RESUMO
Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3beta (GSK-3beta). The increased phosphorylation of GSK-3beta by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3beta in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3beta mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3beta and inhibits its apoptotic activity in neurons.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular , AMP Cíclico/metabolismo , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas , Sulfonamidas , Animais , Apoptose , Química Encefálica , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Fracionamento Celular , Células Cultivadas , Cerebelo/citologia , Colforsina/farmacologia , Meios de Cultura Livres de Soro , AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Immunoblotting , Isoquinolinas/farmacologia , Neurônios/citologia , Neurônios/enzimologia , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Myocyte enhancer factor 2 (MEF2) proteins are important regulators of gene expression during the development of skeletal, cardiac, and smooth muscle. MEF2 proteins are also present in brain and recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B or MEF2C, were phosphorylated with the induction of apoptosis. The putative sites that were phosphorylated during apoptosis are functionally distinct from those previously reported to enhance MEF2 transcription. The increased phosphorylation of MEF2A and MEF2D was followed by decreased DNA binding, reduced transcriptional activity, and caspase-dependent cleavage to fragments containing N-terminal DNA binding domains and C-terminal transactivation domains. Expression of the highly homologous N terminus of MEF2A (1-131 amino acids) antagonized the transcriptional activity and prosurvival effects of a constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that MEF2A and MEF2D are prosurvival factors with high transcriptional activity in postmitotic cerebellar granule neurons. When these neurons are induced to undergo apoptosis by lowering extracellular potassium, MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding and cleavage by a caspase-sensitive pathway to N-terminal fragments lacking the transactivation domains. The degradation of MEF2D and MEF2A and the generation of MEF2 fragments that have the potential to act as dominant-inactive transcription factors lead to apoptotic cell death.
Assuntos
Apoptose , Caspases/metabolismo , Cerebelo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Inibidores de Caspase , Sobrevivência Celular , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Immunoblotting , Fatores de Transcrição MEF2 , Substâncias Macromoleculares , Fatores de Regulação Miogênica , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Potássio/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacosRESUMO
We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Neurônios/fisiologia , Proteínas Oncogênicas , Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Androstadienos/farmacologia , Animais , Apoptose/fisiologia , Northern Blotting , Western Blotting , Bromodesoxiuridina/química , Comunicação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Flavonoides/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunossupressores/farmacologia , Camundongos , Microscopia de Fluorescência , Testes de Precipitina , Proteínas/genética , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/genética , Sirolimo/farmacologia , Wortmanina , Receptor Tirosina Quinase AxlRESUMO
Fetal cell transplantation therapies are being developed for the treatment of a number of neurodegenerative disorders including Parkinson's disease [10-12,21,22,24,36,43]. Massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. We have explored a novel protein kinase pathway for its role in apoptosis of dopamine neurons. We have discovered that inhibitors of p38 MAP kinase (the pyridinyl imidazole compounds: PD169316, SB203580, and SB202190) improve survival of rat dopamine neurons in vitro and after transplantation into hemiparkinsonian rats. In embryonic rat ventral mesencephalic cultures, serum withdrawal led to 80% loss of dopamine neurons due to increased apoptosis. Incubation of the cultures with p38 MAP kinase inhibitors at the time of serum withdrawal prevented dopaminergic cell death by inhibiting apoptosis. In the hemiparkinsonian rat, preincubation of ventral mesencephalic tissue with PD169316 prior to transplantation accelerated behavioral recovery and doubled the survival of transplanted dopamine neurons. We conclude that inhibitors of stress-activated protein kinases improve the outcome of cell transplantation by preventing apoptosis of neurons after grafting.
Assuntos
Transplante de Tecido Encefálico , Sobrevivência Celular/efeitos dos fármacos , Dopamina/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neurônios/transplante , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Inibidores Enzimáticos/farmacologia , Feto , Sobrevivência de Enxerto/fisiologia , Imidazóis/farmacologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/terapia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1beta, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1beta-induced HIV production was suppressed by more than 90% and IL-1beta-induced IL-8 production was suppressed completely, both with IC50 of 0.01 microM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , HIV-1/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Replicação Viral , Adesão Celular , Linhagem Celular , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Interleucina-1/farmacologia , Interleucina-8/biossíntese , Pressão Osmótica , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.
Assuntos
Proteínas de Bactérias , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Actinas/metabolismo , Adenoviridae/genética , Animais , Apoptose , Toxinas Bacterianas/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspase 3 , Caspases/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Citoesqueleto/metabolismo , Ativação Enzimática , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Immunoblotting , Imuno-Histoquímica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Fosforilação , Potássio/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Transdução de Sinais , Transcrição Gênica , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
IGF-I is known to support growth and to prevent apoptosis in neuronal cells. Activation of the nuclear transcription factor cAMP response element-binding protein (CREB) has emerged as a central determinant in neuronal functions. In the present investigation, we examined the IGF-I-mediated phosphorylation and transcriptional activation of CREB in rat pheochromocytoma (PC12) cells, a cellular model for neuronal differentiation, and defined three distinct postreceptor signaling pathways important for this effect including the p38 mitogen-activated protein kinase (MAPK) pathway. CREB phosphorylation at serine 133 and its transcriptional activation as measured by a CREB-specific Gal4-CREB reporter and the neuroendocrine-specific gene chromogranin A was induced 2-3.3-fold by insulin-like growth factor (IGF)-I. This activation was significantly blocked (p < 0.001) by the dominant negative K-CREB or by mutation of the CRE site. IGF-I stimulated chromogranin A gene expression by Northern blot analysis 3.7-fold. Inhibition of MAPK kinase with PD98059, PI 3-kinase with wortmannin, and p38 MAPK with SB203580 blocked IGF-I-mediated phosphorylation and transcriptional activation of CREB by 30-50% (p < 0.001). Constitutively active and dominant negative regulators of the Ras and PI 3-kinase pathways confirmed the contribution of these pathways for CREB regulation by IGF-I. Cotransfection of PC12 cells with p38beta and constitutively active MAPK kinase 6 resulted in enhanced basal as well as IGF-I-stimulated chromogranin A promoter. IGF-I activated p38 MAPK, which was blocked by the inhibitor SB203580. This is the first description of a p38 MAPK-mediated nuclear signaling pathway for IGF-I leading to CREB-dependent neuronal specific gene expression.