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1.
Mol Pharmacol ; 73(2): 451-60, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17978169

RESUMO

Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) alpha response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4alpha- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4alpha increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4alpha mRNA levels by 4-fold; however, the amount of HNF4alpha nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4alpha to the HNF4-RE and that an interaction of GR and HNF4alpha occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4alpha to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4alpha with an HNF4-RE rather than a glucocorticoid response element.


Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Dexametasona/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Oxigenases de Função Mista/biossíntese , Regiões Promotoras Genéticas/fisiologia , Receptores de Glucocorticoides/metabolismo , Regulação para Cima/fisiologia , Hidrocarboneto de Aril Hidroxilases/genética , Células Cultivadas , Citocromo P-450 CYP2A6 , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Células HeLa , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Glucocorticoides/genética , Regulação para Cima/efeitos dos fármacos
2.
Biochem J ; 400(1): 135-41, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16836488

RESUMO

The effect of GSTA1-1 (glutathione S-transferase Alpha 1-1) on JNK (c-Jun N-terminal kinase) activation was investigated in Caco-2 cells in which GSTA1 expression increases with degree of confluency, and in MEF3T3 cells with Tet-Off-inducible GSTA1 expression. Comparison of GSTA1 expression in pre-confluent, confluent and 8-day post-confluent Caco-2 cells revealed progressively increasing mRNA and protein levels at later stages of confluency. Exposure of pre-confluent cells to stress conditions including IL-1beta (interleukin-1beta), H2O2 or UV irradiation resulted in marked increases in JNK activity as indicated by c-Jun phosphorylation. However, JNK activation was significantly reduced in post-confluent cells exposed to the same stresses. Western-blot analysis of GSTA1-1 protein bound to JNK protein pulled down from cellular extracts showed approx. 4-fold higher GSTA1-1-JNK complex formation in post-confluent cells compared with pre-confluent cells. However, stress conditions did not alter the amount of GSTA1-1 bound to JNK. The role of GSTA1-1 in JNK suppression was more specifically revealed in Tet-Off-inducible MEF3T3-GSTA1-1 cells in which GSTA1 overexpression significantly reduced phosphorylation of c-Jun following exposure to IL-1beta, H2O2 and UV irradiation. Finally, the incidence of tumour necrosis factor alpha/butyrate-induced apoptosis was significantly higher in pre-confluent Caco-2 cells expressing low levels of GSTA1 compared with post-confluent cells. These results indicate that GSTA1 suppresses activation of JNK signalling by a pro-inflammatory cytokine and oxidative stress and suggests a protective role for GSTA1-1 in JNK-associated apoptosis.


Assuntos
Apoptose/fisiologia , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de Sinais/fisiologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Butiratos/farmacologia , Células CACO-2 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/genética , Glutationa Transferase/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Marcação In Situ das Extremidades Cortadas , Interleucina-1beta/farmacologia , Isoenzimas/genética , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia
3.
Cancer Lett ; 177(1): 65-74, 2002 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-11809532

RESUMO

Glutathione S-transferase (GST) mu phenotype was assessed in colon tissue from patients with ulcerative colitis and colorectal neoplasms that were positive for GSTM1 genotype. GST mu protein (enzyme linked immunosorbent assay) was absent in 2/9 unaffected colon tissue (22.3%), 4/13 tissues with chronic ulcerative colitis (CUC) (30.7%), 4/11 adenomas (36.4%) and 7/14 adenocarcinomas (50.0%; P

Assuntos
Adenocarcinoma/enzimologia , Adenoma/enzimologia , Neoplasias Colorretais/enzimologia , Glutationa Transferase/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Biomarcadores Tumorais , Western Blotting , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Neoplasias Colorretais/genética , DNA/metabolismo , Primers do DNA/química , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Genótipo , Glutationa Transferase/metabolismo , Humanos , Técnicas Imunoenzimáticas , Fenótipo , Reação em Cadeia da Polimerase
4.
Mol Pharmacol ; 71(1): 201-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17021248

RESUMO

Down-regulation of glutathione transferase A1 (GSTA1) expression has profound implications in cytoprotection against toxic by-products of lipid peroxidation produced during inflammation. We investigated the role of hepatic nuclear factor 1 (HNF-1) in repression of human GSTA1 expression by interleukin (IL)-1beta in Caco-2 cells. In luciferase reporter assays, overexpression of HNF-1alpha increased GSTA1 transcriptional activity via an HNF-1 response element (HRE) in the proximal promoter. In addition, constitutive mRNA levels of GSTA1 and HNF-1alpha rose concurrently in Caco-2 cells with increasing stage of confluence. IL-1beta reduced GSTA1 mRNA levels at all stages of confluence; however, HNF-1alpha mRNA levels were not altered. IL-1beta repressed GSTA1 transcriptional activity, an effect that was abolished by mutating the HRE. Similar results were observed in HT-29 and HepG2 cells. Overexpression of HNF-1alpha did not counteract IL-1beta-mediated repression of GSTA1 transcription either in reporter assays or at the mRNA level. Involvement of the transdominant repressor C isoform of variant HNF-1 (vHNF-1C) in GSTA1 repression was demonstrated, because vHNF-1C overexpression significantly reduced GSTA1 transcriptional activity. Finally, IL-1beta caused concentration-related up-regulation of vHNF-1C mRNA levels and increased binding of vHNF-1C protein to the HRE, whereas HNF-1alpha-HRE complex formation was reduced. These findings indicate that IL-1beta represses GSTA1 transcription via a mechanism involving overexpression of vHNF-1C.


Assuntos
Glutationa Transferase/biossíntese , Fator 1 Nuclear de Hepatócito/genética , Interleucina-1beta/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Repressão Enzimática , Glutationa Transferase/genética , Humanos , Cinética , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional
5.
Drug Metab Dispos ; 30(11): 1186-93, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12386123

RESUMO

The influence of pro-inflammatory cytokines on alpha class glutathione S-transferase A1 and A2 (GSTA1/A2) expression was examined in human colonic epithelial cells (Caco-2) in culture. Dose-dependent reductions in GSTA1/A2 mRNA, protein, and activity levels occurred in Caco-2 cells cultured in conditioned medium (CM) from lipopolysaccharide-stimulated murine monocyte-macrophage cells (RAW 264.7). Neutralizing anti-interleukin-1beta (IL-1beta) antibodies attenuated this repression of GSTA1/A2 expression by CM. Moreover, recombinant human IL-1beta reduced GSTalpha expression at the mRNA, protein, and activity levels in a dose-related fashion. Reduction of GSTA1/A2 mRNA levels by IL-1beta was attenuated by pretreatment with IL-1 receptor antagonist. GSTA1/A2 mRNA half-lives were similar in control and IL-1beta-treated cells, indicating that IL-1beta has no effect on mRNA stability. In reporter gene studies, IL-1beta caused a dose-related reduction of luciferase activity in Caco-2 cells transfected with the full-length GSTA1 promoter-luciferase construct. Using truncated constructs, IL-1beta responsiveness was mapped to a region 286 base pairs upstream to the coding region. Deletion of a hepatic nuclear factor 1 (HNF-1) site in this region abrogated the IL-1beta-mediated repression of GSTA1 promoter activity. These results demonstrate that IL-1beta down-regulates GSTA1/A2 expression in cultured human enterocytes by a transcriptional mechanism involving an HNF-1 site.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/biossíntese , Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Proteínas Nucleares , Northern Blotting , Células CACO-2 , Proteínas de Transporte/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Enterócitos/metabolismo , Glutationa Transferase , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos/genética , RNA/biossíntese , RNA/genética , Fatores de Transcrição/farmacologia , Transfecção
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