RESUMO
Metabotropic glutamate receptor 6, mGluR6, interacts with scaffold proteins and Gßγ subunits via its intracellular C-terminal domain (CTD). The mGluR6 pathway is critically involved in the retinal processing of visual signals. We herein investigated whether the CTD (residues 840-871) was necessary for mGluR6 cell surface localization and G-protein coupling using mGluR6-CTD mutants with immunocytochemistry, surface biotinylation assays, and electrophysiological approaches. We used 293T cells and primary hippocampal neurons as model systems. We examined C-terminally truncated mGluR6 and showed that the removal of up to residue 858 did not affect surface localization or glutamate-induced G-protein-mediated responses, whereas a 15-amino acid deletion (Δ857-871) impaired these functions. However, a 21-amino acid deletion (Δ851-871) restored surface localization and glutamate-dependent responses, which were again attenuated when the entire CTD was removed. The sequence alignment of group III mGluRs showed conserved amino acids resembling an ER retention motif in the CTD. These results suggest that the intracellular CTD is required for the cell surface transportation and receptor function of mGluR6, whereas it may contain regulatory elements for intracellular trafficking and signaling.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato/metabolismo , Aminoácidos/metabolismo , Animais , Biotinilação , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Deleção de Genes , Ácido Glutâmico/farmacologia , Humanos , Mutação/genética , Ratos , Receptores de Glutamato/genética , Transdução de Sinais/genéticaRESUMO
A comprehensive summary of more than 450 estrogenic chemicals including estrogenic endocrine disruptors is provided here to understand the complex and profound impact of estrogen action. First, estrogenic chemicals are categorized by structure as well as their applications, usage and effects. Second, estrogenic signaling is examined by the molecular mechanism based on the receptors, signaling pathways, crosstalk/bypassing and autocrine/paracrine/homeostatic networks involved in the signaling. Third, evaluation of estrogen action is discussed by focusing on the technologies and protocols of the assays for assessing estrogenicity. Understanding the molecular mechanisms of estrogen action is important to assess the action of endocrine disruptors and will be used for risk management based on pathway-based toxicity testing.
Assuntos
Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/toxicidade , Estrogênios/metabolismo , Estrogênios/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , HumanosRESUMO
There is increasing evidence that ATP acts on purinergic receptors and mediates synaptic transmission in the retina. In a previous study, we raised the possibility that P2X-purinoceptors, presumably P2X(2)-purinoceptors in OFF-cholinergic amacrine cells, play a key role in the formation of OFF pathway-specific modulation. In this study, we examined whether the P2Y(1)-purinoceptors can function in cholinergic amacrine cells in the mouse retina since cholinergic amacrine cells in the rat retina express P2Y(1)-purinoceptors. P2Y(1)-purinoceptors were shown to be expressed in dendrites of both ON- and OFF-cholinergic amacrine cells in adults. At postnatal day 7, there was immunoreactivity for P2Y(1)-purinoceptors in the soma of cholinergic amacrine cells. At postnatal day 14, weak immunoreactivity for P2Y(1)-purinoceptors was detected in the dendrites but not in the soma of cholinergic amacrine cells. At postnatal day 21, strong immunoreactivity for P2Y(1)-purinoceptors was detected in dendrites of cholinergic amacrine cells. The expression pattern of P2Y(1)-purinoceptors was not affected by visual experience. We concluded that P2Y(1)-purinoceptors are not involved in the OFF-pathway-specific signal transmission in cholinergic amacrine cells of the mouse retina.
Assuntos
Receptores Purinérgicos P2Y1/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Neurônios Colinérgicos/metabolismo , Camundongos , Camundongos Transgênicos , Transporte Proteico , Ratos , Transdução de Sinais , Transmissão SinápticaRESUMO
Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17ß-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA.
Assuntos
Actinas/metabolismo , Movimento Celular , Cofilina 1/metabolismo , Estradiol/farmacologia , Área Pré-Óptica/efeitos dos fármacos , Caracteres Sexuais , Animais , Animais Recém-Nascidos , Western Blotting , Cofilina 1/genética , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Imuno-Histoquímica , Quinases Lim/genética , Quinases Lim/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Gravidez , Área Pré-Óptica/embriologia , Área Pré-Óptica/metabolismo , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We provide here evidence for a conserved regulatory element for transcription of the ß-family globin genes based on a comparative study of 32 genes from 16 mammals. The element is characterized by the appearance of AA or TT dinucleotides in the A + T-rich region located 200-400 bp upstream of the cap sites. G-tracts 3-5 nucleotides long exist between the A + T-rich region and the conserved transcription factor binding sites (GATA-1 site and the CACCC, CCAAT, and ATA boxes) apparently dividing the regions. The average periodicity of AA or TT dinucleotides in the region from a total of 18 ß-family globin genes from four species was approximately 10 bp, suggesting that the DNA in these regions shows right-handed superhelicity. The proposed biological function of this element is to adjust the spatial positions for the first interaction of the transcription factor(s) which can recognize specific DNA sequences in the presence of packed chromatin.
Assuntos
Sequência Conservada , Regiões Promotoras Genéticas , Globinas beta/genética , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Coelhos , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Estrogen plays critical roles in the neuroendocrine system of adult female rats through separate actions, respectively, in the preoptic area (POA) and the ventromedial nucleus of the hypothalamus (VMH). Seven-week-old rats were treated with/without estrogen after they were ovariectomized, and four estrogen-responsive, neuronal system-related genes, encoding alpha4 neuronal nicotinic acetylcholine receptor (Chrna4), GABA(A) receptor delta (Gabrd), serotonin receptor 6 (Htr6), and GABA transporter 2 (Slc6a13), were investigated by real-time RT-PCR and Western blot analyses to examine their differential regulation by estrogen between the anterior part containing POA and the posterior part containing VMH. We further examined Bax, Bcl2, and Prkce, the former two genes to be involved in the gene expression network of Chrna4 and the latter gene, that of Gabrd. The regulation of Bax and Bcl2 by estrogen differed between the anterior and posterior parts. The results demonstrated differential regulation of these neuronal system-related genes by estrogen between the anterior and posterior parts of the hypothalamus and suggested the roles of gene expression networks for the respective genes in the neuroendocrine system of adult female rats.
Assuntos
Estrogênios/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipotálamo/metabolismo , Animais , Western Blotting , Feminino , Proteínas da Membrana Plasmática de Transporte de GABA/biossíntese , Proteínas da Membrana Plasmática de Transporte de GABA/genética , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genética , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Receptores de Serotonina/biossíntese , Receptores de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of an estrogen receptor (ER) alpha promoter were generated to tag ERalpha-positive neurons in the brain. Two transgenes, one containing sequences for promoter A and DsRed and the other containing sequences for promoter 0/B and EGFP, were injected simultaneously into Wistar rat zygotes. Twenty-two founders with both transgenes were identified. Ten lines of these founders expressed the EGFP tag in the brains of their first filial generation, whereas none similarly expressed the DsRed tag. In two lines selected for the brightness of the EGFP fluorescence in their brains, tagged cells showed essentially the same patterns. Tagged cells were in the preoptic area (POA), bed nucleus of the stria terminalis (BNST), hypothalamic arcuate nucleus and medial amygdala. ERalpha-immunoreactive neurons were identified in all of these structures by immunohistochemistry. In ovariectomized females, approximately 75% of the EGFP-fluorescent cells in the POA-BNST were immunoreactive for ERalpha. In the POA-BNST, ovariectomy increased the number of EGFP-immunopositive cells and estrogen supplementation reversed this effect, indicating that the promoter 0/B is involved in estrogen-induced downregulation of ERalpha. EGFP was also present in cells in the cerebral cortex and hippocampus, which have not previously been associated with endocrine regulation. Conversely, only a few cells were tagged in the hypothalamic ventromedial nucleus, which contained many ERalpha-immunoreactive neurons. This discrepancy could have arisen as a result of differential promoter usage.
Assuntos
Regulação para Baixo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Regiões Promotoras Genéticas , Prosencéfalo , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Masculino , Ovariectomia , Prosencéfalo/anatomia & histologia , Prosencéfalo/metabolismo , Ratos , Ratos WistarRESUMO
The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.
Assuntos
DNA/genética , DNA/metabolismo , Biblioteca Gênica , Nucleossomos/genética , Nucleossomos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Southern Blotting , Galinhas , Clonagem Molecular , DNA/química , Eritrócitos , Globinas/genética , Humanos , Células K562 , Região de Controle de Locus Gênico/genética , Nuclease do Micrococo/metabolismo , Conformação Molecular , Dados de Sequência Molecular , Nucleossomos/química , Ligação ProteicaRESUMO
Adult female rats were ovariectomized and treated with or without estrogen for two weeks. mRNA was obtained from the hypothalamus, uterus, liver, kidney and skeletal muscle and analyzed by Northern blotting and/or RT-PCR. We examined two types of estrogen-responsive genes from rats, neuronal system-related genes (Amphiregulin, AR; Neuropeptide Y-Y1 receptor, NPY-Y1R; Bassoon, BSN; N-Cadherin, N-CADH) and estrogen-susceptible cancer-related genes (C-terminal binding protein interacting protein, CtIP), based on the results of a cDNA microarray analysis which was carried out to profile estrogen-responsive genes in the human breast cancer cell line MCF-7. The N-CADH gene showed identical response to that in MCF-7 cells. In the hypothalamus, all except the AR gene were down-regulated in their expression. In other tissues, the expression showed marked differences: expression of the BSN gene was not detected by either method, and the NPY-Y1R gene showed down-regulation in most tissues except for skeletal muscle. We then analyzed the time course of the estrogen-responsiveness of these genes in several tissues, finding changes in expression patterns especially in skeletal muscle but not in the hypothalamus. Our results show that the estrogen-responsive genes, which were demonstrated simply as either up- or down-regulated in their expression by estrogen in a human cell line using cDNA microarrays, exhibit tissue and temporal-specific expression patterns in adult female rats.
Assuntos
Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fenômenos Fisiológicos do Sistema Nervoso , Oncogenes , Animais , Regulação para Baixo , Feminino , Hipotálamo/metabolismo , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.
Assuntos
Poluentes Ambientais/toxicidade , Estrogênios/toxicidade , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Estrogênio/efeitos dos fármacos , Bioensaio/métodos , Neoplasias da Mama/patologia , Poluentes Ambientais/farmacologia , Estrogênios/farmacologia , Feminino , Humanos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Human and mouse globin genes were separated approximately 200 million years ago but still share homology and synergism in many aspects including DNA structure. We first mapped DNA bend sites in the mouse epsilon-globin gene and found that these sites were distributed in a regular manner except in the coding region and their overall average distance was 650.7 bp. The first bend site upstream of the cap site (MepsilonB-1, -334 to -147 bp) was found to contain A + T-rich sequences and features contributing to DNA curvature by computer analysis. Transcription assays using deletion constructs indicated strong promoter activity up to bp -215 in erythriod K562 cells. Therefore, the MepsilonB-1 site was located immediately upstream of the promoter region. A reporter gene assay using a series of constructs containing the promoter region revealed that the MepsilonB-1 site showed repressor activity, and on replacement of the DNA curvature with one from another source the activity was retained. A similar feature was found in the other conserved B-1 sites in the human, bovine, and rabbit beta-like globin genes, with the exception of an unconserved B-1 site in the chicken beta-globin gene. A common feature of these conserved B-1 sites was not the nucleotide sequences but the DNA curvature. Furthermore, a unique nucleosome phase at the MepsilonB-1 site was likely to be directed by DNA curvature. Based on these results, DNA curvature is one of the major features of these promoter regions which might influence transcription through nucleosome positioning.
Assuntos
DNA/química , Evolução Molecular , Globinas/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sequência Conservada , DNA/genética , Genes Reporter , Globinas/metabolismo , Camundongos , Dados de Sequência Molecular , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.
Assuntos
DNA/genética , Nucleossomos/genética , Sequência de Bases , Cromossomos Humanos , Biblioteca Gênica , Humanos , Células K562 , Sequências Repetitivas de Ácido NucleicoRESUMO
We previously reported five DNA bend sites (ERB-4 to -1, and ERB+1) in the promoter region of the human estrogen receptor alpha (ERalpha) gene [FEBS Lett. 444 (1999) 117]. One of these sites, ERB-2, was accompanied by two half motifs of the estrogen response element (ERE) and several short poly(dA)(.)poly(dT) tracts including an A(4) tract located next to a half ERE motif. This A(4) tract and the 20 bp immediate flanking sequence containing a half ERE motif (T3B) exhibited DNA curvature. Transcription assays using luciferase as a reporter gene indicated that T3B sequence conferred positive estrogen responsiveness. Mutations introduced in this sequence indicated that both bendability and estrogen responsiveness were synergistically associated with the A(4) tract located next to the half ERE motif. This motif and a mutant sequence, GGTTA, had affinity for ERalpha protein, which seems to account for ERalpha protein binding to the region without an ERE motif. These findings suggest that some DNA curvature acts as a transcriptional modulator by modifying the state of ligand effects.