RESUMEN
Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.
Asunto(s)
Caspasas/metabolismo , Mitocondrias/enzimología , Óxido Nítrico Sintasa/metabolismo , Caspasa 3 , Caspasa 9 , Precursores Enzimáticos/metabolismo , Humanos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Señales de Clasificación de Proteína , Transporte de Proteínas , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/fisiología , Células Tumorales Cultivadas , Receptor fas/farmacologíaRESUMEN
We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/enzimología , Óxido Nítrico Sintasa/biosíntesis , Proteínas ras/metabolismo , Animales , Butadienos/farmacología , Diferenciación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Vectores Genéticos/metabolismo , Imidazoles/farmacología , Immunoblotting , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Nitrilos/farmacología , Células PC12 , Piridinas/farmacología , RatasRESUMEN
We have examined nerve growth factor (NGF)-triggered signaling in two NIH3T3 cell lines exogenously expressing the NGF receptor, TrkA. TRK1 cells cease to proliferate and extend long processes in response to NGF, while E25 cells continue to proliferate in the presence of NGF. These two cell lines express similar levels of TrkA and respond to NGF with rapid elevation of mitogen-activated protein kinase (MAPK) activity. MAPK activation is slightly more sustained for E25 cells than for TRK1 cells, although sustained activation of MAPK has been suggested to cause cell-cycle arrest. As judged by NADPH-diaphorase staining, nitric oxide synthase (NOS) activity is increased in TRK1 cells upon exposure to NGF. In contrast, diaphorase staining in E25 cells is unaffected by NGF treatment. Immunocytochemistry shows that levels of the brain NOS (bNOS) isoform are increased in TRK1, but not E25, cells exposed to NGF. Furthermore, Western blots show that NGF elevated cyclin-dependent kinase inhibitor, p21(WAF1), in TRK1 cells only. NGF-induced p21(WAF1) expression, cell-cycle arrest and process extension are abolished by N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS. The inactive enantiomer, D-NAME, did not inhibit these responses. Furthermore, even though E25 cells do not respond to NGF or nitric oxide donors, they do undergo a morphological change in response to NGF plus a nitric oxide donor. Therefore, NOS and p21(WAF1) are induced only in the TrkA-expressing NIH3T3 cell line that undergoes cell-cycle arrest and morphological changes in response to NGF. These results demonstrate that sustained activation of MAPK is not the sole determining factor for NGF-induced cell-cycle arrest and implicate NO in the cascade of events leading to NGF-induced morphological changes and cell-cycle arrest.
Asunto(s)
Ciclo Celular , Óxido Nítrico Sintasa/metabolismo , Receptor trkA/metabolismo , Células 3T3 , Animales , Western Blotting , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Citometría de Flujo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Donantes de Óxido Nítrico/farmacologíaRESUMEN
Mutations of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a protein and lipid phosphatase, have been associated with gliomas, macrocephaly, and mental deficiencies. We have assessed PTEN's role in the nervous system and find that PTEN is expressed in mouse brain late in development, starting at approximately postnatal day 0. In adult brain, PTEN is preferentially expressed in neurons and is especially evident in Purkinje neurons, olfactory mitral neurons, and large pyramidal neurons. To analyze the function of PTEN in neuronal differentiation, we used two well established model systems-pheochromocytoma cells and cultured CNS stem cells. PTEN is expressed during neurotrophin-induced differentiation and is detected in both the nucleus and cytoplasm. Suppression of PTEN levels with antisense oligonucleotides does not block initiation of neuronal differentiation. Instead, PTEN antisense leads to death of the resulting, immature neurons, probably during neurite extension. In contrast, PTEN is not required for astrocytic differentiation. These observations indicate that PTEN acts at multiple sites in the cell, regulating the transition of differentiating neuroblasts to postmitotic neurons.
Asunto(s)
Encéfalo/metabolismo , Neuronas/citología , Neuronas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Células Madre/citología , Proteínas Supresoras de Tumor , Animales , Astrocitos/citología , Astrocitos/metabolismo , Diferenciación Celular , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Embrión de Mamíferos , Genes Supresores de Tumor , Glioma/genética , Glioma/metabolismo , Hipocampo/metabolismo , Humanos , Ratones , Bulbo Olfatorio/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Células PC12 , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/genética , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Células Madre/fisiologíaRESUMEN
During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS) (Peunova, N., and Enikolopov, G. (1995) Nature 375, 68-73). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To determine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53, and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells.
Asunto(s)
Ciclinas/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Óxido Nítrico/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Neuronas/citología , Células PC12 , Regiones Promotoras Genéticas , Ratas , Transducción de SeñalRESUMEN
We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.