PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells
© Roscioni et al. 2009
Received: 16 May 2009
Accepted: 29 September 2009
Published: 29 September 2009
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.
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.
The 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.
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.
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 . Airway smooth muscle cells have recognized as immunomodulatory cells able to synthesize multiple inflammatory mediators such as cytokines, including interleukin-8 (IL-8) [4–6].
IL-8 represents one of the best characterized members of the family of chemokines known to attract and activate leukocytes and plays a major role in the initiation and maintenance of inflammatory responses . In particular, IL-8 is a potent chemoattractant for neutrophils and eosinophils [8, 9], that have been implicated in inflammatory airway diseases . Indeed, enhanced IL-8 has been detected in blood and bronchial mucosa  and in bronchial epithelial cells of patients with asthma , in bronchoalveolar lavage fluid (BALF) of asthmatic and chronic bronchitis patients , in BALF and sputum from patients with COPD [14, 15]. IL-8 levels correlate with the number of airway neutrophils, which are strongly associated with severe asthma and are increased during acute exacerbations of chronic bronchitis . Airway smooth muscle are a rich source of IL-8 . 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).
Bradykinin is a pluripotent nonapeptide generated by plasma and tissue kallikreins, and is upregulated in patients with asthma . It has been reported that bradykinin stimulates the expression of IL-8 in human lung fibroblasts and airway smooth muscle [6, 18]. This response is coupled to activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) [18, 20] and appears to involve cyclooxygenase-dependent and -independent signals [6, 21].
Gs-protein-coupled receptor activation (e.g. β2-adrenergic or prostanoid receptors) modulates the release of cytokines from airway cells , probably via activation of adenylyl cyclase and subsequent increase in intracellular cyclic AMP (cAMP). Importantly, a synergism between bradykinin and the cAMP-elevating agents salmeterol and prostaglandin E2 (PGE2) has been reported at the level of IL-6 production from airway smooth muscle . Although these studies clearly indicate a role for cAMP in pro-inflammatory cytokine production, the engagement of distinct cAMP-regulated effectors has not been yet addressed in the airways. Given the importance of the bradykinin- and the cAMP-driven pathways in both the pathophysiology and the treatment of pulmonary diseases, insights into the cellular mechanisms of their interaction are warranted.
Indeed, increasing evidence suggests that cAMP actively regulates transcription and gene expression events in several airway cells [23, 24], and that such mechanism may regulate local cytokine production in human airway smooth muscle . Until recently, intracellular effects of cAMP have been attributed to the activation of protein kinase A (PKA) and subsequent changes in PKA-mediated protein expression and function . In the last decade, exchange proteins directly activated by cAMP (Epac1 and Epac2) have been identified as cAMP-regulated guanine nucleotide exchange factors for Ras-like GTPases, such as Rap1 and Rap2 . Epac controls a variety of cellular functions including integrin-mediated cell-adhesion , endothelial integrity and permeability , exocytosis and insulin secretion [28, 29]. Epac also signals to ERK although the outcome of this particular signalling appears to depend on the cell type and specific cellular localization of Epac and their effectors [30–33]. Epac has been shown to act alone [34, 35] or to either antagonize [32, 36] or synergize with PKA [37, 38]. Although a role of Epac in lung fibroblasts and airway smooth muscle proliferation has recently been addressed [34, 35, 39], the impact of both PKA and Epac on the production of inflammatory mediators in the airways is presently unknown. Here, we report on novel cAMP-driven molecular mechanisms inducing augmentation of bradykinin-induced release of IL-8 from human airway smooth muscle and we demonstrate that Epac1 and Epac2 act in concert with PKA to modulate this cellular response via signaling to the Ras-like GTPase Rap1 and ERK1/2.
1,4-diamino-2,3-dicyano-1, 4-bis [2-aminophenylthio]butadiene (U0126) and forskolin were purchased from Tocris (Bristol, UK). 6-Bnz-cAMP, 8-pCPT-2'-O-Me-cAMP, Rp-8-CPT-cAMPS, Sp-8-pCPT-2'-O-Me-cAMPS and 8-pCPT-2'-O-Me-cGMP were from BIOLOG Life Science Institute (Bremen, Germany). Fenoterol was from Boehringer Ingelheim (Ingelheim, Germany). Bradykinin, Na3VO4, aprotinin, leupeptin, pepstatin and mouse anti-β-actin antibody (A5441), peroxidase-conjugated goat anti-rabbit (A5420) and peroxidase-conjugated rabbit anti-mouse (A9044) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). The anti-phospho-ERK1/2 (P-ERK1/2) (9101), anti-ERK1/2 (9102) and anti-VASP which also binds to phospho-VASP (P-VASP) (3112) were from Cell Signaling Technology (Beverly, MA). The antibodies against Rap1 (121, sc-65), Rap2 (124, sc-164) and caveolin-1 (N-20, sc-894) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody against Rac-1 (Mab 3735) was from Millipore (Billerica, MA). The mouse monoclonal antibodies against Epac1 and Epac2 were generated and kindly provided by Dr. J. L. Bos . Clostridium difficile toxin B-1470 was kindly provided by Drs C. von Eichel-Streiber and H. Genth. DMEM, FBS, penicillin/streptomycin solution were obtained from GIBCO-BRL Life Technologies (Paisley, UK). Alamar Blue solution was from Biosource (Camarillo, CA), the dyazo die trypan blue from Fluka Chemie (Buchs, Switzerland) and the Pierce BCA protein assay kit from Thermo Scientific (Rockford, IL). siRNA probes were purchased from Dharmacon Inc. (Lafayette, CO) and the transfection vehicle lipofectamine 2000 was from Invitrogen (Carlsbad, CA). The western lightning ECL solution was from PerkinElmer Inc. (Waltman, MA) and the IL-8 ELISA kit from Sanquin (Amsterdam, The Netherlands). All used chemicals were of analytical grade.
Cell culture, toxin treatment, cell number and viability measurements
Human bronchial smooth muscle cell lines, immortalized by stable ectopic expression of human telomerase reverse transcriptase enzyme were used for all the experiments (hTERT-airway smooth muscle cells). The primary human bronchial smooth muscle cells used to generate these cells were prepared as described previously . All procedures were approved by the human Research Ethics Board of the University of Manitoba. As described previously , each cell line was thoroughly characterized to passage 10 and higher. Passage 10 to 25 myocytes, grown on uncoated dishes in DMEM supplemented with antibiotics and 10% FBS, were used. Before each experiments, cells were serum deprived for one day in DMEM supplemented with antibiotics. For toxin B-1470 treatment, cells were treated for 24 hrs with 100 pg/ml toxin B-1470. Toxin-induced glucosylation of Ras-like GTPases was monitored by using a specific anti-Rac1 antibody , and changes in cell morphology were monitored by phase-contrast microscopy, using an Olympus IX50 microscope equipped with a digital image capture system (Color View Soft Imaging System). The toxicity of used drugs as well as their vehicle (DMSO) towards hTERT-airway smooth muscle cells was determined by an Alamar Blue assay. Briefly, cells were incubated with HBSS containing 10% vol/vol Alamar blue solution and then analyzed by fluorimetric analysis. Fluorescence derives from the conversion of Alamar blue into its reduced form by mitochondrial cytochromes and is therefore a measure of the number of cells. Viability was set as 100% in control cells. Viability of cells was also measured by resuspending cells 1:1 in the diazo dye trypan blue, which is absorbed by non viable cells, and the number of blue cells was then measured.
Cells were lysed in 50 mM Tris (pH 7.4) supplemented with 1 mM Na3VO4, 1 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 7 μg/ml pepstatin and then fractioned as described earlier . The protein amount of all the fractions was determined using Pierce protein determination according to the manifacturer's instructions. Membrane, cytosolic and nuclear enriched fractions were subsequently used for detection of Epac1, Epac2, Rap1 and Rap2 expression.
Silencing of Epac1 and Epac2 expression using siRNAs
Cells were transfected with siRNA probes targeted to either Epac1 or Epac2; the target sequences for human Epac1 siRNA mixture were: sense: 5'-CGUGGGAACUCAUGAGAUG-3' (J-007676-05), sense: 5'-GGACCGAGAUGCCCAAUUC-3' (J-007676-06), sense: 5'-GAGCGUCUCUUUGUUGUCA-3' (J-007676-07), sense: 5'CGUGGUACAUUAUCUGGAA-3' (J-007676-08) and for the Epac2 siRNA mixture: sense: 5'-GAACACACCUCUCAUUGAA-3' (J-009511-05), sense: 5'- GGAGAAAUAUCGACAGUAU-3' (J-009511-06), sense: 5'-GCUCAAACCUAAUGAUGUU-3' (J-009511-07), sense: 5'-CAAGUUAGCACUAGUGAAU-3' (J-009511-08). Non-silencing siRNA control was used as a control in all siRNA transfection experiments. Cells were transfected with 200 pmol of appropriate siRNA by using lipofectamine 2000 (1 mg/ml) as vehicle. 6 hrs after transfection, cells were washed with DMEM supplemented with antibiotics to reduce toxicity effects of the transfection reagent. Cells were subsequently analyzed for Epac1 and Epac2 expression, GTP-loading of Rap1 or IL-8 production.
Activation of Rap1, phosphorylation of ERK1/2-VASP and immunoblot analysis
The amount of activated Rap1 and Rap2 was measured with the pull-down technique by using glutathione S-transferase (GST)-tagged RalGDS (Ras-binding domain of the Ral guanine nucleotide dissociation stimulator) as previously described . For the measurement of the phosphorylation of ERK1/2 and VASP, cell were lysed followed by determination of the protein concentration. Equal amounts of protein (or samples) were loaded on 10-15% polyacrylamide gels and analyzed for the protein of interest by using the specific first antibody (dilution Rap1 and Rap2 1:250, P-ERK 1:1000, ERK 1:500, VASP 1:1000) and the secondary HRP-conjugated antibody (dilution 1:2000 anti-rabbit or 1:3000 anti-mouse). Protein bands were subsequently visualized on film using western lightning plus-ECL and quantified by scanning densitometry using TotalLab software (Nonlinear Dynamics, Newcastle, UK). Results were normalized for protein levels by using specific control proteins.
The concentration of IL-8 in the culture medium was determined by ELISA according to the manifacturer's instructions (Sanquin, the Netherlands). Results were normalized for cell number according to Alamar Blue measurement. Basal IL-8 levels ranged between 0.3 and 18,8 pg/ml.
Data were expressed as the mean ± SEM of n determinations. Statistical analysis was performed using the statistical software Prism. Data were compared by using an unpaired or paired two-tailed Student's t test to determine significant differences. p values < 0.05 were considered to be statistically significant.
Cyclic AMP-regulated PKA and Epac augment bradykinin-induced IL-8 release from human airway smooth muscle
Role of Ras-like GTPases in cAMP-dependent bradykinin-induced IL-8 release from human airway smooth muscle
Role of ERK1/2 in cAMP-dependent bradykinin-induced IL-8 release from human airway smooth muscle
PKA and Epac cooperate to activate Rap1 and to augment bradykinin-induced IL-8 release from human airway smooth muscle
The use of cyclic nucleotide analogs as pharmacological tools to study the specific effects of cAMP-driven signaling is now widely accepted . However, studies indicated that various cyclic nucleotide analogs, including 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP might, in addition to their primary effects, also cause elevation of cAMP or cGMP upon inhibition of phosphodiesterases  or act upon production of cAMP hydrolysis products . We did not observe indirect activation of the PKA-dependent effectors such as VASP by any of the Epac-related analogs. Moreover, phosphorylation of VASP by forskolin, fenoterol and 6-Bnz-cAMP was sensitive to the PKA inhibitor Rp-8-CPT-cAMPS. Both the PKA activator 6-Bnz-cAMP and the Epac activators used (8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS) augmented bradykinin-induced IL-8 release in the cells, whereas no alteration of the cellular IL-8 levels was observed with the cGMP analog 8-pCPT-2'-O-Me-cGMP. Hence, it is reasonable to assume that PKA- and Epac-related cyclic nucleotides act via their primary pharmacological targets. Collectively, our results indicate that cAMP-dependent augmentation of bradykinin-induced IL-8 release from hTERT-airway smooth muscle cells is regulated by both PKA and Epac.
In agreement with studies in human airway smooth muscle , the β2-agonist fenoterol and the distinct PKA/Epac-related cyclic nucleotide analogs used in our studies solely alter the release of IL-8 from hTERT-airway smooth muscle in the presence of bradykinin, suggesting that this GPCR ligand might also directly affect the cAMP pathway. Previous studies have shown that bradykinin can increase intracellular levels of cAMP in airway smooth muscle via induction of cyclooxygenase and subsequent production of PGE2 . As we observed phosphorylation of VASP by bradykinin already ≤ 15 minutes, such prostanoid-driven indirect effects may not account for the bradykinin responses observed in our study. Protein kinase C, a major downstream effector of bradykinin, has been reported to activate type II adenylyl cyclase (AC) in intact cells and to elicit activation of basal AC activity . The type II AC isoform is abundantly expressed in airway smooth muscle and is activated by both Gαs and PKC probably leading to synergistic cAMP formation [62, 63]. Moreover, PKC may cooperate in assembling the prostanoid synthetic machinery . In addition, it has been reported that bradykinin inhibits approximately 60% of the total cAMP phosphodiesterase activity in guinea-pig airway smooth muscle . The above mentioned mechanisms could therefore contribute to the increase of cAMP levels by bradykinin in distinct subcellular compartments and subsequently trigger the activation of PKA and Epac in airway smooth muscle.
Here we also focused on the Ras-like GTPase family members Rap1 and Rap2 as the main effectors of Epac being identified [25, 64] and so far the best described in their functional association to Epac . Indeed, both Rap1 and Rap2 are present in hTERT-airway smooth muscle cells in both membrane and cytosolic compartments. Interestingly, activation of PKA and Epac induced GTP-loading of Rap1 in hTERT-airway smooth muscle; both cAMP effectors did not alter basal Rap2 activity. In contrast to Epac1, activation of Rap1 by PKA has been reported to occur mostly indirectly. Evidence suggests that PKA might either activate the Rap1 exchange factor C3G (Crk Src homology domain 3) and Src [51, 54, 65] or inhibit the Rap1-GTPase activating protein . However, it is presently unknown whether such mechanisms are operational in hTERT-airway smooth muscle. To address the role of Ras-like GTPases in bradykinin-induced IL-8 release we used the bacterial toxin B-1470. Toxin B-1470, which is produced by C. difficile strain 1470, inhibits exclusively the Rac protein from the Rho family and, in addition, Rap and Ral proteins from the Ras family of GTPases via glucosylation . Such GTPases are important regulators of cellular adhesion and migration. Indeed, treatment with the toxin induced morphological changes and also caused cell detachment probably associated with inhibition of those GTPases. Toxin treatment only slightly reduced cell number and did not alter cell viability. Importantly, we observed a drastic reduction of bradykinin-induced IL-8 release by PKA and Epac after incubation with Toxin B-1470. Hence, our results suggest that cAMP-dependent augmentation of bradykinin-induced IL-8 requires PKA- and Epac-dependent activation of GTPases, and based on the results presented herein, Rap1 represents a very attractive candidate.
The production and release of IL-8 from airway smooth muscle upon stimulation of pro-inflammatory agonists is regulated by gene transcription and protein expression events . Bradykinin has been shown to modulate the release of IL-8 generally upon activation of distinct signals including ERK1/2 [18, 20]. Phosphorylation of ERK1/2 by bradykinin occurs acutely between 5-30 min in both human airway smooth muscle cells  and human lung fibroblasts . It is generally believed that cAMP modulates transcription and protein expression [23, 67], and its effects have been attributed to the phosphorylation of cAMP response element binding (CREB) protein by PKA and its subsequent binding to the CRE promoter of the specific genes . Although the human IL-8 promoter does contain a CRE region, activation of CREB has not been related to the regulation of IL-8 expression in airway cells. Moreover, recent studies indicate that Epac1 also modulates gene transcription and protein expression by inducing the transcription factors CCAAT/Enhancer-binding Proteins (C/EBPs) in COS-1 cells . Interestingly, both PKA and Epac have been reported to activate ERK1/2 in a cell-type specific manner . Once activated, ERK1/2 signals to the nucleus, promoting transcription of genes usually associated with inflammation and proliferation. Activation of Epac and PKA in hTERT-airway smooth muscle cells increased basal ERK1/2 phosphorylation (1-30 min) and enhanced bradykinin-induced ERK1/2 phosphorylation measured after 10 min. Hence, these findings indicate that ERK1/2 activation may be an important mechanism by which β2-agonists augment IL-8 production in airway smooth muscle. This was confirmed by the fact that treatment with the pharmacologic inhibitor U0126 reduced the IL-8 release by bradykinin alone and even in a more pronounced way, by the combination of bradykinin with both 8-pCPT-2'-O-Me-cAMP and 6-Bnz-cAMP. The fact that toxin B-1470 treatment largely impaired ERK1/2 phosphorylation by PKA and Epac, most likely places ERK1/2 downstream of toxin B-1470-sensitive GTPases.
Previous studies in human lung fibroblasts have shown that Epac1, Epac2 and PKA act independently on distinct cellular functions [34, 35]. For example, the anti-proliferative signalling properties in human lung fibroblasts have been assigned to Epac1, but not to Epac2 [34, 35]. The diverse effects of Epac proteins and PKA could be explained by their different subcellular localization  or downstream effector availability [70, 71]. Indeed, we observed that Epac isoforms Epac1 and Epac2 exhibit different cellular localization in hTERT-airway smooth muscle cells, the former being more expressed at the plasma membrane and the latter in the cytosolic fraction of the cells. However, we here demonstrate that silencing of Epac1 or Epac2 expression in hTERT-airway smooth muscle cells abolished the augmentation of bradykinin-induced IL-8 release by the Epac activator 8-pCPT-2'-O-Me-cAMP, and also largely diminished the enhancement of this cellular response by the PKA activator 6-Bnz-cAMP. These data point at a positive cooperativity between cAMP-regulated Epac1-Epac2 and PKA, which was confirmed by pharmacological approaches using the PKA inhibitor Rp-8-CPT-cAMPS and the combinations of the the PKA and Epac activators. Importantly, activation of Rap1 by either PKA or Epac appeared to be sensitive to inhibition of PKA by Rp-8-CPT-cAMPS or to silencing of Epac1 and Epac2 by siRNA. This results suggest that Epac and PKA work in concert to activate Rap1 and subsequently augment IL-8 release by bradykinin. Our findings might implicate that both Epac isoforms and PKA bind to the same signalling complex which are then directed to the same target(s). Indeed, distinct intracellular cAMP signaling compartments have been recently identified in primary cultures of neonatal cardiac ventriculocytes  and cAMP-responsive multiprotein complexes including PKA and Epac1-Epac2 seem to confer signaling specificity [73, 74]. Thus, our data indicate that in airway smooth muscle both Epac1 and Epac2 act in concert with PKA to modulate pro-inflammatory signaling properties.
We describe novel cAMP-dependent mechanisms to induce augmentation of bradykinin-induced IL-8 release from airway smooth muscle. Evidence is provided that cAMP-regulated Epac1 and Epac2 cooperate with PKA to induce Ras-like GTPases activation (presumably Rap1) and subsequent activation of ERK1/2. Our findings implicate that PKA, Epac1 and Epac2 exert pro-inflammatory signaling properties in human airway smooth muscle depending on the input of distinct GPCR signals. The relevance of these findings is reflected by the importance of bradykinin- and cAMP-mediated signals in airway disease pathogenesis and treatment and opens new avenues for future therapeutic intervention.
We thank J. Schuur and I.S.T. Bos for technical assistance. We also thank Drs. C. von Eichel-Streiber and H. Genth for providing Clostridium difficile toxin B-1470. This work was supported by a Ubbo Emmius Fellowship from the School of Behavioral and Cognitive Neurosciences to S.S. Roscioni and a Rosalind Franklin Fellowship from the University of Groningen to M. Schmidt. R. Gosens is the recipient of a Veni grant (916.86.036) from the Dutch Organisation for Scientific Research (NWO). A.J. Halajko is supported by the Canada Research Chairs Program and Canadian Institutes of Health Research. We are grateful to Dr. W.T. Gerthoffer (University of Nevada-Reno) for preparation of the hTERT-airway smooth muscle cell lines.
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