RESUMEN
Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.
Asunto(s)
Glutatión Transferasa/análisis , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Línea Celular , Humanos , Hígado/enzimología , Isoformas de Proteínas/análisis , Alineación de SecuenciaRESUMEN
An alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1(∗). The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.
Asunto(s)
Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Porcinos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biocatálisis , Clonación Molecular , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica , Glutatión Transferasa/química , Glutatión Transferasa/aislamiento & purificación , Humanos , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Masculino , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.
Asunto(s)
Glutatión Transferasa/química , Isoenzimas/química , Esteroide Isomerasas/química , Secuencia de Aminoácidos , Animales , Catálisis , Femenino , Glutatión Transferasa/metabolismo , Humanos , Isoenzimas/metabolismo , Cinética , Masculino , Datos de Secuencia Molecular , Ovario/metabolismo , Alineación de Secuencia , Esteroide Isomerasas/metabolismo , Porcinos , Testículo/metabolismoRESUMEN
hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.
Asunto(s)
Marcación de Gen , Glutatión Transferasa/genética , Placenta/citología , Placenta/enzimología , Progesterona/biosíntesis , Progesterona/genética , Línea Celular , Inhibidores Enzimáticos/química , Femenino , Marcación de Gen/métodos , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/fisiología , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/genética , Hormonas Esteroides Gonadales/fisiología , Humanos , Placenta/metabolismo , Interferencia de ARN , EstereoisomerismoRESUMEN
We describe a novel cis-element in the 5' coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP)C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides downstream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is observed in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qualitative changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addition, we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.
Asunto(s)
Citostáticos/farmacología , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico/genética , Proteína p53 Supresora de Tumor/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Codón/genética , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Dactinomicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Humanos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Sistemas de Lectura Abierta , Especificidad de Órganos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Sondas ARN/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Glucocorticoids down regulate the inducible nitric oxide synthase (iNOS) gene both transcriptionally and post-transcriptionally. The post-transcriptional events are suggested to involve destabilization of the iNOS transcript although the molecular mechanisms for this effect are not known. Recently, our laboratory demonstrated a lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-induction-dependent interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I and hnRNPL with a destabilizing element contained in the 3'untranslated region (UTR) of iNOS mRNA. The aim of this study was to investigate if dexamethasone, which down regulates iNOS, is able to modulate this protein-mRNA interaction. As expected, dexamethasone inhibited the induction of iNOS by LPS and IFNgamma in RAW 264.7 cells, and destabilized the iNOS mRNA. Dexamethasone also counteracted the LPS/IFNgamma-induced disappearance of a gel shifted iNOS mRNA-protein complex containing hnRNPI and hnRNPL. UV cross-linking and Western blot analyses revealed that the RNA-binding and levels of hnRNPI, which decreased by LPS/IFNgamma treatment, were restored by dexamethasone. The results support our hypothesis that hnRNPI is pivotal in the post-transcriptional regulation of iNOS and strongly suggest that hnRNPI is one of the trans-acting factors mediating the post-transcriptional effects of dexamethasone.
Asunto(s)
Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Óxido Nítrico Sintasa de Tipo II/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacosRESUMEN
Bovine glutathione transferase A1-1 (bGST A1-1) and human GST A3-3 (hGST A3-3) share both high amino acid sequence similarity and selective expression in steroidogenic organs. hGST A3-3 is the most efficient steroid isomerase known in mammals, and is thought to catalyze isomerization reactions in the biosynthesis of steroid hormones. We observed that four out of five residues essential to the high steroid isomerase activity of hGST A3-3 are conserved in bGST A1-1. The bovine GST was cloned, heterologously expressed, and purified to homogeneity. Its specific activity towards classical GST substrates and two steroids, Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, was studied, and the steady-state kinetic parameters with the steroids were determined. We find that bGST A1-1 exhibits enzymatic activities comparable to those of hGST A3-3 towards non-steroid substrates. However, the bovine enzyme had 100 times lower catalytic efficiency in steroid isomerization reactions than the human GST. Nevertheless, bGST A1-1 was found as efficient as bovine 3beta-hydroxysteroid dehydrogenase as a steroid isomerase. We discuss likely reasons for the contrasting steroid isomerase activities of bGST A1-1 and hGST A3-3, and alternative roles of bGST A1-1.
Asunto(s)
Glutatión Transferasa/metabolismo , Isomerasas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , ADN Complementario , Glutatión Transferasa/genética , Humanos , Isomerasas/genética , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The aim of this study was to investigate the role of heterogeneous nuclear ribonucleoprotein I (hnRNPI) and hnRNPL in the regulation of the murine inducible nitric oxide synthase (iNOS) gene during inflammation. Treatment of mice with lipopolysaccharide (LPS)/D-galactosamine, or of RAW 264.7 cells with LPS/interferon-gamma (IFN-gamma), strongly increased iNOS expression while reducing hnRNPI levels and complex formation between hnRNPI/hnRNPL and the 3'-untranslated region (3'-UTR) of iNOS mRNA. Introduction of the iNOS 3'-UTR to a luciferase reporter gene reduced its expression in RAW 264.7 cells. However, when hnRNPI and hnRNPL binding sites were deleted, luciferase expression was recovered. LPS/IFN-gamma increased the luciferase activity of the full-length 3'-UTR construct compared to control, while its effects on the deletion constructs were modest. The results indicate that LPS/IFN-gamma induce iNOS through a mechanism involving hnRNPI and hnRNPL binding to iNOS 3'-UTR. Our data suggest that iNOS mRNA degradation is promoted upon binding of hnRNPI and hnRNPL to a destabilizing region within its 3'-UTR, while inflammatory stimuli causing dissociation of the mRNA-protein complex, yield a more stable transcript. This appears to be particularly significant during extended inflammatory stimuli, resulting in sustained nitric oxide production. The critical event launching this process appears to be the degradation of hnRNPI.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Unión Proteica , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
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 GenéticaRESUMEN
Glutathione transferases (GSTs) constitute a superfamily of detoxifying enzymes with a major role in protecting cellular macromolecules from reactive electrophilic compounds. A growing body of evidence suggests, however, that at least certain glutathione transferases are involved in other essential cellular processes, such as cellular signaling and anabolic pathways. One of them is the human GST A3-3, which is selectively expressed in steroidogenic organs and which efficiently catalyzes the obligatory isomerization of the Delta(5)-ketosteroid precursors in the biosynthesis of progesterone and testosterone. In this chapter, we summarize the current knowledge on human GST A3-3 and describe methods for heterologous expression and functional characterization of the enzyme.
Asunto(s)
Glutatión Transferasa/metabolismo , Isomerasas/metabolismo , Esteroides/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Isomerasas/química , Isomerasas/genética , Datos de Secuencia Molecular , Estructura Molecular , Esteroides/química , Distribución TisularRESUMEN
The human xenobiotic-metabolizing enzyme cytochrome P450, CYP2A6, catalyzes the bioactivation of a number of carcinogens and drugs and is overexpressed in cases of liver diseases, such as cirrhosis, viral hepatitis, and parasitic infestation, and in certain tumor cells. This suggests that CYP2A6 may be a major liver catalyst in pathological conditions. In the present study, we have addressed molecular mechanisms underlying the regulation of the CYP2A6 gene. We present evidence of several proteins present in human hepatocytes that interact specifically with the 3'-untranslated region (UTR) of CYP2A6 mRNA. Biochemical and immunological evidence show that the RNA-protein complex of highest intensity contains the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 or a closely related protein. Mapping of the hnRNP A1 binding site within CYP2A6 3'-UTR reveals that the smallest portion of RNA supporting significant binding consists of 111 central nucleotides of the 3'-UTR. Our studies also indicate that hnRNPA1 from HepG2 cancer cells exhibits modified binding characteristics to the CYP2A6 3'-UTR compared with primary hepatocytes. We found that the level of CYP2A6 mRNA remains high in conditions of impaired transcription in primary human hepatocytes, showing that CYP2A6 expression can be affected post-transcriptionally in conditions of cellular stress. Our results indicate that the post-transcriptional regulation involves interaction of the hnRNP A1 protein with CYP2A6 mRNA. The present data suggest that hnRNPA1 is a critical regulator of expression of the human CYP2A6 gene and support the notion that this P450 isoform may be of particular significance in stressed human liver cells.
Asunto(s)
Regiones no Traducidas 3'/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxigenasas de Función Mixta/genética , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular/patología , Citocromo P-450 CYP2A6 , Dactinomicina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hepatocitos/enzimología , Ribonucleoproteína Nuclear Heterogénea A1 , Humanos , Ratones , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Estructura Secundaria de Proteína , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Contaminantes Ambientales/toxicidad , Oxigenasas de Función Mixta/biosíntesis , Sistema Respiratorio/metabolismo , Animales , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Inducción Enzimática , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Metimazol/toxicidad , Ratones , Ratones Endogámicos DBA , Conducto Nasolagrimal/citología , Conducto Nasolagrimal/efectos de los fármacos , Conducto Nasolagrimal/metabolismo , Nitrilos/toxicidad , Mucosa Olfatoria/citología , Mucosa Olfatoria/efectos de los fármacos , Mucosa Olfatoria/metabolismo , Fenobarbital/toxicidad , Pirazoles/toxicidad , Sistema Respiratorio/citología , Sistema Respiratorio/efectos de los fármacos , Glándulas Salivales/citología , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/metabolismoRESUMEN
The cytochrome P450 (Cyp) 2a5 gene can be upregulated transcriptionally or by mRNA stabilization. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacting with the CYP2A5 mRNA has been shown to be a key post-transcriptional regulator of the Cyp2a5 gene. The aim of this study was to investigate if the transcriptional and post-transcriptional steps of Cyp2a5 expression are linked. This was done by modifying the transcription rate with transcriptional inducers (phenobarbital and cyclic AMP) and inhibitors (actinomycin D and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole) and analyzing the effects upon post-transcriptional events. We found that inhibition of transcription led to relocalization of hnRNP A1 from the nucleus to the cytoplasm, to its strongly increased binding to the cytoplasmic CYP2A5 mRNA and to CYP2A5 mRNA stabilization. In contrast, stimulated transcription resulted in increased binding of nuclear hnRNP A1 to the Cyp2a5 promoter, and overexpression of hnRNP A1 led to stimulated transcription of a Cyp2a5 promoter-driven luciferase recombinant. This strongly suggests that the transcriptional and post-transcriptional stages of Cyp2a5 expression are interrelated and that the nucleocytoplasmic shuttling hnRNP A1 may coordinate these different steps.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas/fisiología , Transcripción Genética , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Secuencia de Bases , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , ADN/análisis , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos DBA , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.
Asunto(s)
Regiones no Traducidas 3'/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxigenasas de Función Mixta/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión/genética , Sitios de Unión/fisiología , Northern Blotting , Western Blotting , Línea Celular , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Genes Reporteros , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Luciferasas/genética , Ratones , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , Eliminación de Secuencia , TransfecciónRESUMEN
Interaction of two members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with the 3'untranslated region (UTR) of the murine inducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study. An iNOS RNA-protein complex is formed using protein extracts from untreated and septic shock treated mouse liver. UV cross-linking reveals that the complex consists of at least two proteins, with apparent molecular masses of 60 and 70 kDa, respectively. The 60-kDa protein binding site lies within a 112-nt pyrimidine-rich sequence, approximately 160 nt from the coding sequence, and the RNA-protein complex can be precipitated by a monoclonal antibody directed against hnRNP I [also named polypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a 43-nt sequence near the 3'end of the 3'UTR and is immunoprecipitated by a monoclonal antibody against hnRNP L. A computer-simulated conformation of the 3'UTR suggests that both binding sites reside in regions easily accessible for a protein. Supershifts of the native RNA-protein complex could only be achieved with anti-hnRNP L, suggesting that within this multiprotein RNA complex, only hnRNP L is exposed to the antibodies, whereas the hnRNP I/PTB is mainly responsible for its interaction with the mRNA. Up-regulation of iNOS by septic shock reduces the RNA-protein complex formation, thus showing that hnRNP I/PTB and hnRNP L binding to the iNOS mRNA is modulated by inflammation. This suggests a novel function for the two previously described proteins as regulators of the iNOS gene.
Asunto(s)
Regiones no Traducidas 3'/metabolismo , Inflamación/metabolismo , Óxido Nítrico Sintasa/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Regiones no Traducidas 3'/química , Animales , Secuencia de Bases , Ribonucleoproteína Heterogénea-Nuclear Grupo L , Ribonucleoproteínas Nucleares Heterogéneas , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Peso Molecular , Complejos Multiproteicos , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Conformación de Ácido Nucleico , Proteína de Unión al Tracto de Polipirimidina , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Choque Séptico/metabolismoRESUMEN
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) functions in the packaging of nascent RNA polymerase II transcripts and participates in a variety of nuclear and cytoplasmic processes that modulate gene expression. The RNA binding characteristics of hnRNP A1 suggest that it can modulate the expression of specific genes, but little is known about its possible targets in vivo. In this article, we show that hnRNP A1 interacts with the transcript of a cytochrome P450 gene, Cyp2a5, induced by xenobiotics and during liver damage. Binding of the hnRNP A1 to CYP2A5 mRNA was demonstrated by immunoprecipitation of the xenobiotic-stimulated (37/39 kDa) CYP2A5 mRNA-protein complex with a monoclonal anti-hnRNP A1 antibody, by partial trypsin digestion of the complex, and by showing that the RNA-protein complex is not formed with protein extracts from cells lacking the hnRNP A1. We also show that a specific hepatotoxic inducer of the Cyp2a5 gene, pyrazole, increases the cytoplasmic levels of hnRNP A1 in vivo. Finally, we show that hnRNP A1 can be overexpressed in mouse primary hepatocytes, leading to an accumulation of the CYP2A5 mRNA. Collectively, these results indicate that the hnRNP A1 is an important regulator of the Cyp2a5 gene.