RESUMEN
PURPOSE: Silicone oil is used as endotamponade in combination with vitrectomy. Thinning of retinal layers and loss of retinal cells under silicone oil use have been found. Here, we investigate the influence of silicone oil on primary microglia cells. METHODS: Primary microglia cells were prepared from the porcine retina. Microglia identity was assessed with Iba1 staining. Silicone oil was emulsified by sonification. Cell morphology and silicone oil uptake were evaluated by light microscopy after Coomassie blue staining. Cytokine secretion was evaluated with ELISA. Toxicity of silicone oil on microglia and toxic effect of silicone oil-treated microglia on neuronal cell line PC12 were evaluated by MTT or WST assay, respectively. RESULTS: Microglia took up silicone oil droplets after 72 h of incubation. Silicone oil induced no toxicity but increased the metabolism in microglial cells. In addition, the secretion of IL-6 and IL-8, but not of IL-1ß or TNF-α, was induced. Silicone oil-treated microglia did not exert any neurotoxic effect on differentiated PC12 cells but induced an increase in metabolism. CONCLUSION: Emulsified silicone oil changes the activity level of microglia and induces the secretion of IL-6 and IL-8. Neurotoxicity is not induced. Further experiments are required to investigate the long-term effect of silicone oil on microglia and their consequent effect on neuronal cells.
Asunto(s)
Endotaponamiento/métodos , Microglía/efectos de los fármacos , Enfermedades de la Retina/cirugía , Aceites de Silicona/administración & dosificación , Vitrectomía/métodos , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Emulsiones/administración & dosificación , Ratas , Desprendimiento de Retina , Enfermedades de la Retina/diagnóstico , Porcinos , Tomografía de Coherencia ÓpticaRESUMEN
Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Neuroblastoma/metabolismo , Tretinoina/farmacología , Western Blotting , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Plásmidos/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Azithromycin is a widely used macrolide antibiotic with sustained and high tissue penetration and intracellular accumulation. While short-term exposure to low-dose azithromycin is usually well tolerated, prolonged treatment can lead to unwanted neurological effects like paresthesia and hearing loss. However, the mechanism causing neurodegeneration is still unknown. Here, we show that even low therapeutically relevant azithromycin concentrations like 1µg/ml decreased cell viability by 15% and induced neurite loss of 47% after 96h in differentiated PC12 cells, which are a well-established model system for neuronal cells. When higher concentrations were used, the drug-induced effects occurred earlier and were more pronounced. Thereby, azithromycin altered tropomyosin-related kinase A (TrkA) signaling and attenuated protein kinase B (Akt) activity, which subsequently induced autophagy. Simultaneously, the antibiotic impaired lysosomal functions by blocking the autophagic flux, and this concurrence reduced cell viability. In good agreement with reversible effects observed in patients, PC12 cells could completely recover if azithromycin was removed after 24h. In addition, the detrimental effects of azithromycin were limited to differentiated cells, as confirmed in the human neuronal model cell line SH-SY5Y. Thus, azithromycin alters cell surface receptor signaling and autophagy in neuronal cells, but does not automatically induce irreversible damage when used in low concentrations and for a short time.
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Azitromicina/efectos adversos , Diferenciación Celular , Neuronas/citología , Neuronas/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/metabolismo , Células PC12 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
The c-Jun N-terminal kinases (JNKs) are important mediators of cell viability and structural integrity in postmitotic neurons, which is required for maintaining synaptic connections and neural plasticity. In the present study, we chose differentiated PC12 cells as a well-characterised neuronal model system to selectively examine the regulation of basal JNK activity by extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We detected a complex interaction between the kinases to prevent cell death and neurite loss. Especially the appropriate level of JNK activation determined cellular survival. Basal activity of ERK1/2 attenuated the potentiation of JNK phosphorylation and thereby the induction of apoptosis. Importantly, when JNK activity was too low, cell viability and the number of neurite-bearing cells also decreased, even though the activation of ERK1/2 was enhanced. In this case, the JNK-mediated survival signals via activating transcription factor-3 (ATF3) were inhibited. Furthermore, the phosphorylation of ERK1/2 induced by the JNK inhibitor SP600125 inhibited the basal activity of Akt, which normally supported cell viability. Thus, controlling JNK activity is crucial to promote survival and neurite stability of differentiated neuronal cells.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuritas/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Antracenos/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.
Asunto(s)
Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , MicroARNs/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Anticonvulsivantes/farmacología , Modelos Animales de Enfermedad , Epilepsia Refractaria/tratamiento farmacológico , Epilepsia Refractaria/metabolismo , Estimulación Eléctrica , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Femenino , Expresión Génica , Células Hep G2 , Hipocampo/efectos de los fármacos , Humanos , Neuroestimuladores Implantables , MicroARNs/genética , Mutación , Fenobarbital/farmacología , Canales de Potasio de Dominio Poro en Tándem/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas Sprague-Dawley , Estado Epiléptico/tratamiento farmacológico , Estado Epiléptico/metabolismo , Quinasas DyrKRESUMEN
Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.
Asunto(s)
MAP Quinasa Quinasa 4/genética , Empalme del ARN , Secuencia de Aminoácidos , Animales , Apoptosis , Proliferación Celular , Clonación Molecular , Humanos , MAP Quinasa Quinasa 4/química , Datos de Secuencia Molecular , Células PC12 , Ratas , Homología de Secuencia de Aminoácido , Transducción de Señal , TransfecciónRESUMEN
The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Disulfuros/química , Humanos , Sanguijuelas , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Neuronas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Sales (Química)/química , Dispersión de Radiación , Homología de Secuencia de AminoácidoRESUMEN
The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7-9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD.The successful and substantial reduction of the severe, TNBS-evoked intestinal damages and clinical symptoms render the JNK-inhibiting peptide XG-102 a powerful therapeutic principle of IBD.
Asunto(s)
Apoptosis/efectos de los fármacos , Colitis Ulcerosa/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Péptidos/farmacología , Análisis de Varianza , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Complejo CD3/metabolismo , Caspasa 3/metabolismo , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/patología , Proteína Ligando Fas/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Ratones , Péptidos/uso terapéutico , Proteínas Proto-Oncogénicas/metabolismo , Ácido Trinitrobencenosulfónico/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Pérdida de Peso/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes.
Asunto(s)
Apoptosis , MAP Quinasa Quinasa 7/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuronas/enzimología , Transducción de Señal , Animales , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neurogénesis , Células PC12 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The functions of mitogen-activated protein kinases (MKKs) 4 and 7 are typically associated with the c-Jun N-terminal kinase (JNK) signaling pathway. Both MKKs synergistically phosphorylate different JNK isoforms and are therefore involved in numerous physiological (e.g. differentiation and proliferation) and pathological (e.g. apoptosis and tumorigenesis) processes. MKK4 and MKK7 share similar molecular characteristics as well as several upstream activators and scaffold proteins. However, their functions are non-redundant and determined by different stimuli, biochemical interactions and differential tissue distribution. The central question is how two MKKs regulate or affect the multiple actions of their JNK substrates. Similar to JNKs, MKK4 and MKK7 can simultaneously exert divergent functions in different cellular compartments and signalosomes. It is also important to realize that the MKK effects are splice variant-specific. The present review not only summarizes the various modes of MKK4 and MKK7 activation and activity, but also their functions. We also extensively describe their impact on JNK signaling, their molecular interactions resulting in the formation of context-specific signalosomes and the functional consequences of JNK deficiency.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Apoptosis/fisiología , HumanosRESUMEN
The c-Jun N-terminal kinases (JNKs) mediate a diversity of physiological and pathophysiological effects. Apart from isoform-specific JNK activation, upstream kinases are supposed to be the relevant regulators, which are involved in the context- and signalosome-depending functions. In the present study we report the cloning and characterization of the novel rat MKK7gamma1, a splice variant of MKK7 with an additional exon in the N-terminal region, in the neuronal pheochromocytoma cell line PC12. Transfected MKK7gamma1 increased basal JNK activity, in particular phosphorylation of JNK2. Consequently, JNK signalling was changed in mRNA-, protein- and activation-levels of JNK targets, such as transcription factors (c-Jun, p53, c-Myc), cell cycle regulators (p21, CyclinD1) and apoptotic proteins (Fas, Bim, Bcl-2, Bcl-xl). These alterations promote the sensitivity of MKK7gamma1-transfected cells towards cell death and repress cell proliferation under normal cell growth conditions. Complexes of JIP-1, MKK7 and JNK2 were the major JNK signalosomes under basal conditions. After stimulation with taxol (5muM) and tunicamycin (1.4mug/ml), MKK7gamma1- but not MKK7beta1-transfection, reduced cell death and even increased cell proliferation. Cellular stress also led to an increased phosphorylation of JNK1 and the almost complete abrogation of complexes of JIP-1, MKK7 and JNK2 in MKK7gamma1-transfected PC12 cells. Summarizing, MKK7gamma1 affects the function and activity of individual JNK isoforms and the formation of their signalosomes. This study demonstrates for the first time that one splice-variant of MKK7 tightly controls JNK signalling and effectively adapts JNK functions to the cellular context.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Antineoplásicos Fitogénicos/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Datos de Secuencia Molecular , Paclitaxel/farmacología , Fosforilación , Isoformas de Proteínas/metabolismo , Ratas , Tunicamicina/farmacología , Regulación hacia ArribaRESUMEN
Deferoxamine mesylate is clinically used as a chelating agent but might induce retinopathy. To evaluate its effect on the retinal pigment epithelium (RPE), porcine RPE cells were stimulated with deferoxamine. Cell death was assessed with trypan blue exclusion assay. To investigate the pathway of cell death, the mitogen-activated protein kinases (MAPKs) Erk, JNK, and p38 were inhibited with U0126, SP600125, and SB203580, respectively. Their activity was determined by Western blot. Deferoxamine induces significant cell death in RPE cells, accompanied by phosphorylation of p38 and Erk. Inhibition of p38 attenuates cell death. In conclusion, deferoxamine is directly toxic on RPE cells, its toxicity depending on p38.
Asunto(s)
Deferoxamina/toxicidad , Quelantes del Hierro/toxicidad , Epitelio Pigmentado de la Retina/patología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Antracenos/uso terapéutico , Western Blotting , Butadienos/uso terapéutico , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/toxicidad , Imidazoles/uso terapéutico , Nitrilos/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Epitelio Pigmentado de la Retina/enzimología , Porcinos , Azul de Tripano , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.
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Isquemia Encefálica/tratamiento farmacológico , Infarto Cerebral/tratamiento farmacológico , Encefalitis/tratamiento farmacológico , Gliosis/tratamiento farmacológico , Degeneración Nerviosa/tratamiento farmacológico , Tacrolimus/farmacología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Isquemia Encefálica/inmunología , Isquemia Encefálica/fisiopatología , Células Cultivadas , Infarto Cerebral/inmunología , Infarto Cerebral/fisiopatología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Técnicas de Cocultivo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalitis/inmunología , Encefalitis/fisiopatología , Gliosis/inmunología , Gliosis/fisiopatología , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tacrolimus/uso terapéuticoRESUMEN
Investigation of the c-Jun N-terminal kinases (JNKs) has mainly focused on their response to stress and their pro-apoptotic effects. In this regard, JNKs are crucial mediators of chemotherapy-induced killing of tumor cells. Importantly, however, JNKs also have physiological functions in cancer involving cell cycle regulation or oncogenesis. Hypothetically, the composition of JNK signalosomes determines the signaling outcome which,in turn, implies a multitude of different, sometimes opposing and interfering functions. In the present study,the well-characterized human neuroblastoma cell line SH-SY5Y served as a model system to separate physiological and pro-apoptotic JNK actions in the response to the cytoskeleton-interfering substances colchicine, cytochalasin D and taxol. Basically, JNKs mediated both cell death and proliferation. Using the chemical JNK inhibitor SP600125 as well as compartment-specific JNK-inhibiting constructs and dominant negative isoform mutants, we show that the nuclear subgroup of JNK2 is the dominant effector in colchicine and taxol-induced apoptosis, while cell cycle promotion is mediated by both cytoplasmic and nuclear JNK2.In contrast, cytochalasin D-triggered apoptosis is independent of JNK signaling. Interestingly, the data of the present study demonstrate for the first time that both cell protective (cell cycle progression) and destructive mechanisms (apoptosis) are simultaneously controlled by a single JNK isoform in the same cell system even under the influence of one stimulus. This has implications for the therapeutic application of JNK inhibitors and cytoskeleton-interfering substances in oncologic disorders.
Asunto(s)
Citoprotección , Neuroblastoma/enzimología , Neuroblastoma/patología , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colchicina/farmacología , Citocalasina D/farmacología , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 9 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Modelos Biológicos , Neuroblastoma/genética , Paclitaxel/farmacología , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
In response to injury, peripheral neuronal cells initiate complex signalling cascades to promote survival and regeneration. In the present study, we used a model of experimental injury in the rat pheochromocytoma cell line PC12 to investigate receptor signals that lead to neurite outgrowth. Nerve growth factor (NGF) dose-dependently induced sprouting and the expression of the NGF receptors Trk tyrosine kinase receptor (TrkA) and p75 neurotrophin receptor (p75(NTR)) as well as Fas and Fas ligand. Neurite regeneration was decreased by chemical inhibition of TrkA, but not p75(NTR), and by the Fas inhibitor protein Fas-Fc. The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNKs) were activated in response to NGF and both significantly contributed to neurite re-growth. Interestingly, otherwise apoptotic Fas ligation supported neuronal recovery exclusively via JNKs and promoted sprouting parallel to NGF. These findings suggest a novel signal integration from the NGF and Fas pathways in the JNK axis of MAPK signalling, where JNKs function as "physiological" mediators of normally apoptotic signals.
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Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Regeneración Nerviosa/fisiología , Neuritas , Receptor fas/fisiología , Animales , Western Blotting , Activación Enzimática , Proteína Ligando Fas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Células PC12 , Ratas , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptor fas/metabolismoRESUMEN
The c-Jun N-terminal kinases (JNKs) are important regulators of physiological and pathological processes in the central and peripheral nervous system. In general, JNKs are considered as mediators of neuronal degeneration in response to stress and injury. However, recent data have provided substantial evidence that JNKs are also essential for physiological and regenerative signalling in neurons. This review summarizes the importance of JNKs for neurite formation and outgrowth, brain development, dendritic architecture and regeneration of nerve fibers after injury. We discuss putative mechanisms which control the bipartite actions of individual JNK isoforms for neuronal death and repair after nerve fiber injury with a particular focus on the role of the transcription factor c-Jun.
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Encéfalo/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Fibras Nerviosas/fisiología , Regeneración Nerviosa/fisiología , Neuronas/fisiología , Animales , HumanosRESUMEN
We have investigated the breakpoints in a male child with pharmacoresistant epileptic encephalopathy and a de novo balanced translocation t(Y;4)(q11.2;q21). By fluorescence in situ hybridisation, we have identified genomic clones from both chromosome 4 and chromosome Y that span the breakpoints. Precise mapping of the chromosome 4 breakpoint indicated that the c-Jun N-terminal kinase 3 (JNK3) gene is disrupted in the patient. This gene is predominantly expressed in the central nervous system, and it plays an established role in both neuronal differentiation and apoptosis. Expression studies in the patient lymphoblastoid cell line show that the truncated JNK3 protein is expressed, i.e. the disrupted transcript is not immediately subject to nonsense-mediated mRNA decay, as is often the case for truncated mRNAs or those harbouring premature termination codons. Over-expression studies with the mutant protein in various cell lines, including neural cells, indicate that both its solubility and cellular localisation differ from that of the wild-type JNK3. It is plausible, therefore, that the presence of the truncated JNK3 disrupts normal JNK3 signal transduction in neuronal cells. JNK3 is one of the downstream effectors of the GTPase-regulated MAP kinase cascade, several members of which have been implicated in cognitive function. In addition, two known JNK3-interacting proteins, beta-arrestin 2 and JIP3, play established roles in neurite outgrowth and neurological development. These interactions are likely affected by a truncated JNK3 protein, and thereby provide an explanation for the link between alterations in MAP kinase signal transduction and brain disorders.
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Encefalopatías/genética , Sistema Nervioso Central/metabolismo , Epilepsia/genética , Proteína Quinasa 10 Activada por Mitógenos/genética , Secuencia de Bases , Western Blotting , Cromosomas Humanos Par 4 , Cromosomas Humanos Y , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Índice de Severidad de la Enfermedad , Translocación GenéticaRESUMEN
The c-Jun N-terminal kinases (JNKs), which are essential regulators of physiological and pathological processes, are involved in several diseases including diabetes, atherosclerosis, stroke, and Parkinson's and Alzheimer's diseases. Inhibition of JNKs suppresses pathological features of these diseases but the many physiological functions of these enzymes argue against the use of sustained, systemic, nonspecific inhibition in the treatment of these diseases. For example, deletion of the gene that encodes JNK1 prevents insulin resistance but disrupts neuronal cytoarchitecture and initiates the pathology of Alzheimer's disease. Thus, it is not sufficient to inhibit selectively either JNKs or individual isoforms of JNK. Instead, the aim is to inhibit the damaging actions of JNK. This can be achieved using peptides that selectively block molecular domains of individual JNK signaling complexes (exclusively) that form under pathological conditions. To date, peptide inhibitors of JNK have been successful in protecting against ischemia-induced brain damage and insulin resistance following obesity. In this review, we discuss novel pharmacological strategies to inhibit JNK and the limitations of these strategies.
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Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Carbazoles/uso terapéutico , Humanos , Indoles/uso terapéutico , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Sistema de Señalización de MAP QuinasasRESUMEN
After injury, peripheral neuronal cells initiate complex signaling cascades to promote survival and regeneration. In the present study, we have identified the mitogen-activated protein kinase (MAPK) isoforms which are necessary for nerve growth factor (NGF)-induced neurite regrowth after injury of differentiated PC12 cells. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the usually pro-apoptotic c-Jun N-terminal kinase 2 (JNK2) are crucial for neurite regrowth, while p38 plays no role in this context. Surprisingly, the MEK1 inhibitors PD 98059 and U 0126 blocked both ERK1/2 and JNK phosphorylation, indicating a novel form of balancing MAPK cascade cross-talk. Results from RNAi experiments excluded direct ERK/JNK interactions. We identified the upstream kinase MEKK1 as an activator of both the ERK1/2 and JNK2 pathways, whereby the ERK1/2 kinase MEK1 and the JNK kinase MKK7 bind to MEKK1 in a competing fashion. Our findings suggest an important role of JNK2 and MAPK pathway cross-talk in neurite regeneration.