RESUMEN
Mitogen activated protein kinase phosphatase-1 (MKP-1) has emerged as an important protein mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies, especially proteasome inhibitors. In this in vitro study, we utilized the breast cancer epithelial cell lines MCF-7 and MDA-MB-231, in comparison to MCF-10A control cells, to examine the impact of MKP-1 on breast cancer cell growth and repression by proteasome inhibitors. We confirm that proteasome inhibitors MG-132 and bortezomib induce MKP-1 protein upregulation and we show that one of the ways in which bortezomib increases MKP-1 in breast cancer cells, in addition to inhibition of ubiquitin-proteasome system, is via upregulation of MKP-1 mRNA expression in p38 MAPK-mediated manner. Notably, these effects are specific to cancer cells, as bortezomib activated p38 MAPK and induced MKP-1 in MCF-7 and MDA-MB-231 breast cancer cells, but not in control cells (MCF-10A). We took a dual approach toward targeting MKP-1 to show that bortezomib-induced effects are enhanced. Firstly, treatment with the non-specific MKP-1 inhibitor triptolide reduces breast cancer cell growth and augments proteasome inhibitor-induced effects. Secondly, specific knock-down of MKP-1 with siRNA significantly repressed cell viability by reduced cyclin D1 expression, and enhanced repression of cancer cell growth by proteasome inhibitors. Taken together, these results indicate that removing the unwanted (MKP-1-inducing) effects of bortezomib significantly improves the efficacy of proteasome inhibition in breast cancer cells. Thus, future development of drugs targeting MKP-1 offer promise of combination therapies with reduced toxicity and enhanced cell death in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas/genética , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , TransfecciónRESUMEN
The bioactive sphingolipid sphingosine 1-phosphate (S1P) is found in increased amounts in the airways of asthmatics. S1P can regulate airway smooth muscle functions associated with asthmatic inflammation and remodeling, including cytokine secretion. To date however, whether S1P induces secretion of an important chemokine responsible for neutrophilia in airway inflammation--IL-8--was unexplored. The aim of this study was to investigate whether S1P induces IL-8 gene expression and secretion to enhance neutrophil chemotaxis in vitro, as well as examine the molecular mechanisms responsible for repression by the corticosteroid dexamethasone. We show that S1P upregulates IL-8 secretion from ASM cells and enhance neutrophil chemotaxis in vitro. The corticosteroid dexamethasone significantly represses IL-8 mRNA expression and protein secretion in a concentration- and time-dependent manner. Additionally, we reveal that S1P-induced IL-8 secretion is p38 MAPK and ERK-dependent and that these key phosphoproteins act on the downstream effector mitogen- and stress-activated kinase 1 (MSK1) to control secretion of the neutrophil chemoattractant cytokine IL-8. The functional relevance of this in vitro data was demonstrated by neutrophil chemotaxis assays where S1P-induced effects can be significantly attenuated by pretreatment with dexamethasone, pharmacological inhibition of p38 MAPK- or ERK-mediated pathways, or by knocking down MSK-1 with siRNA. Taken together, our study reveals the molecular pathways responsible for IL-8 secretion from ASM cells in response to S1P and indicates ways in which the impact on IL-8-driven neutrophilia may be lessened.
Asunto(s)
Interleucina-8/metabolismo , Lisofosfolípidos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Esfingosina/análogos & derivados , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , FN-kappa B/metabolismo , Esfingosina/farmacologíaRESUMEN
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.
Asunto(s)
Corticoesteroides/farmacología , Fosfatasa 1 de Especificidad Dual/efectos de los fármacos , Interleucina-6/biosíntesis , Lisofosfolípidos/farmacología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Músculo Liso/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Esfingosina/análogos & derivados , AMP Cíclico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Humanos , Interleucina-6/metabolismo , Músculo Liso/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Esfingosina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Bronquios/citología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fosfatasa 1 de Especificidad Dual/biosíntesis , Lisofosfolípidos/farmacología , Miocitos del Músculo Liso/enzimología , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenilil Ciclasas/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Fosforilación/efectos de los fármacos , Esfingosina/farmacología , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.
Asunto(s)
Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopéptidos/farmacología , Receptor Toll-Like 2/agonistas , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba , Transporte Activo de Núcleo Celular , Bronquios/inmunología , Bronquios/patología , Bronquios/fisiología , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Expresión Génica , Humanos , Inflamación/metabolismo , Interleucina-6/genética , Interleucina-8/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Neutrófilos , Cultivo Primario de Células , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
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.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/genética , Retroalimentación Fisiológica , Miocitos del Músculo Liso/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Bronquios/citología , Bronquios/metabolismo , Diterpenos/farmacología , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 1 de Especificidad Dual/deficiencia , Compuestos Epoxi/farmacología , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Imidazoles/farmacología , Inflamación/genética , Inflamación/metabolismo , Interleucina-8/biosíntesis , Miocitos del Músculo Liso/citología , Fenantrenos/farmacología , Cultivo Primario de Células , Regiones Promotoras Genéticas , Piridinas/farmacología , Estabilidad del ARN/efectos de los fármacos , ARN Interferente Pequeño/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
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.