RESUMEN
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
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
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
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
Several of the complications seen in patients with both type I and type II diabetes mellitus are associated with alterations in the expression of matrix metalloproteinases. To identify the cis-acting elements that mediate the stimulatory effect of insulin on collagenase-1 (matrix metalloproteinase-1) gene transcription a series of collagenase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into HeLa cells. Multiple promoter elements, including an Ets and activator protein-1 (AP-1) motif, were required for the effect of insulin. The AP-1 motif appears to be a target for insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene, whereas the data suggest that the Ets motif acts to enhance the effect of insulin mediated through the AP-1 motif. Multiple promoter elements were also required for the stimulatory effect of phorbol esters on collagenase-CAT gene transcription, and the AP-1 motif was also a target for phorbol ester signaling. However, the cis-acting elements required for the effects of insulin and phorbol esters were not identical. Moreover, phorbol esters were a much more potent inducer of collagenase-CAT gene transcription than insulin, a difference that may be explained by selective effects of insulin and phorbol esters on AP-1 expression.
Asunto(s)
Colagenasas/genética , Insulina/farmacología , Ésteres del Forbol/farmacología , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión , Células CHO , Cloranfenicol O-Acetiltransferasa/genética , Cricetinae , Genes Reporteros , Células HeLa , Humanos , Metaloproteinasa 1 de la Matriz , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacosRESUMEN
In liver and kidney, the terminal step in the gluconeogenic pathway is catalyzed by glucose-6-phosphatase (G-6-Pase). This enzyme is actually a multicomponent system, the catalytic subunit of which was recently cloned. Numerous reports have also described the presence of G-6-Pase activity in islets, although the role of G-6-Pase in this tissue is unclear. Arden and associates have described the cloning of a novel cDNA that encodes an islet-specific G-6-Pase catalytic subunit-related protein (IGRP) (Arden SD, Zahn T, Steegers S, Webb S, Bergman B, O'Brien RM, Hutton JC: Molecular cloning of a pancreatic islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP). Diabetes 48:531-542, 1999). We screened a mouse BAC library with this cDNA to isolate the IGRP gene, which spans approximately 8 kbp of genomic DNA. The exon/intron structure of the IGRP gene has been mapped and, as with the gene encoding the liver/kidney G-6-Pase catalytic subunit, it is composed of five exons. The sizes of these exons are 254 (I), 110 (II), 112 (III), 116 (IV), and 1284 (V) bp, similar to those of the G-6-Pase catalytic subunit gene. Two interspecific backcross DNA mapping panels were used to unambiguously localize the IGRP gene (map symbol G6pc-rs) to the proximal portion of mouse chromosome 2. The IGRP gene transcription start site was mapped by primer extension analysis, and the activity of the IGRP gene promoter was analyzed in both the islet-derived HIT cell line and the liver-derived HepG2 cell line. The IGRP and G-6-Pase catalytic subunit gene promoters show a reciprocal pattern of activity, with the IGRP promoter being approximately 150-fold more active than the G-6-Pase promoter in HIT cells.
Asunto(s)
Mapeo Cromosómico , Glucosa-6-Fosfatasa/genética , Islotes Pancreáticos/metabolismo , Regiones Promotoras Genéticas , Proteínas/genética , Animales , Secuencia de Bases , Carcinoma Hepatocelular , Exones , Biblioteca de Genes , Marcadores Genéticos , Humanos , Intrones , Riñón/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas , Ratones , Datos de Secuencia Molecular , Proteínas/química , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyl-transferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.
Asunto(s)
Insulina/farmacología , Isoenzimas/biosíntesis , Hígado/efectos de los fármacos , Malato Deshidrogenasa/biosíntesis , Regiones Promotoras Genéticas , Transcripción Genética/genética , Animales , Colagenasas/genética , Citosol/enzimología , Isoenzimas/genética , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/patología , Malato Deshidrogenasa/genética , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transfección , Células Tumorales CultivadasRESUMEN
Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. Transcription of the gene encoding the glucose-6-phosphatase catalytic subunit (G6Pase) is stimulated by cAMP and glucocorticoids whereas insulin strongly inhibits both this induction and basal G6Pase gene transcription. Previously, we have demonstrated that the maximum repression of basal G6Pase gene transcription by insulin requires two distinct promoter regions, designated A (from -271 to -199) and B (from -198 to -159). Region B contains an insulin response sequence because it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. By contrast, region A fails to mediate an insulin response in a heterologous context, and the mutation of region B within an otherwise intact promoter almost completely abolishes the effect of insulin on basal G6Pase gene transcription. Therefore, region A is acting as an accessory element to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. Such an arrangement is a common feature of cAMP and glucocorticoid-regulated genes but has not been previously described for insulin. A combination of fusion gene and protein-binding analyses revealed that the accessory factor binding region A is hepatocyte nuclear factor-1. Thus, despite the usually antagonistic effects of cAMP/glucocorticoids and insulin, all three agents are able to use the same factor to enhance their action on gene transcription. The potential role of G6Pase overexpression in the pathophysiology of MODY3 and 5, rare forms of diabetes caused by hepatocyte nuclear factor-1 mutations, is discussed.
Asunto(s)
Proteínas de Unión al ADN , Glucosa-6-Fosfatasa/genética , Insulina/farmacología , Proteínas Nucleares , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cartilla de ADN , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Células Tumorales CultivadasRESUMEN
Transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase) is stimulated by glucocorticoids and strongly repressed by insulin. We have explored the signaling pathways by which insulin mediates the repression of G6Pase transcription in H4IIE cells. Wortmannin, a phosphatidylinositide 3-kinase (PtdIns 3-kinase) inhibitor blocked the repression of G6Pase mRNA expression by insulin. However, both rapamycin, which inhibits p70S6 kinase activation, and PD98059, an inhibitor of mitogen-activated protein kinase activation, were without effect. Insulin inhibited dexamethasone-induced luciferase expression from a transiently transfected plasmid that places the luciferase gene under the control of the G6Pase promoter. This effect of insulin was mimicked by the overexpression of a constitutively active PtdIns 3-kinase but not by a constitutively active protein kinase B. Taken together, these data demonstrate that PtdIns 3-kinase activation is both necessary and at least partly sufficient for the repression of G6Pase expression by insulin, but neither mitogen-activated protein kinase nor p70S6 kinase are involved. In addition, activation of protein kinase B alone is not sufficient for repression of the G6Pase gene. These results imply the existence of a novel signaling pathway downstream of PtdIns 3 kinase that is involved in the regulation of G6Pase expression by insulin.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucosa-6-Fosfatasa/genética , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Dexametasona/farmacología , Genes Reporteros/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/metabolismo , Ratas , Proteínas Represoras/fisiología , Transducción de Señal/fisiología , Transcripción Genética/genética , Transfección/genética , Células Tumorales Cultivadas , WortmaninaRESUMEN
Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways. The transcription of the gene encoding the catalytic subunit of G6Pase is stimulated by glucocorticoids, whereas insulin strongly inhibits both basal G6Pase gene transcription and the stimulatory effect of glucocorticoids. To identify the insulin response sequence (IRS) in the G6Pase promoter through which insulin mediates its action, we have analyzed the effect of insulin on the basal expression of mouse G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes transiently expressed in hepatoma cells. Deletion of the G6Pase promoter sequence between -271 and -199 partially reduces the inhibitory effect of insulin, whereas deletion of additional sequence between -198 and -159 completely abolishes the insulin response. The presence of this multicomponent IRS may explain why insulin potently inhibits basal G6Pase-CAT expression. The G6Pase promoter region between -198 and -159 contains an IRS, since it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. This region contains three copies of the T(G/A)TTTTG sequence, which is the core motif of the phosphoenolpyruvate carboxykinase (PEPCK) gene IRS. This suggests that a coordinate increase in both G6Pase and PEPCK gene transcription is likely to contribute to the increased hepatic glucose production characteristic of patients with non-insulin-dependent diabetes mellitus.
Asunto(s)
Dexametasona/farmacología , Glucosa-6-Fosfatasa/biosíntesis , Glucosa-6-Fosfatasa/genética , Insulina/farmacología , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/biosíntesis , Inducción Enzimática/efectos de los fármacos , Represión Enzimática/efectos de los fármacos , Cinética , Ratones , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxiquinasa (GTP)/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de SecuenciaRESUMEN
Fifteen archival human osteosarcoma specimens were examined by in situ hybridization for the expression of human and mouse transforming growth factor-beta (isoforms 1, 2, and 3), c-fos, and metalloproteinase (stromelysin-3 and matrilysin). Osteosarcoma subtypes were confirmed by review of patients' radiographs, histopathology, and age at diagnosis. The outcome and method of treatment were documented. The subtypes of osteosarcoma consisted of nine conventional osteosarcomas and two each of fibroblastic, telangiectatic, and post-radiation osteosarcomas. Each specimen was histologically examined under light microscopy, and then adjacent paraffin sections were assayed with sense and anti-sense RNA probes by in situ hybridization. The probes localized to the neoplastic cells, confirming the methodology of the technique. Human transforming growth factor-beta 1 had the most uniform binding affinity to the osteosarcomas examined and was more specific in binding than mouse transforming growth factor-beta 1. Specific mRNA encoding for the transforming growth factor-beta s, c-fos, and metalloproteinases are detectable in patterns within osteosarcoma cells, and collectively, their expression parallels the different histopathologic subtypes. The less differentiated subtypes (telangiectatic and post-radiation osteosarcomas) expressed the fewest molecular markers. Osteosarcoma is a heterogeneous tumor. Differential expression of matrilysin in osteosarcoma is the first reported detection of metalloproteinase activity in human skeletal sarcoma.
Asunto(s)
Neoplasias Óseas/genética , Proteínas de Neoplasias/biosíntesis , Osteosarcoma/genética , Adulto , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Niño , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación in Situ , Metaloproteinasa 3 de la Matriz , Metaloproteinasa 7 de la Matriz , Metaloendopeptidasas/biosíntesis , Ratones , Proteínas de Neoplasias/genética , Neoplasias Inducidas por Radiación/genética , Neoplasias Primarias Secundarias/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Sondas ARN , ARN Neoplásico/análisis , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.