RESUMO
Inhaled ß-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with ß-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of ß-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased ß-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, ß-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which ß-agonists exert their relaxant effects.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Sistema Respiratório/metabolismo , Linhagem Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Músculo Liso/citologia , Sistema Respiratório/citologiaRESUMO
Inhaled ß-agonists are effective airway smooth muscle (ASM)-relaxing agents that help reverse bronchoconstriction in asthma, but their ability to affect the aberrant ASM growth that also occurs with asthma is poorly understood. ß-Agonists exhibit PKA-dependent antimitogenic effects in several cell types. However, recent studies suggest that Epac, and not PKA, mediates the antimitogenic effect of cAMP in both ASM and fibroblasts. This study aims to clarify the role of PKA in mediating the effect of G(s)-coupled receptors on human ASM growth. Pretreatment of ASM cultures with ß-agonists albuterol, isoproterenol, or salmeterol (100 nM to 10 µM) caused a significant (â¼ 25-30%) inhibition of EGF-stimulated ASM thymidine incorporation and cell proliferation, whereas a much greater inhibition was observed from pretreatment with PGE(2) (75-80%). However, all agents were ineffective in cells expressing GFP chimeras of either PKI (a PKA inhibitor) or a mutant PKA regulatory subunit relative to the control cells expressing GFP. The antimitogenic efficacy of PGE(2) in inhibiting control cultures was associated with greater ability to stimulate sustained PKA activation and greater inhibition of late-phase promitogenic p42/p44 and PI3K activities. These findings suggest that therapeutic approaches enabling superior PKA activation in ASM will be most efficacious in deterring ASM growth.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Albuterol/análogos & derivados , Albuterol/farmacologia , Broncodilatadores/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Isoproterenol/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Xinafoato de Salmeterol , Fatores de Tempo , Traqueia/citologia , TransfecçãoRESUMO
Glucocorticoids (GCs) and protein kinase A (PKA)-activating agents (beta-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma--excessive ASM growth--are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1 beta and TNF-alpha mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2-dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1 beta stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1 beta, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein-coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of beta-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Androstadienos/farmacologia , Antiasmáticos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucocorticoides/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Fator de Crescimento Epidérmico/metabolismo , Fluticasona , Perfilação da Expressão Gênica/métodos , Humanos , Interleucina-1beta/metabolismo , Miócitos de Músculo Liso/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Traqueia/enzimologia , TransfecçãoRESUMO
Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/genética , Humanos , Músculo Liso/enzimologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.
Assuntos
Corticosteroides/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/fisiologia , Mitógenos/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citocinas/genética , Humanos , Interleucina-1beta/genética , Interleucina-1beta/fisiologia , Mitógenos/genéticaRESUMO
The interleukin-1 receptor associated kinases (IRAKs) sit at the bottle neck for the Toll-like-receptor (TLR) mediated signal transduction process controlling host innate immune response. However, the exact role and regulation of IRAKs are still in the early stage and not fully understood. This review intends to summarize the recent advancement in this important topic and point out areas that need further intensive investigation.