RESUMEN
Sesquiterpene lactones, derived from Mexican-Indian medicinal plants, are known to have potent anti-inflammatory properties but the mechanisms of this effect are not completely understood. Recent data demonstrated that sesquiterpene lactones were potent inhibitors of the pro-inflammatory transcription factor NF-kappaB. Because activation of NF-kappaB is involved in the regulation of the chemokine interleukin 8 (IL-8), we hypothesized that the sesquiterpene lactones, isohelenin and parthenolide, would inhibit IL-8 gene expression in cultured human respiratory epithelium. Incubating A549 cells with tumour necrosis factor alpha (TNF-alpha) induced IL-8 mRNA expression and secretion of immunoreactive IL-8. Pretreatment with either isohelenin or parthenolide inhibited TNF-alpha-mediated IL-8 gene expression in a concentration-dependent manner. Pretreatment with either compound inhibited TNF-alpha mediated activation of the IL-8 promoter and TNF-alpha-mediated nuclear translocation of NF-kappaB. In addition, pretreatment with isohelenin or parthenolide inhibited TNF-alpha-mediated degradation of the NF-kappaB inhibitory protein, I-kappaBalpha. We conclude that sesquiterpene lactones are potent in vitro inhibitors of IL-8 gene expression in cultured human respiratory epithelium. The most proximal mechanism of inhibition appears to involve inhibition of I-kappaBalpha degradation. Stabilization of cytoplasmic I-kappaBalpha leads to inhibition of NF-kappaB nuclear translocation and of subsequent IL-8 promoter activation. The ability of sesquiterpene lactones to modulate IL-8 gene expression may explain, in part, their anti-inflammatory effects.
Asunto(s)
Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas I-kappa B , Interleucina-8/genética , Plantas Medicinales/química , Sesquiterpenos/farmacología , Transporte Biológico , Proteínas de Unión al ADN/metabolismo , Epitelio/metabolismo , Humanos , Pulmón/citología , Pulmón/inmunología , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Interleukin (IL)-8 is an important mediator of acute lung injury. Hyperoxia induces IL-8 production in some cell types, but its effect on IL-8 gene expression in respiratory epithelium is not well described. In addition, IL-8 gene expression resulting from the combined effects of hyperoxia and proinflammatory cytokines has not been well characterized. We treated cultured respiratory epithelial-like cells (A549 cells) with hyperoxia alone, tumor necrosis factor (TNF)-alpha alone, or the combination of TNF-alpha and hyperoxia and evaluated IL-8 gene expression. Hyperoxia alone had a minimal effect on IL-8 gene expression, and TNF-alpha alone increased IL-8 gene expression in a time-dependent manner. In contrast, the combination of TNF-alpha and hyperoxia synergistically increased IL-8 gene expression as measured by ELISA (TNF-alpha alone for 24 h = 769 +/- 89 pg/ml vs. hyperoxia + TNF-alpha for 24 h = 1, 189 +/- 89 pg/ml) and Northern blot analyses. Experiments involving IL-8 promoter-reporter assays, electromobility shift assays, and Western blot analyses demonstrated that hyperoxia augmented TNF-alpha-mediated activation of the IL-8 promoter by a nuclear factor (NF)-kappaB-dependent mechanism and increased the duration of NF-kappaB nuclear translocation after concomitant treatment with TNF-alpha. Additional reporter gene assays demonstrated, however, that increased activation of NF-kappaB does not fully account for the synergistic effect of hyperoxia and that the NF-IL-6 site in the IL-8 promoter is also required for the synergistic effect of hyperoxia. We conclude that hyperoxia alone has a minimal effect on IL-8 gene expression but synergistically increases IL-8 gene expression in the presence of TNF-alpha by a mechanism involving cooperative interaction between the transcription factors NF-kappaB and NF-IL-6.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Expresión Génica/efectos de los fármacos , Hiperoxia/genética , Interleucina-8/genética , Factores de Transcripción , Factor de Necrosis Tumoral alfa/farmacología , Sitios de Unión/genética , Proteína delta de Unión al Potenciador CCAAT , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Genes Reporteros/genética , Humanos , Interleucina-8/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Mutación/fisiología , FN-kappa B/metabolismo , FN-kappa B/fisiología , Proteínas Nucleares/fisiología , Plásmidos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Recent data indicate that the heat shock response inhibits nuclear translocation of the proinflammatory transcription factor NF-kappaB. Under basal conditions NF-kappaB is retained in the cytoplasm by an inhibitory protein called I-kappaB which exists as two major isoforms: I-kappaBalpha and I-kappaBbeta. Induction of the heat shock response in BEAS-2B cells, a human cell line representative of bronchial epithelium, increased expression of I-kappaBalpha mRNA in a time-dependent manner. Coincubation with actinomycin-D inhibited heat shock-mediated expression of I-kappaBalpha mRNA. Transient transfection assays with a plasmid containing the reporter gene firefly luciferase, under the control of the human I-kappaBalpha promoter, demonstrated that heat shock activated the I-kappaBalpha promoter. Heat shock-mediated induction of I-kappaBalpha was associated with inhibition of NF-kappaB activation. We conclude that heat shock increases I-kappaBalpha mRNA expression in BEAS-2B cells by activating the I-kappaBalpha promoter, and propose that heat shock-mediated up-regulation of I-kappaBalpha is a potential mechanism by which the heat shock response inhibits proinflammatory responses in lung cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas I-kappa B , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Calor , Humanos , Inflamación/prevención & control , Luciferasas/genética , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Nitric oxide (NO) is an important regulator and effector molecule in various inflammatory disease states. High output of NO during inflammation is generated by the inducible isoform of nitric oxide synthase (iNOS). Sesquiterpene lactones are derived from Mexican-Indian medicinal plants and are known to have potent anti-inflammatory properties. The mechanisms by which sesquiterpene lactones exert their anti-inflammatory effects are not fully understood. In the current studies we determined if the sesquiterpene lactones, parthenolide and isohelenin, modulate iNOS gene expression in cultured rat aortic smooth muscle cells (RASMC) treated with lipopolysaccharide and interferon-gamma. Treatment with parthenolide or isohelenin inhibited NO production and iNOS mRNA expression in a concentration-dependent manner. Transient transfection studies with an iNOS promoter-luciferase reporter plasmid demonstrated that parthenolide and isohelenin also inhibited activation of the iNOS promoter. Inhibition of iNOS promoter activation was associated with inhibition of both I-kappaBalpha degradation and nuclear translocation of NF-kappaB. Neither parthenolide nor isohelenin induced the heat shock response in RASMC. We conclude that sesquiterpene lactones inhibit iNOS gene expression by a mechanism involving stabilization of the I-kappaBalpha/NF-kappaB complex. This effect is not related to induction of the heat shock response. The ability of sesquiterpene lactones to inhibit iNOS gene expression may account, in part, for their anti-inflammatory effects.
Asunto(s)
Proteínas I-kappa B , Lactonas/farmacología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Sesquiterpenos/farmacología , Animales , Antiinflamatorios/farmacología , Aorta/citología , Aorta/enzimología , Compartimento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Músculo Liso Vascular/enzimología , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Ratas , Sesquiterpenos/toxicidadRESUMEN
Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.
Asunto(s)
Catalasa/genética , Glutatión Peroxidasa/genética , Proteínas HSP70 de Choque Térmico/biosíntesis , Oxígeno/toxicidad , Superóxido Dismutasa/genética , Adenocarcinoma , Adenosina Trifosfato/metabolismo , Catalasa/biosíntesis , Supervivencia Celular , Citomegalovirus/genética , Glutatión Peroxidasa/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Humanos , Hiperoxia , Cinética , Peroxidación de Lípido , Neoplasias Pulmonares , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Superóxido Dismutasa/biosíntesis , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-alpha induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-alpha-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-alpha-mediated activation of the RANTES promoter. Inhibition of NF-kappaB nuclear translocation with isohelenin inhibited TNF-alpha-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-kappaB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-kappaB inhibitory protein, I-kappaBalpha but did not significantly inhibit phosphorylation of I-kappaBalpha. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-kappaB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-kappaB nuclear translocation involves stabilization of I-kappaBalpha, without significantly affecting phosphorylation of I-kappaBalpha.
Asunto(s)
Quimiocina CCL5/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Respuesta al Choque Térmico/genética , Proteínas I-kappa B , Pulmón/metabolismo , Adenocarcinoma , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Neoplasias Pulmonares , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Fosforilación , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Induction of heat shock proteins (HSPs) confers protection against a variety of cytotoxic agents. We hypothesized that induction of HSPs would protect cultured human respiratory epithelium against nitric oxide (NO)-mediated injury. Incubation of a human bronchial epithelial cell line (BEAS-2B cells) at 43 degrees C for 1.5 h induced expression of several HSPs. Prior induction of HSPs was associated with protection against the NO-donors S-nitroso-N-acetyl penicillamine and 3-morpholinsydnonimine. Protection was evident as improved short term survival and improved ability of cells to recover and proliferate after exposure to NO. Prior induction of HSPs also attenuated NO-mediated decreases in cellular ATP levels, but did not decrease nitrotyrosine formation. Specific overexpression of HSP-70 by plasmid-directed gene transfer protected murine respiratory epithelial cells against S-nitroso-N-acetyl penicillamine. We conclude that in cultured human respiratory epithelium induction of HSPs confers protection against NO-mediated cytotoxicity, possibly by preservation of cellular energetics. We also suggest that HSP-70 may play a specific role in protection.