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

<p<Abstract</p< <p<Background</p< <p<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 G<sub<q</sub<-and G<su...
Ausführliche Beschreibung

<p<Abstract</p< <p<Background</p< <p<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 G<sub<q</sub<-and G<sub<s</sub<-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.</p< <p<Methods</p< <p<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 <it<Clostridium difficile </it<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.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-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'-<it<O</it<-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.</p< <p<Conclusion</p< <p<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.</p< Ausführliche Beschreibung

Autor*in:	Halayko Andrew J [verfasserIn] Gosens Reinoud [verfasserIn] Elzinga Carolina RS [verfasserIn] Menzen Mark H [verfasserIn] Kistemaker Loes EM [verfasserIn] Roscioni Sara S [verfasserIn] Meurs Herman [verfasserIn] Schmidt Martina [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2009

Übergeordnetes Werk:	In: Respiratory Research - BMC, 2003, 10(2009), 1, p 88
Übergeordnetes Werk:	volume:10 ; year:2009 ; number:1, p 88

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1186/1465-9921-10-88

Katalog-ID:	DOAJ066084261