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Published on: June 25, 2012
PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells
Sara S Roscioni1, Loes E M Kistemaker, Mark H Menzen
1Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands. S.S.Roscioni@rug.nl
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.
Conclusion:
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.
Insights
Airway smooth muscle cells release interleukin-8 (IL-8) contributing to pulmonary disease. Protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) modulate this release through Rap1 signaling.
Area of Science:
- Cellular and Molecular Biology
- Pulmonary Medicine
- Immunology
Background:
- Airway smooth muscle cells (ASMCs) contribute to pulmonary disease pathogenesis by secreting inflammatory mediators like interleukin-8 (IL-8).
- Interleukin-8 (IL-8) production in ASMCs is regulated by Gq- and Gs-coupled receptors.
- The roles of cyclic AMP (cAMP) effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2), in bradykinin-induced IL-8 release were investigated.
Purpose of the Study:
- To elucidate the molecular mechanisms by which PKA and Epac1/Epac2 regulate bradykinin-induced IL-8 release in human ASMCs.
- To determine the involvement of Rap1 GTPase and extracellular signal-regulated kinases (ERK1/2) in this signaling pathway.
Main Methods:
- Human ASMC cell line used for experiments.
- Interleukin-8 (IL-8) release measured by ELISA.
- Pharmacological activators and inhibitors of PKA, Epac, and GTPases were employed.
- Western blot and siRNA were used to assess protein expression and function.
- GTP-loading of Rap1 and Rap2 evaluated using pull-down assays.
Main Results:
- Bradykinin-induced IL-8 release was augmented by fenoterol, a PKA activator, and Epac activators.
- PKA and Epac activators increased IL-8 release and induced Rap1 GTP-loading.
- Inhibition of PKA or Epac, along with Rap1 activation, significantly reduced IL-8 release.
- Pharmacological inhibitors of GTPases and ERK1/2 also reduced IL-8 release.
Conclusions:
- PKA, Epac1, and Epac2 act synergistically to regulate ASMC inflammatory responses.
- This modulation occurs via signaling pathways involving the Ras-like GTPase Rap1 and ERK1/2.
- Findings provide insights into the molecular mechanisms underlying airway inflammation in pulmonary diseases.
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