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 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. 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.


Background
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].
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 [7]. In particular, IL-8 is a potent chemoattractant for neutrophils and eosinophils [8,9], that have been implicated in inflammatory airway diseases [10]. Indeed, enhanced IL-8 has been detected in blood and bronchial mucosa [11] and in bronchial epithelial cells of patients with asthma [12], in bronchoalveolar lavage fluid (BALF) of asthmatic and chronic bronchitis patients [13], 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 [16]. Airway smooth muscle are a rich source of IL-8 [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).
Bradykinin is a pluripotent nonapeptide generated by plasma and tissue kallikreins, and is upregulated in patients with asthma [19]. 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 signalregulated protein kinases 1 and 2 (ERK1/2) [18,20] and appears to involve cyclooxygenase-dependent and -independent signals [6,21]. G s -protein-coupled receptor activation (e.g. β 2 -adrenergic or prostanoid receptors) modulates the release of cytokines from airway cells [6], 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 E 2 (PGE 2 ) has been reported at the level of IL-6 production from airway smooth muscle [22]. 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 [21]. 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 [23]. 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 [25]. Epac controls a variety of cellular functions including integrin-mediated cell-adhesion [26], endothelial integrity and permeability [27], 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][31][32][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 Raslike GTPase Rap1 and ERK1/2.

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 [41]. All procedures were approved by the human Research Ethics Board of the University of Manitoba. As described previously [42], 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. Toxininduced glucosylation of Ras-like GTPases was monitored by using a specific anti-Rac1 antibody [43], 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.

Cell fractionation
Cells were lysed in 50 mM Tris (pH 7.4) supplemented with 1 mM Na 3 VO 4 , 1 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 7 μg/ml pepstatin and then fractioned as described earlier [44]. 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: (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 Stransferase (GST)-tagged RalGDS (Ras-binding domain of the Ral guanine nucleotide dissociation stimulator) as previously described [45]. 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.

IL-8 assay
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.

Statistical analysis
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 bradykinininduced IL-8 release from human airway smooth muscle
Given the importance of IL-8 in airway inflammatory processes [7], we examined the role of the cAMP-elevating agent β 2 -agonist fenoterol in bradykinin-induced IL-8 release from hTERT-airway smooth muscle cells. As illustrated in Fig. 1A, bradykinin induced an increase in the release of IL-8 from the cells. The concentration of 10 μM bradykinin appeared to be most effective (~2-fold increase, P < 0.001) and was chosen for further experiments. The β 2 -agonist fenoterol at the concentration of 1 μM further enhanced bradykinin-induced IL-8 release of about 2 fold (P < 0.05), whereas it did not alter basal IL-8 production (Fig. 1B). These data suggest that bradykinininduced IL-8 release from hTERT-airway smooth muscle cells may be augmented by cAMP signaling.
Me-cGMP did not alter basal IL-8 release (Fig. 3B). Collectively, these data indicate that augmentation of bradykinin-induced IL-8 release from hTERT-airway smooth muscle cells is regulated by cAMP, likely through both PKA and Epac.
To further validate our findings, we analyzed the phosphorylation of VASP, known to be phosphorylated at Ser-157, a PKA-specific site [49], by using a VASP-specific antibody that recognizes both phospho-VASP (upper band) and total VASP (lower band). Phosphorylation of VASP was not altered by any of the Epac-related cAMP compounds being studied (each 100 μM) (Fig. 4A). In contrast, 1 μM fenoterol, 100 μM forskolin and 500 μM 6-Bnz-cAMP induced VASP phosphorylation (Fig. 4A). In addition, treatment of the cells with the pharmacological selective PKA inhibitor Rp-8-CPT-cAMPS blocked phosphorylation of VASP by 6-Bnz-cAMP (P < 0.05) and largely reduced VASP phosphorylation by forskolin (P = 0.067) and fenoterol (P < 0.05) (Fig. 4B). Bradykinin also induced VASP phosphorylation (P = 0.055) (Fig. 4B). All together, these data indicate that the cyclic nucleotides used in our study specifically activate their primary pharmacological targets PKA and Epac, and thereby induce augmentation of bradykinin-induced IL-8 release from hTERT-airway smooth muscle cells.

Role of Ras-like GTPases in cAMP-dependent bradykinininduced IL-8 release from human airway smooth muscle
PKA and Epac have been reported to modulate GTP-loading of the Ras-like GTPase Rap1 and Rap2 [45,50,51]. In hTERT-airway smooth muscle cells, Rap1 and Rap2 were both present at membrane-associated and cytosolic compartments (Fig. 5). As shown in Fig. 5A, activation of Epac by 8-pCPT-2'-O-Me-cAMP induced about a 2-fold increase in GTP-loading of Rap1 in hTERT-airway smooth muscle cells (P < 0.01). Activation of PKA by 6-Bnz-cAMP activated Rap1 by about 1,5-fold (P < 0.05) (Fig. 5A). In contrast, activation of Epac or PKA did not induce GTPloading of Rap2 (Fig. 5B). To study whether activation of Ras-like GTPases by cAMP is required for the augmentation of bradykinin-induced IL-8 release, cells were treated Effects of cyclic nucleotide analogs and cAMP-elevating agents on VASP phosphorylation with Clostridium difficile toxin B-1470 known to inactivate Ras family members, including Rap1 [52]. We analyzed cell morphology and immunoreactivity of the toxin-substrate GTPase Rac1 to monitor the functionality of toxin B-1470 [43]. Treatment of the cells with 100 pg/ml toxin B-1470 profoundly altered cell morphology, as demonstrated by the occurrence of a high number of rounded cells (Fig. 6A). Toxin B-1470 also completely abolished Rac1 immunoreactivity under any experimental condition studied (not shown). Although hTERT-airway smooth muscle cells were toxin B-1470 sensitive, toxin treatment lowered cell number only of about 20% (Fig.   6B, upper panel) and did not alter cell viability (not shown). Importantly, toxin treatment completely reversed the augmentation of bradykinin-induced IL-8 release by 8-pCPT-2'-O-Me-cAMP and 6-Bnz-cAMP (each P < 0.05), without affecting IL-8 release by bradykinin alone (Fig.  6B, lower panel). As we show that PKA and Epac induce GTP-loading of Rap1 and that inhibition of Ras-like GTPases, including Rap1, largely affect augmentation of bradykinin-induced IL-8 release by both PKA and Epac, our data point at Rap1 as an important modulator of this response.

PKA and Epac cooperate to activate Rap1 and to augment bradykinin-induced IL-8 release from human airway smooth muscle
Studies on the molecular mechanisms of cAMP-related signaling demonstrate that the classical cAMP effector PKA acts alone or in concert with the novel cAMP sensor Epac [25,56,57]. To study whether cAMP-regulated PKA Results represent mean ± SEM of separate experiments (n = 3-9). *P < 0.05, **P < 0.01, ***P < 0.001 compared to unstimulated control; § P < 0.05, § § P < 0.01, § § § P < 0.001 compared to basal condition.  9B). To further validate PKA and Epac cooperative effects, we used different approaches to specifically inhibit the two cAMP-driven effectors and we studied the impact of these inhibitions on GTP-loading of Rap1 and IL-8 release from hTERT-airway smooth muscle cells.
As shown before, Rp-8-CPT-cAMPS acts as a specific inhibitor of PKA in hTERT-airway smooth muscle cells. Interestingly, treatment of cells with Rp-8-CPT-cAMPS reduced GTP-loading of Rap1 by both 8-pCPT-2'-O-Me-cAMP and 6-Bnz-cAMP (Fig. 10A). In addition, in the presence of Rp-8-CPT-cAMPS, augmentation of bradykinin-induced IL-8 release by the PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP was largely diminished (P < 0.05), whereas basal and bradykinininduced IL-8 release were not significantly altered (Fig.  10B). These data suggest that PKA and Epac pathways work in concert both at the level of Rap1 activation and the downstream production of IL-8.
At present, highly specific pharmacological inhibitors of individual Epac isoforms, Epac1 and Epac2, are not available [46]. Thus, to more precisely study the role of Epac1 and Epac2 in specific functions, siRNA is generally used to suppress their endogenous expression [31,[34][35][36]. As illustrated in Fig. 11A, the siRNA approaches were effective in reducing expression of membrane-associated Epac1 and cytosolic Epac2 of about ~40% (P < 0.01 for Epac1 and for Epac2) leaving the expression of the cell fraction-specific marker proteins caveolin-1 and β-actin unaffected. Silencing of Epac1 and Epac2 was most efficient 72 hrs after transfection, indicating that the proteins exhibit a slow turn-over rate in hTERT-airway smooth muscle cells. As illustrated in Fig. 11B, silencing of Epac1 and Epac2 did not only reduced GTP-loading of Rap1 by 8-pCPT-2'-O-Me-cAMP, but also its activation by 6-Bnz-cAMP. In addition, silencing of Epac1 and Epac2 severely impaired augmentation of bradykinin-induced IL-8 release by 8-pCPT-2'-O-Me-cAMP (each P < 0.05), whereas basal and bradykinin-induced IL-8 release were again not significantly changed (Fig. 11C). Intriguingly, silencing of Epac1 also significantly reduced augmentation of bradykinin-induced IL-8 release by 6-Bnz-cAMP (P < 0.05) (Fig. 11C). Silencing of cellular Epac2 appeared to modestly reduce the PKA-mediated IL-8 response, although this effect was not significant (P = 0.075) (Fig.  11C). Taken together, these data indicate that cAMP-regulated PKA and Epac (Epac1 and Epac2) are interconnected with regard to the activation of Rap1 and the cellular production of IL-8 in hTERT-airway smooth muscle cells.

Discussion
Bradykinin is known to enhance the expression of several cytokines in airway smooth muscle [18,58]. cAMP-elevating agents also modulate release of cytokines from airway sources including airway smooth muscle [59]. For example, prostaglandin E 2 (PGE 2 ) was shown to increase IL-8 production in airway smooth muscle cells [6]. Interestingly, salmeterol and PGE 2 have been reported to augment bradykinin-induced production of IL-6 by airway smooth muscle [22], but the cAMP-regulated targets responsible for this cellular response have not been identified. Here we report on novel cAMP-dependent mechanisms that augment bradykinin-induced release of IL-8 from airway smooth muscle. We demonstrate that augmentation of bradykinin-induced IL-8 production by cAMP signaling requires the cooperative action of PKA and Epac, leading subsequently to the activation of Raslike GTPases such as Rap1 and ERK1/2 (Fig. 12).
The use of cyclic nucleotide analogs as pharmacological tools to study the specific effects of cAMP-driven signaling is now widely accepted [46]. 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 [47] or act upon production of cAMP hydrolysis products [48]. 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 In agreement with studies in human airway smooth muscle [22], the β 2 -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 PGE 2 [60]. 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 [61]. The type II AC isoform is abundantly expressed in airway smooth muscle and is activated by both Gα s and PKC probably leading to synergistic cAMP formation [62,63]. Moreover, PKC may cooperate in assembling the prostanoid synthetic machinery [60]. In addition, it has been reported that bradykinin inhibits approximately 60% of the total cAMP phosphodiesterase activity in guinea-pig airway smooth muscle [20]. 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 [25]. Indeed, both Rap1 Impact of Epac silencing on Rap1 activation and bradykinin-induced IL-8 release . **P < 0.01, ***P < 0.001 compared to unstimulated control; § P < 0.05, § § P < 0.01 compared to control siRNA.
and Rap2 are present in hTERT-airway smooth muscle cells in both membrane and cytosolic compartments. Interestingly, activation of PKA and Epac induced GTPloading 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 [66]. However, it is presently unknown whether such mechanisms are operational in hTERT-airway smooth muscle. To address the role of Ras-like GTPases in bradykinininduced 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 [52]. 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 bradykinininduced 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 [21]. 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 [22] and human lung fibroblasts [18]. 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 [67]. 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 [68]. Interestingly, both PKA and Epac have been reported to activate ERK1/ 2 in a cell-type specific manner [51]. Once activated, ERK1/2 signals to the nucleus, promoting transcription of genes usually associated with inflammation and prolifer-ation. 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 β 2 -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-1470sensitive 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 [69] 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 bradykinininduced 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 [72] 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.
Augmentation of bradykinin-induced IL-8 release in hTERT-airway smooth muscle cells by Epac and PKA Figure 12 Augmentation of bradykinin-induced IL-8 release in hTERT-airway smooth muscle cells by Epac and PKA. Activation of ERK1/2 is mediated via different GPCRs. The β 2 -agonist fenoterol acts on G s -coupled receptors inducing cAMP elevation via activation of adenylyl cyclase (AC) while forskolin directly activates AC. cAMP activates two distinct cellular effectors: PKA and Epac, followed by activation of Ras-like GTPases, such as Rap1, and ERK1/2, and subsequently induction of specific transcription factors resulting in the production of IL-8. Bradykinin also elicits ERK1/2 phosphorylation most likely via activation of G q -coupled receptors. The dotted line represents a potential pathway which has not been fully addressed in our study. ⊥ indicates inactivation and → indicates activation, see text for further details.

Conclusion
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.