk receptors by neurotrophins (NTs) results in phosphorylation of beta-catenin at residue Y654, and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or expression of a Y654F mutant blocks these effects. beta-catenin phosphorylated at Y654 colocalizes with the cytoskeleton at growth cones. However, HGF, which also increases axon growth and branching, induces beta-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant-negative DeltaN-TCF4 abolishes the effects of HGF in axon growth and branching, but not that of NTs. We conclude that NT- and HGF-signalling differentially phosphorylate beta-catenin, targeting this protein to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and -independent mechanisms. These results place beta-catenin downstream of growth-factor-RTK signalling in axon differentiation.
Assuntos
Axônios/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Morfogênese/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , beta Catenina/metabolismo , Animais , Axônios/metabolismo , Axônios/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Fator de Crescimento de Hepatócito/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/embriologia , Hipocampo/metabolismo , Humanos , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Ratos , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosina/metabolismoRESUMO
Differentiated cardiomyocytes are resistant to caspase-dependent cell death; however, the mechanisms involved are still uncertain. We previously reported that low Apaf1 expression partially accounts for cardiomyocyte resistance to apoptosis. Here, we extend the knowledge on the molecular basis of cardiac resistance to caspase activation by showing that the whole caspase-dependent pathway is silenced during heart development. Experimental ischemia triggers caspase activation in embryonic cardiomyocytes and proliferating fibroblasts, but not in neonatal and adult cardiomyocytes. Ischemia induces the release of the proapoptotic factors cytochrome c, truncated-AIF, and EndoG from mitochondria in postnatal cardiomyocytes in the absence of caspase activation. On the one hand, lentiviral-driven knockdown of EndoG shows that this gene is essential for ischemia-induced DNA degradation in neonatal cardiomyocytes, but not in proliferating fibroblasts; on the other hand, the AIF gene is essential for high molecular DNA cleavage in fibroblasts, but not in postmitotic cardiomyocytes, where it plays a prosurvival role during reoxygenation. These results show the switch from caspase-dependent to caspase-independent death pathways after cardiac cell differentiation, and disclose the relevance of EndoG in the caspase-independent DNA processing of differentiated cardiomyocytes.
Assuntos
Caspases/fisiologia , DNA/química , Endodesoxirribonucleases/fisiologia , Coração/embriologia , Miócitos Cardíacos/metabolismo , Animais , Apoptose , Caspases/metabolismo , Fragmentação do DNA , Endodesoxirribonucleases/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Isquemia Miocárdica , Ratos , Ratos Sprague-DawleyRESUMO
Neurotrophins (Nt) and their tyrosine kinase Trk receptors play an essential role in the development and maintenance of the complex vertebrate nervous system. Invertebrate genome sequencing projects have suggested that the Nt/Trk system is a vertebrate innovation. We describe the isolation and characterisation of the amphioxus Trk receptor, AmphiTrk. Its ancestral link to vertebrate Trk receptors is supported by phylogenetic analysis and domain characterisation. The genomic structure of AmphiTrk strongly suggests that a ProtoTrk gene emerged by means of exon-shuffling prior to the cephalochordate/vertebrate split. We also examined the physiological response of AmphiTrk to vertebrate neurotrophins, and found that despite 500 million years of divergence, AmphiTrk transduces signals mediated by NGF, BDNF, NT3 and NT4. Markedly, AmphiTrk is able to activate survival and differentiation pathways, but fails to activate the PLCgamma pathway, which is involved in synaptic plasticity in higher vertebrates. AmphiTrk is expressed during amphioxus embryogenesis in sensory neural precursors in the epidermis, which possesses single migratory cells. We propose that the duplication and divergence of the Nt/Trk system, in tandem with recruitment of the PLCgamma pathway, may have provided the genetic basis for a key aspect of vertebrate evolution: the complexity of the nervous system.
Assuntos
Cordados não Vertebrados/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Southern Blotting , Cordados não Vertebrados/embriologia , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/embriologia , Análise de Sequência de Proteína , Vertebrados/metabolismoRESUMO
Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.
Assuntos
Cálcio/metabolismo , Fatores de Crescimento Neural/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Western Blotting , Calmodulina/química , Morte Celular , Sobrevivência Celular , Embrião de Galinha , Ativação Enzimática , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Fatores de Crescimento Neural/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Sefarose/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.