RESUMO
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.
Assuntos
Corticosteroides/farmacologia , Fosfatase 1 de Especificidade Dupla/efeitos dos fármacos , Interleucina-6/biossíntese , Lisofosfolipídeos/farmacologia , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Músculo Liso/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Esfingosina/análogos & derivados , AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Humanos , Interleucina-6/metabolismo , Músculo Liso/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingosina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Inflammasomes have emerged as playing key roles in inflammation and innate immunity. A growing body of evidence has suggested that the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Inflammasome activation results, in part, in pro-IL-1ß processing and the secretion of the proinflammatory cytokine IL-1ß. Because asthma exacerbations are associated with elevated concentrations of secreted IL-1ß, we addressed whether the NLRP3 inflammasome is activated under in vitro conditions that mimic infectious exacerbations in asthma. Primary cultures of airway smooth muscle (ASM) cells were treated with infectious stimuli (mimicked using the Toll-like receptor-2 agonist Pam3CSK4, a synthetic bacterial lipopeptide). Whereas Pam3CSK4 robustly up-regulated ASM cytokine expression in response to TNF-α and significantly enhanced IL-1ß mRNA expression, we were unable to detect IL-1ß in the cell supernatants. Thus, IL-1ß was not secreted and therefore was unable to act in an autocrine manner to promote the amplification of ASM inflammatory responses. Moreover, Toll-like receptor-2 ligation did not enhance NLRP3 or caspase-1 expression in ASM cells, and NLRP3 and caspase-1 protein were not present in the ASM layer of tracheal sections from human donors. In conclusion, these data demonstrate that the enhanced synthetic function of ASM cells, induced by infectious exacerbations of airway inflammation, is NLRP3 inflammasome-independent and IL-1ß-independent. Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Músculo Liso/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Humanos , Inflamassomos/genética , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.
Assuntos
Brônquios/citologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosfatase 1 de Especificidade Dupla/biossíntese , Lisofosfolipídeos/farmacologia , Miócitos de Músculo Liso/enzimologia , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adenilil Ciclases/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Esfingosina/farmacologia , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 µg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.
Assuntos
Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima , Transporte Ativo do Núcleo Celular , Brônquios/imunologia , Brônquios/patologia , Brônquios/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Neutrófilos , Cultura Primária de Células , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
Asthma is a chronic inflammatory condition. Inhibition of the ubiquitin-proteasome system offers promise as a anti-inflammatory strategy, being responsible for the degradation of key proteins involved in crucial cellular functions, including gene expression in inflammation (e.g. inhibitory IkappaB-alpha and the endogenous MAPK deactivator - MKP-1). As MKP-1 inhibits MAPK-mediated pro-remodeling functions in human airway smooth muscle (ASM; a pivotal immunomodulatory cell in asthma) in this study we investigate the effect of the proteasome inhibitor MG-132 on MKP-1 and evaluate the anti-inflammatory effect of MG-132 on cytokine secretion from ASM cells. Examining the time-course of induction of MKP-1 mRNA and protein by MG-132 (10microM) we show that MKP-1 mRNA was first detected at 30min, increased to significant levels by 4h, resulting in a 12.6+/-1.5-fold increase in MKP-1 mRNA expression by 24h (P<0.05). MKP-1 protein levels corroborate the mRNA results. Investigating the effect of MG-132 on secretion of the cytokine IL-6 we show that while short-term pretreatment with MG-132 (30min) partially reduced TNFalpha-induced IL-6 via inhibition of IkappaB-alpha degradation and the NF-kappaB pathway, longer-term proteasome inhibition (up to 24h) robustly upregulated MKP-1 and was temporally correlated with repression of p38-mediated IL-6 secretion from ASM cells. Moreover, utilizing a cytokine array we show that MG-132 represses the secretion of multiple cytokines implicated in asthma. Taken together, our results demonstrate that MG-132 upregulates MKP-1 and represses cytokine secretion from ASM and highlight the potential of the proteasome as a therapeutic target in asthma.
Assuntos
Citocinas/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Miócitos de Músculo Liso/metabolismo , Inibidores de Proteassoma , Mucosa Respiratória/anatomia & histologia , Asma/imunologia , Inibidores de Cisteína Proteinase/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leupeptinas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Mitogen-activated protein kinase phosphatase 1 (MKP-1) represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting ß2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting ß2-agonists (salmeterol and formoterol), alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone) in primary human airway smooth muscle (ASM) cells in vitro. ß2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting ß2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion). As IL-6 expression (like MKP-1) is cAMP/adenylate cyclase-mediated, the long-acting ß2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, ß2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα). Conversely, as IL-8 is not cAMP-responsive, ß2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting ß2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Androstadienos/farmacologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fluticasona , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP/química , Transportador 1 de Cassete de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Airway smooth muscle (ASM) plays an important immunomodulatory role in airway inflammation in asthma. In our previous in vitro studies in ASM cells delineating the pro-inflammatory mitogen-activated protein kinase (MAPK) signaling pathways activated by tumor necrosis factor α (TNFα), we observed that TNFα concomitantly induces the rapid, but transient, upregulation of the anti-inflammatory protein-mitogen-activated protein kinase phosphatase 1 (MKP-1). As this was suggestive of a negative feedback loop, the aim of this study was to investigate the molecular mechanisms of MKP-1 upregulation by TNFα and to determine whether MKP-1 is a negative feedback effector that represses MAPK-mediated pro-inflammatory signaling pathways and cytokine secretion in ASM cells. Herein, we show that TNFα increases MKP-1 mRNA expression and protein upregulation in a p38 MAPK-dependent manner. TNFα does not increase MKP-1 transcription (measured by MKP-1 promoter activity); rather, we found that TNFα-induced MKP-1 mRNA stability is regulated by the p38 MAPK pathway. Inhibiting MKP-1 upregulation (with triptolide) demonstrated the precise temporal control exerted on MAPK signaling by MKP-1. In the absence of MKP-1, downstream phosphoprotein targets of MAPKs (such as MSK-1 and histone H3) are not turned off at the right time, allowing pro-inflammatory pathways to continue in an unrestrained manner. This is confirmed by knocking-down MKP-1 by siRNA where enhanced secretion of the neutrophil chemoattractant cytokine-interleukin 8 was detected in the absence of MKP-1. Thus, by activating p38 MAP kinase, TNFα concomitantly upregulates the MAPK deactivator MKP-1 to serve as an important negative feedback effector, limiting the extent and duration of pro-inflammatory MAPK signaling and cytokine secretion in ASM cells.
Assuntos
Fosfatase 1 de Especificidade Dupla/genética , Retroalimentação Fisiológica , Miócitos de Músculo Liso/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Brônquios/citologia , Brônquios/metabolismo , Diterpenos/farmacologia , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/deficiência , Compostos de Epóxi/farmacologia , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Imidazóis/farmacologia , Inflamação/genética , Inflamação/metabolismo , Interleucina-8/biossíntese , Miócitos de Músculo Liso/citologia , Fenantrenos/farmacologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Piridinas/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
SB203580 is the prototypical p38 MAPK inhibitor; however it cannot be used clinically due to liver toxicity. We developed a structural analogue of SB203580 - ML3403 - with equal in vitro and ex vivo p38alpha MAPK inhibition as SB203580, but with reduced activity towards liver cytochrome P450 enzymes. In addition, we developed a selective p38alpha MAPK inhibitor - CP41. The aim of this study is to compare the anti-inflammatory activity of ML3403 and CP41, with SB203580. We compare and contrast the ability of the p38 MAPK inhibitors to repress tumour necrosis factor alpha (TNFalpha)-induced interleukin 6 (IL-6) and interleukin 8 (IL-8) mRNA expression and protein secretion from airway smooth muscle cells. We also examined and compared the binding affinities of ML3403 and SB203580 to the active and inactive p38alpha MAPK. We demonstrate that ML3403 binds to both active and inactive p38 MAPK with high affinity and that it inhibits p38 MAPK-mediated airway smooth muscle synthetic function to an equivalent degree with SB203580. CP41 was not able to reduce IL-6 and IL-8 secretion in airway smooth muscle cells; a function of its higher IC(50) against p38alpha MAPK when compared to SB203580 and ML3403. We show that p38 MAPK-mediated pro-inflammatory pathways in airway smooth muscle cells can be inhibited by ML3403. The anti-inflammatory activity is equivalent to the prototypical p38 MAPK inhibitor SB203580. Our results implicate a future pharmacotherapeutic strategy towards reducing inflammation in asthma and airway remodelling.