RESUMEN

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

Asunto(s)

Citocromo P-450 CYP2A6/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Citocromo P-450 CYP2A6/genética , Daño del ADN , Ensayo de Cambio de Movilidad Electroforética , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba

RESUMEN

BACKGROUND: Transcription factor GA-binding protein (GABP) is suggested to be involved in the formation of the neuromuscular junctions by regulating the transcription of synapse genes. Interestingly, neurons and podocytes share molecular and functional similarities that led us to investigate the expression and function of GABP in podocytes and its role in transcriptional regulation of nephrin, the key molecule of the podocyte slit diaphragm that is essential for normal glomerular ultrafiltration. METHODS: The expression and localization of GABP in the rat and human kidney as well as in human embryonic kidney A293 cells and undifferentiated and differentiated human podocytes were analysed by immunoblotting and immunostaining. The role of GABP in activating the nephrin promoter was investigated by reporter gene assay and site-directed mutagenesis of the GABP-binding elements, and the interaction of GABP with the nephrin promoter was analysed by chromatin immunoprecipitation. The function of GABP in podocytes was studied by knocking down GABPα in differentiated human podocytes using lentiviral shRNA targeting GABPα. RESULTS: GABP is expressed in the nuclei in rat and human glomeruli. In addition, in A293 cells and undifferentiated and differentiated human podocytes, GABP highly enriches in the nucleus. GABP activates and binds nephrin proximal promoter and Ets sites are essential for this activity. Knock-down of GABPα stimulates apoptosis in cultured podocytes. CONCLUSIONS: The results show that GABP is expressed in podocytes and is involved in the regulation of nephrin gene expression. Furthermore, GABP may be important in the maintenance of podocyte function by regulating apoptosis.

Asunto(s)

Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Podocitos/metabolismo , Animales , Apoptosis , Western Blotting , Caspasa 3/metabolismo , Células Cultivadas , Inmunoprecipitación de Cromatina , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Factor de Transcripción de la Proteína de Unión a GA/antagonistas & inhibidores , Factor de Transcripción de la Proteína de Unión a GA/genética , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Podocitos/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , Ratas , Ratas Sprague-Dawley , Secuencias Reguladoras de Ácidos Nucleicos/genética

RESUMEN

BACKGROUND: Nephrin and Neph3 are homologous molecules expressed in the podocyte slit diaphragms that are essential for normal glomerular ultrafiltration. Nephrin and Neph3 genes form a bidirectional gene pair suggesting that they may share key features in their regulation. We investigated if nephrin and Neph3 genes have similar mechanisms in their transcriptional regulation focussing on transcription factor Wilms' tumour 1 (WT1) and nuclear factor-κB (NF-κB) and DNA methylation. METHODS: Transcriptional regulation of nephrin and Neph3 by WT1 and NF-κB was analysed by overexpression studies, reporter gene assay and chromatin immunoprecipitation using A293 cells and cultured podocytes. The interaction between WT1 and NF-κB was studied by co-immunoprecipitation. The effect of NF-κB activator tumour necrosis factor-α (TNF-α) with or without NF-κB pathway inhibitor (BAY 11-7082) on nephrin and Neph3 messenger RNA (mRNA) expression and on cellular distribution of NF-κB was determined by quantitative polymerase chain reaction (PCR) and immunostaining, respectively. The role of DNA methylation in regulating nephrin and Neph3 genes was studied by demethylating agent (5-aza-2'-deoxycytidine) treatment and quantitative PCR. RESULTS: WT1 and NF-κB interact with nephrin and Neph3 promoter and cooperatively regulate nephrin and Neph3. The cooperation was further supported by the physical interaction between WT1 and NF-κB. TNF-α increased nephrin and Neph3 mRNA expression and this effect was mediated by NF-κB. Furthermore, DNA methylation played a role in silencing nephrin and Neph3 expression in a cell-type and differentiation stage-dependent manner. CONCLUSION: These results provide novel insights into the transcriptional regulation of nephrin and Neph3 genes and indicate that nephrin and Neph3 share the same mechanisms in their regulation.

Asunto(s)

Metilación de ADN/fisiología , Inmunoglobulinas/fisiología , Riñón/fisiología , Proteínas de la Membrana/fisiología , FN-kappa B/fisiología , Transcripción Genética/fisiología , Proteínas WT1/fisiología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Células Cultivadas , Decitabina , Humanos , Inmunoglobulinas/genética , Técnicas In Vitro , Riñón/citología , Proteínas de la Membrana/genética , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , Nitrilos/farmacología , Podocitos/citología , Podocitos/fisiología , Interferencia de ARN/fisiología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Sulfonas/farmacología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

BACKGROUND: Neph3 (filtrin) is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. Neph3 protein shows homology and structural similarity to Neph1, Neph2 and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. The exact function of Neph3 in the kidney is not known but we have previously shown that the level of Neph3 mRNA is decreased in proteinuric diseases. This suggests that Neph3 may play a role in the pathogenesis of kidney damage, and emphasizes the need to analyze the regulatory mechanisms of Neph3 gene. In this study we investigated the transcriptional regulation of Neph3 gene by identifying transcription factors that control Neph3 expression. RESULTS: We cloned and characterized approximately 5 kb fragment upstream of the Neph3 gene. Neph3 proximal promoter near the transcription start site was found to be devoid of TATA and CAAT boxes, but to contain a highly GC-rich area. Using promoter reporter gene constructs, we localized the main activating regulatory region of Neph3 gene in its proximal promoter region from -105 to -57. Within this region, putative transcription factor binding sites for NF-kappaB and Sp1 were found by computational analysis. Mutational screening indicated that NF-kappaB and Sp1 response elements are essential for the basal transcriptional activity of the Neph3 promoter. Co-transfection studies further showed that NF-kappaB and Sp1 regulate Neph3 promoter activity. In addition, overexpression of NF-kappaB increased endogenous Neph3 gene expression. Chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both NF-kappaB and Sp1 interact with the Neph3 promoter. CONCLUSION: Our results show that NF-kappaB and Sp1 are key regulators of Neph3 expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of Neph3 gene.

Asunto(s)

Regulación de la Expresión Génica , Inmunoglobulinas/genética , Proteínas de la Membrana/genética , FN-kappa B/metabolismo , Podocitos/metabolismo , Factor de Transcripción Sp1/metabolismo , Línea Celular , Humanos , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/genética , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta , Factor de Transcripción Sp1/genética

RESUMEN

The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1alpha and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1alpha expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1alpha expression vector demonstrated that PGC-1alpha is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4alpha response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1alpha binds, together with HNF-4alpha, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1alpha mediates the expression of Cyp2a5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4alpha. This strongly suggests that PGC-1alpha is the major factor mediating the fasting response of CYP2A5.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/biosíntesis , Ayuno/metabolismo , Hepatocitos/enzimología , Transactivadores/metabolismo , Xenobióticos/metabolismo , Adenoviridae/genética , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Técnicas de Cultivo de Célula , Células Cultivadas , GMP Cíclico/metabolismo , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Inducción Enzimática , Vectores Genéticos , Factor Nuclear 4 del Hepatocito/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Transducción de Señal , Factores de Tiempo , Transactivadores/genética , Factores de Transcripción , Activación Transcripcional , Transfección , Regulación hacia Arriba

RESUMEN

The aryl hydrocarbon receptor nuclear translocator (ARNT) belongs to the basic-helix-loop-helix (bHLH) transcription factors and regulates several genes as heterodimers with other bHLH proteins. ARNT is also able to homodimerize, but no mammalian target genes for the homodimer have been shown. We identified a palindromic E-box element in the 5' regulatory region of the murine cytochrome P450 (Cyp) 2a5 gene that was found to be important for Cyp2a5 transcription in primary hepatocytes, and was found by chromatin immunoprecipitation assays to interact with ARNT. Electrophoretic mobility-shift assay experiments with in vitro translated ARNT showed binding without heterodimerization partner, indicating binding as a homodimer. Transfection studies in wild-type and ARNT-deficient Hepa-1 cells revealed that ARNT expression is necessary for full activity of the Cyp2a5 promoter. In the liver-specific Arnt-null mouse line, the level of hepatic CYP2A5 mRNA was decreased significantly. Co-transfection studies with an ARNT expression vector lacking the transactivation domain (TAD) demonstrated that the ARNT TAD is needed for Cyp2a5 activation, which suggests that ARNT transactivates Cyp2a5 as a homodimer. In primary hepatocytes, the mRNA levels of both CYP2A5 and ARNT splice variant 1 were increased during cultivation. Upstream stimulatory factors 1 and 2a were also able to bind to the same E-box as ARNT, indicating that there may be competition for DNA binding between these factors. Indeed, the upstream stimulatory factors activated the Cyp2a5 promoter through the E-box only in the presence of hepatocyte nuclear factor-4alpha, while ARNT transactivation was independent of hepatocyte nuclear factor-4alpha. In conclusion, these results indicate that ARNT controls Cyp2a5 transcription and thus, for the first time, suggest active involvement of the ARNT homodimer in mammalian gene regulation.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/química , Translocador Nuclear del Receptor de Aril Hidrocarburo/fisiología , Regulación de la Expresión Génica , Oxigenasas de Función Mixta/química , Factores Estimuladores hacia 5'/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/química , Línea Celular Tumoral , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Dimerización , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , ARN Mensajero/metabolismo , Transcripción Genética , Activación Transcripcional

RESUMEN

We have previously shown that cadmium, a metal that alters cellular redox status, induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2+/+) mice but not in the knockout (Nrf2-/-) mice. In the present studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels, with a substantial increase observed within 3 h of exposure. Immunoblotting showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure. Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis of the promoter localized the Nrf2 responsive region to an area from -2656 to -2339 base pair. Computer-based sequence analysis identified two putative stress response elements (StRE) within the region at positions -2514 to -2505 and -2386 to -2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that interaction of the more proximal StRE with Nrf2 was stimulated by CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5 promoter-luciferase reporter plasmids abolished Nrf2-mediated induction. Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by directly binding to the StRE in the 5'-flanking region of the gene. This acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified under the control of the StRE-Nrf2 pathway with a potential role in adaptive response to cellular stress.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica , Oxigenasas de Función Mixta/genética , Factor 2 Relacionado con NF-E2/fisiología , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Western Blotting , Cloruro de Cadmio/metabolismo , Cloruro de Cadmio/farmacología , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Ensayo de Cambio de Movilidad Electroforética , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos DBA , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/métodos , Mutación , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Sitio de Iniciación de la Transcripción

RESUMEN

We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica , Oxigenasas de Función Mixta/genética , Receptores de Hidrocarburo de Aril/fisiología , Animales , Hidrocarburo de Aril Hidroxilasas/análisis , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Translocador Nuclear del Receptor de Aril Hidrocarburo , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Proteínas de Unión al ADN/fisiología , Inducción Enzimática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Oxigenasas de Función Mixta/análisis , Oxigenasas de Función Mixta/biosíntesis , Dibenzodioxinas Policloradas/farmacología , ARN Mensajero/análisis , Factores de Transcripción/fisiología , Transcripción Genética

RESUMEN

The cytochrome P4502a5 (Cyp2a5) gene is expressed principally in liver and olfactory mucosa. In the present study, the transcriptional mechanisms of hepatocyte-specific expression of Cyp2a5 were studied in mouse primary hepatocytes. The Cyp2a5 5'-flanking region -3033 to +10 was cloned in front of a luciferase reporter gene and transfected into hepatocytes. Deletion analysis revealed two major activating promoter regions localized at proximal 271 bp and at a more distal area from -3033 to -2014 bp. The proximal activation region was characterized further by DNase I footprinting, and a single clear footprint was detected in the studied area centred over a sequence similar to the NF-I (nuclear factor I)-binding site. The binding of NF-I was confirmed using an EMSA (electrophoretic mobility-shift assay). A putative HNF-4 (hepatocyte nuclear factor 4)-binding site was localized at the proximal promoter by computer analysis of the sequence, and HNF-4alpha was shown to interact with the site using an EMSA. The functional significance of HNF-4 and NF-I binding to the Cyp2a5 promoter was evaluated by site-directed mutagenesis of the binding motifs in reporter constructs. Both mutations strongly decreased transcriptional activation by the Cyp2a5 promoter in primary hepatocytes, and double mutation almost completely abolished transcriptional activity. Also, the functionality of the distal activation region was found to be dependent on the intact HNF-4 and NF-I sites at the proximal promoter. In conclusion, these results indicate that HNF-4 and NF-I play major roles in the constitutive regulation of hepatic expression of Cyp2a5.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/genética , Proteínas de Unión al ADN/fisiología , Hepatocitos/enzimología , Oxigenasas de Función Mixta/genética , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases/genética , Sitios de Unión , Células COS/enzimología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Mapeo Cromosómico/métodos , Clonación Molecular/métodos , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Huella de ADN/métodos , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 4 del Hepatocito , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Factores de Transcripción/metabolismo