PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells.
BACKGROUND
Airway 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.


METHODS
IL-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.


RESULTS
The beta2-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.


CONCLUSIONS
Collectively, 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.
Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands. S.S.Roscioni@rug.nl
Sara S
SS
Roscioni
Loes E M
LE
Kistemaker
Mark H
MH
Menzen
Carolina R S
CR
Elzinga
Reinoud
R
Gosens
Andrew J
AJ
Halayko
Herman
H
Meurs
Martina
M
Schmidt
D001920:Bradykinin / Q000494:pharmacology*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D016209:Interleukin-8; C116122:RAPGEF3 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D001920:Bradykinin