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Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells
Life Sciences 80 (2007) 1409 – 1414 www.elsevier.com/locate/lifescie
Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells Yasunari Kanda ⁎, Yasuhiro Watanabe Department of Pharmacology, National Defense Medical College, 3-2, Namiki, Tokorozawa, Saitama 359-8513, Japan Received 27 September 2006; accepted 26 December 2006
Abstract Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentrationdependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers. © 2007 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Extracellular signal-regulated kinase; Epidermal growth factor receptor; Nicotine; Vascular endothelial growth factor; Vascular smooth muscle cells
Introduction Epidemiological studies have established that cigarette smoke is a major risk factor for accelerated atherosclerosis (Jonas et al., 1992). However, the cellular basis for this association has not yet been elucidated. The proliferation and migration of vascular smooth muscle cells (VSMC) have been reported to play a key role in the progression of atherosclerosis (Ross, 1993). In the proposed model, following the injury of endothelial cells, the VSMC migrate from the tunica media to the intima and proliferate under the stimulation of several growth factors. Vascular endothelial growth factor (VEGF) has been identified as a mitogen that promotes vascular endothelial cell proliferation (Leung et al., 1989). It is well established that VEGF plays a key role in angiogenesis (Neufeld et al., 1999). ⁎ Corresponding author. Tel.: +81 42 995 1484; fax: +81 42 996 5191. E-mail address: [email protected] (Y. Kanda). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.12.033
Although VEGF has been demonstrated to be relatively specific for endothelial cells, several agonists, such as thrombin, have been shown to release VEGF in VSMC (Bassus et al., 2001). Cigarette smoke contains over 4000 different compounds (Hoffman and Wynder, 1986), with nicotine being one of the most important. Nicotine is thought to induce the release of catecholamine from sympathetic nerve endings. Furthermore, it is known that the subsequent activation of α-adrenoceptors causes the contraction of VSMC and an increase in blood pressure (Toda et al., 1995). Recently, α subunits of the nicotinic acetylcholine receptor (nAChR) have been detected in VSMC (Bruggmann et al., 2003). However, the functional role of the nAChR in VSMC is not fully understood. Extracellular signal-regulated kinase (ERK) is a member of the family of mitogen-activated protein kinases (MAPK) and has been demonstrated to play an important role in transmitting extracellular signals into various cellular responses such as proliferation and migration (English et al., 1999). We have previously reported that ERK phosphorylation stimulated by G
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protein-coupled receptor is mediated through the epidermal growth factor receptor (EGFR) transactivation pathway in VSMC (Kanda et al., 2001). Although nicotine has recently been demonstrated to activate ERK in VSMC (Di Luozzo et al., 2005), the mechanisms by which nicotine activates ERK is not well characterized. In this study, we report here that exposure to nicotine results in VEGF release through the nAChR in VSMC. Furthermore, VEGF release induced by nicotine is mediated via the EGFRERK pathway in VSMC. This might explain the progression of atherosclerosis in cigarette smokers. Methods
Rockford, IL). The samples were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by electroblotting (15 V, 90 min). After blocking in 5% skimmed milk in phosphatebuffered saline containing 0.2% Tween 20 (PBS-T), the membranes were reacted with specific antibodies for 1.5 h at room temperature. The blots were washed with PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution; Calbiochem-Novabiochem, La Jolla, CA) for 1 h at room temperature. After washing with PBS-T, the signal was detected by enhanced chemiluminescence (ECL detection kit; Amersham Pharmacia Biotech, Buckinghamshire, UK).
Cell culture ERK phosphorylation Rat aortic VSMC were prepared from 8-week-old Sprague– Dawley rats by using the explant method as described previously (Nishio and Watanabe, 1997). The rats were housed individually in a temperature-controlled environment on a 12h light:12-h dark-cycle with the lights on from 0700 to 1900 and given ad libitum access to food and water. All the procedures involving animal preparation were approved by the National Defense Medical College Animal Committee. The identity of the isolated cells was confirmed by immunostaining with αsmooth muscle actin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air in 100-mm dishes. The growth medium used was Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), 100 units/ml of penicillin (Gibco BRL, Gaitherburg, MD), and 100 μg/ml of streptomycin (Gibco BRL). The medium was changed twice a week. Passages 2–6 were used for the experiments. VEGF assay The VSMC were serum-starved for 72 h in serum-free DMEM. The cells were then stimulated with nicotine in serumfree DMEM for 48 h. The supernatant was collected and the VEGF in the medium was measured using a rat VEGF ELISA kit (R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. Cell lysis and immunoblotting Cell lysis and immunoblotting were performed as previously described (Kanda and Watanabe, 2005). The serum-starved cells were incubated with appropriate stimulants for the indicated times at 37 °C and lysed in a buffer [50 mM Tris, pH7.4, 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM sodium fluoride, 2 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 10 μg/ ml aprotinin, and 10 μg/ml leupeptin] on ice for 30 min. After the lysed cells were centrifuged at 15,000 ×g for 20 min at 4 °C, the supernatant was collected and the protein concentration was determined using a BCA Protein Assay Reagent Kit (Pierce,
The cell lysates were prepared as described above. ERK phosphorylation was analyzed by immunoblotting with an antiphospho-ERK antibody (Cell Signaling Technology, Beverly, MA) as described previously (Kanda et al., 2001). EGFR phosphorylation EGFR phosphorylation was analyzed as previously reported (Kanda et al., 2001). Briefly, the cell lysates were precleared with protein G-sepharose beads (Amersham Pharmacia Biotech, Buckinghamshire, UK) and incubated with anti-EGFR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated to sepharose beads overnight at 4 °C. The immunoprecipitants were immunoblotted with anti-phosphotyrosine monoclonal antibody (PY20) (Santa Cruz Biotechnology). Materials Nicotine and hexamethonium were from Wako Pure Chemical Inductries (Osaka, Japan). Epibatidine was from Sigma-Aldrich (St. Louis, MO). AG1478, PD98059 and αBungarotoxin were from Calbiochem-Novabiochem. U0126 was from Promega (Madison, WI). All other reagents were of analytical grades and obtained from commercial sources. Statistical analysis The values are expressed as the arithmetic means ± S.D. The statistical analysis of the data was performed by means of a oneway analysis of variance (ANOVA), followed by Scheffe's test when the F ratios were significant (P b 0.05). Results Effect of nicotine on VEGF release in VSMC To determine whether nicotine directly influences VSMC, we examined whether nicotine induces the production of VEGF in rat VSMC. We found that nicotine induced VEGF release in a time- and concentration-dependent manner (Fig. 1). As shown
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phosphorylation. Nicotine induced a transient phosphorylation of ERK (Fig. 3A). U0126, which is a selective inhibitor of MAPK/ERK kinase (MEK) that activates ERK, inhibited nicotine-induced ERK phosphorylation (Fig. 3B). Nicotineinduced ERK phosphorylation was inhibited by hexamethonium (Fig. 3C), confirming that the ERK phosphorylation induced by nicotine is mediated via an nAChR. As shown in Fig. 4, U0126 inhibited the nicotine-induced VEGF release in a dose-dependent manner. However, it had no effect on the basal level. Taken together, these data suggest that nicotine induces the release of VEGF via the ERK-dependent pathway in VSMC.
Fig. 1. Effect of nicotine on VEGF release in VSMC. (A) Serum-starved VSMC were incubated in the presence or absence of nicotine (10 μM) for the indicated times. (B) VSMC were incubated for 48 h in the presence of the indicated concentration of nicotine. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
in Fig. 2A, the nicotine-induced VEGF release was inhibited by hexamethonium (a non-selective nAChR antagonist) but not by α-bungarotoxin (an α7 subunit-selective nAChR antagonist), suggesting that the effect of nicotine is mediated through an nAChR other than the α7 subunit. In addition, we tested whether the effects of nicotine were mimicked by epibatidine, another nAChR agonist that is structurally different from nicotine. As shown in Fig. 2B, epibatidine induced VEGF release in the cells and hexamethonium inhibited the effect of epibatidine. These results suggest that nicotine induces the release of VEGF through the nAChR in VSMC. Involvement of ERK in nicotine-induced VEGF release To examine whether ERK is involved in nicotine-induced VEGF release, we tested the effect of nicotine on ERK
Fig. 2. Effects of nAChR antagonists on nicotine-induced VEGF release in VSMC. (A) Serum-starved VSMC were incubated with hexamethonium (Hex, 10 μM) or α-bungarotoxin (Bun, 1 μM) for 30 min. The cells were then incubated in the presence of nicotine (Nic, 10 μM) for 48 h. (B) Serumstarved VSMC were incubated with hexamethonium (10 μM) for 30 min. After incubation, the cells were incubated in the presence of epibatidine (Epib, 10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
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Involvement of EGFR in nicotine-induced VEGF release We have previously reported that thrombin-induced ERK phosphorylation is mediated through an EGFR-dependent pathway in rat A10 VSMC (Kanda et al., 2001). To determine whether this pathway is involved in nicotine-induced VEGF release, we examined the effect of AG1478, a selective inhibitor of EGFR kinase (Levitzki and Gazit, 1995), on the EGFR receptor phosphorylation, ERK phosphorylation and VEGF release in VSMC. As shown in Fig. 5A, nicotine induced EGFR phosphorylation and AG1478 inhibited the nicotine-induced
Fig. 3. Nicotine induced ERK phosphorylation in VSMC. (A) Serum-starved VSMC were incubated with nicotine (10 μM) for the indicated times. (B) After pretreatment with U0126 (1 μM) for 30 min, the cells were incubated in the presence of nicotine (10 μM) for 5 min. (C) The cells were incubated with hexamethonium (10 μM) or α-bungarotoxin (1 μM) for 30 min. After incubation, the cells were incubated with nicotine (10 μM) for 5 min. ERK phosphorylation was determined by immunoblotting with anti-phospho-ERK antibody. Values represent the means ± S.D. from three independent experiments.
Fig. 4. Effect of U0126 on nicotine-induced VEGF release in VSMC. Serumstarved VSMC were pretreated with various concentrations of U0126 for 30 min, and then incubated in the presence of nicotine (10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
Fig. 5. Effect of AG1478 on nicotine-induced EGFR phosphorylation, ERK phosphorylation and VEGF release in VSMC. (A) Serum-starved VSMC were incubated with or without an EGFR kinase inhibitor (AG1478; AG) for 30 min and stimulated with nicotine (10 μM) for 5 min. EGFR was immunoprecipitated from cell lysates and analyzed by immunoblotting with either anti-phosphotyrosine (PY20) (top panel) or anti-EGFR polyclonal antibody (bottom panel). (B) Serumstarved VSMC were incubated with or without AG1478 for 30 min and stimulated with nicotine (10 μM) for 5 min. ERK phosphorylation was determined by immunoblotting with anti-phosho-ERK antibody. The results shown are representative of three independent experiments. (C) Serum-starved VSMC were pretreated with AG1478 (1 μM) for 30 min and incubated in the presence of nicotine (10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
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EGFR phosphorylation. Although AG1478 inhibited the nicotine-induced ERK phosphorylation (Fig. 5B) and VEGF release (Fig. 5C), it had no effect on the basal level. These data suggest that nicotine stimulates VEGF release through the EGFR-ERK pathway in VSMC. Discussion In the present study, we demonstrate that exposure to nicotine results in VEGF release via the nAChR in rat VSMC. Moreover, nicotine induced VEGF release through an EGFRERK transactivation pathway. Our data demonstrate that nicotine might stimulate its nAChR, which is presumed to be expressed and functional in VSMC. Nicotine has been considered to act on VSMC in an indirect manner. It has been suggested that nicotine induces the release of catecholamine from sympathetic nerve endings and that the subsequent activation of α-adrenoceptors causes the contraction of VSMC (Toda et al., 1995). Consistent with our data, there are several studies that have focused on the direct vascular action of nicotine. Bruggmann et al. reported the presence of multiple α subunits of nAChR in VSMC by using RT-PCR (Bruggmann et al., 2003). Li et al. reported that the α7 subunit of nAChR mediates the migration of VSMC derived from brain basilar arteries (Li et al., 2004). Using a pharmacological approach, we demonstrate that the subtype of nAChR that produces VEGF is different to the α7 subunit. Although other subtype-selective nAChR antagonists are not commercially available, RNAi techniques might prove useful for determining the subtype. We are currently investigating the subtype of nAChR that is involved in VEGF release in VSMC. We have previously reported that EGFR can be activated by thrombin, a G protein-coupled receptor agonist, in rat A10 VSMC (Kanda et al., 2001). We found that nicotine also activates the same transactivation pathway (Fig. 5). Since nAChR is an ionotropic receptor that forms ion channels in plasma membranes and has no intrinsic kinase activity, the mechanism by which nicotine phosphorylates EGFR is not understood. Two different pathways have been proposed depending on whether or not transactivation involves the release of an EGFR ligand (Gschwind et al., 2001). One possible pathway is involved in the shedding of heparin-binding EGF (HB-EGF). Prenzel et al. have recently reported that HB-EGF acts in a paracrine manner to activate EGFR (Prenzel et al., 1999). Furthermore, the same mechanism has been demonstrated to be utilized in cigarette smoke-induced mucin production (Shao et al., 2004). The other proposed pathway operates in a ligand-independent manner. An increase in intracellular calcium has been demonstrated to be involved in the angiotensin IIstimulated EGFR transactivation pathway in VSMC (Saito and Berk, 2001). Furthermore, nicotine induces Ca2+ influx and stimulates the ERK pathway in neuronal cells (Nakayama et al., 2002). It remains to be determined whether an EGFR ligand or intracellular calcium is involved in the induction of EGFR transactivation in VSMC. Our data demonstrate that the EGFR transactivation pathway plays a role in VEGF release in VSMC. The physiological role
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of this pathway has been suggested to be involved in proliferation in a variety of cells (Gschwind et al., 2001). As far as we examined, nicotine does not induce mitogenic activity in VSMC (unpublished data). Nicotine has recently been reported to induce migration via ERK in VSMC (Di Luozzo et al., 2005). Moreover, VSMC have been demonstrated to migrate via VEGF release (Wang et al., 2004). Nicotine might induce a migratory response via EGFR in our cells. The plasma concentration of nicotine in habitual smokeless tobacco users is reported to range between 10 nM and 10 μM (Gritz et al., 1981). In our studies, the effects of nicotine can be observed at concentrations of 1 μM, suggesting that the concentrations of nicotine used here are similar with the blood concentrations of nicotine in tobacco users. Whereas we demonstrate the involvement of nicotine, there are numerous other molecules in cigarette smoke that might affect VSMC. Further studies will be needed to address the effect of constituents other than nicotine on VSMC. In conclusion, we have demonstrated that exposure to nicotine results in VEGF release in VSMC. Nicotine has a direct effect through its nAChR and the EGFR-ERK pathway in VSMC. VEGF release might be related to the development and progression of atherosclerosis in cigarette smokers. Acknowledgements This work was supported in part by a grant from the Smoking Research Foundation to Y.W. References Bassus, S., Herkert, O., Kronemann, N., Gorlach, A., Bremerich, D., Kirchmaier, C.M., Busse, R., Schini-Kerth, V.B., 2001. Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species. Arteriosclerosis, Thrombosis, and Vascular Biology 21 (9), 1550–1555. Bruggmann, D., Lips, K.S., Pfeil, U., Haberberger, R.V., Kummer, W., 2003. Rat arteries contain multiple nicotinic acetylcholine receptor α-subunits. Life Sciences 72 (18–19), 2095–2099. Di Luozzo, G., Pradhan, S., Dhadwal, A.K., Chen, A., Ueno, H., Sumpio, B.E., 2005. Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration. Atherosclerosis 178 (2), 271–277. English, J., Pearson, G., Wilsbacher, J., Swantek, J., Karandikar, M., Xu, S., Cobb, M.H., 1999. New insights into the control of MAP kinase pathways. Experimental Cell Research 253 (1), 255–270. Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van Vunakis, H., Jarvik, M.E., 1981. Plasma nicotine and cotine concentrations in habitual smokeless tobacco users. Clinical Pharmacology and Therapeutics 30 (2), 201–209. Gschwind, A., Zwick, E., Prenzel, N., Leserer, M., Ullrich, A., 2001. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transactivation. Oncogene 20 (13), 1594–1600. Hoffman, D., Wynder, E.L., 1986. Chemical constituents and bioactivity of tobacco smoke. International Agency for Research on Cancer. World Health Organization, Oxford University Press, London. pp. 145–165. Jonas, M.A., Oates, J.A., Ockene, J.K., Hennekens, C.H., 1992. Statement on smoking and cardiovascular disease for health care professionals. Circulation 86 (5), 1664–1669. Kanda, Y., Watanabe, Y., 2005. Thrombin-induced glucose transport via Src-p38 MAPK pathway in vascular smooth muscle cells. British Journal of Pharmacology 146 (1), 60–67.
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Kanda, Y., Mizuno, K., Kuroki, Y., Watanabe, Y., 2001. Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway. British Journal of Pharmacology 132 (8), 1657–1664. Leung, D.W., Cachianes, G., Kuang, W.J., Goeddel, D.V., Ferrara, N., 1989. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246 (4935), 1306–1309. Levitzki, A., Gazit, A., 1995. Tyrosine kinase inhibition: an approach to drug development. Science 267 (5205), 1782–1788. Li, S., Zhao, T., Xin, H., Ye, L.H., Zhang, X., Tanaka, H., Nakamura, A., Kohama, K., 2004. Nicotinic acetylcholine receptor alpha7 subunit mediates migration of vascular smooth muscle cells toward nicotine. Journal of Pharmacological Sciences 94 (3), 334–338. Nakayama, H., Numakawa, T., Ikeuchi, T., 2002. Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12 h cells. Journal of Neurochemistry 83 (6), 1372–1379. Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z., 1999. Vascular endothelial growth factor (VEGF) and its receptors. FASEB Journal 13 (1), 9–22. Nishio, E., Watanabe, Y., 1997. The involvement of reactive oxygen species and arachidonic acid in α1-adrenoceptor-induced smooth muscle cell proliferation and migration. British Journal of Pharmacology 121 (4), 665–670.
Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., Ullrich, A., 1999. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402 (6764), 884–888. Ross, R., 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362 (6423), 801–809. Saito, Y., Berk, B.C., 2001. Transactivation: a novel signaling pathway from angiotensin II to tyrosine kinase receptors. Journal of Molecular and Cellular Cardiology 33 (1), 3–7. Shao, M.X., Nakanaga, T., Nadel, J.A., 2004. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. American Journal of Physiology 287 (2), L420–L427. Toda, N., Yoshida, K., Okamura, T., 1995. Involvement of nitroxidergic and noradrenergic nerves in the relaxation of dog and monkey temporal veins. Journal of Cardiovascular Pharmacology 25 (5), 741–747. Wang, Z., Castresana, M.R., Newman, W.H., 2004. Reactive oxygen speciessensitive p38 MAPK controls thrombin-induced migration of vascular smooth muscle cells. Journal of Molecular and Cellular Cardiology 36 (1), 49–56.
Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells Yasunari Kanda ⁎, Yasuhiro Watanabe Department of Pharmacology, National Defense Medical College, 3-2, Namiki, Tokorozawa, Saitama 359-8513, Japan Received 27 September 2006; accepted 26 December 2006
Abstract Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentrationdependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers. © 2007 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Extracellular signal-regulated kinase; Epidermal growth factor receptor; Nicotine; Vascular endothelial growth factor; Vascular smooth muscle cells
Introduction Epidemiological studies have established that cigarette smoke is a major risk factor for accelerated atherosclerosis (Jonas et al., 1992). However, the cellular basis for this association has not yet been elucidated. The proliferation and migration of vascular smooth muscle cells (VSMC) have been reported to play a key role in the progression of atherosclerosis (Ross, 1993). In the proposed model, following the injury of endothelial cells, the VSMC migrate from the tunica media to the intima and proliferate under the stimulation of several growth factors. Vascular endothelial growth factor (VEGF) has been identified as a mitogen that promotes vascular endothelial cell proliferation (Leung et al., 1989). It is well established that VEGF plays a key role in angiogenesis (Neufeld et al., 1999). ⁎ Corresponding author. Tel.: +81 42 995 1484; fax: +81 42 996 5191. E-mail address: [email protected] (Y. Kanda). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.12.033
Although VEGF has been demonstrated to be relatively specific for endothelial cells, several agonists, such as thrombin, have been shown to release VEGF in VSMC (Bassus et al., 2001). Cigarette smoke contains over 4000 different compounds (Hoffman and Wynder, 1986), with nicotine being one of the most important. Nicotine is thought to induce the release of catecholamine from sympathetic nerve endings. Furthermore, it is known that the subsequent activation of α-adrenoceptors causes the contraction of VSMC and an increase in blood pressure (Toda et al., 1995). Recently, α subunits of the nicotinic acetylcholine receptor (nAChR) have been detected in VSMC (Bruggmann et al., 2003). However, the functional role of the nAChR in VSMC is not fully understood. Extracellular signal-regulated kinase (ERK) is a member of the family of mitogen-activated protein kinases (MAPK) and has been demonstrated to play an important role in transmitting extracellular signals into various cellular responses such as proliferation and migration (English et al., 1999). We have previously reported that ERK phosphorylation stimulated by G
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protein-coupled receptor is mediated through the epidermal growth factor receptor (EGFR) transactivation pathway in VSMC (Kanda et al., 2001). Although nicotine has recently been demonstrated to activate ERK in VSMC (Di Luozzo et al., 2005), the mechanisms by which nicotine activates ERK is not well characterized. In this study, we report here that exposure to nicotine results in VEGF release through the nAChR in VSMC. Furthermore, VEGF release induced by nicotine is mediated via the EGFRERK pathway in VSMC. This might explain the progression of atherosclerosis in cigarette smokers. Methods
Rockford, IL). The samples were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by electroblotting (15 V, 90 min). After blocking in 5% skimmed milk in phosphatebuffered saline containing 0.2% Tween 20 (PBS-T), the membranes were reacted with specific antibodies for 1.5 h at room temperature. The blots were washed with PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution; Calbiochem-Novabiochem, La Jolla, CA) for 1 h at room temperature. After washing with PBS-T, the signal was detected by enhanced chemiluminescence (ECL detection kit; Amersham Pharmacia Biotech, Buckinghamshire, UK).
Cell culture ERK phosphorylation Rat aortic VSMC were prepared from 8-week-old Sprague– Dawley rats by using the explant method as described previously (Nishio and Watanabe, 1997). The rats were housed individually in a temperature-controlled environment on a 12h light:12-h dark-cycle with the lights on from 0700 to 1900 and given ad libitum access to food and water. All the procedures involving animal preparation were approved by the National Defense Medical College Animal Committee. The identity of the isolated cells was confirmed by immunostaining with αsmooth muscle actin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air in 100-mm dishes. The growth medium used was Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS), 100 units/ml of penicillin (Gibco BRL, Gaitherburg, MD), and 100 μg/ml of streptomycin (Gibco BRL). The medium was changed twice a week. Passages 2–6 were used for the experiments. VEGF assay The VSMC were serum-starved for 72 h in serum-free DMEM. The cells were then stimulated with nicotine in serumfree DMEM for 48 h. The supernatant was collected and the VEGF in the medium was measured using a rat VEGF ELISA kit (R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. Cell lysis and immunoblotting Cell lysis and immunoblotting were performed as previously described (Kanda and Watanabe, 2005). The serum-starved cells were incubated with appropriate stimulants for the indicated times at 37 °C and lysed in a buffer [50 mM Tris, pH7.4, 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM sodium fluoride, 2 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 10 μg/ ml aprotinin, and 10 μg/ml leupeptin] on ice for 30 min. After the lysed cells were centrifuged at 15,000 ×g for 20 min at 4 °C, the supernatant was collected and the protein concentration was determined using a BCA Protein Assay Reagent Kit (Pierce,
The cell lysates were prepared as described above. ERK phosphorylation was analyzed by immunoblotting with an antiphospho-ERK antibody (Cell Signaling Technology, Beverly, MA) as described previously (Kanda et al., 2001). EGFR phosphorylation EGFR phosphorylation was analyzed as previously reported (Kanda et al., 2001). Briefly, the cell lysates were precleared with protein G-sepharose beads (Amersham Pharmacia Biotech, Buckinghamshire, UK) and incubated with anti-EGFR polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated to sepharose beads overnight at 4 °C. The immunoprecipitants were immunoblotted with anti-phosphotyrosine monoclonal antibody (PY20) (Santa Cruz Biotechnology). Materials Nicotine and hexamethonium were from Wako Pure Chemical Inductries (Osaka, Japan). Epibatidine was from Sigma-Aldrich (St. Louis, MO). AG1478, PD98059 and αBungarotoxin were from Calbiochem-Novabiochem. U0126 was from Promega (Madison, WI). All other reagents were of analytical grades and obtained from commercial sources. Statistical analysis The values are expressed as the arithmetic means ± S.D. The statistical analysis of the data was performed by means of a oneway analysis of variance (ANOVA), followed by Scheffe's test when the F ratios were significant (P b 0.05). Results Effect of nicotine on VEGF release in VSMC To determine whether nicotine directly influences VSMC, we examined whether nicotine induces the production of VEGF in rat VSMC. We found that nicotine induced VEGF release in a time- and concentration-dependent manner (Fig. 1). As shown
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phosphorylation. Nicotine induced a transient phosphorylation of ERK (Fig. 3A). U0126, which is a selective inhibitor of MAPK/ERK kinase (MEK) that activates ERK, inhibited nicotine-induced ERK phosphorylation (Fig. 3B). Nicotineinduced ERK phosphorylation was inhibited by hexamethonium (Fig. 3C), confirming that the ERK phosphorylation induced by nicotine is mediated via an nAChR. As shown in Fig. 4, U0126 inhibited the nicotine-induced VEGF release in a dose-dependent manner. However, it had no effect on the basal level. Taken together, these data suggest that nicotine induces the release of VEGF via the ERK-dependent pathway in VSMC.
Fig. 1. Effect of nicotine on VEGF release in VSMC. (A) Serum-starved VSMC were incubated in the presence or absence of nicotine (10 μM) for the indicated times. (B) VSMC were incubated for 48 h in the presence of the indicated concentration of nicotine. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
in Fig. 2A, the nicotine-induced VEGF release was inhibited by hexamethonium (a non-selective nAChR antagonist) but not by α-bungarotoxin (an α7 subunit-selective nAChR antagonist), suggesting that the effect of nicotine is mediated through an nAChR other than the α7 subunit. In addition, we tested whether the effects of nicotine were mimicked by epibatidine, another nAChR agonist that is structurally different from nicotine. As shown in Fig. 2B, epibatidine induced VEGF release in the cells and hexamethonium inhibited the effect of epibatidine. These results suggest that nicotine induces the release of VEGF through the nAChR in VSMC. Involvement of ERK in nicotine-induced VEGF release To examine whether ERK is involved in nicotine-induced VEGF release, we tested the effect of nicotine on ERK
Fig. 2. Effects of nAChR antagonists on nicotine-induced VEGF release in VSMC. (A) Serum-starved VSMC were incubated with hexamethonium (Hex, 10 μM) or α-bungarotoxin (Bun, 1 μM) for 30 min. The cells were then incubated in the presence of nicotine (Nic, 10 μM) for 48 h. (B) Serumstarved VSMC were incubated with hexamethonium (10 μM) for 30 min. After incubation, the cells were incubated in the presence of epibatidine (Epib, 10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
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Involvement of EGFR in nicotine-induced VEGF release We have previously reported that thrombin-induced ERK phosphorylation is mediated through an EGFR-dependent pathway in rat A10 VSMC (Kanda et al., 2001). To determine whether this pathway is involved in nicotine-induced VEGF release, we examined the effect of AG1478, a selective inhibitor of EGFR kinase (Levitzki and Gazit, 1995), on the EGFR receptor phosphorylation, ERK phosphorylation and VEGF release in VSMC. As shown in Fig. 5A, nicotine induced EGFR phosphorylation and AG1478 inhibited the nicotine-induced
Fig. 3. Nicotine induced ERK phosphorylation in VSMC. (A) Serum-starved VSMC were incubated with nicotine (10 μM) for the indicated times. (B) After pretreatment with U0126 (1 μM) for 30 min, the cells were incubated in the presence of nicotine (10 μM) for 5 min. (C) The cells were incubated with hexamethonium (10 μM) or α-bungarotoxin (1 μM) for 30 min. After incubation, the cells were incubated with nicotine (10 μM) for 5 min. ERK phosphorylation was determined by immunoblotting with anti-phospho-ERK antibody. Values represent the means ± S.D. from three independent experiments.
Fig. 4. Effect of U0126 on nicotine-induced VEGF release in VSMC. Serumstarved VSMC were pretreated with various concentrations of U0126 for 30 min, and then incubated in the presence of nicotine (10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means ± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
Fig. 5. Effect of AG1478 on nicotine-induced EGFR phosphorylation, ERK phosphorylation and VEGF release in VSMC. (A) Serum-starved VSMC were incubated with or without an EGFR kinase inhibitor (AG1478; AG) for 30 min and stimulated with nicotine (10 μM) for 5 min. EGFR was immunoprecipitated from cell lysates and analyzed by immunoblotting with either anti-phosphotyrosine (PY20) (top panel) or anti-EGFR polyclonal antibody (bottom panel). (B) Serumstarved VSMC were incubated with or without AG1478 for 30 min and stimulated with nicotine (10 μM) for 5 min. ERK phosphorylation was determined by immunoblotting with anti-phosho-ERK antibody. The results shown are representative of three independent experiments. (C) Serum-starved VSMC were pretreated with AG1478 (1 μM) for 30 min and incubated in the presence of nicotine (10 μM) for 48 h. VEGF release in the media was determined by ELISA as described in Methods. Values represent the means± S.D. from three independent experiments. ⁎P b 0.05 as compared with the respective control.
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EGFR phosphorylation. Although AG1478 inhibited the nicotine-induced ERK phosphorylation (Fig. 5B) and VEGF release (Fig. 5C), it had no effect on the basal level. These data suggest that nicotine stimulates VEGF release through the EGFR-ERK pathway in VSMC. Discussion In the present study, we demonstrate that exposure to nicotine results in VEGF release via the nAChR in rat VSMC. Moreover, nicotine induced VEGF release through an EGFRERK transactivation pathway. Our data demonstrate that nicotine might stimulate its nAChR, which is presumed to be expressed and functional in VSMC. Nicotine has been considered to act on VSMC in an indirect manner. It has been suggested that nicotine induces the release of catecholamine from sympathetic nerve endings and that the subsequent activation of α-adrenoceptors causes the contraction of VSMC (Toda et al., 1995). Consistent with our data, there are several studies that have focused on the direct vascular action of nicotine. Bruggmann et al. reported the presence of multiple α subunits of nAChR in VSMC by using RT-PCR (Bruggmann et al., 2003). Li et al. reported that the α7 subunit of nAChR mediates the migration of VSMC derived from brain basilar arteries (Li et al., 2004). Using a pharmacological approach, we demonstrate that the subtype of nAChR that produces VEGF is different to the α7 subunit. Although other subtype-selective nAChR antagonists are not commercially available, RNAi techniques might prove useful for determining the subtype. We are currently investigating the subtype of nAChR that is involved in VEGF release in VSMC. We have previously reported that EGFR can be activated by thrombin, a G protein-coupled receptor agonist, in rat A10 VSMC (Kanda et al., 2001). We found that nicotine also activates the same transactivation pathway (Fig. 5). Since nAChR is an ionotropic receptor that forms ion channels in plasma membranes and has no intrinsic kinase activity, the mechanism by which nicotine phosphorylates EGFR is not understood. Two different pathways have been proposed depending on whether or not transactivation involves the release of an EGFR ligand (Gschwind et al., 2001). One possible pathway is involved in the shedding of heparin-binding EGF (HB-EGF). Prenzel et al. have recently reported that HB-EGF acts in a paracrine manner to activate EGFR (Prenzel et al., 1999). Furthermore, the same mechanism has been demonstrated to be utilized in cigarette smoke-induced mucin production (Shao et al., 2004). The other proposed pathway operates in a ligand-independent manner. An increase in intracellular calcium has been demonstrated to be involved in the angiotensin IIstimulated EGFR transactivation pathway in VSMC (Saito and Berk, 2001). Furthermore, nicotine induces Ca2+ influx and stimulates the ERK pathway in neuronal cells (Nakayama et al., 2002). It remains to be determined whether an EGFR ligand or intracellular calcium is involved in the induction of EGFR transactivation in VSMC. Our data demonstrate that the EGFR transactivation pathway plays a role in VEGF release in VSMC. The physiological role
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of this pathway has been suggested to be involved in proliferation in a variety of cells (Gschwind et al., 2001). As far as we examined, nicotine does not induce mitogenic activity in VSMC (unpublished data). Nicotine has recently been reported to induce migration via ERK in VSMC (Di Luozzo et al., 2005). Moreover, VSMC have been demonstrated to migrate via VEGF release (Wang et al., 2004). Nicotine might induce a migratory response via EGFR in our cells. The plasma concentration of nicotine in habitual smokeless tobacco users is reported to range between 10 nM and 10 μM (Gritz et al., 1981). In our studies, the effects of nicotine can be observed at concentrations of 1 μM, suggesting that the concentrations of nicotine used here are similar with the blood concentrations of nicotine in tobacco users. Whereas we demonstrate the involvement of nicotine, there are numerous other molecules in cigarette smoke that might affect VSMC. Further studies will be needed to address the effect of constituents other than nicotine on VSMC. In conclusion, we have demonstrated that exposure to nicotine results in VEGF release in VSMC. Nicotine has a direct effect through its nAChR and the EGFR-ERK pathway in VSMC. VEGF release might be related to the development and progression of atherosclerosis in cigarette smokers. Acknowledgements This work was supported in part by a grant from the Smoking Research Foundation to Y.W. References Bassus, S., Herkert, O., Kronemann, N., Gorlach, A., Bremerich, D., Kirchmaier, C.M., Busse, R., Schini-Kerth, V.B., 2001. Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species. Arteriosclerosis, Thrombosis, and Vascular Biology 21 (9), 1550–1555. Bruggmann, D., Lips, K.S., Pfeil, U., Haberberger, R.V., Kummer, W., 2003. Rat arteries contain multiple nicotinic acetylcholine receptor α-subunits. Life Sciences 72 (18–19), 2095–2099. Di Luozzo, G., Pradhan, S., Dhadwal, A.K., Chen, A., Ueno, H., Sumpio, B.E., 2005. Nicotine induces mitogen-activated protein kinase dependent vascular smooth muscle cell migration. Atherosclerosis 178 (2), 271–277. English, J., Pearson, G., Wilsbacher, J., Swantek, J., Karandikar, M., Xu, S., Cobb, M.H., 1999. New insights into the control of MAP kinase pathways. Experimental Cell Research 253 (1), 255–270. Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van Vunakis, H., Jarvik, M.E., 1981. Plasma nicotine and cotine concentrations in habitual smokeless tobacco users. Clinical Pharmacology and Therapeutics 30 (2), 201–209. Gschwind, A., Zwick, E., Prenzel, N., Leserer, M., Ullrich, A., 2001. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transactivation. Oncogene 20 (13), 1594–1600. Hoffman, D., Wynder, E.L., 1986. Chemical constituents and bioactivity of tobacco smoke. International Agency for Research on Cancer. World Health Organization, Oxford University Press, London. pp. 145–165. Jonas, M.A., Oates, J.A., Ockene, J.K., Hennekens, C.H., 1992. Statement on smoking and cardiovascular disease for health care professionals. Circulation 86 (5), 1664–1669. Kanda, Y., Watanabe, Y., 2005. Thrombin-induced glucose transport via Src-p38 MAPK pathway in vascular smooth muscle cells. British Journal of Pharmacology 146 (1), 60–67.
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