RESUMO
Wild type PC12 pheochromocytoma cells that had been infected with a Wnt-1-carrying virus and thus express Wnt-1 (PC12/Wnt-1) are known to acquire the same flat cell phenotype as that of spontaneously occurring PC12 flat cell variants except that the latter do not presently express Wnt-1. Flat cell variants of PC12 cells exhibit markedly altered morphology and gene expression. In order to assess the possibility that the spontaneously occurring flat cell variants could have been induced in wild type PC12 cells by previous transient expression of the cell's endogenous Wnt-1, we have isolated PC12/Wnt-1 cells expressing little or no Wnt-1. In spite of absent Wnt-1 expression, they retained their flat cell morphology, glutamate/aspartate transporter activity, increased neu mRNA levels and lack of both norepinephrine transporter activity and nerve growth factor-induced differentiation. Thus, Wnt-1 expression is not required to maintain the flat cell phenotype. However, we identified one gene, ret, whose mRNA level in PC12 was not only increased by Wnt-1 expression, but whose increased mRNA level was also dependent on continual Wnt-1 expression. This finding suggests that the induction of ret by Wnt-1 can be used to elucidate the Wnt-induced signalling pathway in mammalian cells.
Assuntos
Proteínas de Drosophila , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/biossíntese , Proteínas de Peixe-Zebra , Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias das Glândulas Suprarrenais , Sistema X-AG de Transporte de Aminoácidos , Animais , Ácido Aspártico , Sequência de Bases , Transporte Biológico , Diferenciação Celular , Cricetinae , Primers do DNA , Dopamina/metabolismo , Variação Genética , Humanos , Dados de Sequência Molecular , Células PC12 , Feocromocitoma , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptor ErbB-2/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Wnt , Proteína Wnt1RESUMO
C6 glioma cells take up aspartate and glutamate by a Na(+)-dependent transporter. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved regions of previously cloned glutamate transporters, we isolated from these cells a partial cDNA clone with a sequence of the neuronal type EAAC1 glutamate transporter. The cells express a 4.4 kb message that hybridizes to this cDNA, and they do not express either of the previously described glial type glutamate transporters, GLT-1 or GLAST. The cells were sensitive to the toxic aspartate analog alanosine, which enters the cells by a glutamate transporter. Several human brain tumors examined, including some astrocytic tumors, expressed the EAAT3 glutamate transporter, which is the human homolog of the rodent EAAC1 transporter. Some of the tumors also expressed the other types of glutamate transporter.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Encefálicas/metabolismo , Expressão Gênica/genética , Glioma/metabolismo , Células Tumorais Cultivadas/metabolismo , Sequência de Aminoácidos , Sistema X-AG de Transporte de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Ratos , Fatores de TempoRESUMO
BACKGROUND: Transporters, ion pumps, and ion channels are membrane proteins that regulate selective permeability and maintain ionic gradients across cell membranes. Mutations in CACNA1A encoding a neuronal calcium channel and ATP1A2 encoding an ion pump cause episodic ataxia, hemiplegic migraine, and seizures. Mutant gene products of both CACNA1A and ATP1A2 may affect neurotransmission of glutamate, the most abundant excitatory amino acid neurotransmitter. METHODS: We examined our patient population with episodic ataxia and hemiplegic migraine but with no mutation in either CACNA1A or ATP1A2. We looked for mutations in SLC1A3, which encodes the glutamate transporter excitatory amino acid transporter (EAAT) 1 that is important in removing glutamate from the synaptic cleft. RESULTS: A patient with episodic ataxia, seizures, migraine, and alternating hemiplegia has a heterozygous mutation in SLC1A3 that is not present in his asymptomatic parents and controls. Expression studies of the mutant EAAT1 showed decreased expression of the protein with a markedly reduced capacity for glutamate uptake. When coexpressed, the mutant EAAT1 decreased the activity of wild-type EAAT1 but not of two other transporters EAAT2 or EAAT3, suggesting that mutant EAAT1 specifically multimerizes with wild-type EAAT1 to exert its dominant negative effect. CONCLUSION: Our data show that a heterozygous mutation in EAAT1 can lead to decreased glutamate uptake, which can contribute to neuronal hyperexcitability to cause seizures, hemiplegia, and episodic ataxia.
Assuntos
Ataxia/genética , Transportador 1 de Aminoácido Excitatório/genética , Predisposição Genética para Doença/genética , Ácido Glutâmico/metabolismo , Hemiplegia/genética , Convulsões/genética , Animais , Ataxia/metabolismo , Ataxia/fisiopatologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Química Encefálica/genética , Edema Encefálico/genética , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Células COS , Criança , Chlorocebus aethiops , Análise Mutacional de DNA , Feminino , Testes Genéticos , Hemiplegia/metabolismo , Hemiplegia/fisiopatologia , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Linhagem , Convulsões/metabolismo , Convulsões/fisiopatologiaRESUMO
The regulation of glial and neuronal Na+-dependent glutamate/aspartate transporters is of interest because abnormal glutamate transport may be responsible for certain neurological diseases. Because expression of the Wnt-1 protooncogene results in induction of the glial-type glutamate transporter GLAST in PC12 neuron-like cells, we have evaluated the effect of Wnt-1-induced signaling on glutamate transporter expression in rat C6 glioma cells. C6 cells are known normally to express EAAC1, a neuronal glutamate transporter, but not the GLAST or the GLT-1 glutamate transporter. C6 cells that ectopically expressed Wnt-1 contained a GLT-1 RNA species similar in size (>10 kb) to the GLT-1 transcript present in rat brain, and they also contained a previously unreported 3.3-kb GLT-1 RNA species. Both GLT-1 RNAs contain large parts of the coding region. However, the 3.3-kb GLT-1 species contains at least one small deletion within the coding region. The Wnt-1-expressing C6 cells contained little, if any, GLT-1 protein as determined by immunological techniques. We suggest that one or both of the GLT-1 RNA species induced by Wnt-1 either fail to be translated or yield abnormal translation products that are quickly degraded. Wnt-1-expressing C6 cells may thus represent a novel in vitro system for studying GLT-1 transporter expression at the transcriptional and/or posttranscriptional levels.