Abstract
BackgroundAirway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.MethodsIL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.ResultsThe β2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.ConclusionCollectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.
Highlights
Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8)
To study whether cyclic AMP (cAMP)-regulated effectors protein kinase A (PKA) and Epac participate in this response, we analyzed the role of the cAMP analogs 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP known to preferentially activate PKA or Epac, respectively [46,47]
To validate the data obtained with the Epac activator 8-pCPT-2'-O-Me-cAMP, 8-pCPT-2'-O-Me-cGMP, a cGMP analogue with substitutions identical to those in 8pCPT-2'-O-Me-cAMP which is known to neither activate crFeAilgeMuaPsre-eeol1efvIaLt-i8ng agent fenoterol augments bradykinin-induced cAMP-elevating agent fenoterol augments bradykinin-induced release of IL-8. hTERT-airway smooth muscle cells were stimulated for 18 hrs with the indicated concentrations of bradykinin (A)
Summary
Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). We study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. Asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases characterized by structural and functional changes of the airways [1,2]. The underlying pathogenic processes of asthma and COPD include the production and release of chemokines and cytokines by inflammatory and structural cells [3]. Airway smooth muscle cells have recognized as immunomodulatory cells able to synthesize multiple inflammatory mediators such as cytokines, including interleukin-8 (IL-8) [4,5,6]. The gene expression of IL-8 is tightly regulated by inflammatory and pro-contractile agonists [6,17,18] acting on the large superfamily of G-protein-coupled receptors (GPCRs)
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