RESUMEN
OBJECTIVE: The aim of this project was to test the hypothesis that redox factor 1 (Ref-1) was a critical upstream determinant of NF-kappaB-dependent survival signaling pathways in the vessel wall. METHODS AND RESULTS: Aortas from hemizygous transgenic mice harboring a single allele of Ref-1 exhibited a significant loss in NF-kappaB DNA binding activity. The NF-kappaB-dependent survival gene A20 was significantly downregulated in aortas of hemizygous Ref-1 mice, whereas IAP-2 was unchanged. Overexpression of A20 rescued cells from tumor necrosis factor (TNF)-induced apoptosis, suggesting that the loss of A20 in Ref-1 hemizygotes may be a rate-determining step in endothelial cell fate. Deletion of the previously defined redox-sensitive or the AP endonuclease domains of Ref-1 significantly decreased NF-kappaB transcriptional activation and endothelial cell survival. Furthermore, TNF-induced apoptosis was significantly potentiated in endothelial cells after delivery of Morpholino antisense oligodeoxynucleotides targeted to Ref-1. Loss of the redox-sensitive domain blocked the ability of Ref-1 to reduce p50; however, loss of the endonuclease domain did not effect p50 reduction, suggesting alternative mechanisms of action of Ref-1 on NF-kappaB activity. CONCLUSIONS: These findings establish a role for Ref-1 as an upstream determinant of NF-kappaB and A20-dependent signaling and endothelial survival in the vessel wall.
Asunto(s)
Apoptosis/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/deficiencia , Células Endoteliales/patología , FN-kappa B/metabolismo , Animales , Aorta , Bovinos , Línea Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/fisiología , Endotelio Vascular/patología , Ratones , Ratones Endogámicos C57BL , Homología de Secuencia de AminoácidoRESUMEN
NF-kappaB is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. During the screening of a plant extract library of traditional Chinese herbal medicines, we found that NF-kappaB activity was potently inhibited by andrographolide (Andro), an abundant component of the plant Andrographis that has been commonly used as a folk remedy for alleviation of inflammatory disorders in Asia for millennia. Mechanistically, it formed a covalent adduct with reduced cysteine (62) of p50, thus blocking the binding of NF-kappaB oligonucleotide to nuclear proteins. Andro suppressed the activation of NF-kappaB in stimulated endothelial cells, which reduced the expression of cell adhesion molecule E-selectin and prevented E-selectin-mediated leukocyte adhesion under flow. It also abrogated the cytokine- and endotoxin-induced peritoneal deposition of neutrophils, attenuated septic shock, and prevented allergic lung inflammation in vivo. Notably, it had no suppressive effect on IkappaBalpha degradation, p50 and p65 nuclear translocation, or cell growth rates. Our results thus reveal a unique pharmacological mechanism of Andro's protective anti-inflammatory actions.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Cisteína/metabolismo , Diterpenos/farmacología , Medicamentos Herbarios Chinos/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/aislamiento & purificación , Unión Competitiva/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Diterpenos/aislamiento & purificación , Diterpenos/metabolismo , Evaluación Preclínica de Medicamentos , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/metabolismo , Selectina E/biosíntesis , Selectina E/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores de Crecimiento/farmacología , Células HL-60 , Humanos , Hipersensibilidad/patología , Hipersensibilidad/prevención & control , Proteínas I-kappa B/antagonistas & inhibidores , Proteínas I-kappa B/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Células 3T3 NIH , Infiltración Neutrófila/efectos de los fármacos , Sondas de Oligonucleótidos/antagonistas & inhibidores , Sondas de Oligonucleótidos/metabolismo , Oxidación-Reducción , Peritonitis/patología , Peritonitis/prevención & control , Unión Proteica/efectos de los fármacos , Choque Séptico/prevención & control , Factor de Transcripción ReIARESUMEN
Recent studies show that NF-kappa B activator 1 (Act1) functions as an important adapter molecule for CD40-mediated signaling in epithelial cells. To explore the physiological function of the CD40-Act1 axis, we studied the regulation of gene expression of CD40 and Act1 both in vivo and in cell culture models. Although CD40 and Act1 are up-regulated in mouse lung upon LPS stimulation, IL-1 plus IFN-alpha, -beta, or -gamma synergistically up-regulate both CD40 and Act1 gene expression in human epithelial A549 cells. Cycloheximide superinduces the Act1 mRNA, whereas actinomycin D completely abolishes the Act1 mRNA, indicating that the induction of Act1 mRNA is at the transcriptional level and does not require protein synthesis. Promoter sequence analyses identified putative IFN regulatory factor (IRF)-1, C/EBP-beta, and AP-1 transcription factor binding sites in the Act1 promoter. Although mutation of any of the three sites abolished the promoter activity, Abs against IRF-1 and C/EBP-beta, but not AP-1, blocked the formation of the DNA-binding complex induced by IL-1 plus IFN-beta stimulation, suggesting cooperative action between IRF-1 and C/EBP-beta in mediating Act1 promoter activity. Importantly, LPS-induced gene expression of CD40 and Act1 in the mouse lung is abolished in IRF-1(-/-) mice, indicating an essential role of transcription factor IRF-1 in the coordinated regulation of these two genes during airway inflammation. The induced expression of the CD40-Act1 axis by inflammatory cytokines in epithelial cells probably plays an important role in priming these cells for their response to CD40 ligand during airway inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antígenos CD40/fisiología , Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/fisiología , Interferón gamma/fisiología , Pulmón/inmunología , Pulmón/patología , Fosfoproteínas/fisiología , Regulación hacia Arriba/inmunología , Animales , Secuencia de Bases , Proteína beta Potenciadora de Unión a CCAAT/genética , Antígenos CD40/biosíntesis , Antígenos CD40/genética , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Línea Celular , Citocinas/farmacología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células HT29 , Células HeLa , Humanos , Inflamación/genética , Inflamación/inmunología , Factor 1 Regulador del Interferón , Interferón gamma/genética , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Regiones Promotoras Genéticas/inmunología , Secuencias Reguladoras de Ácidos Nucleicos/inmunología , Factor de Transcripción AP-1/genética , Células Tumorales Cultivadas , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral , Regulación hacia Arriba/genéticaRESUMEN
Act1 (also called CIKS) is a recently identified molecule, which activates NF-kappaB and AP-1. Here, we identified alternatively spliced Act1 that lacked the exon 2 encoding the first nine amino acids in the amino terminus of the protein. Compared to full-length Act1, this truncated Act1 appeared to be equally active. We demonstrated further that only the spliced Act1 was detected in cDNA libraries derived from human fetal brain, liver, leukocytes, and bone marrow. In contrast, both the spliced and full-length Act1 templates were detected in a variety of human cancer cell lines. The expression of both the spliced and full-length transcripts was detected at 4-h time point, following the treatment of endothelial cells with tumor necrosis factor-alpha, interleukin-1beta or bacterial endotoxin. Notably, the dominant amounts of the spliced Act1 over the full-length Act1 were amplified from both the cancer cell mRNAs and the stimulated endothelial cell mRNAs. Taken together with the act1 chromosome localization at the 6q21 subregion, our findings indicate that the newly identified alternatively spliced Act1 is a major transcript of the molecule and that Act1 may play important roles in oncogenesis.