RESUMO
Nuclear factor of activated T cells 5 (NFAT5) has been implicated in regulating several genes that are thought to be neuroprotective in ischemic injury. Because of the embryonic lethality of NFAT5 knockout (NFAT5(-/-)) mice, the heterozygous (NFAT5(+/-)) mice were used to study the in vivo role of NFAT5 in hypoxia/ischemia (H/I) condition. The NFAT5(+/-) mice exhibited more severe neurological deficits, larger infarct area and edema formation associated with increased aquaporin 4 expressions in the brain. Under in vitro H/I condition, increased apoptotic cell death was found in NFAT5(-/-) neurons. Moreover, SMIT, a downstream to NFAT5, was upregulated in NFAT5(+/+) neurons, while the SMIT level could not be upregulated in NFAT5(-/-) neurons under H/I condition. The elevation of reactive oxygen species generation in NFAT5(-/-) neurons under H/I condition further confirmed that NFAT5(-/-) neurons were more susceptible to oxidative stress. The present study demonstrated that activation of NFAT5 and its downstream SMIT induction is important in protecting neurons from ischemia-induced oxidative stress.
Assuntos
Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Morte Celular/genética , Morte Celular/fisiologia , Neurônios/patologia , Fatores de Transcrição/deficiência , Animais , Barreira Hematoencefálica/fisiologia , Western Blotting , Células Cultivadas , Soluções Hipertônicas , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Infarto da Artéria Cerebral Média/patologia , Ataque Isquêmico Transitório/patologia , L-Lactato Desidrogenase/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pressão Osmótica , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Simportadores/biossíntese , Simportadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: Osmotic Response Element-Binding Protein (OREBP), also known as TonEBP or NFAT5, is a unique transcription factor. It is hitherto the only known mammalian transcription factor that regulates hypertonic stress-induced gene transcription. In addition, unlike other monomeric members of the NFAT family, OREBP exists as a homodimer and it is the only transcription factor known to bind naked DNA targets by complete encirclement in vitro. Nevertheless, how OREBP interacts with target DNA, also known as ORE/TonE, and how it elicits gene transcription in vivo, remains unknown. METHODOLOGY: Using hypertonic induction of the aldose reductase (AR) gene activation as a model, we showed that OREs contained dynamic nucleosomes. Hypertonic stress induced a rapid and reversible loss of nucleosome(s) around the OREs. The loss of nucleosome(s) was found to be initiated by an OREBP-independent mechanism, but was significantly potentiated in the presence of OREBP. Furthermore, hypertonic induction of AR gene was associated with an OREBP-dependent hyperacetylation of histones that spanned the 5' upstream sequences and at least some exons of the gene. Nevertheless, nucleosome loss was not regulated by the acetylation status of histone. SIGNIFICANCE: Our findings offer novel insights into the mechanism of OREBP-dependent transcriptional regulation and provide a basis for understanding how histone eviction and transcription factor recruitment are coupled.
