RESUMEN
AIMS: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. RESULTS: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. INNOVATION: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. CONCLUSION: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.
Asunto(s)
Factor 7 de Crecimiento de Fibroblastos/metabolismo , Peroxiredoxina VI/biosíntesis , Activación Transcripcional/genética , Animales , Elementos de Respuesta Antioxidante/genética , Dexametasona/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxiredoxina VI/metabolismo , Alveolos Pulmonares/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: [Arg8]-vasopressin (AVP) activates 3 G-protein-coupled receptors: V1A, V2, and V1B. The AVP-V1A receptor is the primary AVP receptor in the heart; however, its role in cardiac homeostasis is controversial. To better understand AVP-mediated signaling in the heart, we created a transgenic mouse with controlled overexpression of the V1A receptor. METHODS AND RESULTS: The V1A receptor transgene was placed under the control of the tetracycline-regulated, cardiac-specific α-myosin heavy chain promoter (V1A-TG). V1A-TG mice had a normal cardiac function phenotype at 10 weeks of age; however, by 24 weeks of age, tetracycline-transactivating factor/V1A-TG mouse hearts had reduced cardiac function, cardiac hypertrophy, and dilatation of the ventricular cavity. Contractile dysfunction was also observed in isolated adult cardiac myocytes. When V1A receptor transgene was induced to be expressed in adult mice (V1A-TG(Ind)), left ventricular dysfunction and dilatation were also seen, albeit at a later time point. Because the V1A receptor mediates cell signaling through Gα(q) protein, we blocked Gα(q) signaling by crossing tetracycline-transactivating factor/V1A mice with transgenic mice that expressed a small inhibitory peptide against Gα(q). Gα(q) blockade abrogated the development of the heart failure phenotype in tetracycline-transactivating factor/V1A-TG mice. The heart failure phenotype could be reversed by administration of doxycycline. CONCLUSIONS: Our results demonstrate a role for V1A-mediated signaling in the development of heart failure and support a role for V1A blockade in the treatment of patients with elevated levels of vasopressin.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Vasopresinas/genética , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/metabolismo , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Fenotipo , Embarazo , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Prdx6 (peroxiredoxin 6), a bifunctional protein with both GSH peroxidase and PLA(2) (phospholipase A(2)) [aiPLA(2) (acidic calcium-independent PLA(2))] activities, is responsible for the metabolism of lung surfactant phospholipids. We propose that the aiPLA(2) activity of the enzyme is regulated through phosphorylation. Incubation of isolated rat alveolar type II cells (AECII) with PMA, a PKC (protein kinase C) agonist, had no effect on Prdx6 expression but led to approximately 75% increase in aiPLA(2) activity that was abolished by pretreatment of cells with the MAPK (mitogen-activated protein kinase) inhibitors, SB202190 or PD98059. Prdx6 phosphorylation after incubation of AECII with PMA was demonstrated by autoradiography after immunoprecipitation with either anti-phosphothreonine o-phosphoserine antibodies. in vitro, several active isoforms of ERK (extracellular-signal-regulated kinase) and p38 phosphorylated Prdx6, resulting in an 11-fold increase in aiPLA(2) activity. The increased activity was calcium-independent and was abolished by the aiPLA(2) inhibitors, surfactant protein A and hexadecyl-3-trifluorethylglycero-sn-2-phospho-methanol (MJ33). The peroxidase activity of Prdx6 was unaffected by phosphorylation. Mass spectroscopic analysis of in vitro phosphorylated Prdx6 showed a unique phosphorylation site at Thr-177 and mutation of this residue abolished protein phosphorylation and the increase in MAPK-mediated activity. These results show that the MAPKs can mediate phosphorylation of Prdx6 at Thr-177 with a consequent marked increase in its aiPLA(2) activity.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peroxiredoxina VI/metabolismo , Fosfolipasas A2/metabolismo , Animales , Extractos Celulares , Electroforesis en Gel Bidimensional , Endocitosis/efectos de los fármacos , Humanos , Inmunoprecipitación , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación/genética , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/enzimología , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacología , Treonina/metabolismoRESUMEN
Peroxiredoxin 6 (Prdx6) is a unique antioxidant enzyme that can reduce phospholipid and other hydroperoxides. A549 cells, a human lung-derived cell line, express both Prdx6 and Nrf2, a transcription factor that binds to antioxidant-response elements (AREs) and promotes expression of antioxidant genes. Treatment of A549 cells with 500 microM H(2)O(2) increased Prdx6 mRNA levels 2.5-fold, whereas treatment with 400 microM H(2)O(2) or 200 microM tert-butylhydroquinone (t-BHQ) triggered a corresponding 2.5-fold increase in reporter gene activity in A549 cells transfected with the pSEAP2:Basic vector (BD Bioscience), containing 1524 nucleotides of the human Prdx6 promoter region. Deletion of a consensus ARE sequence present between positions 357 and 349 before the start of transcription led to a striking decrease in both basal and H(2)O(2)- or t-BHQ-induced activation in A549 cells and H(2)O(2)-induced activation in primary rat alveolar type II cells. Cotransfection with Nrf2 stimulated the Prdx6 promoter in an ARE-dependent manner, whereas it was negatively regulated by Nrf3. siRNA targeting Nrf2 down-regulated reporter gene expression, whereas siRNA targeting the Nrf2 repressor, Keap1, up-regulated it. Binding of Nrf2 to the ARE sequence in chromatin was confirmed by PCR after chromatin immunoprecipitation. These data demonstrate that the ARE within the Prdx6 promoter is a key regulator of basal transcription of the Prdx6 gene and of its inducibility under conditions of oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Células Epiteliales/fisiología , Estrés Oxidativo/fisiología , Peroxiredoxina VI/metabolismo , Activación Transcripcional/fisiología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Línea Celular , Ensamble y Desensamble de Cromatina , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/genética , Células Epiteliales/citología , Retroalimentación Fisiológica , Humanos , Peróxido de Hidrógeno/farmacología , Hidroquinonas/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Pulmón/citología , Masculino , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Peroxiredoxina VI/genética , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Eliminación de Secuencia , Activación Transcripcional/efectos de los fármacos , TransfecciónRESUMEN
Receptor-mediated signaling is commonly associated with multiple functions, including the production of reactive oxygen species. However, whether mitochondrion-derived superoxide (mROS) contributes directly to physiological signaling is controversial. Here we demonstrate a previously unknown mechanism in which physiologic Ca(2+)-evoked mROS production plays a pivotal role in endothelial cell (EC) activation and leukocyte firm adhesion. G protein-coupled receptor (GPCR) and tyrosine kinase-mediated inositol 1,4,5-trisphosphate-dependent mitochondrial Ca(2+) uptake resulted in NADPH oxidase-independent mROS production. However, GPCR-linked mROS production did not alter mitochondrial function or trigger cell death but rather contributed to activation of NF-kappaB and leukocyte adhesion via the EC induction of intercellular adhesion molecule 1. Dismutation of mROS by manganese superoxide dismutase overexpression and a cell-permeative superoxide dismutase mimetic ablated NF-kappaB transcriptional activity and facilitated leukocyte detachment from the endothelium under simulated circulation following GPCR- but not cytokine-induced activation. These results demonstrate that mROS is the downstream effector molecule that translates receptor-mediated Ca(2+) signals into proinflammatory signaling and leukocyte/EC firm adhesion.
Asunto(s)
Calcio/metabolismo , Células Endoteliales/citología , Leucocitos/citología , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Pollos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/ultraestructura , Humanos , Inflamación , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/efectos de los fármacos , Ratones , Mitocondrias/enzimología , Mitocondrias/patología , Mitocondrias/ultraestructura , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Receptor PAR-1/metabolismo , Superóxidos/metabolismo , Trombina/farmacologíaRESUMEN
Connective tissue growth factor (CTGF/CCN2) is an immediate early gene-encoded polypeptide modulating cell growth and collagen synthesis. The importance of CTGF/CCN2 function is highlighted by its disregulation in fibrotic disorders. In this study, we investigated the regulation and signaling pathways that are required for various stimuli of intracellular signaling events to induce the expression of the endogenous CTGF/CCN2 gene in smooth muscle cells. Incubation with the bioactive lysolipid sphingosine 1-phosphate (S1P) produced a threefold increase, whereas stimulation with either fetal bovine serum or anisomycin induced an even stronger activation (eightfold) of CTGF/CCN2 expression. Using a combination of pathway-specific inhibitors and mutant forms of signaling molecules, we found that S1P- and fetal bovine serum-induced CTGF/CCN2 expression were dependent on both RhoA GTPase and p38 mitogen-activated protein kinase transduction pathways, whereas the effects of anisomycin largely involved p38 and phosphatidyl inositol 3-kinase signaling mechanisms. However, activation via these signaling events was absolutely dependent on actin cytoskeleton integrity. In particular, RhoA-dependent regulation of the CTGF/CCN2 gene was concomitant to increased polymerization of actin microfilaments resulting in decreased G- to F-actin ratio and appeared to be achieved at the transcriptional level. The p38 signaling pathway was RhoA-independent and led to CTGF/CCN2 mRNA stabilization. Use of actin-binding drugs showed that the actual physical state of monomeric G-actin is a critical determinant for CTGF/CCN2 gene induction. These data indicate that distinct cytoskeletally based signaling events within the intracellular signaling machinery affect either transcriptionally or post-transcriptionally the expression of the CTGF/CCN2 gene in smooth muscle cells.