BACKGROUND AND PURPOSEAirway remodelling is a consequence of long-term inflammation and MAPKs are key signalling molecules that drive pro-inflammatory pathways. The endogenous MAPK deactivator -MAPK phosphatase 1 (MKP-1) -is a critical negative regulator of the myriad pro-inflammatory pathways activated by MAPKs in the airway. EXPERIMENTAL APPROACHHerein we investigated the molecular mechanisms responsible for the upregulation of MKP-1 in airway smooth muscle (ASM) by the corticosteroid dexamethasone and the b2-agonist formoterol, added alone and in combination. KEY RESULTSMKP-1 is a corticosteroid-inducible gene whose expression is enhanced by long-acting b2-agonists in an additive manner. Formoterol induced MKP-1 expression via the b2-adrenoceptor and we provide the first direct evidence (utilizing overexpression of PKIa, a highly selective PKA inhibitor) to show that PKA mediates b2-agonist-induced MKP-1 upregulation. Dexamethasone activated MKP-1 transcription in ASM cells via a cis-acting corticosteroid-responsive region located between -1380 and -1266 bp of the MKP-1 promoter. While the 3′-untranslated region of MKP-1 contains adenylate + uridylate elements responsible for regulation at the post-transcriptional level, actinomycin D chase experiments revealed that there was no increase in MKP-1 mRNA stability in the presence of dexamethasone, formoterol, alone or in combination. Rather, there was an additive effect of the asthma therapeutics on MKP-1 transcription. CONCLUSIONS AND IMPLICATIONSTaken together, these studies allow us a greater understanding of the molecular basis of MKP-1 regulation by corticosteroids and b2-agonists and this new knowledge may lead to elucidation of optimized corticosteroid-sparing therapies in the future. AbbreviationsAd5, adenoviral serotype 5; ASM, airway smooth muscle; CREB, cyclic-AMP response element binding protein; GC, glucocorticoid; GRE, GC-responsive element; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; MKP-1, MAPK phosphatase 1; MOI, multiplicity of infection; 3'-UTR, 3′-untranslated region

Background and purpose: Due to their potent bronchodilator properties, b2-adrenoceptor agonists are a mainstay of therapy in asthma. However, the effects of b2-adrenoceptor agonists on inflammation are less clear. Accordingly, we have investigated the effects of b2-adrenoceptor agonists on inflammatory mediator release. Experimental approach: Transcription factor activation, and both release and mRNA expression of IL-6 and IL-8 were examined by luciferase reporter assay, ELISA and real-time RT-PCR in bronchial human epithelial BEAS-2B cells or primary human bronchial epithelial cells grown at an air-liquid interface. Key results: Pre-incubation with b2-adrenoceptor agonists (salbutamol, salmeterol, formoterol) augmented the release and mRNA expression of IL-6 and IL-8 induced by IL-1b and IL-1b plus histamine, whereas NF-kB-dependent transcription was significantly repressed, and AP-1-dependent transcription was unaffected. These effects were mimicked by other cAMPelevating agents (PGE2, forskolin). Enhancement of cytokine release by b2-adrenoceptor agonists also occurred in primary bronchial epithelial cells. Addition of dexamethasone with salmeterol repressed IL-6 and IL-8 release to levels that were similar to the repression achieved in the absence of salmeterol. IL-6 release was enhanced when salmeterol was added before, concurrently or after IL-1b plus histamine stimulation, whereas IL-8 release was only enhanced by salmeterol addition prior to stimulation. Conclusions and implications:Enhancement of IL-6 and IL-8 release may contribute to the deleterious effects of b2-adrenoceptor agonists in asthma. As increased inflammatory mediator expression is prevented by the addition of glucocorticoid to the b2-adrenoceptor, our data provide further mechanistic support for the use of combination therapies in asthma management.