Functional Analysis of the Proximal 5′-Flanking Region of the N-Methyl-D-aspartate Receptor Subunit Gene, NMDAR1

Bai, Guang; Kusiak, John W.

doi:10.1074/jbc.270.13.7737

Cited by 77 publications

(111 citation statements)

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“…However non-hormone response element containing genes, such as the AP1 response element (Paech et al 1997), can also respond to the estrogen receptor ligand (Katzenellenbogen et al 1996). An SP1 motif, which is similar to the AP1 motif, has been found in the NMDA receptor subunit 1 (Bai and Kusiak 1993;Bai and Kusiak 1995) and 2B (Suchanek et al 1995) promoters. Nevertheless, further investigations with gel shift and DNA footprinting assays are required to clearly answer this potential mechanism of action.…”

Section: Nmda Receptors Mrna Levelsmentioning

confidence: 85%

Estrogen-like Activity of Tamoxifen and Raloxifene on NMDA Receptor Binding and Expression of its Subunits in Rat Brain

Cyr

Thibault

Morissette

et al. 2001

Neuropsychopharmacology

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Hormonal specificity of modulation of N-methyl-D-aspartate (NMDA) receptors was investigated by comparing the effects of estradiol with tamoxifen or raloxifene, which display different responses in breast, bone, and uterus. Two weeks ovariectomy in rats decreasedIt has been shown that the female sex steroid estradiol exerts profound effects on neuronal differentiation during development (Toran-Allerand et al. 1983) and recently, accumulating evidence support also a modulatory role of estrogens in the normal maintenance of brain function during aging (Simpkins et al. 1994). Beneficial effects of estradiol in mood disorders (i.e., depression, pre-menstrual syndrome, post-natal depression), as well as mental diseases, such as schizophrenia and Gille de la Tourette's syndrome, have been reported (Di Paolo 1994;Fink et al. 1998). Moreover, a beneficial role of estradiol in neurodegenerative disFrom the Oncology and Molecular Endocrinology Research Center, Laval University Medical Center, (CHUL), and Faculty of Pharmacy, Laval University, Sainte-Foy, Quebec, Canada, G1V 4G2.Address correspondence to: Dr. Thérèse Di Paolo, Oncology and Molecular Endocrinology Research Center, Laval University Medical Center, CHUQ, 2705, Laurier Boulevard, Sainte-Foy, Quebec, Canada G1V 4G2.Received July 5, 2000; revised December 20, 2000; accepted January 3, 2001. N EUROPSYCHOPHARMACOLOGY 2001 -VOL . 25 , NO . 2 SERMs Modulate NMDA Receptors 243 eases, such as Alzheimer has been recently suggested (Tang et al. 1996;Henderson 1997;Inestrosa et al. 1998). However, the mechanisms by which these effects of estrogens are yet to be fully characterized. N-methyl-D-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptor, have been implicated, as mediators, in some effects of estradiol on morphological plasticity and related physiological and cognitive processes in the brain (Moriyoshi et al. 1991). However, only few studies have directly investigated the role of estradiol in regulating NMDA receptors in rat and, in addition, these studies have focused on the effects of estradiol on hippocampal NMDA receptors (Woolley and McEwen 1992;Gazzaley et al. 1996;Woolley et al. 1997). We have recently investigated the effects of estradiol on the NMDA receptors agonist sites in brain regions implicated in mental disorders, such as frontal cortex, striatum, and nucleus accumbens (Cyr et al. 2000a). Our experiments showed that ovariectomy, as well as estradiol have regional specific effects since, for example, ovariectomy decreases NMDA receptor specific binding in hippocampal CA1 and dentate gyrus regions, whereas it has no effect in frontal cortex. In contrast, estradiol increases NMDA specific binding in the hippocampal CA1 and the dentate gyrus, but it decreases this binding in the frontal cortex (Cyr et al. 2000a).The present study sought to determine whether changes observed previously at the protein level after hormonal withdrawal due to ovariectomy or after estradiol treatment reflect changes at the level of gene transcription...

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Section: Nmda Receptors Mrna Levelsmentioning

confidence: 85%

Estrogen-like Activity of Tamoxifen and Raloxifene on NMDA Receptor Binding and Expression of its Subunits in Rat Brain

Cyr

Thibault

Morissette

et al. 2001

Neuropsychopharmacology

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“…Co-transfection of luciferase and ␤-galactosidase constructs and assay of reporters were done as described previously (37,38). In some experiments, cells were used for RNA extraction 2 days after co-transfection.…”

Section: Methodsmentioning

confidence: 99%

“…DNA-Protein Interaction Analysis-Crude nuclear extractions were prepared from cells as previously described (37). DNA sequences used as probes in electrophoretic mobility shift assays (EMSA) are as follows: GC-box-a, 5Ј-GCCTCCGGGGTGGGCGACTGC; GC-box-b, 5Ј-CCGGG-GAGCGGGCGGAGAGCGTGGT; GC-Box-c, 5Ј-GCATCCTGGGCGGG-TGTGTGC.…”

Section: Methodsmentioning

confidence: 99%

“…Cell Culture, Transfection, and Reporter Gene Assay-PC12, HeLa, HEK293, and P19 cells were maintained and transfected as described previously (37,38) except that LipofectAMIN2000 (Invitrogen) was used according to the manufacturer's instruction. Co-transfection of luciferase and ␤-galactosidase constructs and assay of reporters were done as described previously (37,38).…”

Section: Methodsmentioning

confidence: 99%

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Functional Analysis of the Rat N-Methyl-D-aspartate Receptor 2A Promoter

Liu

Zhuang

Hoffman

et al. 2003

Journal of Biological Chemistry

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N-Methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) is an important modulatory component of the NMDA subtype of glutamate receptors. To investigate the transcription mechanism of the NR2A gene, we cloned the 5 -flanking sequence from a rat genomic library. RNA mapping with rat brain RNA revealed two sets of major and several minor transcription start points in a single exon of 1140 bp. Reporter gene and mutation studies indicated that core promoter activity resided in exon 1, whereas the 5 -flanking sequence up to 1.5 kb showed no significant impact on promoter activity. Fragments containing minor transcription start points were able to drive a reporter gene in transfected cells and produce nascent RNAs in an in vitro transcription system. All fragments tested showed more promoter activity in dissociated neurons of the rat embryonic cerebrocortex and cell lines expressing NR2A mRNA than that in glial cultures and non-neuronal cells. Within exon 1 there are three GC-box elements that displayed distinct binding affinity to both Sp1-and Sp4-like factors. Overexpression of Sp1 or Sp4, but not Sp3, significantly increased the activity of the promoter containing these elements. Inclusion of exon 2 and 3 sequences, which contain five short open-reading frames, attenuated promoter-driven reporter activity more than 3-fold but attenuated the level of reporter mRNA less than 1.4-fold. Our results suggest that the core promoter of the rat NR2A gene requires exon 1, that Sp factors positively regulate this core promoter, and that a post-transcriptional mechanism may negatively regulate expression of the gene.

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“…PC12 pheochromocytoma cells were cultured in DMEM supplemented with 5% FCS and 5% HS, as described. 21 The luciferase reporter plasmids, constructed with both alleles, were transiently transfected into the cells using the TransFast lipofection reagent (Promega). The Renilla luciferase expression plasmid, pRL-TK, was co-transfected as an internal standard.…”

Section: Dual-luciferase Gene Reporter Assaymentioning

confidence: 99%

Identification of a novel variant of the human NR2B gene promoter region and its possible association with schizophrenia

2002

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N-methyl-D-aspartate (NMDA) receptor dysfunction is involved in the pathogenesis of schizophrenia. We determined the nucleotide sequence of the 5′-upstream region of the human NMDA receptor 2B (NR2B) subunit gene and identified a novel T-200G variant located in one of the Sp1 binding sites. To investigate the effect of this variant on the transcriptional activity of the hNR2B gene, we performed gene reporter assays using PC12 pheochromocytoma cells transiently transfected with luciferase reporter plasmids. In the absence of nerve growth factor (NGF), luciferase activities did not significantly differ between the two alleles and the control plasmid. However, luciferase reporter activity of the T allele was significantly up-regulated compared to that of the G allele in the presence of NGF (P = 0.0013), indicating that this polymorphic site is a critical region for NR2B gene regulation through NGF-induced Sp1-binding. A case control study showed that the frequency of the G allele (P = 0.0164) was significantly higher in 100 schizophrenics than in 100 controls. These findings suggest that the T-200G variant causes dysfunction of NMDA receptors consisting of the NR2B subunit and may be involved in the development of schizophrenia. Replication studies of independent samples and family-based association studies are necessary to further evaluate the significance of our findings.

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Functional Analysis of the Proximal 5′-Flanking Region of the N-Methyl-D-aspartate Receptor Subunit Gene, NMDAR1

Cited by 77 publications

References 50 publications

Estrogen-like Activity of Tamoxifen and Raloxifene on NMDA Receptor Binding and Expression of its Subunits in Rat Brain

Estrogen-like Activity of Tamoxifen and Raloxifene on NMDA Receptor Binding and Expression of its Subunits in Rat Brain

Functional Analysis of the Rat N-Methyl-D-aspartate Receptor 2A Promoter

Identification of a novel variant of the human NR2B gene promoter region and its possible association with schizophrenia

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