RESUMEN
The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Segregación Cromosómica , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Humanos , Ratones , Ratones Mutantes , Mitosis/genética , Neoplasias/genética , Neoplasias/patología , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genéticaRESUMEN
The Death Receptor 3 (DR3)/Tumour Necrosis Factor-like cytokine 1A (TL1A) axis stimulates effector T cells and type 2 innate lymphocytes (ILC2) that trigger cytokine release and drive disease pathology in several inflammatory and autoimmune diseases, including murine models of acute allergic lung inflammation (ALI). The aim of this study was to elucidate the role of DR3 in chronic ALI compared to acute ALI, using mice genetically deficient in the DR3 gene (DR3ko). Results showed DR3 expression in the lungs of wild-type mice was up-regulated following induction of acute ALI and this increased expression was maintained in chronic disease. DR3ko mice were resistant to cellular accumulation within the alveolar passages in acute, but not chronic ALI. However, DR3ko mice displayed reduced immuno-histopathology and goblet cell hyperplasia; hallmarks of the asthmatic phenotype; in chronic, but not acute ALI. These data suggest DR3 is a potential therapeutic target, involved in temporally distinct aspects of ALI progression and pathogenesis.
Asunto(s)
Células Caliciformes/patología , Hipersensibilidad/inmunología , Pulmón/inmunología , Linfocitos/inmunología , Neumonía/inmunología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/metabolismo , Enfermedad Aguda , Animales , Células Cultivadas , Enfermedad Crónica , Progresión de la Enfermedad , Femenino , Hiperplasia , Hipersensibilidad/fisiopatología , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía/fisiopatología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/genética , Células Th2/inmunologíaRESUMEN
The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.
Asunto(s)
Apoptosis/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Inflamación/inmunología , Neutrófilos/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Animales Modificados Genéticamente , Benzoatos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Humanos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Morfolinos/genética , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Pez Cebra/genéticaRESUMEN
Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1 protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Metiltransferasas/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Bronquios/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interferón gamma/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Lisina/metabolismo , Metilación/efectos de los fármacos , Metiltransferasas/química , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.
Asunto(s)
Antiinflamatorios/farmacología , Benzamidas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Indazoles/farmacología , Receptores de Glucocorticoides/metabolismo , Androstadienos/química , Androstadienos/farmacología , Antiinflamatorios/química , Benzamidas/química , Benzoquinonas/farmacología , Dexametasona/química , Dexametasona/farmacología , Fluticasona , Células HeLa , Humanos , Enfermedades del Sistema Inmune , Indazoles/química , Lactamas Macrocíclicas/farmacología , Ligandos , Terapia Molecular Dirigida , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Transporte de Proteínas , Receptores de Glucocorticoides/agonistas , Activación Transcripcional/efectos de los fármacosRESUMEN
The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.
Asunto(s)
Isomerasa de Peptidilprolil/fisiología , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional , Línea Celular , Dexametasona/farmacología , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Coactivador 3 de Receptor Nuclear/fisiología , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Fosforilación , Regiones Promotoras Genéticas , Estabilidad Proteica , Receptores de Glucocorticoides/química , Proteínas Represoras/metabolismoRESUMEN
Diurnal variation in inflammatory and immune function is evident in the physiology and pathology of humans and animals, but molecular mechanisms and mediating cell types that provide this gating remain unknown. By screening cytokine responses in mice to endotoxin challenge at different times of day, we reveal that the magnitude of response exhibited pronounced temporal dependence, yet only within a subset of proinflammatory cytokines. Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. Loss of circadian gating was coincident with suppressed rev-erbα expression, implicating this nuclear receptor as a potential link between the clock and inflammatory pathways. This finding was confirmed in vivo and in vitro through genetic and pharmacological modulation of REV-ERBα activity. Circadian gating of endotoxin response was lost in rev-erbα(-/-) mice and in cultured macrophages from these animals, despite maintenance of circadian rhythmicity within these cells. Using human macrophages, which show circadian clock gene oscillations and rhythmic endotoxin responses, we demonstrate that administration of a synthetic REV-ERB ligand, or genetic knockdown of rev-erbα expression, is effective at modulating the production and release of the proinflammatory cytokine IL-6. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin, and identifies REV-ERBα as the key link between the clock and immune function. REV-ERBα may therefore represent a unique therapeutic target in human inflammatory disease.
Asunto(s)
Ritmo Circadiano/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/inmunología , Interleucina-6/inmunología , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/inmunología , Factores de Transcripción ARNTL/genética , Análisis de Varianza , Animales , Endotoxinas/toxicidad , Humanos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Factores de TiempoRESUMEN
It is characteristic of asthma that symptoms worsen overnight, particularly in the early hours of the morning. Nocturnal symptoms in asthma are common and are an important indicator for escalation of treatment. An extensive body of research has demonstrated that nocturnal symptoms of cough and dyspnea are accompanied by circadian variations in airway inflammation and physiologic variables, including airflow limitation and airways hyper-responsiveness. The molecular apparatus that underpins circadian variations, controlled by so called 'clock' genes, has recently been characterised. Clock genes control circadian rhythms both centrally, in the suprachiasmatic nucleus of the brain and peripherally, within every organ of the body. Here, we will discuss how clock genes regulate circadian rhythms. We will focus particularly on the peripheral lung clock and the peripheral immune clock and discuss how these might relate to both the pathogenesis and treatment of asthma.
Asunto(s)
Asma/fisiopatología , Relojes Circadianos/genética , Pulmón/fisiopatología , Factores de Transcripción ARNTL/genética , Antiasmáticos/administración & dosificación , Broncoscopía , Proteínas CLOCK/genética , Cronoterapia de Medicamentos , HumanosRESUMEN
BACKGROUND: We have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1ß stimulation. We analysed signalling transduction and global gene expression after IL-1ß stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models. RESULTS: IL-1ß significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1ß stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1ß stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1ß stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1ß following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose. CONCLUSIONS: These results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.
RESUMEN
As our understanding of inflammatory resolution increases, drugs that trigger proresolution pathways may become significant in treating chronic inflammatory diseases. However, anti-inflammatory drugs are traditionally tested during the first hours of onset (i.e., to dampen leukocyte and edema formation), and their ability to trigger proresolution processes has never been investigated. Moreover, there is no model available to screen for putative proresolving agents. In this study, we present a new strategy to identify therapeutics for their ability to switch inflammation off and restore homeostasis. Injecting 1.0 mg of zymosan i.p. causes transient inflammation characterized by polymorphonuclear neutrophil clearance and dominated by recently described resolution-phase macrophages along with an innate-type lymphocyte repopulation, the latter being a marker of tissue homeostasis. In contrast, 10 mg of zymosan elicits an aggressive response characterized by classically activated macrophages leading to systemic inflammation and impaired lymphocyte repopulation. Although this latter model eventually resolves, it nonetheless represents inflammation in the clinically relevant setting of polymorphonuclear neutrophil/classically activated macrophage dominance driving a cytokine storm. Treating such a reaction therapeutically with proresolution drugs provides quantifiable indices of resolution--polymorphonuclear neutrophil/macrophage clearance, macrophage phenotype switching (classically activated to resolution phase), and repopulation with resolution-phase lymphocytes--cardinal signs of inflammatory resolution and homeostasis in the peritoneum. As an illustration, mice bearing peritonitis induced by 10 mg of zymosan were given ibuprofen, resolvin E1, a prostaglandin D(2) receptor 1 agonist, dexamethasone, rolipram, or azithromycin, and their ability to trigger resolution and homeostasis in this new inflammatory setting was investigated. We present the first model for testing drugs with targeted proresolution properties using quantifiable parameters of inflammatory resolution and homeostasis.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Mediadores de Inflamación/administración & dosificación , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células Cultivadas , Homeostasis/efectos de los fármacos , Homeostasis/inmunología , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/farmacología , Inyecciones Intraperitoneales , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/patología , Peritonitis/inmunología , Peritonitis/patología , Zimosan/administración & dosificaciónRESUMEN
Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-ß. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting ß2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications.
Asunto(s)
Bronquios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Glucocorticoides/toxicidad , Mediadores de Inflamación/metabolismo , Interferón Tipo I/metabolismo , Rhinovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/toxicidad , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/virología , Quimioterapia Combinada , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Fumarato de Formoterol/toxicidad , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas/genética , Proteínas/metabolismo , Rhinovirus/crecimiento & desarrollo , Rhinovirus/inmunología , Transducción de SeñalRESUMEN
The liver X receptors (LXRalpha/beta) are orphan nuclear receptors that are expressed in a large number of cell types and have been shown to have anti-inflammatory properties. Nuclear receptors have previously proved to be amenable targets for small molecular mass pharmacological agents in asthma, and so the effect of an LXR ligand was assessed in models of allergic airway inflammation. LXR agonist, GW 3965, was profiled in rat and mouse models of allergic asthma. In the Brown Norway rats, GW 3965 (3-30 mg/kg) was unable to reduce the bronchoalveolar lavage eosinophilia associated with this model and had no impact on inflammatory biomarkers (eotaxin and IL-1beta). The compound did significantly stimulate ABCA-1 (ATP-binding cassette A1) mRNA expression, indicating that there was adequate exposure/LXR activation. In the mouse model, the LXR ligand surprisingly increased airway reactivity, an effect that was apparent in both the Ag and nonchallenged groups. This increase was not associated with a change in lung tissue inflammation or number of mucus-containing cells. There was, however, a marked increase in airway smooth muscle thickness in both treated groups. We demonstrated an increase in contractile response to exogenous methacholine in isolated airways taken from LXR agonist-treated animals compared with the relevant control tissue. We corroborated these findings in a human system by demonstrating increased proliferation of cultured airway smooth muscle. This phenomenon, if evidenced in man, would indicate that LXR ligands may directly increase airway reactivity, which could be detrimental, especially in patients with existing respiratory disease and with already compromised lung function.
Asunto(s)
Asma/inmunología , Asma/metabolismo , Benzoatos/administración & dosificación , Bencilaminas/administración & dosificación , Hiperreactividad Bronquial/metabolismo , Proteínas de Unión al ADN/agonistas , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Regulación hacia Arriba/inmunología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Asma/patología , Asma/fisiopatología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Relación Dosis-Respuesta Inmunológica , Humanos , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/inmunología , Receptores Nucleares Huérfanos , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Ratas , Ratas Endogámicas BN , Receptores Citoplasmáticos y Nucleares/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
p38 mitogen-activated protein kinase (MAPK) signaling is known to be increased in chronic obstructive pulmonary disease (COPD) macrophages. We have studied the effects of the p38 MAPK inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]-pyrimidin-2-yl]amino}ethyl)guanidine (SB706504) and dexamethasone on COPD macrophage inflammatory gene expression and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. Lipopolysaccharide (LPS)-stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AMs) were cultured with dexamethasone and/or SB706504. MDMs were used for gene array and protein studies, whereas tumor necrosis factor (TNF) alpha protein production was measured from AMs. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared with SB706504 alone (-2.04) or dexamethasone (-3.39). Twenty-three genes were insensitive to the effects of both drugs, including interleukin (IL)-1beta, IL-18, and chemokine (CC motif) ligand (CCL) 5. In addition, the chromosome 4 chemokine cluster members, CXCL1, CXCL2, CXCL3, and CXCL8, were all glucocorticoid-resistant. SB706504 significantly inhibited LPS-stimulated TNFalpha production from COPD and smoker AMs, with near-maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS-regulated genes, including IL-1beta, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.
Asunto(s)
Quimiocinas/metabolismo , Dexametasona/uso terapéutico , Expresión Génica/efectos de los fármacos , Guanidinas/uso terapéutico , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Enfermedad Pulmonar Obstructiva Crónica/prevención & control , Pirimidinonas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Interacciones Farmacológicas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/inducido químicamente , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Inhaled corticosteroids are highly effective in the treatment of asthma and also show efficacy in chronic obstructive pulmonary disease (COPD). Considerable effort continues to be focused on improvement of their pharmacology and pharmacokinetic properties. Corticosteroids act through the glucocorticoid receptor, one of a family of ligand activated transcription factors. These proteins are highly tractable to drug discovery, and are targeted by a number of existing therapeutic agents. The possibility that family members other than the glucocorticoid receptor might have therapeutic benefit in lung disease has received increasing attention. Recent studies using ligands for the vitamin D3 receptor, the peroxisome proliferator-activated receptors, and the liver X receptor have demonstrated significant potential for these novel targets in the treatment of lung disease.
Asunto(s)
Enfermedades Pulmonares/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/fisiología , Humanos , Inflamación/prevención & control , Receptores X del Hígado , Enfermedades Pulmonares/etiología , Receptores Nucleares Huérfanos , PPAR gamma/agonistas , PPAR gamma/fisiología , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de Glucocorticoides/fisiología , Receptores X Retinoide/agonistas , Receptores X Retinoide/fisiologíaRESUMEN
Inhaled corticosteroids are highly effective agents for the treatment of asthma and chronic obstructive pulmonary disease. Although local delivery to the lung has led to significant progress towards minimising side effects associated with systemic exposure, additional opportunities exist for further enhancement of the therapeutic index. Recent developments in the molecular and structural understanding of corticosteroid biology have made it increasingly clear that novel corticosteroids with intrinsically different pharmacology can be designed.
Asunto(s)
Corticoesteroides , Asma/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores de Glucocorticoides , Corticoesteroides/farmacología , Corticoesteroides/fisiología , Corticoesteroides/uso terapéutico , Animales , Humanos , Ligandos , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/fisiología , Relación Estructura-ActividadRESUMEN
Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available 'cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Cumarinas/química , Cumarinas/farmacología , Evaluación Preclínica de Medicamentos , Pez Cebra/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cromolin Sódico/química , Cromolin Sódico/farmacología , Furocumarinas/química , Furocumarinas/farmacología , Inflamación/patología , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.
Asunto(s)
Abietanos/farmacología , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Inflamación/tratamiento farmacológico , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Pez Cebra , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Larva , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Investigación Biomédica Traslacional , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/inmunología , Pez Cebra/metabolismoRESUMEN
REV-ERBα has emerged as an important target for regulation of circadian rhythm and its associated physiology. Herein, we report on the optimization of a series of REV-ERBα agonists based on GSK4112 (1) for potency, selectivity, and bioavailability. (1) Potent REV-ERBα agonists 4, 10, 16, and 23 are detailed for their ability to suppress BMAL and IL-6 expression from human cells while also demonstrating excellent selectivity over LXRα. Amine 4 demonstrated in vivo bioavailability after either iv or oral dosing.
Asunto(s)
Aminas/síntesis química , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/agonistas , Aminas/química , Aminas/farmacología , Animales , Disponibilidad Biológica , Proteínas Portadoras/metabolismo , Línea Celular , Ritmo Circadiano , Glicina/análogos & derivados , Glicina/síntesis química , Glicina/química , Glicina/farmacología , Humanos , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas de Unión al ARN , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
Drug discovery scientists, faced with the myriad challenges involved in developing novel therapeutics as medicines, have tended to overlook the question of the most beneficial time to administer the drug. Recent developments in our understanding of circadian biology and the availability of tools to characterise the molecular clock indicate that time and duration of dosing may have profound consequences for the efficacy and safety of new and existing therapeutic agents. Progress in the field also suggests that many key physiological mechanisms are remarkably dependent on the circadian clock. It has also become clear that a number of diseases with important unmet medical need display marked circadian variation in their symptoms and severity. These discoveries now reveal opportunities for new therapeutic strategies to be developed that act by modulation of biological rhythms. These novel therapeutic approaches are likely to be facilitated by the continuing development of chemical probes and synthetic ligands targeted to an increasing number of the key proteins that regulate the molecular clock.
Asunto(s)
Relojes Circadianos , Cronoterapia de Medicamentos , Descubrimiento de Drogas/tendencias , Preparaciones Farmacéuticas/administración & dosificación , Animales , Humanos , Ratones , Preparaciones Farmacéuticas/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a smoking related inflammatory airway disease in which macrophages play a key role. Previously we have shown that cigarette smoke extract (CSE) causes suppression of macrophage inflammatory mediators, with the exception of IL-8. We now investigate the effects of dexamethasone on these gene expression changes. Monocyte derived macrophages (MDMs) were cultured with CSE and dexamethasone. Microarray analysis was used to assess inflammatory mediator regulation, with qPCR and ELISA also performed for selected cytokines. The major effect of CSE was down-regulation of inflammatory genes (11 probe sets). For CSE regulated genes (n=13), the median fold change with CSE alone was -2.84 and with dexamethasone alone was -2.97. Both treatments combined caused the greatest suppression of gene expression; -4.47. qPCR also showed that IL-1beta, GM-CSF and IL-6 mRNA levels were significantly reduced by CSE and further suppressed by dexamethasone. qPCR and ELISA showed that IL-8 levels were increased by CSE, with suppression by dexamethasone. We show that CSE suppressed the expression of some inflammatory genes whilst up-regulating IL-8. Dexamethasone further suppressed gene expression when combined with CSE. The combined effect of GC and CSE causes suppression of the macrophage innate immune response.