RESUMEN
Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells (VSMC). This process is dependent on nuclear factor-kappa B (NF-kappaB) activation and is suppressed by glucocorticoids. The aim of this study was to investigate the molecular mechanisms of inhibition of iNOS expression by the synthetic glucocorticoid, dexamethasone (DEX), in rat VSMC. Treatment of VSMC with LPS plus interferon-gamma (LPS/IFN) caused activation of NF-kappaB and the iNOS promoter. LPS/IFN induced iNOS mRNA and NO synthesis. DEX markedly depressed LPS/IFN-stimulated iNOS mRNA expression and NO production. DEX also suppressed LPS/IFN-stimulated activity of a 1.7-kb iNOS promoter, indicating that the inhibition of iNOS expression by DEX occurs at the level of transcription. NF-kappaB activation by LPS/IFN was repressed by DEX. The inhibition of NF-kappaB by DEX exhibited dose-dependent kinetics, which corresponded to DEX suppression of iNOS promoter activation, iNOS mRNA expression, and NO production. However, activation of activator protein-1 (AP-1), which is also contained in the iNOS promoter, was not enhanced by LPS/IFN or inhibited by DEX. Thus, glucocorticoids appear to block iNOS expression, at least in part, through inhibition of NF-kappaB activation, which results in decreased NO production.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Músculo Liso Vascular/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Animales , Aorta Torácica , Células Cultivadas , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Expresión Génica , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Masculino , Mifepristona/farmacología , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.
Asunto(s)
Aldehídos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Desarrollo de Músculos , Músculo Liso Vascular/citología , Músculo Liso Vascular/crecimiento & desarrollo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/genéticaRESUMEN
Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.
Asunto(s)
División Celular/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Proteína Quinasa C/metabolismo , Albúmina Sérica/farmacología , Animales , Línea Celular , ADN/biosíntesis , ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Productos Finales de Glicación Avanzada , Humanos , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Maleimidas/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional/efectos de los fármacos , Albúmina Sérica GlicadaRESUMEN
A cyclopentenone-type prostaglandin, 15-deoxy-Delta(12, 14)-prostaglandin J(2) (15-d-PGJ(2)), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ(2)-treated cells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production was decreased in untreated cells according to duration of culture. PGD(2) and PGJ(2), which are precursors to 15-d-PGJ(2), exhibited an effect similar to 15-d-PGJ(2). However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-gamma, and WY-14643, a specific PPAR-alpha ligand, lacked this effect. 15-d-PGJ(2) and its precursors, but not the thiazolidinediones, induced gene expression for heme oxygenase-1 (HO-1), a stress-related protein, and strongly inhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production. Cyclopentenone-type PGs have been recently shown to inhibit nuclear factor-kappaB (NF-kappaB) activation via a direct and PPAR-independent inhibition of inhibitor-kappaB kinase, suggesting that, in human thyrocytes, such PGs may inhibit IL-1-induced NO production, possibly via an inhibition of NF-kappaB activation. On the other hand, sodium arsenite, a known activator of the stress response pathway, induced HO-1 mRNA expression but lacked a promoting effect on TG production. Thus 15-d-PGJ(2) and its precursors appear to facilitate TG production via a PPAR-independent mechanism and through a different pathway from the cellular stress response that is available to cyclopentenone-type PGs. Our findings reveal a novel role of these PGs associated with thyrocyte differentiation.