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1.
Exp Anim ; 66(2): 75-89, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27980293

RESUMO

The aryl hydrocarbon receptor (AHR) is a pivotal chemical sensor that transduces extrinsic and intrinsic signals into cellular responses. AHR was originally thought to be involved in not only drug metabolism but also carcinogenic and toxicological responses against environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons. However, recent studies demonstrate that the AHR plays multiple intrinsic roles in host defense and homeostasis as well, including immunity, stem cell maintenance, and cell differentiation, upon binding with an increasing number of newly defined dietary, cellular, and microbe-derived ligands. In addition, AHR is a convergence point for several signaling cascades, which may be involved in the diverse diseases caused by binding of the persistent ligand TCDD with extremely high affinity to AHR. A comprehensive understanding of physiological and pathological processes initiated by endogenous AHR agonists and antagonists may allow for the therapeutic regulation of AHR activity. Thus, the AHR can be a valuable diagnostic marker and therapeutic target for human diseases.


Assuntos
Homeostase , Receptores de Hidrocarboneto Arílico/imunologia , Receptores de Hidrocarboneto Arílico/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Dioxinas/metabolismo , Humanos , Ligantes , Terapia de Alvo Molecular , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Transdução de Sinais , Células-Tronco/citologia
2.
Sci Rep ; 6: 23820, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27068235

RESUMO

Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígeno CD11c/análise , Colite/patologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células Apresentadoras de Antígenos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Técnicas de Cocultura , Deleção de Genes , Regulação da Expressão Gênica , Imunidade Inata , Camundongos Endogâmicos C57BL , Organoides , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Via de Sinalização Wnt
3.
Carcinogenesis ; 34(7): 1620-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23455376

RESUMO

The aryl hydrocarbon receptor (AhR) plays a suppressive role in cecal carcinogenesis by CUL4B/AhR-mediated ubiquitylation and degradation of ß-catenin, which is activated by xenobiotics and natural ligands. AhR-deficient (AhR(-)(/-)) mice develop cecal tumors with severe inflammation. To elucidate whether the tumors develop autonomously in AhR(-/-) mice due to impaired ß-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR(-/-) mice and compound mutant mice lacking genes for AhR and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which plays an essential role in caspase-1 activation in inflammasomes. Both GF AhR(-/-) and AhR(-/-)•ASC(-/-) mice showed considerably reduced tumor development compared with that in AhR(-/-) mice albeit in a 'cancer-prone' state with aberrant ß-catenin accumulation. Blocking of the interleukin (IL)-1ß signaling pathway by treatment with a caspase-1 inhibitor, YVAD, reduced cecal tumorigenesis in AhR(-/-) mice. Signal transducers and activators of transcription 3 (STAT3) activation was detected in the cecal epithelium of the AhR(-/-) mice due to enhanced IL-6 production. An inhibitor of the STAT3 signaling pathway, AG490 suppressed the tumor formation. ASC-mediated inflammation was also found to play a critical role in tumor development in Apc(Min/+) mice, a mouse model of familial adenomatous polyposis. Collectively, these results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in the intestinal tumorigenesis of mice and suggest a possible chemical therapeutic intervention, including AhR ligands and inhibitors of the inflammation pathway.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Neoplasias do Ceco/patologia , Inflamação/patologia , Receptores de Hidrocarboneto Arílico/metabolismo , Polipose Adenomatosa do Colo/imunologia , Polipose Adenomatosa do Colo/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Neoplasias do Ceco/imunologia , Linhagem Celular , Ativação Enzimática , Feminino , Vida Livre de Germes , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Cross-Talk , Receptores de Hidrocarboneto Arílico/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , beta Catenina/imunologia , beta Catenina/metabolismo
4.
J Steroid Biochem Mol Biol ; 127(1-2): 102-7, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21397018

RESUMO

The arylhydrocarbon receptor (AhR) is a ligand-dependent transcription factor mediating the adverse effects of dioxins. Although cross-talk of dioxins with estrogen and androgen signaling pathways are well described, the underlying molecular mechanisms have been largely elusive. Recent studies showed that modulation of estrogen/androgen signaling by dioxins is exerted in part through direct association of AhR with estrogen (ER) or androgen (AR) receptors. Agonist-bound AhR and ERα work as a functional unit to regulate expression of target genes. In addition to such genomic actions, AhR mediates non-genomic actions of AhR-ligands through the assembly of a CUL4B-based ubiquitin ligase complex and promotes the degradation of ERα and AR. These findings reveal the roles of the ubiquitin system in sensing and biological response to environmental chemicals, in which AhR acts as a ubiquitin ligase component to enhance the destruction of specific substrates.


Assuntos
Dioxinas/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo , Androgênios/farmacologia , Animais , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/agonistas , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismo
5.
J Dermatol Sci ; 58(3): 211-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20478695

RESUMO

BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. When environmental pollutants, including chemical carcinogens, bind to AhR, the receptor translocates to nucleus and transcriptionally activates target genes including drug metabolizing enzymes such as P450s. Recent studies have shown that AhR mediates various responses, including cellular growth, differentiation, immune system and development. OBJECTIVE: In this study, we investigated the physiological function of AhR in skin. METHODS: Distribution of AhR in murine skin was examined by immunohistochemistry. Expression of a target gene which is transcriptionally activated by AhR is analysed by RT-PCR. RESULTS: We found that AhR co-localizes with the transcriptional repressor B lymphocyte maturation protein 1 (Blimp1) in sebaceous gland. In this report, we show that expression of Blimp1 is induced by treatment with AhR ligands, such as methylcolanthrene (MC) in sebocyte and keratinocyte cell lines. Exposure to ultraviolet B, which has been reported to generate AhR ligand intracellularly, also increased Blimp1 mRNA. This ligand-dependent induction of Blimp1 requires the expression of both AhR and ARNT, since transfection of siRNA specific to either AhR or ARNT significantly reduced Blimp1 mRNA in response to MC. Analysis using kinase inhibitors revealed that ligand-dependent induction of Blimp1, but not that of CYP1A1, is inhibited by staurosporine. TPA, a potent activator of protein kinase C, increased Blimp1 mRNA but not CYP1A1. CONCLUSION: These data indicate that Blimp1 is a novel AhR-target gene in epidermal keratinocyte and sebocyte.


Assuntos
Queratinócitos/fisiologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Glândulas Sebáceas/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Linfócitos B/fisiologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Células Epidérmicas , Epiderme/fisiologia , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Fator 1 de Ligação ao Domínio I Regulador Positivo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Sebáceas/citologia , Estaurosporina/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-20075607

RESUMO

The aryl hydrocarbon receptor (AhR) was originally identified as a ligand-activated transcription factor that is involved in the induction of xenobiotic-metabolizing Cytochrome P4501A1 (CYP1A1). For several decades, AhR has been studied in relation to toxicology and pharmacology. With recent discoveries on novel AhR functions, AhR research has expanded into multiple aspects of physiology, such as reproduction, innate immunity and tumor suppression. In this review, we summarize and discuss recent progress in mechanistic and functional studies on AhR with particular emphasis on physiological processes.


Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Hidrocarboneto Arílico/imunologia , Reprodução , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
7.
Mol Cell Biol ; 29(24): 6391-400, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19822660

RESUMO

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is known to mediate a wide variety of pharmacological and toxicological effects caused by polycyclic aromatic hydrocarbons. Recent studies have revealed that AhR is involved in the normal development and homeostasis of many organs. Here, we demonstrate that AhR knockout (AhR KO) mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, mainly due to the dysfunction of their macrophages. In response to LPS, bone marrow-derived macrophages (BMDM) of AhR KO mice secreted an enhanced amount of interleukin-1beta (IL-1beta). Since the enhanced IL-1beta secretion was suppressed by supplementing Plasminogen activator inhibitor-2 (Pai-2) expression through transduction with Pai-2-expressing adenoviruses, reduced Pai-2 expression could be a cause of the increased IL-1beta secretion by AhR KO mouse BMDM. Analysis of gene expression revealed that AhR directly regulates the expression of Pai-2 through a mechanism involving NF-kappaB but not AhR nuclear translocator (Arnt), in an LPS-dependent manner. Together with the result that administration of the AhR ligand 3-methylcholanthrene partially protected mice with wild-type AhR from endotoxin-induced death, these results raise the possibility that an appropriate AhR ligand may be useful for treating patients with inflammatory disorders.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Choque Séptico/induzido quimicamente , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Citocinas/imunologia , Perfilação da Expressão Gênica , Humanos , Interleucina-1beta/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Choque Séptico/imunologia
8.
Proc Natl Acad Sci U S A ; 106(32): 13481-6, 2009 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-19651607

RESUMO

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of beta-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent beta-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc(Min/+) mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Intestinos/patologia , Lesões Pré-Cancerosas/patologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Neoplasias do Ceco/metabolismo , Neoplasias do Ceco/patologia , Mucosa Intestinal/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Lesões Pré-Cancerosas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Hidrocarboneto Arílico/deficiência , Transdução de Sinais , Ubiquitinação , beta Catenina/metabolismo
9.
Biochem Pharmacol ; 77(4): 588-96, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18983832

RESUMO

Because aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, its nuclear translocation in response to ligands may be directly linked to transcriptional activation of target genes. We have investigated the biological significance of AhR from the perspective of its subcellular localization and revealed that AhR possesses a functional nuclear localization signal (NLS) as well as a nuclear export signal (NES) which controls the distribution of AhR between the cytoplasm and nucleus. The intracellular localization of AhR is regulated by phosphorylation of amino acid residues in the vicinity of the NLS and NES. In cell culture systems, cell density affects not only its intracellular distribution of AhR, but also its transactivation activity of the target genes such as transcriptional repressor Slug, which is important for the induction of epithelial-mesenchymal transitions. These effects of AhR observed in cultured cells are proposed to be reflected on the in vivo response such as morphogenesis and tumor formation. This review summarizes recent work on the control mechanism of AhR localization and progress in understanding the physiological role of AhR in the skin. We propose that AhR is involved in normal skin formation during fetal development as well as in pathological states such as epidermal wound healing and skin carcinogenesis.


Assuntos
Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias Cutâneas/metabolismo , Fenômenos Fisiológicos da Pele , Pele/metabolismo , Animais , Contagem de Células , Humanos , Dibenzodioxinas Policloradas/toxicidade , Transporte Proteico , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/fisiologia , Pele/lesões , Pele/patologia , Neoplasias Cutâneas/patologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
10.
Arch Biochem Biophys ; 464(2): 207-12, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17481570

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that functions as an intracellular mediator in the xenobiotic signaling pathway. Although a number of studies have examined AhR-mediated CYP1A1 induction in detail, recent studies of AhR-null mice have revealed that AhR plays important regulatory roles in the normal homeostasis and development of animals. In this short review, we summarize the present state of knowledge about the molecular mechanisms of AhR-mediated CYP1 induction, and we also focus on recent advances in the study of the physiological functions of AhR.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Modelos Biológicos , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/fisiologia
11.
Exp Cell Res ; 312(18): 3585-94, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16952353

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. We previously showed that AhR localizes predominantly in the cytoplasm under high cell densities of a keratinocytes cell line, HaCaT, but accumulates in the nucleus at low cell densities. In the current report, we show that the Slug, which is a member of the snail/slug family of zinc finger transcriptional repressors critical for induction of epithelial-mesenchymal transitions (EMT), is activated transcriptionally in accordance with nuclear accumulation of AhR. By reporter assay of the promoter of the Slug gene, gel shift and chromatin immunoprecipitation analyses showed AhR directly binds to xenobiotic responsive element 5 at -0.7 kb of the gene. AhR-targeted gene silencing by small interfering RNA duplexes led to the abolishment of not only CYP1A1 but also Slug induction by 3-methycholanthrene. The Slug was co-localized to the AhR at the wound margins of HaCaT cells, where apparent nuclear distribution of AhR and Slug was observed. The induced Slug was associated with reduction of an epithelial marker of cytokeratin-18 and with an increase in the mesenchymal marker, fibronectin. Taken together, these findings suggest that AhR participated in Slug induction, which, in turn, regulates cellular physiology including cell adhesion and migration.


Assuntos
Regulação da Expressão Gênica , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Genes Reporter , Humanos , Camundongos , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
12.
Int J Oncol ; 29(3): 689-93, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16865286

RESUMO

A t(11;22)(q24;q12) translocation is present in 90% of Ewing's sarcoma, and results in the formation of the EWS-FLI1 fusion gene encoding an oncogenic transcription factor. To clarify the function of chimeric EWS-FLI1 proteins, an identification of a nuclear localization signal (NLS) in the EWS, FLI1 and EWS-FLI1 proteins is important because the chimeric oncoprotein may lose or gain NLS function different from native proteins resulting in different subcellular localization, and in deregulated gene expression. Furthermore, some studies reported that patients with one type of fusion gene ('type 1') had better overall survival than those with other types, suggesting that functional differences may be present among various fusion proteins. There has been only one study reporting a NLS in EWS, but none reporting those in FLI1 and EWS-FLI1. To clarify the molecular mechanisms of Ewing tumor development, we first identified the NLSs of EWS and FLI1. We allocated the NLS to amino acid residues 632-656 near the C-terminal region of EWS that is different from the previous study, and identified two NLSs of FLI1, NLS1 (63-90) in the N-terminal domain and NLS2 (319-360) in the 3'-ETS domain. In addition, the present study showed that all of the EWS-FLI1 fusion proteins completely reside in the nucleus without affecting the frequency of nuclear localization among variants, suggesting that NLS2 of FLI1 was used for nuclear translocation of various EWS-FLI1 fusion proteins.


Assuntos
Neoplasias Ósseas/metabolismo , Núcleo Celular/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Transporte Proteico , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sarcoma de Ewing/metabolismo , Fatores de Transcrição/fisiologia , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa/metabolismo , Humanos , Proteínas Nucleares , Plasmídeos , Estrutura Terciária de Proteína , Proteína EWS de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
13.
Biochem Biophys Res Commun ; 331(4): 902-8, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15882963

RESUMO

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21Cip1, which was abolished by pretreatment with actinomycin D. A p38 MAPK specific inhibitor, SB203580, blocked the increase of p21Cip1 mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21Cip1 mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21Cip1.


Assuntos
Divisão Celular/fisiologia , Células Intersticiais do Testículo/citologia , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Sequência de Bases , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Primers do DNA , Células Intersticiais do Testículo/enzimologia , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Receptores de Hidrocarboneto Arílico/genética
14.
Biochem Biophys Res Commun ; 317(2): 545-50, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-15063792

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which plays a role as an intracellular mediator of the xenobiotic signaling pathway. We previously identified the minimum nuclear localization signal (NLS) of AhR(13-39): it is composed of two basic amino acid segments, AhR(13-16:RKRR) and AhR(37-39:KRH). In this study, we showed that the two protein kinase C (PKC) sites of Ser-12 and Ser-36 are located one amino acid upstream from each of the two segments, and that a ligand-dependent nuclear import of AhR is inhibited by substitution of aspartic acid for Ser-12 (S12D) or Ser-36 (S36D), which mimics the negative charge of phosphorylation. This observation was supported by microinjection analysis, an in vitro nuclear transport assay, and a luciferase reporter assay, suggesting a two-step mechanism in the ligand-dependent nuclear translocation of AhR.


Assuntos
Núcleo Celular/metabolismo , Fígado/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Cães , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Relação Estrutura-Atividade
15.
J Biol Chem ; 279(18): 19209-16, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-14985336

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR contains signals for both nuclear localization and nuclear export (NES). The objective of this study was to demonstrate how AhR intracellular distribution was regulated physiologically in cells. We found that cell density, but not the cell cycle, influenced the subcellular distribution of AhR in a keratinocyte cell line, HaCaT: AhR was predominantly nuclear at sparse cell densities, both nuclear and cytoplasmic at subconfluence, and predominantly cytoplasmic at confluence. Stable transfectants of HaCaT carrying a reporter gene fused with xenobiotic responsive element showed an association between xenobiotic responsive element-mediated transcription and AhR relocalization. Leptomycin B promoted nuclear accumulation of AhR irrespective of cell density, suggesting that this alteration may be because of a change of the regulation of the nuclear export of AhR. We found that Ser-68 in the NES of AhR was phosphorylated after nuclear accumulation of activated AhR and the nuclear export of a chimeric GST-AhR-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid, which mimics the negative charge of phosphorylation. This novel cell density-dependent AhR relocalization was affected by exposure to SB203580, okadaic acid, and low Ca(2+) concentrations. These findings strongly suggest that cell density regulates the intracellular localization and function of AhR, because of modulation of nuclear export activity. The p38 MAPK-mediated phosphorylation of the NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization.


Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Ligação a DNA , Queratinócitos/citologia , Receptores de Hidrocarboneto Arílico/metabolismo , Ativação Transcricional , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Compartimento Celular , Contagem de Células , Ciclo Celular , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Sinais Direcionadores de Proteínas , Transporte Proteico , Receptores de Hidrocarboneto Arílico/biossíntese , Fatores de Transcrição , Cicatrização , Proteínas Quinases p38 Ativadas por Mitógeno
16.
Mol Endocrinol ; 17(6): 994-1004, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12610109

RESUMO

Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax-1, NR0B1) is an orphan nuclear receptor that represses transcription by Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1). Observations on human diseases and the phenotypes of mice, in which the corresponding genes have been disrupted, have elucidated essential roles of these two nuclear receptors in differentiation of steroidogenic tissues. However, little is known about how the functions of these factors are regulated. Here we have examined their subcellular localization and have clarified the molecular mechanisms regulating subcellular localization of Dax-1. Prompted by the finding that nuclear localization of Dax-1 correlates with the presence of Ad4BP/SF-1 in the early stages of pituitary development, we have tested the possibility that interaction between the two factors is essential for the nuclear localization of Dax-1. In vitro studies with cultured cells demonstrated that an interaction involving the LXXLL motifs in the N-terminal repeat region of Dax-1 plays a key role in its subcellular localization. In addition, we found that a mutant form of DAX-1 (L466R), from a patient with adrenal hypoplasia congenita, was defective in nuclear localization in spite of having an intact N terminus. Taken together, the results reveal that the subcellular localization of Dax-1 is influenced by the presence of Ad4BP/SF-1, and that two regions of Dax-1 have important roles for this process.


Assuntos
Motivos de Aminoácidos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Estrutura Terciária de Proteína/fisiologia , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/fisiologia , Receptor Nuclear Órfão DAX-1 , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Fushi Tarazu , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HeLa , Proteínas de Homeodomínio , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Hipófise/embriologia , Hipófise/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/genética , Proteínas Repressoras/genética , Fator Esteroidogênico 1 , Relação Estrutura-Atividade , Frações Subcelulares/fisiologia , Fatores de Transcrição/genética
17.
Mol Pharmacol ; 63(3): 524-31, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12606758

RESUMO

The steroid and xenobiotic receptor (SXR) is an orphan nuclear receptor that plays a key role in the regulation of xenobiotic response by controlling the expression of drug metabolizing and clearance enzymes. We observed that pregnane X receptor (PXR), the mouse ortholog of SXR, was retained in the cytoplasm of hepatic cells of untreated mice, whereas PXR was translocated to the nucleus after administration of a ligand, pregnenolone 16 alpha-carbonitrile. To understand the molecular mechanisms underlying the xenochemical-dependent nuclear translocation of SXR, we identified the signal sequence of SXR that regulates its nuclear translocation; using an in vitro expression system, we allocated the nuclear localization signal (NLS) to amino acid residues 66 to 92 within the DNA binding domain of SXR. The NLS of SXR is characterized as the bipartite type, and is recognized by the three molecular species of importin alpha: Rch1 (PTAC58), NPI1, and Qip1, in the presence of PTAC97 of importin beta to target the nuclear pore. The nuclear translocation of SXR was observed as an essential regulatory event for transcription of its target genes such as CYP3A4. These results strongly suggest that the molecular mechanism of the nuclear import of SXR was different from that of another xenosensor, the constitutively active receptor, whose translocation into the nucleus is mediated by a leucine-rich xenochemical response signal in its ligand binding domain.


Assuntos
Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Fragmentos de Peptídeos/metabolismo , Receptor de Pregnano X , Proteínas Recombinantes de Fusão/farmacologia , Frações Subcelulares , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , alfa Carioferinas/metabolismo
18.
J Biochem ; 131(1): 79-85, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11754738

RESUMO

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that acts in concert with the AhR nuclear translocator (ARNT). Subcellular localization and transcriptional activation of target genes are mainly regulated by ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We have previously reported that AhR migrates in cells as a nucleocytoplasmic shuttling protein mediated by its nuclear localization and export signals. A short sequence motif LxxLL (L is leucine and x is any amino acid) found in transcriptional co-activators has been reported to mediate the binding to liganded nuclear receptors. The role of the two LxxLL motifs, AhR[50-54] and [224-228], has now been analyzed by determining the localization of AhR and its transcriptional activity with Leu to Ala mutations in full-length AhR. Immunocytostaining revealed that mutation of the motif at AhR[50-54] promotes the efficiency of nuclear localization in the absence of ligand without altering HSP90 and ARA9 binding or nuclear export activity. Furthermore, this mutation decreases the transcriptional activity of the AhR/ARNT system, which is likely due to the suppression of AhR/ARNT/XRE complex formation. Another LxxLL motif at AhR[224-227] affects neither the subcellular localization nor transcriptional activity. These results indicate that the LxxLL motif at AhR[50-54] is important for the regulation of AhR activity.


Assuntos
Receptores de Hidrocarboneto Arílico/genética , Ativação Transcricional , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Células COS/metabolismo , Chlorocebus aethiops , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Leucina/química , Leucina/genética , Leucina/fisiologia , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Frações Subcelulares/química
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