Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes

European Journal of Pharmacology 535 (2006) 253 – 262 www.elsevier.com/locate/ejphar

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes Reinoud Gosens ⁎, Mechteld M. Grootte Bromhaar, Harm Maarsingh, Anita ten Damme, Herman Meurs, Johan Zaagsma, S. Adriaan Nelemans Department of Molecular Pharmacology, University Centre for Pharmacy, University of Groningen, Deusinglaan 1, 9713 AV Groningen, The Netherlands Received 31 October 2005; received in revised form 25 January 2006; accepted 27 January 2006 Available online 9 March 2006

Abstract This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factorinduced [3H]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B2 receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide I; specific for conventional and novel PKCs) and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Gö 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B2 receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA. Published by Elsevier B.V. Keywords: Airway remodeling; Asthma; G protein coupled receptor; PDGF (Platelet-derived growth factor); TGF-β (Transforming growth factor-β)

1. Introduction Bradykinin is a nonapeptide generated by kallikreinmediated breakdown of kininogens during inflammatory responses. It is involved in a variety of (patho)physiological responses in the airways, including microvascular leakage, bronchoconstriction, mucus secretion and pain perception (Barnes et al., 1998). Bradykinin may play a role in asthma, since asthmatics display exaggerated bronchoconstrictor responses to bradykinin when compared to healthy controls (Polosa and Holgate, 1990). Moreover, kallikrein levels are increased in the bronchoalveolar lavage fluid of asthmatic subjects after allergen challenge (Christiansen et al., 1992). The bradykinin B2 receptor antagonist HOE 140 (D-Arg–Arg–Pro– ⁎ Corresponding author. Tel.: +31 50 363 3323; fax: +31 50 363 6908. E-mail address: [email protected] (R. Gosens). 0014-2999/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.ejphar.2006.01.065

Hyp–Gly–Thi–Ser–D–Tic–Oic–Arg) has been shown to improve airway function of asthmatics (Akbary et al., 1996) and is known to attenuate allergen-induced microvascular leakage and bronchoconstriction in guinea pigs (Bertrand et al., 1993; Mashito et al., 1999; Ricciardolo et al., 1994). Asthma is also characterized by structural alterations in the airways. Thus, cross-sections of pulmonary airways of asthmatics reveal a thickened airway smooth muscle layer (Ebina et al., 1993). Growth factors may in part be responsible for this process by increasing cell number (hyperplasia) or size (hypertrophy) through the activation of receptor tyrosine kinases. In addition, G protein coupled receptor agonists can be mitogenic for cultured airway smooth muscle cells and/or can augment growth factor induced proliferation (Ediger and Toews, 2000). For example, cholinergic signaling (Gosens et al., 2003, 2005), tachykinins (Noveral and Grunstein, 1995), inflammatory mediators such as histamine (Panettieri et al., 1990) and

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leukotriene D4 (Panettieri et al., 1998) have been reported to be pro-mitogenic, alone or in combination with growth factors. Since activation of pro-mitogenic signaling pathways such as the p42/p44 mitogen activated protein (MAP) kinase pathway by the bradykinin B2 receptor has been described in airway smooth muscle (Huang et al., 2003), it could be envisaged that bradykinin induces airway smooth muscle proliferation or proliferation synergy in combination with growth factors. Bradykinin is known not to induce human (Panettieri et al., 1995) and bovine (Malarkey et al., 1995) airway smooth muscle proliferation, presumably because of the inability to induce sustained p42/p44 MAP kinase activation. However, weakly mitogenic or even non-mitogenic G protein coupled receptor activation can be sufficient to potentiate receptor tyrosine kinase-induced growth (Ediger and Toews, 2000; Gosens et al., 2003). Therefore we investigated the effects of bradykinin on bovine tracheal smooth muscle in combination with peptide growth factors. We found that, while bradykinin is not mitogenic by itself, it augments growth factor-induced mitogenic responses through activation of bradykinin B2 receptors and with the involvement of conventional protein kinase C (PKC) isozymes.

refreshed every 48–72 h. Cell cultures were allowed to grow and, upon confluency, were passaged further at a 1 : 2 split ratio, by means of trypsinization. Cultured cells were used for experiments in passage 1–3. 2.3. [Ca2+]i-measurements Measurements of [Ca2+]i were carried out according to Hoiting et al. (1996). Briefly, cells were detached from the flask bottom by trypsinization and washed three times in Krebs– Ringer–Henseleit buffer (composition in mM: NaCl 125.0, KCl 6.0, MgCl2 2.5, CaCl2 1.2, NaH2PO4 1.2, HEPES 25.0 and glucose 11.0, pH 7.4), supplemented with 2% bovine serum albumin. Next, cells were loaded with the fluorescent dye Fura2/AM (3μM) for 30 min at 20 °C. Fura-2/AM loaded cells were washed, diluted to a concentration of 1.106 cells/ml and were used for experiments within 2–4 h following the loading procedure. Measurements were carried out at 37 °C, during which Fura-2 emitted fluorescence was measured at excitation wavelengths of 340 and 380nm and an emission wavelength of 510nm with a Perkin-Elmer Spectrometer (LS-50B). [Ca2+]i was calculated every 0.2s according to Grynkiewicz et al. (1985).

2. Methods 2.4. [3H]Thymidine-incorporation 2.1. Isolation of bovine tracheal smooth muscle cells Bovine tracheae were obtained from local slaughterhouses and transported to the laboratory in Krebs–Henseleit (KH) buffer of the following composition (mM): NaCl 117.5, KCl 5.60, MgSO4 1.18, CaCl2 2.50, NaH2PO4 1.28, NaHCO3 25.00 and glucose 5.50, pregassed with 5% CO2 and 95% O2; pH 7.4. After dissection of the smooth muscle layer and removal of mucosa and connective tissue, tracheal smooth muscle was chopped using a McIlwain tissue chopper, three times at a setting of 300 μm and three times at a setting of 100 μm. Tissue particles were washed two times with Dulbecco's Modification of Eagle's Medium (DMEM), supplemented with NaHCO3 (7 mM), HEPES (10 mM), sodium pyruvate (1 mM), nonessential amino acid mixture (1 : 100), gentamicin (45 μg/ml), penicillin (100 U/ml), streptomycin (100 μg/ml), amphotericin B (1.5 μg/ml) and 0.5% fetal bovine serum. Enzymatic digestion was performed using the same medium, supplemented with collagenase P (0.75 mg/ml), papain (1mg/ml) and soybean trypsin inhibitor (1 mg/ml). During digestion, the suspension was incubated in an incubator shaker (Innova 4000) at 37°C, 55 rpm for 20 min, followed by a 10 min period of shaking at 70 rpm. After filtration of the obtained suspension over 50 μm gauze, cells were washed three times in DMEM, supplemented as above, containing 10% fetal bovine serum. 2.2. Cell culture After isolation, bovine tracheal smooth muscle cells were seeded in culture flasks at a density of 1.106 cells/ml. Cultured cells were kept viable in medium containing 10% fetal bovine serum at 37°C in a humidified 5% CO2-incubator. Medium was

Bovine tracheal smooth muscle cells were plated in 24 well cluster plates at a density of 30,000 cells per well in 10% fetal bovine serum containing medium at 37 °C in a humidified 5% CO2-incubator. After 48 h, cells were washed two times with sterile phosphate buffered saline (PBS, composition (mM) NaCl, 140.0; KCl, 2.6; KH2PO4, 1.4; Na2HPO4·2H2O, 8.1; pH 7.4) and made quiescent by incubation in serum-free medium, supplemented with apo-transferrin (5 μg/ml), ascorbate (100 μM) and insulin (1μM) for 72 h. Cells were then washed with PBS and stimulated with mitogens in serum-free medium for 28 h, the last 24 h in the presence of [3H]thymidine (0.25 μCi/ml). To study the synergistic effects of bradykinin and growth factors, these agonists were added to the wells simultaneously. After two washes with PBS at room temperature and one with ice-cold 5% trichloroacetic acid (cells were treated with this trichloroacetic acid-solution on ice for 30 min) the acid-insoluble fraction was dissolved in 1ml NaOH (1M). Incorporated [3H]thymidine was quantified by liquid-scintillation counting. When applied, kinase inhibitors were added to the cells 30 min before stimulation with mitogens. 2.5. Proliferation assay Bovine tracheal smooth muscle cell cultures were prepared as described above, after which cells were incubated with mitogens for 2days in serum-free medium. Thereafter, cells were washed 2 times with Hank's Balanced Salt Solution (HBSS), and incubated with HBSS containing 5% v/v Alamar Blue® solution (Biosource, Camarillo, CA, USA) for 2 h. Conversion of Alamar Blue® into its reduced form by mitochondrial cytochromes was then assayed by dual wavelength

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spectrophotometry at wavelengths of 562 and 630 nm. Preliminary experiments (data not shown) confirmed that the degree of Alamar Blue® conversion is proportional to cell number, as indicated by the manufacturer.

for unpaired observations, where appropriate. The statistical analyses performed are indicated in the figure legends. Differences were considered to be statistically significant when P b 0.05.

2.6. Activation of p42/p44 MAP kinase

2.8. Materials

Bovine tracheal smooth muscle cells were plated in 6-well cluster plates at a density of 120,000 cells/well in medium, containing 10% fetal bovine serum. After 48 h, cells were washed two times with sterile PBS and made quiescent by incubation in serum-free medium, supplemented with apotransferrin (5μg/ml), ascorbate (100 μM) and insulin (1 μM) for 72 h. Cells were then washed with PBS and stimulated with agonists in serum-free medium. At different time-points, cells were washed twice in ice-cold PBS and lysed in 0.5 ml of homogenization buffer (composition in mM: NaCl 150.0, Tris HCl 10.0, 2-glycerophosphoric acid 5.0, EGTA 2.0, dithiothreitol 2.0, phenylmethylsulphonylfluoride 1.0, Na3VO4 1.0, NaF 1.0, pH 7.5), containing 0.5μg/ml leupeptin, 2μg/ml aprotinin and 1% w/v Triton X-100. Cell lysates were stored at − 80 °C until further use. Protein content was determined according to Bradford (1976). Homogenates containing equal amounts of protein per lane were then subjected to immunoblot analysis using antibodies that recognize the phosphorylated forms of p42/p44 MAPK (Thr202/Tyr204). The antibodies were visualized using enhanced chemiluminescence, Photographs of the blots were then scanned and analyzed by densitometry (Totallab™; Nonlinear Dynamics, Newcastle, UK).

Dulbecco's modification of Eagle's Medium (DMEM) was obtained from ICN Biomedicals (Costa Mesa, CA, U.S.A.). Fetal bovine serum, NaHCO3 solution (7.5%), HEPES solution (1M), sodium pyruvate solution (100 mM), non-essential amino acid mixture, gentamycin solution (10 mg/ml), penicillin/ streptomycin solution (5000 U/ml / 5000 μg/ml), amphotericin B solution (250 μg/ml) (Fungizone) and trypsin/EDTA were obtained from Gibco BRL Life Technologies (Paisley, UK). Epidermal growth factor (EGF, human recombinant), insulin (from bovine pancreas), transforming growth factor β1 (TGFβ1, from human platelets), Phorbol 12-myristate 13acetate (PMA), apo-transferrin (human), bradykinin, HOE 140 ( D –Arg–Arg–Pro–Hyp–Gly–Thi–Ser– D –Tic–Oic–Arg), aprotinin, leupeptin, soybean trypsin inhibitor and fura-2/AM were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [Methyl-3H]thymidine (specific activity 25Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). Papain and collagenase P were from Roche Diagnostics (Mannheim, Germany). Anti-phospho-p42/p44 MAP kinase (rabbit polyclonal) and horseradish peroxidase-linked goat anti-rabbit polyclonal were from Cell Signaling Technology (Beverly, MA, U.S.A.). GF 109203X (Bisindolylmaleimide I), U 0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) and PD 98059 (2-(2-Amino-3-methoxyphenyl)-4H-1benzopyran-4-one) were obtained from Tocris Cookson Ltd. (Bristol, UK), and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole) from Calbiochem (La Jolla, CA, U.S.A.). All other chemicals were of analytical grade.

2.7. Data analysis All data represent means ± S.E.M from n separate experiments. The statistical significance of differences between data was determined by a Student's t-test for unpaired observations or by two-way ANOVA, followed by a post hoc Student's t-test

Fig. 1. Bradykinin augments growth factor-induced DNA-synthesis and cell proliferation. The effects of bradykinin (10μM) on DNA-synthesis (A) and cell proliferation (B) were assayed in bovine tracheal smooth muscle cell cultures, both in the absence (white bars) and presence (striped bars) of peptide growth factors. Specifications of the growth factors and concentrations used: epidermal growth factor (EGF) 10ng/ml; platelet-derived growth factor (PDGF) 1 ng/ml; transforming growth factor-β1 (TGFβ1) 2ng/ml. Data shown represent the means ± S.E.M. of eighteen to twenty-seven experiments, obtained using six to nine animals. ⁎P b 0.05 compared to basal; #P b 0.05 compared to absence of bradykinin (Student's t-test for unpaired observations).

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3. Results 3.1. Bradykinin responsiveness in cultured bovine tracheal smooth muscle cells Since cell culture may affect expression or functional coupling of G protein coupled receptors to their effectors in airway smooth muscle cells, the functional presence of Gq-coupled bradykinin receptors was confirmed by measurements of bradykinin-induced changes in intracellular [Ca2+] in cultured (passage 1–3) bovine tracheal smooth muscle cells, using the fluorescent dye Fura2AM. In all cell cultures tested, bradykinin (10μM) induced a strong transient increase in intracellular [Ca2+], that averaged 3.0 ± 0.4-fold of basal [Ca2+] (basal value: 132 ± 20nM; P b 0.01). 3.2. Effects of bradykinin on cell proliferation The putative effects of bradykinin on airway smooth muscle cell cycle progression were evaluated using measurements of DNA synthesis ([3H]thymidine incorporation, Fig. 1A) and cell number (Alamar Blue® assay, Fig. 1B) in cultured bovine tracheal smooth muscle cells. A high concentration of bradykinin (10μM) did not increase [3H]thymidine incorporation by itself. In addition, DNA-synthesis in response to the relatively weak mitogen TGFβ1 (2ng/ml) was not significantly enhanced by bradykinin. In contrast, bradykinin markedly induced the DNAsynthesis responses of the more effective peptide growth factors EGF (10ng/ml) and platelet-derived growth factor (PDGF; 1ng/ ml; Fig. 1A). Similar effects of bradykinin, both by itself and in combination with EGF and PDGF were found in a cell proliferation assay (Fig. 1B). In these experiments, the potentiating effect of bradykinin on TGF β1 was statistically significant. Further experimentation demonstrated that bradykinin concentration-dependently augmented EGF (10 ng/ml)-induced responses (EC50 = 24 nM). At the highest concentration of

Fig. 3. Role of bradykinin B2 receptors in synergism between bradykinin (BK) and EGF. [3H]thymidine incorporation in cultured bovine tracheal smooth muscle cells was measured in response to bradykinin (10 μM), EGF (10 ng/ml) and their combination in the absence (white bars) and presence (striped bars) of HOE 140 (1 μM). Data shown represent the means ± S.E.M. of twelve experiments, obtained using four different animals. ⁎P b 0.05 (Student's t-test for unpaired observations).

bradykinin investigated (10 μM), the EGF-response was augmented 1.8 ± 0.1-fold (P b 0.01; Fig. 2A). Moreover, the responses to increasing concentrations of EGF were consistently augmented by bradykinin, with no effect on sensitivity to EGF (Fig. 2B). Since cyclo-oxygenase products have been reported to be involved in bradykinin-induced effects, such as cytokine production from smooth muscle cells (Huang et al., 2003; Pang and Knox, 1998), we studied the effects of indomethacin (3 μM) in an additional set of experiments. The bradykinin-induced increase of the EGF-response, amounting 1.8 ± 0.1-fold and 1.9 ± 0.4-fold in the absence and presence of indomethacin, respectively, was not affected however. 3.3. Role of bradykinin B2 receptors To establish the bradykinin B2 receptor nature of the observed effects of bradykinin, cells were treated with the potent bradykinin B2 receptor antagonist HOE 140 (1μM). The presence of HOE 140 completely prevented the synergistic mitogenic response induced by bradykinin (10μM) and EGF (10ng/ml, Fig. 3). 3.4. Role of calcium

Fig. 2. Bradykinin dose-dependently augments EGF-induced DNA-synthesis in bovine tracheal smooth muscle cells. (A) Concentration–effect curves of bradykinin-induced [3H]thymidine incorporation in the absence (open symbols) and presence (closed symbols) of EGF (10ng/ml). (B) Concentration–effect curves of EGF-induced [3H]thymidine incorporation in the absence (open symbols) and presence (closed symbols) of bradykinin (10μM). Data shown represent the means ± S.E.M. of fifteen to eighteen experiments, obtained using five to six animals.

We next investigated if the functional interaction between EGF and bradykinin could be explained by a synergistic effect at the level of [Ca2+]i. Therefore, bradykinin-induced increases in [Ca2+]i were measured both in the absence and presence of EGF (10 ng/ml). EGF did not induce any change in baseline [Ca2+]i and did not affect the transient or plateau response to bradykinin (Fig. 4). 3.5. Role of protein kinase C Since the bradykinin B2 receptor is Gq protein coupled, it is possible that the effects of bradykinin on EGF responses are

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synergistic effects of bradykinin on EGF-induced DNAsynthesis. In further support of a role for PKC, the PKC activator PMA potentiated the mitogenic responses to EGF in a synergistic fashion (P b 0.05, Fig. 6). In contrast to bradykinin, however, PMA induced a marked increase in [3H]thymidine incorporation by itself, which was concentration-dependent (pEC50 = 7.5 ± 0.4; maximal effect (Emax) = 2.7 ± 0.7-fold of basal, P b 0.05). Interestingly, both the basal mitogenic effects of PMA (0.1 μM) and its synergistic interaction with EGF (10 ng/ml) were completely insensitive to the conventional PKC inhibitor Gö 6976 (300 nM), whereas GF 109203X (10 μM) abrogated both responses (P b 0.05, Fig. 6). Fig. 4. EGF and bradykinin do not induce synergistic effects on [Ca2+]i. Average baseline [Ca2+]i (basal) and bradykinin-induced peak and sustained increases in [Ca2+]i were measured in cultured bovine tracheal smooth muscle cells. Measurements were performed both in the absence (white bars) and presence (striped bars) of 10ng/ml EGF, applied 30s before the addition of bradykinin. Data represent means ± S.E.M. of five experiments.

downstream of PKC activation. To test this hypothesis, two specific PKC-inhibitors were used: GF 109203X (10 μM) and Gö 6976 (300 nM). At these concentrations, GF 109203X inhibits both conventional (in bovine tracheal smooth muscle: α, βI and βII ) and novel (in bovine tracheal smooth muscle: δ, ε and ζ) PKC isozymes, whereas Gö 6976 inhibits conventional PKCs specifically (Martiny-Baron et al., 1993; Webb et al., 1997). Gö 6976 may also inhibit PKCμ (Gschwendt et al., 1996), though the expression of this isozyme is yet to be demonstrated in bovine tracheal smooth muscle. Interestingly, pretreatment with either inhibitor inhibited the synergistic mitogenic response induced by bradykinin and EGF (Fig. 5). Of note, GF 109203X potentiated the EGF-induced response significantly (P b 0.05), whereas basal and BK-induced responses were not affected. Collectively, these results point to a need for conventional PKC isozyme activation in the

3.6. Role of p42/p44 MAP kinase The pro-mitogenic effect of phorbol esters has been reported to be mediated through PKC-mediated p42/p44 MAP kinase activation (Orsini et al., 1999). Indeed, PMA-induced mitogenic responses were abrogated by the MAP kinase kinase inhibitor PD 98059 (30 μM, Fig. 7) and PMA treatment significantly activated the p42/p44 MAP kinase pathway in a sustained fashion (Fig. 8). Although bradykinin also activated p42/p44 MAP kinase in these cells, this effect was transient (Fig. 8). Interestingly, the transient p42/p44 MAP kinase induction by bradykinin could be inhibited by both Gö 6976 and GF 109203X, indicating its dependence on conventional PKC isozymes (Fig. 9). These results indicate that the differential induction of airway smooth muscle DNA-synthesis by PMA and bradykinin correlates with their differential ability to regulate the sustained activation of p42/p44 MAP kinase, which is required for effective cell cycle progression. The role of p42/p44 MAP kinase was further investigated using inhibitors of MAP kinase kinase, known to effectively prevent activation of the p42/p44 MAP kinase pathway (Ge et

Fig. 5. Role of PKC in synergism between bradykinin (BK) and EGF. [3H]thymidine incorporation in cultured bovine tracheal smooth muscle cells was measured in response to bradykinin (10 μM), EGF (10 ng/ml) and their combination in the absence (white bars) or presence of Gö 6976 (300nM, striped bars) or GF 109203X (10 μM, hatched bars). Data shown represent the means ± S.E.M. of twelve to twenty-four experiments, obtained using four to eight different animals. Statistical significance of treatments was calculated by two-way ANOVA, followed by Bonferroni's post hoc test. ⁎P b 0.05; #P b 0.05 compared to absence of Gö 6976; n.s. not significant.

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Fig. 6. PKC activation increases EGF-induced DNA-synthesis. [3H]thymidine incorporation in cultured bovine tracheal smooth muscle cells was measured in response to PMA (0.1μM), EGF (10 ng/ml) and their combination in the absence (white bars) or presence of Gö 6976 (300nM, striped bars) or GF 109203X (10 μM, hatched bars). Data shown represent the means ± S.E.M. of twelve to fifteen experiments, obtained using four to five different animals. Statistical significance of treatments was calculated by two-way ANOVA, followed by Bonferroni's post hoc test. ⁎P b 0.05; n.s. not significant; #P b 0.05 compared to absence of GF 109203X.

al., 2002; Karpova et al., 1997). The inhibitors U 0126 (3 μM) and PD 98059 (30 μM), applied at selective concentrations, diminished both basal and EGF-induced [3H]thymidine incorporation (Fig. 7). In addition, the synergistic responses observed for the combination of EGF and bradykinin were abolished. However, combined treatment with EGF and bradykinin did not activate the p42/p44 MAP kinase pathway synergistically, neither in its early activation phase, nor after 2 h. The combination of PMA and EGF did not activate the p42/p44 MAP kinase pathway synergistically either, irrespective of the time-point studied (Fig. 8). Collectively, these results indicate

that p42/p44 MAP kinase activation by EGF is not further induced by PKC. 4. Discussion The results presented in this study demonstrate that bradykinin is mitogenic for bovine tracheal smooth muscle cells when applied in combination with the peptide growth factors EGF or PDGF. In combination with the weaker mitogenic stimulus TGFβ1, the synergistic effect of bradykinin was less pronounced. The high potency of bradykinin in

Fig. 7. Role of p42/p44 MAP kinase in DNA-synthesis induced by bradykinin (BK), PMA and EGF. [3H]thymidine incorporation in cultured bovine tracheal smooth muscle cells was measured in response to bradykinin (10μM), PMA (0.1μM), EGF (10ng/ml) and their combination in the absence (white bars) or presence of U 0126 (3μM, striped bars) or PD 98059 (30μM, hatched bars). Data shown represent the means ± S.E.M. of twelve experiments, obtained using four different animals. Statistical significance of treatments was calculated by two-way ANOVA, followed by Bonferroni's post hoc test ⁎P b 0.05 compared to basal. N.D. no data. #P b 0.05 compared to absence of inhibitor.

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Fig. 8. Time-dependent p42/p44 MAP kinase activation by bradykinin (BK), PMA and EGF. p42/p44 MAP kinase activation in cultured bovine tracheal smooth muscle cells was measured in response to bradykinin (10μM), PMA (0.1μM), EGF (10ng/ml) and their combination. The phosphorylation of p42/ p44 MAP kinase was assayed both in its early phase (A; 2–5min) and late phase (B; 2h). Data shown represent the means ± S.E.M. of five to ten experiments. ⁎P b 0.05 (Student's t-test for unpaired observations).

augmenting EGF-induced responses (EC50 = 24 nM) is in agreement with other effects of bradykinin on airway smooth muscle, such as contraction of human bronchial preparations (EC50 = 20nM) (Molimard et al., 1994) and IL-6 release in human airway smooth muscle cells (EC50 = 26nM) (Huang et al., 2003). As for these other effects of bradykinin, the bradykinin receptor involved in the synergistic responses was of the B2 subtype, shown using the bradykinin B2 receptor selective antagonist HOE 140. Although functional B1 receptors are expressed in airway smooth muscle (Marsh and Hill, 1994; Zhang et al., 2004), this preference is explained by the much greater affinity of bradykinin for B2 receptors compared to that for B1 receptors. Cyclo-oxygenase products have been implicated in various effects of bradykinin in human airway smooth muscle cells: the cyclo-oxygenase inhibitor indomethacin partially inhibits bradykinin-induced interleukin-6-production (Huang et al., 2003) and almost completely inhibits bradykinin-induced interleukin-8 production (Pang and Knox, 1998). In addition, epithelium-dependent relaxation of guinea pig tracheal smooth muscle induced by bradykinin is, in part, caused by bradykinininduced production of prostaglandin E2 (Bramley et al., 1990). Furthermore, bradykinin-induced, PKC dependent prostaglandin E2 production has been reported to cause cAMP production in airway smooth muscle cells, limiting p42/p44 MAP kinase activation by bradykinin (Moughal et al., 1995; Pyne et al.,

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1995). These effects are, however, not likely to be of relevance to the bradykinin effects in the current model: first, prostaglandin E2 is antiproliferative for guinea pig tracheal (Florio et al., 1994) and human airway smooth muscle cells (Johnson et al., 1995). Therefore, it seems highly unlikely that prostaglandin E2 mediates the observed increase in DNA-synthesis in combination with EGF in our study. Second, indomethacin did not reduce, or increase, the mitogenic response to bradykinin, or its synergistic interaction with EGF. Third, PKC inhibition reduced, and failed to potentiate the bradykinin-induced synergy with EGF, and finally, both PKC inhibitors (Gö 6976 and GF 109203X) used in this study largely prevented bradykinin-induced p42/p44 MAP kinase activation (Fig. 9). Collectively, this indicates that cyclo-oxygenase products are not involved in the bradykinin-effects in this study, and that the inability of bradykinin to induce airway smooth muscle proliferation by itself is not related to its parallel effects on PGE2 production. Bradykinin was not mitogenic by itself as also observed by others (Malarkey et al., 1995; Panettieri et al., 1995). Generally, G protein coupled receptor agonists respond with only minor or even completely absent mitogenic responses unless used as a comitogen with more effective peptide growth factors. For example, histamine (Krymskaya et al., 2000), leukotriene D4 (Panettieri et al., 1998), 5-hydroxytryptamine (unpublished observations), methacholine (Gosens et al., 2003) and endothelin-1 (Panettieri et al., 1996) are known to be mitogenic for airway smooth muscle cells in combination with growth factors only. This does not apply for all G protein coupled receptor agonists however, since thrombin is extremely effective on its own (Panettieri et al., 1995). The ability to induce sustained activation of the p42/p44 MAP kinase pathway is needed to trigger cell division, since only a transient increase in p42/p44 MAP kinase activation is observed after non-mitogenic G protein coupled receptor stimulation (Orsini et al., 1999). The transient p42/p44 MAP kinase activation induced by bradykinin and the sustained p42/p44 MAP kinase activation induced by PMA in

Fig. 9. p42/p44 MAP kinase activation by bradykinin (BK) is dependent on conventional PKC isozymes. p42/p44 MAP kinase activation in cultured bovine tracheal smooth muscle cells was measured in response to bradykinin (10μM), both in the absence and presence of Gö 6976 (300nM, ‘Gö’) or GF 109203X (10μM, ‘GF’) as indicated. The phosphorylation of p42/p44 MAP kinase was assayed after 5min of stimulation and expressed relative to that induced by EGF (10ng/ml). Data shown represent the means ± S.E.M. of four experiments. ⁎P b 0.05 compared to control; #P b 0.05 compared to bradykinin (Student's ttest for unpaired observations).

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our study are therefore in line with their respective effects observed on [3H]thymidine incorporation. Potentiation of EGF-induced DNA synthesis by bradykinin occurs via activation of PKC as demonstrated using GF 109203X. Gö 6976, which specifically inhibits conventional PKC isozymes (Martiny-Baron et al., 1993), was equally effective showing that bradykinin-induced synergy with EGF is fully dependent on these conventional PKC isozymes. In contrast, the mitogenic response to PMA was sensitive to GF 109203X only, indicating that this effect does not require conventional PKC isozymes. The mitogenic signaling pathways activated by PMA are likely sufficiently strong to overcome the inhibition of conventional PKCs in the presence of Gö 6976. It is highly unlikely that PMA would not activate these conventional isozymes. It appears therefore that different classes of PKC-isozymes differentially induce the induction of mitogenic responses in airway smooth muscle cells. The effectiveness of GF 109203X (conventional and novel PKCs, (Martiny-Baron et al., 1993)) compared to Gö 6976 suggests that novel PKC isozymes are particularly effective. In agreement with their respective effects on DNA-synthesis, PMA but not bradykinin induced a sustained activation of p42/ p44 MAP kinase. PMA-induced DNA-synthesis was also dependent on the activation of p42/p44 MAP kinase enzymes, as demonstrated using PD 98059. Interestingly, both PD 98059 and U 0126 significantly reduced basal thymidine incorporation. This probably reflects the induction of apoptosis, or a reduction in cell adherence. Despite of the clear functional involvement of p42/p44 MAP kinase in the mitogenic responses (determined using PD 98059 and U 0126), the p42/p44 MAP kinase enzymes were not activated synergistically, neither in its early phase, nor in its late phase by the combinations of bradykinin and EGF or PMA and EGF. In line with this observation, others have not shown such synergistic activation of p42/p44 MAP kinase for combinations of various G protein coupled receptor agonists with EGF in human airway smooth muscle cells (Ediger et al., 2003; Krymskaya et al., 2000). However, synergistic activation of the p42/p44 MAP kinase pathway has been proposed as an explanation for synergism between G protein coupled receptor agonists and growth factors in case of endothelin-1 and EGF in guinea pig airway smooth muscle and for ATP and insulin in coronary arterial smooth muscle (Agazie et al., 2001; Fujitani and Bertrand, 1997). It is of interest what mechanisms could mediate the synergy downstream of PKC activation. Synergistic effects of bradykinin and EGF on calcium increases (Pandiella and Meldolesi, 1989) are not likely to be important, as EGF is without effect on [Ca2+]i in these cells and does not potentiate Ca2+ responses to bradykinin (Fig. 4). EGF receptor transactivation is not likely either; though generally considered an important mechanism of action for mitogenic G protein coupled receptor agonists (Yang et al., 2005; Zwick et al., 1999), in airway smooth muscle, G protein coupled receptor agonists are consistently without effect, including thrombin, histamine and carbachol (Krymskaya et al., 2000). Rather, cooperative regulation of airway smooth muscle growth by G protein coupled receptor agonists and growth factors acting on receptor tyrosine kinases appears to be dependent on

mechanisms involving phosphatidylinositol 3-kinase, Akt and p70 S6kinase (Billington et al., 2005; Krymskaya et al., 2000). Although PKC inhibition is without effect on thrombin and EGFinduced synergy in airway smooth muscle (Billington et al., 2005), PMA-induced mitogenic responses were completely abrogated by phosphatidylinositol 3-kinase inhibition (LY294002, data not shown). PKC (both conventional and novel)-dependent regulation of p70 S6kinase has been described (Romanelli et al., 1999; Wang et al., 2003; Yutsudo et al., 2005). Future studies dissecting the role of PKC isozymes in p70S6kinase activation in these cells are clearly warranted. EGF-induced mitogenic responses were not inhibited by GF 109203X or Gö 6976. Remarkably, GF 109203X even significantly potentiated the EGF-induced response. These results are entirely consistent with observations made by others that showed similar effects of PKC inhibition, using calphostin C and staurosporine, on insulin-and PDGF-induced DNA-synthesis in bovine tracheal smooth muscle cells (Lew et al., 1997). PKC isoform-specific effects may also explain this discrepancy: growth factors activate the inhibitory PKCδ in bovine tracheal smooth muscle cells as a negative feedback mechanism (Page et al., 2002). In conclusion, this study has shown that, in bovine tracheal smooth muscle cells, bradykinin is not mitogenic by itself but augments DNA synthesis and proliferation in combination with the peptide growth factors EGF and PDGF and to a lesser extent TGFβ1. The effects of bradykinin on EGF augmentation were mediated through bradykinin B2 receptor stimulation and subsequent activation of conventional PKC isozymes. The observation that bradykinin potentiates proliferation induced by these growth factors in airway smooth muscle may be relevant for the pathophysiology of asthma, as bradykinin is an important inflammatory mediator involved in this disease (Barnes et al., 1998). Persistent stimulation of airway smooth muscle proliferation through such a mechanism during chronic inflammation may contribute to the increased airway smooth muscle mass seen in asthmatics. Acknowledgement This work was financially supported by the Netherlands Asthma Foundation, grant NAF 99.53. References Agazie, Y.M., Bagot, J.C., Trickey, E., Halenda, S.P., Wilden, P.A., 2001. Molecular mechanisms of ATP and insulin synergistic stimulation of coronary artery smooth muscle growth. Am. J. Physiol, Heart Circ. Physiol. 280, H795–H801. Akbary, A.M., Wirth, K.J., Scholkens, B.A., 1996. Efficacy and tolerability of Icatibant (Hoe 140) in patients with moderately severe chronic bronchial asthma. Immunopharmacology 33, 238–242. Barnes, P.J., Chung, K.F., Page, C.P., 1998. Inflammatory mediators of asthma: an update. Pharmacol. Rev. 50, 515–596. Bertrand, C., Nadel, J.A., Yamawaki, I., Geppetti, P., 1993. Role of kinins in the vascular extravasation evoked by antigen and mediated by tachykinins in guinea pig trachea. J. Immunol. 151, 4902–4907. Billington, C.K., Kong, K.C., Bhattacharyya, R., Wedegaertner, P.B., Panettieri Jr., R.A., Chan, T.O., Penn, R.B., 2005. Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth. Biochemistry 44, 14595–14605.

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