RESUMEN
Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.
Asunto(s)
Inhibidores de Caspasas , Cerebelo/metabolismo , Ácido Glutámico/metabolismo , Interleucina-10/metabolismo , Neuronas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Caspasa 3 , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Glutámico/farmacología , Interleucina-10/farmacología , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , FN-kappa B/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Técnicas de Placa-Clamp , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
In C6-2B astrocytoma cells the Beta NGF content and secretion rate are increased by isoproterenol activation of beta-adrenergic receptors (Schwartz and Costa, 1977). Utilizing poly (A+) RNA hybridization analysis with a cRNA probe for mouse Beta NGF it was found that isoproterenol activation of C6-2B cells produces also a 4 fold increase of the content of messenger RNA encoding Beta NGF. This increase is specifically antagonized by 1-propanolol, but not by phentolamine. Furthermore, addition of dibutyryl-cAMP induces an increase of Beta NGF mRNA content similar to that obtained with isoproterenol. These results are consistent with the hypothesis that regulation of Beta NGF synthesis in neuroglial cells may be modulated by beta-adrenergic receptor activation.
Asunto(s)
Glioma/metabolismo , Factores de Crecimiento Nervioso/metabolismo , ARN Mensajero/biosíntesis , Receptores Adrenérgicos beta/fisiología , Células Tumorales Cultivadas/metabolismo , Animales , Astrocitoma/metabolismo , Isoproterenol/farmacología , ARN Mensajero/aislamiento & purificación , RatasRESUMEN
Northern blot hybridization analysis was used to study regulation of nerve growth factor receptor (NGFR) mRNA content by glucocorticoids. Treatment for 6 h with dexamethasone (1 microM) caused a 40% decrease of NGFR mRNA content in PC12 cells and a 60% decrease in C6-2B glioma cells which was time and dose dependent. Dexamethasone (1 microM/kg) administered s.c. for two days to 21-day-old rats, elicits a 60% decrease in NGFR mRNA content in septum. These results suggest that the expression of NGFR gene in the brain could be inhibited by endogenous glucocorticoids. Whether dexamethasone inhibits NGFR gene expression by directly affecting cis-regulatory elements in the promoter regions of the gene remains to be elucidated.
Asunto(s)
Encéfalo/metabolismo , Dexametasona/farmacología , Factores de Crecimiento Nervioso/metabolismo , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Neoplasias de las Glándulas Suprarrenales , Animales , Encéfalo/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Glioma , Cinética , Feocromocitoma , ARN Mensajero/efectos de los fármacos , Ratas , Receptores de Factor de Crecimiento NerviosoRESUMEN
This study aimed to identify the CD24 and CD44 immunophenotypes within invasive ductal breast carcinoma (IDC) subgroups defined by immunohistochesmistry markers and to determine its influence on prognosis as well as its association with the expression of Ki-67, cytokeratins (CK5 and CK18) and claudin-7. Immunohistochemical expression of CD44 and CD24 alone or in combination was investigated in 95 IDC cases arranged in a tissue microarray (TMA). The association with subgroups defined as luminal A and B; HER2 rich and triple negative, or with the other markers and prognosis was analyzed. CD44+/CD24- and CD44-/CD24+ were respectively present in 8.4% and 16.8% of the tumors, a lack of both proteins was detected in 6.3%, while CD44+/CD24+ was observed in 45.3% of the tumors. Although there was no significant correlation between subgroups and different phenotypes, the CD44+/CD24- phenotype was more common in the basal subgroups but absent in HER2 tumors, whereas luminal tumors are enriched in CD44-/CD24+ and CD44+/CD24+ cells. The frequency of CD44+/CD24- or CD44-/CD24+ was not associated with clinical characteristics or biological markers. There was also no significant association of these phenotypes with the event free (DFS) and overall survival (OS). Single CD44+ was evident in 57.9% of the tumors and was marginally associated to grading and not to any other tumor characteristics as well as OS and DFS. CD24+ was positive in 74.7% of the tumors, showing a significant association with estrogen receptor, progesterone receptor and Ki-67 and a marginal association with CK18 and claudin-7. Expression of claudin-7 and Ki-67 did not associate with the cancer subgroups, while a positive association between CK18 and the luminal subgroups was found (P=0.03). CK5, CK18 and Ki-67 expression had no influence in OS or DFS. Single CD24+ (P=0.07) and claudin-7 positivity (P=0.05) were associated with reduced time of recurrence, suggesting a contribution of these markers to aggressiveness of breast cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Antígeno CD24/biosíntesis , Carcinoma Ductal de Mama/metabolismo , Claudinas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Antígeno CD24/análisis , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/patología , Claudinas/análisis , Femenino , Humanos , Receptores de Hialuranos/análisis , Receptores de Hialuranos/biosíntesis , Inmunohistoquímica , Estimación de Kaplan-Meier , Antígeno Ki-67/análisis , Antígeno Ki-67/biosíntesis , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Análisis de Matrices TisularesRESUMEN
C6-2B rat astrocytoma cells were used to test whether the increase of cellular nerve growth factor (NGF) content and secretion induced by isoproterenol treatment are associated with an increase in the content of mRNA that encodes NGF (NGF mRNA). Incubation of cells with isoproterenol (10 microM) for different time periods produced an increase of NGF mRNA content (3- to 5-fold over control) reaching maximum levels at 3 hr and lasting up to 24 hr. The isoproterenol effect on NGF mRNA was antagonized by the beta adrenergic receptor antagonist I-propranolol (10 microM) but not by phentolamine (10 microM), an alpha adrenergic receptor antagonist. When C6-2B astrocytoma cells were exposed for a short time to isoproterenol (10 microM; 10 or 20 min) followed by a washout period of 3 hr, the NGF mRNA content was increased by about 2-fold. The increase of NGF mRNA was obtained also with 10 microM prostaglandin E1 and this effect was potentiated by 100 microM of 3-isobutyl-1-methylxanthine. Inasmuch as both isoproterenol and prostaglandin E1 increase cyclic AMP content, one can surmise that cyclic AMP is involved in the stimulation of NGF mRNA accumulation. Whether cyclic AMP directly activates NGF gene transcription or activates an intermediate step, however, cannot be assessed by the present experiments.
Asunto(s)
Astrocitoma/metabolismo , Factores de Crecimiento Nervioso/genética , ARN Mensajero/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Prostaglandina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Alprostadil/farmacología , Animales , Línea Celular , Isoproterenol/farmacología , Masculino , Ratones , Propranolol/farmacología , Transcripción GenéticaRESUMEN
Evidence has accumulated to suggest that the NMDA glutamate receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in culture are vulnerable to NMDA-induced neuronal excitotoxicity. In these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA. However, little is known about the molecular mechanisms underlying the protective properties of these trophic factors. Using cultured rat cerebellar granule cells, we investigated whether BDNF and FGF2 prevent NMDA toxicity by downregulating NMDA receptor function. Western blot and RNase protection analyses were used to determine the expression of the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent decrease in the expression of NR2A and NR2C subunits. Because NMDA receptor activation leads to increased intracellular Ca2+ concentration ([Ca2+]i), we studied the effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C synthesis on the NMDA-evoked Ca2+ responses by single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced the NMDA-mediated [Ca2+]i increase with a time dependency that correlates with their ability to decrease NR2A and NR2C subunit expression, suggesting that these trophic factors also induce a functional downregulation of the NMDA receptor. Because sustained [Ca2+]i is believed to be causally related to neuronal injury, we suggest that BDNF and FGF2 may protect cerebellar granule cells against excitotoxicity by altering the NMDA receptor-Ca2+ signaling via a downregulation of NMDA receptor subunit expression.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Cerebelo/citología , Regulación hacia Abajo/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Calcio/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Factor 2 de Crecimiento de Fibroblastos/genética , N-Metilaspartato/farmacología , Neuronas/química , Neuronas/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
Regulation of the cytosolic free Ca2+ concentration by nerve growth factor was investigated in C6-2B glioma cells newly expressing the high affinity nerve growth factor receptor trkA, using Fura-2 fluorescence ratio imaging. In these cells, nerve growth factor (50 ng/ml) evoked a novel approximately 3-fold increase in cytosolic free Ca2+ concentration, while no measurable Ca2+ response was observed in wild type or mock-transfected cells lacking a functional trkA receptor. K-252a, a tyrosine kinase inhibitor which prevents nerve growth factor-mediated responses in C6-2B cells expressing trkA, also blocked the rise in cytosolic free Ca2+ concentration by nerve growth factor. Moreover, basic fibroblast growth factor, which in these cells elicits biochemical changes similar to nerve growth factor, failed to affect cytosolic free Ca2+ concentration, further supporting the specificity of nerve growth factor/trkA receptor in mediating a Ca2+ response. While insensitive to chelation of extracellular Ca2+, the response was abolished following depletion of Ca2+ stores or blockade of intracellular Ca2+ release, providing strong evidence that intracellular Ca2+ is the main source for nerve growth factor-evoked cytosolic free Ca2+ concentration increase. Nerve growth factor increased the cytosolic free Ca2+ concentration also in NIH3T3 cells overexpressing trkA but devoid of p75 nerve growth factor receptor. Our data suggest that trkA but not p75 is required for nerve growth factor-evoked Ca2+ signaling.
Asunto(s)
Calcio/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células 3T3 , Animales , Carbazoles/farmacología , Línea Celular , Citosol , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Alcaloides Indólicos , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas/genética , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Receptor trkA , Receptores de Factor de Crecimiento Nervioso/genética , Transducción de Señal , TransfecciónRESUMEN
The chain of events that results in increased production of nerve growth factor (NGF) following beta-adrenergic receptor (BAR) stimulation has been investigated in the C6-2B rat astrocytoma cell line. Exposure of these cells to the BAR agonist isoproterenol elicits the following cascade of events: (i) increase of cAMP content; (ii) increase of c-Fos mRNA content; (iii) accumulation of c-Fos protein immunoreactivity in the nucleus; (iv) increase of NGF mRNA content. The increase in c-Fos mRNA and its translation product are early events (15 and 40 min, respectively) and precede the accumulation of NGF mRNA, which peaks at 3 hr. The increase in the two mRNAs appears interrelated because cycloheximide inhibits the accumulation of c-Fos protein and NGF mRNA elicited by isoproterenol. Moreover, the accumulation of nuclear c-Fos protein and NGF mRNA induced by BAR stimulation is reduced by 2-aminopurine, an inhibitor of c-Fos mRNA induction. These data suggest that, in C6-2B astrocytoma cells, the nuclear accumulation of c-Fos protein is required for the induction of NGF mRNA expression by BAR stimulation.