RESUMEN
AIMS/HYPOTHESIS: We previously identified the G6PC2 locus as a strong determinant of fasting plasma glucose (FPG) and showed that a common G6PC2 intronic single nucleotide polymorphism (SNP) (rs560887) and two common G6PC2 promoter SNPs (rs573225 and rs13431652) are highly associated with FPG. However, these promoter SNPs have complex effects on G6PC2 fusion gene expression, and our data suggested that only rs13431652 is a potentially causative SNP. Here we examine the effect of rs560887 on G6PC2 pre-mRNA splicing and the contribution of an additional common G6PC2 promoter SNP, rs2232316, to the association signal. METHODS: Minigene analyses were used to characterise the effect of rs560887 on G6PC2 pre-mRNA splicing. Fusion gene and gel retardation analyses characterised the effect of rs2232316 on G6PC2 promoter activity and transcription factor binding. The genetic association of rs2232316 with FPG variation was assessed using regression adjusted for age, sex and BMI in 4,220 Europeans with normal FPG. RESULTS: The rs560887-G allele was shown to enhance G6PC2 pre-mRNA splicing, whereas the rs2232316-A allele enhanced G6PC2 transcription by promoting Foxa2 binding. Genetic analyses provide evidence for association of the rs2232316-A allele with increased FPG (ß = 0.04 mmol/l; p = 4.3 × 10(-3)) as part of the same signal as rs560887, rs573225 and rs13431652. CONCLUSIONS/INTERPRETATION: As with rs13431652, the in situ functional data with rs560887 and rs2232316 are in accord with the putative function of G6PC2 in pancreatic islets, and suggest that all three are potentially causative SNPs that contribute to the association between G6PC2 and FPG.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus/genética , Glucosa-6-Fosfatasa/genética , Polimorfismo de Nucleótido Simple , Alelos , Diabetes Mellitus/sangre , Ayuno , Femenino , Regulación de la Expresión Génica , Genotipo , Células HeLa , Humanos , Masculino , Regiones Promotoras Genéticas , Empalme del ARN , ARN Mensajero/metabolismoAsunto(s)
Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Complicaciones del Embarazo/diagnóstico , Complicaciones del Embarazo/genética , Adulto , Femenino , Humanos , Hiperaldosteronismo/enzimología , Embarazo , Complicaciones del Embarazo/enzimología , Esteroide 11-beta-Hidroxilasa/genéticaRESUMEN
AIMS/HYPOTHESIS: The glucose-6-phosphatase catalytic subunit (G6PC) plays a key role in hepatic glucose production by catalysing the final step in gluconeogenesis and glycogenolysis. Peroxisome proliferator activated receptor gamma coactivator-1alpha (PGC-1alpha) stimulates mouse G6pc-luciferase fusion gene expression through hepatocyte nuclear factor-4alpha (HNF-4alpha), which binds an element located between -76 and -64 in the promoter. The aim of this study was to compare the regulation of mouse G6pc and human G6PC gene expression by PGC-1alpha. METHODS: PGC-1alpha action was analysed by transient transfection and gel retardation assays. RESULTS: In H4IIE cells, PGC-1alpha alone failed to stimulate human G6PC-luciferase fusion gene expression even though the sequence of the -76 to -64 HNF-4alpha binding site is perfectly conserved in the human promoter. This difference could be explained, in part, by a 3 bp sequence variation between the mouse and human promoters. Introducing the human sequence into the mouse G6pc promoter reduced PGC-1alpha-stimulated fusion gene expression, whereas the inverse experiment, in which the mouse sequence was introduced into the human G6PC promoter, resulted in the generation of a G6PC-luciferase fusion gene that was now induced by PGC-1alpha. This critical 3 bp region is located immediately adjacent to a consensus nuclear hormone receptor half-site that is perfectly conserved between the mouse G6pc and human G6PC promoters. Gel retardation experiments revealed that this 3 bp region influences the affinity of HNF-4alpha binding to the half-site. CONCLUSIONS/INTERPRETATION: These observations suggest that PGC-1alpha may be more important in the control of mouse G6pc than human G6PC gene expression.
Asunto(s)
Variación Genética , Glucosa-6-Fosfatasa/genética , Proteínas de Choque Térmico/metabolismo , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Secuencia Conservada , Activación Enzimática , Genes Reporteros , Glucosa-6-Fosfatasa/metabolismo , Humanos , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Plásmidos , Subunidades de Proteína/genética , Alineación de Secuencia , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. METHODS: To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. RESULTS: No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (-14%) and female (-11%) G6pc2 (-/-) mice, while female G6pc2 (-/-) mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. CONCLUSIONS/INTERPRETATION: These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible.
Asunto(s)
Autoantígenos/química , Eliminación de Gen , Glucosa-6-Fosfatasa/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas/metabolismo , Alelos , Animales , Peso Corporal , Catálisis , Dominio Catalítico , Femenino , Glucosa-6-Fosfato/metabolismo , Humanos , Masculino , Ratones , Ratones TransgénicosRESUMEN
The initial stages of diabetic nephropathy are characterized, in part, by expansion of the mesangial matrix and thickening of the glomerular basement membrane which are caused by increased extracellular matrix (ECM) protein synthesis and reduced degradation, a consequence of decreased matrix metalloproteinase (MMP) activity. These changes have been largely attributed to the effects of hyperglycemia such that the potential contribution of impaired insulin action to alterations in the ECM have not been studied in detail. We have shown here that insulin stimulates collagenase-1 fusion gene transcription in the MES 13 mesangial-derived cell line. Multiple collagenase-1 promoter elements are required for the full stimulatory effect of insulin but the action of insulin appears to be mediated through an activator protein-1 (AP-1) motif. Thus, mutation of this AP-1 motif abolishes insulin-stimulated collagenase fusion gene transcription and, in isolation, this AP-1 motif can mediate a stimulatory effect of insulin on the expression of a heterologous fusion gene. This suggested that the other collagenase-1 promoter elements that are required for the full stimulatory effect of insulin probably bind accessory factors that enhance the effect of insulin mediated through the AP-1 motif. In MES 13 cells, the AP-1 motif is bound by Fra-1, Fra-2, Jun B and Jun D. Stimulation of collagenase-1 fusion gene transcription by insulin requires activation of the mitogen-activated protein kinase (MEK) pathway since inhibition of MEK-1 and -2 blocks this effect. The potential significance of these observations with respect to a role for insulin in the pathophysiology of diabetic glomerulosclerosis is discussed.
Asunto(s)
Colagenasas/genética , Mesangio Glomerular/enzimología , Insulina/fisiología , Sistema de Señalización de MAP Quinasas , Factor de Transcripción AP-1/fisiología , Transcripción Genética/fisiología , Animales , Fusión Artificial Génica , Secuencia de Bases , Línea Celular , Cartilla de ADN , Mesangio Glomerular/citología , Humanos , Ratones , Plásmidos , Regiones Promotoras GenéticasRESUMEN
Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The open reading frame of this UGRP cDNA encodes a protein (346 amino acids (aa); Mr 38,755) that shares 36% overall identity (56% similarity) with the mouse G6Pase catalytic subunit (357 aa; Mr 40,454). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis and does not contain a carboxy-terminal di-lysine endoplasmic reticulum retention signal. UGRP mRNA was detected by RNA blot analysis in every mouse tissue examined with the highest expression in heart, brain, testis and kidney. Database analysis showed that the mouse UGRP gene is composed of six exons, spans approximately 4.2 kbp of genomic DNA and is located on chromosome 11 along with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the mouse UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.
Asunto(s)
Dominio Catalítico/genética , ADN Complementario/genética , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfato/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromosomas/genética , Clonación Molecular , Glucosa-6-Fosfatasa/metabolismo , Células HeLa , Humanos , Islotes Pancreáticos/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Músculos/metabolismo , Alineación de Secuencia , Distribución TisularRESUMEN
Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a human cDNA and gene encoding a ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The ORF of this UGRP cDNA encodes a protein (346 amino acids (aa); M(r) 38 709) which shares 36% overall identity to the human G6Pase catalytic subunit (357 aa; M(r) 40 487). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis. UGRP mRNA was detected by RNA blot analysis in every tissue examined with the highest expression in muscle. Database analysis showed that the human UGRP gene is composed of six exons, spans approximately 5.4 kbp of genomic DNA and is located on chromosome 17q21 with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Subunidades de Proteína/genética , Secuencia de Aminoácidos , Cromosomas Humanos Par 17 , Humanos , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Distribución TisularRESUMEN
The regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6-P) transporter gene expression by insulin in conscious dogs in vivo and in tissue culture cells in situ were compared. In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6-P transporter mRNA was unchanged. In contrast, a 5-h period of hyperinsulinemia resulted in a suppression of both G-6-Pase catalytic subunit and G-6-P transporter gene expression. Similarly, insulin suppressed G-6-Pase catalytic subunit and G-6-P transporter gene expression in H4IIE hepatoma cells. However, the magnitude of the insulin effect was much greater on G-6-Pase catalytic subunit gene expression and was manifested more rapidly. Furthermore, cAMP stimulated G-6-Pase catalytic subunit expression in H4IIE cells and in primary hepatocytes but had no effect on G-6-P transporter expression. These results suggest that the relative control strengths of the G-6-Pase catalytic subunit and G-6-P transporter in the G-6-Pase reaction are likely to vary depending on the in vivo environment.
Asunto(s)
Antiportadores/genética , Regulación de la Expresión Génica/fisiología , Glucosa-6-Fosfatasa/genética , Insulina/fisiología , Proteínas de Transporte de Monosacáridos/genética , Animales , Secuencia de Bases , Glucemia/metabolismo , Catálisis , Células Cultivadas , Ciclofilina A/genética , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Hiperinsulinismo , Insulina/farmacología , Islotes Pancreáticos/fisiología , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Subunidades de Proteína , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Porcinos , Transcripción Genética/efectos de los fármacosRESUMEN
Insulin regulates the expression of more than 150 genes, indicating that this is a major action of this hormone. At least eight distinct consensus insulin response sequence (IRSs) have been defined through which insulin can regulate gene transcription. These include the serum response element, the activator protein 1 ('AP-1') motif, the Ets motif, the E-box motif and the thyroid transcription factor 2 ('TTF-2') motif. All of these IRSs mediate stimulatory effects of insulin on gene transcription. In contrast, an element with the consensus sequence T(G/A)TTT(T/G)(G/T), which we refer to as the phosphoenolpyruvate carboxykinase (PEPCK)-like motif, mediates the inhibitory effect of insulin on transcription of the genes encoding PEPCK, insulin-like-growth-factor-binding protein 1 (IGFBP-1), tyrosine aminotransferase and the glucose-6-phosphatase (G6Pase) catalytic subunit. The forkhead transcription factor FKHR has recently been shown to bind this PEPCK-like IRS motif and a model has been proposed in which insulin inhibits gene transcription by stimulating the phosphorylation and nuclear export of FKHR. Our results suggest that this model is consistent with the action of insulin on transcription of the gene encoding IGFBP-1 but not that of the G6Pase catalytic subunit. Thus, even though the IRSs in both promoters seem identical, they are functionally distinct. In addition, in the G6Pase catalytic subunit promoter, hepatocyte nuclear factor 1 ('HNF-1'), acts as an accessory factor to enhance the effect of insulin mediated through the IRS.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glucosa-6-Fosfatasa/genética , Insulina/fisiología , Animales , Secuencia de Consenso , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Subunidades de Proteína , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Transducción de Señal , Transcripción Genética/fisiologíaRESUMEN
Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Secuencia Conservada , Glucosa-6-Fosfatasa/química , Humanos , Técnicas para Inmunoenzimas , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas/química , ARN Mensajero/análisis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse betaTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in betaTC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in betaTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.
Asunto(s)
Glucosa-6-Fosfatasa , Regiones Promotoras Genéticas/genética , Huella de Proteína , Proteínas/genética , Factores de Transcripción , Animales , Fusión Artificial Génica , Secuencia de Bases/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cricetinae , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Genes Reporteros , Factor Nuclear 3-alfa del Hepatocito , Factor Nuclear 3-beta del Hepatocito , Insulinoma/genética , Insulinoma/patología , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/fisiología , Regiones Promotoras Genéticas/fisiología , Proteínas/química , EstereoisomerismoRESUMEN
Glucose-6-phosphatase is a multicomponent system that catalyzes the terminal step in gluconeogenesis. To examine the effect of the cAMP signal transduction pathway on expression of the gene encoding the mouse glucose-6-phosphatase catalytic subunit (G6Pase), the liver-derived HepG2 cell line was transiently co-transfected with a series of G6Pase-chloramphenicol acetyltransferase fusion genes and an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase A (PKA). PKA markedly stimulated G6Pase-chloramphenicol acetyltransferase fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple cis-acting elements were required for this response. One of these elements was mapped to the G6Pase promoter region between -114 and -99, and this sequence was shown to bind hepatocyte nuclear factor (HNF)-6. This HNF-6 binding site was able to confer a stimulatory effect of PKA on the expression of a heterologous fusion gene; a mutation that abolished HNF-6 binding also abolished the stimulatory effect of PKA. Further investigation revealed that PKA phosphorylated HNF-6 in vitro. Site-directed mutation of three consensus PKA phosphorylation sites in the HNF-6 carboxyl terminus markedly reduced this phosphorylation. These results suggest that the stimulatory effect of PKA on G6Pase fusion gene transcription in HepG2 cells may be mediated in part by the phosphorylation of HNF-6.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosa-6-Fosfatasa/metabolismo , Proteínas de Homeodominio/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cloranfenicol O-Acetiltransferasa/metabolismo , AMP Cíclico/metabolismo , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Genes Reporteros , Factor Nuclear 6 del Hepatocito , Humanos , Cinética , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
Human IgE antibodies from nine allergic subjects were found to react with poly-L-lysine (PLL) and other polyamines. Radioimmunoassay inhibition studies indicated that the two amino groups, but not the carboxyl, in lysine contributed to the antibody binding and 4-aminomethyl-1,8-octanediamine, a compound containing three primary amino groups, was a better inhibitor than compounds containing only two primary amino groups. Ethylamine showed weak but clear inhibition indicating that even a single amino group could bind to the antibody combining site. Substituted ethanolamine and quaternary ammonium compounds were well recognized by some sera but with others, substitution hampered recognition. Inhibition studies with compounds containing an amino and a carboxyl group at different distances revealed that an adjacent carboxyl group interfered with recognition of the amino group by some IgE antibodies. IgE binding to PLL was examined at different pHs and ionic strengths. Binding was greatest at pH 5-6 to 8 and decreased markedly outside this range. Ionic strengths higher than 0.3 M significantly diminished the binding. These results indicated that binding of specific antibody to polyamine was due to electrostatic interactions of positively charged amino groups in the polyamine with the antibody combining site. These results may be relevant to mechanisms underlying recognition of some allergens in some atopic conditions.
Asunto(s)
Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/inmunología , Poliaminas/inmunología , Polilisina/inmunología , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Concentración Osmolar , Unión Proteica , Relación Estructura-ActividadRESUMEN
In liver and kidney, the terminal step in gluconeogenesis is catalyzed by glucose-6-phosphatase. To examine the effect of the cAMP signal transduction pathway on transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into either the liver-derived HepG2 or kidney-derived LLC-PK cell line. Co-transfection of an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA) markedly stimulated G6Pase-CAT fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple regions are required for this PKA response in both the HepG2 and LLC-PK cell lines. A sequence in the G6Pase promoter that resembles a cAMP response element is required for the full PKA response in both HepG2 and LLC-PK cells. However, in LLC-PK cells, but not in HepG2 cells, a hepatocyte nuclear factor-1 (HNF-1) binding site was critical for the full induction of G6Pase-CAT expression by PKA. Changing this HNF-1 motif to that for the yeast transcription factor GAL4 reduces the PKA response in LLC-PK cells to the same degree as deleting the HNF-1 site. However, co-transfection of this mutated construct with chimeric proteins comprising the GAL4-DNA binding domain ligated to the coding sequence for HNF-1alpha, HNF-1beta, HNF-3, or HNF-4 completely restored the PKA response. Thus, we hypothesize that, in LLC-PK cells, HNF-1 is acting as an accessory factor to enhance PKA signaling through the cAMP response element by altering G6Pase promoter conformation or accessibility rather than specifically affecting some component of the PKA signal transduction pathway.
Asunto(s)
AMP Cíclico/metabolismo , Proteínas de Unión al ADN/fisiología , Glucosa-6-Fosfatasa/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Proteínas Nucleares , Factores de Transcripción/fisiología , Transcripción Genética , Animales , Secuencia de Bases , Dominio Catalítico , Línea Celular , Glucosa-6-Fosfatasa/genética , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Datos de Secuencia Molecular , Transducción de Señal , PorcinosRESUMEN
BACKGROUND: CD8 T cells are important immunoregulatory cells in animal models of allergic disease, but their role in human allergic immune responses has not been defined. With the development of novel immunotherapeutic reagents, it is clearly important to ascertain whether CD8 T-cell responses are altered following conventional allergen-specific immunotherapy (SIT) and hence targets for future developments/strategies. OBJECTIVE: To study the allergen-specific cytokine release of freshly isolated CD8 T cells from the blood of separate groups of house dust mite- (HDM) allergic patients, patients post-SIT and control nonatopic donors. METHODS: CD8 T cells were isolated by positive selection with immunomagnetic beads and cultured with the affinity purified major mite allergen Der p 1 or with different mitogens, using irradiated autologous peripheral blood mononuclear cells as antigen-presenting cells (APCs). Supernatants were collected at a number of time points and assayed by ELISA for the cytokines interleukin (IL) -4, IL-5 and interferon-gamma (IFNgamma). RESULTS: CD8 T cells stimulated with Der p 1 produced significant quantities of IFNgamma with cells from HDM-allergic subjects releasing considerably more IFNgamma than cells from nonatopic subjects, an average of 804 +/- 283 pg/mL of supernatant compared with 30.2 +/- 18.8 pg/mL (P = 0.006). The cytokine was detected in cultures of 16/17 of the allergic subjects and 4/7 of the nonatopic. CD8 T cells from 6/10 patients who had received HDM-SIT released IFNgamma at an average of 363 +/- 202 pg/mL, which was less than the allergic group but still higher than the nonatopic (P = 0.05). Equivalent levels of IFNgamma were detected when the cells were stimulated with the mitogen PHA and this was the same in all groups. Reliable allergen-specific release of significant quantities of IL-4 or IL-5 was not detected from CD8 T cells. CONCLUSION: Allergen-specific IFNgamma is produced at far greater levels from CD8 T cells of HDM-sensitive allergic patients than from nonatopic control individuals and this level is reduced following SIT.
Asunto(s)
Alérgenos/inmunología , Linfocitos T CD8-positivos/inmunología , Conjuntivitis Alérgica/inmunología , Desensibilización Inmunológica , Interferón gamma/biosíntesis , Monocitos/inmunología , Hipersensibilidad Respiratoria/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos Dermatofagoides , Conjuntivitis Alérgica/terapia , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/inmunología , Humanos , Interleucinas/biosíntesis , Activación de Linfocitos , Ácaros , Hipersensibilidad Respiratoria/terapiaRESUMEN
BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.
Asunto(s)
Alérgenos/inmunología , Glicoproteínas/inmunología , Hipersensibilidad Respiratoria/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos , Antígenos Dermatofagoides , Relación Dosis-Respuesta Inmunológica , Eosinofilia/patología , Inmunización , Inmunoglobulina E/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ácaros/inmunología , Moco/citología , Pruebas de Provocación Nasal , Sistema Respiratorio/patología , Linfocitos T/inmunologíaRESUMEN
Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, we have investigated whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. We have previously shown that the accessory factor binding region A is hepatocyte nuclear factor-1, and we show here that the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa-6-Fosfatasa/genética , Insulina/farmacología , Regiones Promotoras Genéticas , Elementos de Respuesta , Factores de Transcripción/genética , Animales , Secuencia de Bases , Dominio Catalítico , Cloranfenicol O-Acetiltransferasa/genética , Secuencia Conservada , Glucosa-6-Fosfatasa/química , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/efectos de los fármacosRESUMEN
Associations were sought between the presence of allergic sensitivity to Der p 1, a major allergen from the house dust mite, and the HLA-DPB1 genotype. Whilst allergic patients did not differ from controls in DPB1 allelic distribution, there was a correlation of DPB1*11011 with strongly reactive T-cell proliferative responses to Der p 1 and high titre specific IgE to Dermatophagoides pteronyssinus.
Asunto(s)
Alelos , Polvo/efectos adversos , Antígenos HLA-DP/genética , Hipersensibilidad/genética , Ácaros/inmunología , Alérgenos/genética , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides , Genotipo , Glicoproteínas/genética , Glicoproteínas/inmunología , Antígenos HLA-DP/inmunología , Cadenas beta de HLA-DP , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Cadenas beta de HLA-DQ , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Cadenas HLA-DRB1 , Humanos , Inmunofenotipificación , Linfocitos T/inmunologíaRESUMEN
Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.