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Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells
Pharmacological Research 61 (2010) 188–192
Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells Yoshihiko Chiba ∗ , Michiko Todoroki, Miwa Misawa Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
a r t i c l e
i n f o
Article history: Received 14 September 2009 Received in revised form 30 September 2009 Accepted 16 October 2009 Keywords: Interleukin-4 STAT6 RhoA Bronchial smooth muscle cell Airway hyperresponsiveness
a b s t r a c t Interleukin-4 (IL-4) is believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. In the present study, the effects of IL-4 on the expression of RhoA protein, a monomeric GTP-binding protein that contributes to the contraction of smooth muscle, were determined in cultured human bronchial smooth muscle cells (hBSMCs). Incubation of hBSMCs with IL-4 (100 ng/mL) caused a distinct phosphorylation of signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-4, indicating that IL-4 is capable of activating signal transduction in the hBSMCs directly. IL-4 also caused a significant increase in the expression level of RhoA protein: the peak of the upregulation of RhoA protein was observed at 12–24 h after the IL-4 treatment. Both the phosphorylation of STAT6 and the upregulation of RhoA protein induced by IL-4 were inhibited by the co-incubation with AS1517499, a selective inhibitor of STAT6, in a concentration-dependent fashion. These findings suggest that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics, might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The dramatic increase in the number of asthma cases over the last decades is of great concern for public health in the world [1]. Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive diseases, including bronchial asthma. This abnormality is an important sign of the disease, although the pathophysiological variations leading to the hyperresponsiveness are unclear now. It has been suggested that one of the factors that contribute to the exaggerated airway narrowing in asthmatics is an abnormality of the properties of airway smooth muscle [2,3]. Rapid relief from airway limitation in asthmatic patients by -stimulant inhalation [4–7] may also suggest an involvement of augmented airway smooth muscle contraction in the airway obstruction. Thus, it may be important for development of asthma therapy to understand changes in the contractile signaling of airway smooth muscle cells associated with the disease.
Abbreviations: BSM, bronchial smooth muscle; AHR, airway hyperresponsiveness; MLC, myosin light chain; IL, interleukin; STAT, signal transducer and activator of transcription; hEGF, human epidermal growth factor; hFGF-b, human fibroblast growth factor-basic; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ANOVA, analysis of variance. ∗ Corresponding author. Tel.: +81 3 5498 5786; fax: +81 3 5498 5787. E-mail address: [email protected] (Y. Chiba). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.10.003
Recently, an importance of RhoA, a monomeric GTP-binding protein, and its downstream Rho-kinases have been demonstrated in the contraction of smooth muscles, including human bronchial smooth muscle (BSM) [8]. The RhoA/Rho-kinase pathway is involved in the agonist-induced Ca2+ sensitization of contraction in various types of smooth muscles. When the pathway was activated by contractile agonists, the activity of myosin light chain (MLC) phosphatase reduces and the level of phosphorylated MLC then increases, resulting in an augmentation of smooth muscle contraction. Interestingly, the RhoA-mediated Ca2+ sensitization of BSM contraction is augmented in experimental asthma models of rats [9] and mice [10]. An upregulation of RhoA has also been demonstrated in BSMs of these animal models of allergic bronchial asthma [9–11]. It is thus possible that the RhoA/Rho-kinase signaling might be augmented in BSMs of patients with allergic bronchial asthma. The RhoA/Rho-kinase pathway has now been proposed as a novel target for the treatment of AHR in asthma [12,13]. Interleukin-4 (IL-4), one of the T-helper 2 (Th2) cytokines, is believed to play a role in asthma [14–17]. An increased expression of IL-4 has been demonstrated in bronchoalveolar lavage fluid after segmental allergen challenge to asthmatic patients [14]. IL-4 promotes eosinophilic airway inflammation by increasing eotaxin expression and inhibiting eosinophil apoptosis [15]. IL-4 induces mucus hypersecretion [16] that contributes to airway obstruction. Interestingly, IL-4 also acts on airway smooth muscle directly, and
Y. Chiba et al. / Pharmacological Research 61 (2010) 188–192
has an ability to cause hyperresponsiveness of airway smooth muscle [17]. We thus hypothesized that IL-4 may be one of the causes of the RhoA upregulation in BSMs observed in animal models of antigen-induced AHR [10,11]. In the present study, the effect of IL4 on the expression level of RhoA protein was examined in human BSM cells (hBSMCs). 2. Materials and methods 2.1. Cell culture Normal human bronchial smooth muscle cells (hBSMCs; Cambrex Bio Science Walkersville, Inc., Walkersville, MD) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum, 0.5 ng/mL human epidermal growth factor (hEGF), 5 g/mL insulin, 2 ng/mL human fibroblast growth factorbasic (hFGF-b), 50 g/mL gentamicin and 50 ng/mL amphotericin B. Cells were maintained at 37 ◦ C in a humidified atmosphere (5% CO2 ), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 7–9) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at a density of 3500 cells/cm2 and, when 80–85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human IL-4 (PeproTech EC, Ltd., London, UK). A selective inhibitor of signal transducer and activator of transcription 6 (STAT6), AS1517499 (30 or 100 nM; kindly provided from Astellas Pharma Inc., Tokyo, Japan), or its vehicle (0.3% DMSO) was treated 30 min before the addition of IL-4 (100 ng/mL). At the indicated time after the IL-4 treatment, cells were washed with PBS, immediately collected and disrupted with 1× SDS sample buffer (250 L/well), and used for Western blot analyses.
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3. Results 3.1. Effects of IL-4 on RhoA protein expression in cultured hBSMCs To investigate the direct effects of IL-4 on bronchial smooth muscle cells, cultured hBSMCs were treated with recombinant human IL-4 under the serum-free condition as described in Section 2. Our previous study [18] demonstrated that the hBSMCs used expressed IL-4 receptor ␣ (IL4R␣) and IL-13 receptor ␣ chains (IL13R␣1) and STAT6, a major signal transducer activated by IL4 [19–21]. IL-4 is thus capable of activating signal transduction in hBSMCs directly. To confirm this, tyrosine phosphorylation of STAT6 in the IL-4-stimulated hBSMCs was determined by Western blotting with specific antibody against 641-phosphotyrosine-
2.2. Western blot analyses Protein samples were subjected to 15% (for RhoA) or 7.5% SDS-PAGE (for the others) and the proteins were then electrophoretically transferred to a PVDF membrane. After blocking with 3% skim milk (for RhoA) or 1% BlockAceTM (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan; for the others), the PVDF membrane was incubated with the primary antibody. The primary antibodies used in the present study were polyclonal rabbit antiRhoA (1:2500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-STAT6 (1:1000 dilution; Santa Cruz Biotechnology, Inc.), and anti-phospho-STAT6 (1:1000 dilution; Santa Cruz Biotechnology, Inc.) antibodies. Then the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:2500 dilution; Amersham Biosciences, Co., Piscataway, NJ), detected by an enhanced chemiluminescent system (Amersham Biosciences, Co.) and analyzed by a densitometry system. Detection of housekeeping gene was also performed on the same membrane by using monoclonal mouse anti-GAPDH (1:10,000 dilution; Chemicon International, Inc., Temecula, CA) and horseradish peroxidaseconjugated sheep anti-mouse IgG (1:2500 dilution; Amersham Biosciences, Co.) to confirm the same amount of proteins loaded. 2.3. Statistical analyses All the data were expressed as the mean with S.E. statistical significance of difference was determined by unpaired Student’s t-test or two-way analysis of variance (ANOVA) with post hoc Bonferroni/Dunn (StatView for Macintosh ver. 5.0, SAS Institute, Inc., NC). A value of p < 0.05 was considered significant.
Fig. 1. Effects of interleukin-4 (IL-4) on the expression and phosphorylation of signal transducer and activator of transcription 6 (STAT6) in cultured human bronchial smooth muscle cells (hBSMCs). The hBSMCs were incubated with IL-4 (100 ng/mL) for the indicated time, and total protein samples were analyzed by immunoblottings. (A) Representative Western blots of phosphorylated STAT6 (upper), total STAT6 (middle) and GAPDH (lower). The bands were analyzed by a densitometer and pSTAT6/STAT6 and STAT6/GAPDH were calculated in each protein sample, and the data are summarized in (B) and (C) respectively. Each column represents the mean ± SEM from three independent experiments. *P < 0.05 and ***P < 0.001 versus control (Cont) by Bonferroni/Dunn’s test.
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Fig. 2. Effects of interleukin-4 (IL-4) on RhoA protein expression in cultured human bronchial smooth muscle cells (hBSMCs). The hBSMCs were incubated with IL-4 (100 ng/mL) for the indicated time, and total protein samples were analyzed by immunoblottings. (A) Representative Western blots. The bands were analyzed by a densitometer and normalized by the intensity of corresponding GAPDH band, and the data are summarized in (B). Each column represents the mean ± SEM from six independent experiments. *P < 0.05 versus control (Cont) by Bonferroni/Dunn’s test.
STAT6. As shown in Fig. 1, the protein expression of STAT6 was detected in the cultured hBSMCs. When the cells were treated with IL-4, a distinct phosphorylation of STAT6 was observed. The timecourse study revealed that the peak of STAT6 phosphorylation was at 1 hr after the IL-4 treatment (Fig. 1A). The expression level of total STAT6 protein was not affected by the IL-4 treatment (Fig. 1B). Fig. 2 shows the effect of IL-4 on the expression of RhoA protein in the cultured hBSMCs. As a result, the RhoA expression was significantly increased in an incubation time-dependent manner. The peak of upregulation of RhoA protein was observed at 12–24 h after the IL-4 treatment (Fig. 2). 3.2. Effects of AS1517499 on IL-4-induced upregulation of RhoA protein To determine the ability of AS1517499 to inhibit the activation of STAT6 induced by IL-4, the effect of AS1517499 on STAT6 phosphorylation observed at 1 h after the treatment with IL-4 (100 ng/mL) was examined in the hBSMCs. As shown in Fig. 3A, immunoblot analyses revealed that AS1517499 significantly inhibited the IL4-induced STAT6 phosphorylation in a concentration-dependent manner. The STAT6 phosphorylation induced by IL-4 was blocked almost completely when cells were co-treated with 100 nM AS1517499 (Fig. 3A). Thus, AS1517499 can inhibit the activation of STAT6 by blocking its phosphorylation. The upregulation of RhoA protein observed at 24 h after the treatment with IL-4 was also inhibited by AS1517499 in a concentration-dependent manner (Fig. 3B). 4. Discussion Airway smooth muscle is an important effector tissue regulating bronchomotor tone. It has been suggested that modulation of airway smooth muscle by inflammatory mediators such as cytokines may play a crucial role in the development of airway hyperresponsiveness (AHR) [22], one of the characteristic features of
Fig. 3. Inhibitory effects of an inhibitor of signal transducer and activator of transcription 6 (STAT6), AS1517499, on the interleukin-4 (IL-4)-induced phosphorylation of STAT6 and upregulation of RhoA protein in cultured human bronchial smooth muscle cells (hBSMCs). (A) Representative Western blots of phosphorylated STAT6, total STAT6, RhoA and GAPDH. The bands were analyzed by a densitometer and pSTAT6/STAT6 and RhoA/GAPDH were calculated in each protein sample, and the data are summarized in (B) and (C) respectively. The hBSMCs were incubated with IL-4 (100 ng/mL) or its vehicle (left columns in B and C) for 1 h (B) or 24 h (C) in the absence (0 nM) or presence (30 or 100 nM) of AS1517499. Each column represents the mean ± SEM from six independent experiments. ***P < 0.001 versus no IL-4 treatment (left column, respectively), and # P < 0.05, ## P < 0.01 and ### P < 0.001 versus the group that were treated with IL-4 (100 ng/mL) in the absence of AS1517499 (the second column from the left, respectively) by Bonferroni/Dunn’s test.
patients with allergic bronchial asthma. In animal models of allergic bronchial asthma, an increased contractility of isolated BSM to contractile agonists has been found in rats [23] and mice [24]. The augmented BSM contraction induced by antigen challenge has reportedly been associated with an upregulation of RhoA [9–11],
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a small GTPase that is involved in the agonist-induced Ca2+ sensitization of smooth muscle contraction [25,26]. An importance of RhoA and its downstream Rho-kinases was also demonstrated in contraction of human BSM [8]. Here, we show that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. Currently, treatment of the hBSMCs with IL-4 caused a phosphorylation of STAT6 (Fig. 1). The IL-4-mediated events, including an activation of STAT6, are generated by its binding to the IL-4 receptors, the type I IL-4 receptor (a dimer composed of the IL4R␣ chain and the common gamma chain of the IL-2 receptor) and/or the type II IL-4 receptor (a dimer consisting of the IL4R␣ and IL13R␣1 chains). In nonhematopoietic cells including the airway smooth muscle cells, the type II IL-4 receptor is suggested as the main receptor for IL-4 [27,28]. Our previous study also demonstrated the expression of IL4R␣ and IL13R␣1 chains in the cultured hBSMCs [18]. It is thus possible that IL-4 can act on the hBSMCs directly and activate its signal transduction, the Janus kinases (JAKs)/STAT6 pathway [19–21]. In the present study, AS1517499 was used as an inhibitor of STAT6 activation. AS1517499 is a novel selective STAT6 inhibitor synthesized on the basis of the structure of a reported STAT6 inhibitor, TMC-264, discovered from the fungus Phoma [29,30]. Nagashima et al. [31] synthesized a series of 2-{[2(4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities using a STAT6 reporter assay in cells stably transfected with an IL-4-responsive luciferase reporter plasmid. Among these compounds, AS1517499 showed a potent STAT6 inhibition with a 50% inhibitory concentration (IC50 ) of 21 nM [31]. AS1517499 also inhibited the IL-4-induced Th2 cell differentiation of mouse spleen T cells with an IC50 value of 2.3 nM without influencing the IL-12-induced Th1 cell differentiation [31]. Although the exact mechanism of inhibition of STAT6 by AS1517499 is unclear now, the parent compound TMC-264 is known to inhibit both tyrosine phosphorylation of STAT6, with an IC50 value of 1.6 M (1600 nM), and the complex formation of phosphorylated STAT6 with its recognition DNA sequence [30]. The present study suggests that mode of action of AS1517499 is at least the inhibition of phosphorylation of STAT6 (Fig. 3A). One of the important findings of the current study is that IL-4 is capable of inducing an upregulation of RhoA protein (Fig. 2). There is increasing evidence that RhoA is involved in the contraction of airway smooth muscle, and that an augmented contraction due to the upregulated RhoA protein is a cause of AHR [8–10,26]. Although the mechanism of upregulation of RhoA in the diseased bronchial smooth muscle is not fully understood to date, the observation indicates that IL-4, which is upregulated in the airways of allergic bronchial asthmatics [14,32,33], is one of the candidate factors responsible for the event. Indeed, neutralizing IL-4 with anti-IL-4 antibodies prevented the development of antigen-induced AHR in mice [34]. In addition, mice in which targeted deletion of IL-4 was performed fail to develop the AHR [35]. The current observation that the IL-4-induced upregulation of RhoA protein was inhibited by the co-incubation with AS1517499 suggests that this event is mediated by an activation of STAT6 in the cultured hBSMCs. Although the exact mechanism of RhoA transcription in BSMCs is not fully understood, the upstream genomic DNA sequence of human RhoA contains several STATs binding sites, e.g., −77 (from the transcription start) to −69 (score 85.6), −270 to −261 (score 86.5), −417 to −409 (score 78.8) and −518 to −510 (score 84.6), when analyzed using the TFSEARCH program (http://mbs.cbrc.jp/research/db/TFSEARCH.html). In addition, a STAT6-dependent upregulation of RhoA was reported in hBSMCs that were stimulated with IL-13 [18], and IL-13 can also utilize IL4R␣–IL13R␣1 receptor complex for its signaling [28].
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In conclusion, the current findings indicate that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics [14,32,33] might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. Conflict of interest statement None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgements We thank Yuka Narushima and Tomoko Minemura for their technical assistance. This work was partly supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 2006;355:2226–35. [2] Seow CY, Schellenberg RR, Pare PD. Structural and functional changes in the airway smooth muscle of asthmatic subjects. Am J Respir Crit Care Med 1998;158:S179–86. [3] Martin JG, Duguet A, Eidelman DH. The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease. Eur Respir J 2000;16:349–54. [4] Sears MR. Asthma treatment: inhaled beta-agonists. Can Respir J 1998;5(Suppl. A):54A–9A. [5] Rodrigo GJ. Inhaled therapy for acute adult asthma. Curr Opin Allergy Clin Immunol 2003;3:169–75. [6] O’Byrne PM. Acute asthma intervention: insights from the STAY study. J Allergy Clin Immunol 2007;119:1332–6. [7] Papi A, Caramori G, Adcock IM, Barnes PJ. Rescue treatment in asthma. More than as-needed bronchodilation. Chest 2009;135:1628–33. [8] Yoshii A, Iizuka K, Dobashi K, Horie T, Harada T, Nakazawa T, et al. Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization. Am J Respir Cell Mol Biol 1999;20:1190–200. [9] Chiba Y, Takada Y, Miyamoto S, Mitsui-Saito M, Karaki H, Misawa M. Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol 1999;127:597–600. [10] Chiba Y, Ueno A, Shinozaki K, Takeyama H, Nakazawa S, Sakai H, et al. Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice. Respir Res 2005;6 [Art. No. 4]. [11] Chiba Y, Sakai H, Wachi H, Sugitani H, Seyama Y, Misawa M. Upregulation of rhoA mRNA in bronchial smooth muscle of antigen-induced airway hyperresponsive rats. J Smooth Muscle Res 2003;39:221–8. [12] Gosens R, Schaafsma D, Nelemans SA, Halayko AJ. Rho-kinase as a drug target for the treatment of airway hyperresponsiveness in asthma. Mini Rev Med Chem 2006;6:339–48. [13] Schaafsma D, Gosens R, Zaagsma J, Halayko AJ, Meurs H. Rho kinase inhibitors: a novel therapeutical intervention in asthma? Eur J Pharmacol 2008;585:398–406. [14] Batra V, Musani AI, Hastie AT, Khurana S, Carpenter KA, Zangrilli JG, et al. Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts. Clin Exp Allergy 2004;34: 437–44. [15] Steinke JW, Borish L. Th2 cytokines and asthma. Interleukin-4: its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin4 receptor antagonists. Respir Res 2001;2:66–70. [16] Dabbagh K, Takeyama K, Lee HM, Ueki IF, Lausier JA, Nadel JA. IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo. J Immunol 1999;162:6233–7. [17] Bryborn M, Adner M, Cardell LO. Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogen-activated protein kinase pathways. Clin Exp Allergy 2004;34:1291–8. [18] Chiba Y, Nakazawa S, Todoroki M, Shinozaki K, Sakai H, Misawa M. Interleukin13 augments bronchial smooth muscle contractility with an up-regulation of RhoA protein. Am J Respir Cell Mol Biol 2009;40:159–67. [19] Chatila TA. Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends Mol Med 2004;10:493–9.
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[20] Hebenstreit D, Wirnsberger G, Horejs-Hoeck J, Duschl A. Signaling mechanisms, interaction partners, and target genes of STAT6. Cytokine Growth Factor Rev 2006;17:173–88. [21] Kuperman DA, Schleimer RP. Interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma. Curr Mol Med 2008;8:384–92. [22] Amrani Y, Panettieri Jr RA. Modulation of calcium homeostasis as a mechanism for altering smooth muscle responsiveness in asthma. Curr Opin Allergy Clin Immunol 2002;2:39–45. [23] Chiba Y, Misawa M. Alteration in Ca2+ availability involved in antigen-induced airway hyperresponsiveness in rats. Eur J Pharmacol 1995;278:79–82. [24] Chiba Y, Ueno A, Sakai H, Misawa M. Hyperresponsiveness of bronchial but not tracheal smooth muscle in a murine model of allergic bronchial asthma. Inflamm Res 2004;53:636–42. [25] Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83:1325–58. [26] Chiba Y, Misawa M. The role of RhoA-mediated Ca2+ sensitization of bronchial smooth muscle contraction in airway hyperresponsiveness. J Smooth Muscle Res 2004;40:155–67. [27] Murata T, Taguchi J, Puri RK, Mohri H. Sharing of receptor subunits and signal transduction pathway between the IL-4 and IL-13 receptor system. Int J Hematol 1999;69:13–20. [28] Laporte JC, Moore PE, Baraldo S, Jouvin MH, Church TL, Schwartzman IN, et al. Direct effects of interleukin-13 on signaling pathways for physiological responses in cultured human airway smooth muscle cells. Am J Respir Crit Care Med 2001;164:141–8.
[29] Sakurai M, Nishio M, Yamamoto K, Okuda T, Kawano K, Ohnuki T. TMC-264, a novel antiallergic heptaketide produced by the fungus Phoma sp. TC 1674. Org Lett 2003;5:1083–5. [30] Sakurai M, Nishio M, Yamamoto K, Okuda T, Kawano K, Ohnuki T. TMC-264, a novel inhibitor of STAT6 activation produced by Phoma sp. TC 1674. J Antibiot (Tokyo) 2003;56:513–9. [31] Nagashima S, Yokota M, Nakai E, Kuromitsu S, Ohga K, Takeuchi M, et al. Synthesis and evaluation of 2-{[2-(4-hydroxyphenyl)-ethyl]amino}pyrimidine5-carboxamide derivatives as novel STAT6 inhibitors. Bioorg Med Chem 2007;15:1044–55. [32] Ackerman V, Marini M, Vittori E, Bellini A, Vassali G, Mattoli S. Detection of cytokines and their cell sources in bronchial biopsy specimens from asthmatic patients. Relationship to atopic status, symptoms, and level of airway hyperresponsiveness. Chest 1994;105:687–96. [33] Truyen E, Coteur L, Dilissen E, Overbergh L, Dupont LJ, Ceuppens JL, et al. Evaluation of airway inflammation by quantitative Th1/Th2 cytokine mRNA measurement in sputum of asthma patients. Thorax 2006;61: 202–8. [34] Corry DB, Folkesson HG, Warnock ML, Erle DJ, Matthay MA, Wiener-Kronish JP, et al. Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity. J Exp Med 1996;183: 109–17. [35] Kips JC, Brusselle GG, Joos GF, Peleman RA, Devos RR, Tavernier JH, et al. Importance of interleukin-4 and interleukin-12 in allergeninduced airway changes in mice. Int Arch Allergy Immunol 1995;107: 115–8.
Contents lists available at ScienceDirect
Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells Yoshihiko Chiba ∗ , Michiko Todoroki, Miwa Misawa Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
a r t i c l e
i n f o
Article history: Received 14 September 2009 Received in revised form 30 September 2009 Accepted 16 October 2009 Keywords: Interleukin-4 STAT6 RhoA Bronchial smooth muscle cell Airway hyperresponsiveness
a b s t r a c t Interleukin-4 (IL-4) is believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. In the present study, the effects of IL-4 on the expression of RhoA protein, a monomeric GTP-binding protein that contributes to the contraction of smooth muscle, were determined in cultured human bronchial smooth muscle cells (hBSMCs). Incubation of hBSMCs with IL-4 (100 ng/mL) caused a distinct phosphorylation of signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-4, indicating that IL-4 is capable of activating signal transduction in the hBSMCs directly. IL-4 also caused a significant increase in the expression level of RhoA protein: the peak of the upregulation of RhoA protein was observed at 12–24 h after the IL-4 treatment. Both the phosphorylation of STAT6 and the upregulation of RhoA protein induced by IL-4 were inhibited by the co-incubation with AS1517499, a selective inhibitor of STAT6, in a concentration-dependent fashion. These findings suggest that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics, might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The dramatic increase in the number of asthma cases over the last decades is of great concern for public health in the world [1]. Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive diseases, including bronchial asthma. This abnormality is an important sign of the disease, although the pathophysiological variations leading to the hyperresponsiveness are unclear now. It has been suggested that one of the factors that contribute to the exaggerated airway narrowing in asthmatics is an abnormality of the properties of airway smooth muscle [2,3]. Rapid relief from airway limitation in asthmatic patients by -stimulant inhalation [4–7] may also suggest an involvement of augmented airway smooth muscle contraction in the airway obstruction. Thus, it may be important for development of asthma therapy to understand changes in the contractile signaling of airway smooth muscle cells associated with the disease.
Abbreviations: BSM, bronchial smooth muscle; AHR, airway hyperresponsiveness; MLC, myosin light chain; IL, interleukin; STAT, signal transducer and activator of transcription; hEGF, human epidermal growth factor; hFGF-b, human fibroblast growth factor-basic; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ANOVA, analysis of variance. ∗ Corresponding author. Tel.: +81 3 5498 5786; fax: +81 3 5498 5787. E-mail address: [email protected] (Y. Chiba). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.10.003
Recently, an importance of RhoA, a monomeric GTP-binding protein, and its downstream Rho-kinases have been demonstrated in the contraction of smooth muscles, including human bronchial smooth muscle (BSM) [8]. The RhoA/Rho-kinase pathway is involved in the agonist-induced Ca2+ sensitization of contraction in various types of smooth muscles. When the pathway was activated by contractile agonists, the activity of myosin light chain (MLC) phosphatase reduces and the level of phosphorylated MLC then increases, resulting in an augmentation of smooth muscle contraction. Interestingly, the RhoA-mediated Ca2+ sensitization of BSM contraction is augmented in experimental asthma models of rats [9] and mice [10]. An upregulation of RhoA has also been demonstrated in BSMs of these animal models of allergic bronchial asthma [9–11]. It is thus possible that the RhoA/Rho-kinase signaling might be augmented in BSMs of patients with allergic bronchial asthma. The RhoA/Rho-kinase pathway has now been proposed as a novel target for the treatment of AHR in asthma [12,13]. Interleukin-4 (IL-4), one of the T-helper 2 (Th2) cytokines, is believed to play a role in asthma [14–17]. An increased expression of IL-4 has been demonstrated in bronchoalveolar lavage fluid after segmental allergen challenge to asthmatic patients [14]. IL-4 promotes eosinophilic airway inflammation by increasing eotaxin expression and inhibiting eosinophil apoptosis [15]. IL-4 induces mucus hypersecretion [16] that contributes to airway obstruction. Interestingly, IL-4 also acts on airway smooth muscle directly, and
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has an ability to cause hyperresponsiveness of airway smooth muscle [17]. We thus hypothesized that IL-4 may be one of the causes of the RhoA upregulation in BSMs observed in animal models of antigen-induced AHR [10,11]. In the present study, the effect of IL4 on the expression level of RhoA protein was examined in human BSM cells (hBSMCs). 2. Materials and methods 2.1. Cell culture Normal human bronchial smooth muscle cells (hBSMCs; Cambrex Bio Science Walkersville, Inc., Walkersville, MD) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum, 0.5 ng/mL human epidermal growth factor (hEGF), 5 g/mL insulin, 2 ng/mL human fibroblast growth factorbasic (hFGF-b), 50 g/mL gentamicin and 50 ng/mL amphotericin B. Cells were maintained at 37 ◦ C in a humidified atmosphere (5% CO2 ), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 7–9) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at a density of 3500 cells/cm2 and, when 80–85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human IL-4 (PeproTech EC, Ltd., London, UK). A selective inhibitor of signal transducer and activator of transcription 6 (STAT6), AS1517499 (30 or 100 nM; kindly provided from Astellas Pharma Inc., Tokyo, Japan), or its vehicle (0.3% DMSO) was treated 30 min before the addition of IL-4 (100 ng/mL). At the indicated time after the IL-4 treatment, cells were washed with PBS, immediately collected and disrupted with 1× SDS sample buffer (250 L/well), and used for Western blot analyses.
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3. Results 3.1. Effects of IL-4 on RhoA protein expression in cultured hBSMCs To investigate the direct effects of IL-4 on bronchial smooth muscle cells, cultured hBSMCs were treated with recombinant human IL-4 under the serum-free condition as described in Section 2. Our previous study [18] demonstrated that the hBSMCs used expressed IL-4 receptor ␣ (IL4R␣) and IL-13 receptor ␣ chains (IL13R␣1) and STAT6, a major signal transducer activated by IL4 [19–21]. IL-4 is thus capable of activating signal transduction in hBSMCs directly. To confirm this, tyrosine phosphorylation of STAT6 in the IL-4-stimulated hBSMCs was determined by Western blotting with specific antibody against 641-phosphotyrosine-
2.2. Western blot analyses Protein samples were subjected to 15% (for RhoA) or 7.5% SDS-PAGE (for the others) and the proteins were then electrophoretically transferred to a PVDF membrane. After blocking with 3% skim milk (for RhoA) or 1% BlockAceTM (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan; for the others), the PVDF membrane was incubated with the primary antibody. The primary antibodies used in the present study were polyclonal rabbit antiRhoA (1:2500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-STAT6 (1:1000 dilution; Santa Cruz Biotechnology, Inc.), and anti-phospho-STAT6 (1:1000 dilution; Santa Cruz Biotechnology, Inc.) antibodies. Then the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:2500 dilution; Amersham Biosciences, Co., Piscataway, NJ), detected by an enhanced chemiluminescent system (Amersham Biosciences, Co.) and analyzed by a densitometry system. Detection of housekeeping gene was also performed on the same membrane by using monoclonal mouse anti-GAPDH (1:10,000 dilution; Chemicon International, Inc., Temecula, CA) and horseradish peroxidaseconjugated sheep anti-mouse IgG (1:2500 dilution; Amersham Biosciences, Co.) to confirm the same amount of proteins loaded. 2.3. Statistical analyses All the data were expressed as the mean with S.E. statistical significance of difference was determined by unpaired Student’s t-test or two-way analysis of variance (ANOVA) with post hoc Bonferroni/Dunn (StatView for Macintosh ver. 5.0, SAS Institute, Inc., NC). A value of p < 0.05 was considered significant.
Fig. 1. Effects of interleukin-4 (IL-4) on the expression and phosphorylation of signal transducer and activator of transcription 6 (STAT6) in cultured human bronchial smooth muscle cells (hBSMCs). The hBSMCs were incubated with IL-4 (100 ng/mL) for the indicated time, and total protein samples were analyzed by immunoblottings. (A) Representative Western blots of phosphorylated STAT6 (upper), total STAT6 (middle) and GAPDH (lower). The bands were analyzed by a densitometer and pSTAT6/STAT6 and STAT6/GAPDH were calculated in each protein sample, and the data are summarized in (B) and (C) respectively. Each column represents the mean ± SEM from three independent experiments. *P < 0.05 and ***P < 0.001 versus control (Cont) by Bonferroni/Dunn’s test.
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Fig. 2. Effects of interleukin-4 (IL-4) on RhoA protein expression in cultured human bronchial smooth muscle cells (hBSMCs). The hBSMCs were incubated with IL-4 (100 ng/mL) for the indicated time, and total protein samples were analyzed by immunoblottings. (A) Representative Western blots. The bands were analyzed by a densitometer and normalized by the intensity of corresponding GAPDH band, and the data are summarized in (B). Each column represents the mean ± SEM from six independent experiments. *P < 0.05 versus control (Cont) by Bonferroni/Dunn’s test.
STAT6. As shown in Fig. 1, the protein expression of STAT6 was detected in the cultured hBSMCs. When the cells were treated with IL-4, a distinct phosphorylation of STAT6 was observed. The timecourse study revealed that the peak of STAT6 phosphorylation was at 1 hr after the IL-4 treatment (Fig. 1A). The expression level of total STAT6 protein was not affected by the IL-4 treatment (Fig. 1B). Fig. 2 shows the effect of IL-4 on the expression of RhoA protein in the cultured hBSMCs. As a result, the RhoA expression was significantly increased in an incubation time-dependent manner. The peak of upregulation of RhoA protein was observed at 12–24 h after the IL-4 treatment (Fig. 2). 3.2. Effects of AS1517499 on IL-4-induced upregulation of RhoA protein To determine the ability of AS1517499 to inhibit the activation of STAT6 induced by IL-4, the effect of AS1517499 on STAT6 phosphorylation observed at 1 h after the treatment with IL-4 (100 ng/mL) was examined in the hBSMCs. As shown in Fig. 3A, immunoblot analyses revealed that AS1517499 significantly inhibited the IL4-induced STAT6 phosphorylation in a concentration-dependent manner. The STAT6 phosphorylation induced by IL-4 was blocked almost completely when cells were co-treated with 100 nM AS1517499 (Fig. 3A). Thus, AS1517499 can inhibit the activation of STAT6 by blocking its phosphorylation. The upregulation of RhoA protein observed at 24 h after the treatment with IL-4 was also inhibited by AS1517499 in a concentration-dependent manner (Fig. 3B). 4. Discussion Airway smooth muscle is an important effector tissue regulating bronchomotor tone. It has been suggested that modulation of airway smooth muscle by inflammatory mediators such as cytokines may play a crucial role in the development of airway hyperresponsiveness (AHR) [22], one of the characteristic features of
Fig. 3. Inhibitory effects of an inhibitor of signal transducer and activator of transcription 6 (STAT6), AS1517499, on the interleukin-4 (IL-4)-induced phosphorylation of STAT6 and upregulation of RhoA protein in cultured human bronchial smooth muscle cells (hBSMCs). (A) Representative Western blots of phosphorylated STAT6, total STAT6, RhoA and GAPDH. The bands were analyzed by a densitometer and pSTAT6/STAT6 and RhoA/GAPDH were calculated in each protein sample, and the data are summarized in (B) and (C) respectively. The hBSMCs were incubated with IL-4 (100 ng/mL) or its vehicle (left columns in B and C) for 1 h (B) or 24 h (C) in the absence (0 nM) or presence (30 or 100 nM) of AS1517499. Each column represents the mean ± SEM from six independent experiments. ***P < 0.001 versus no IL-4 treatment (left column, respectively), and # P < 0.05, ## P < 0.01 and ### P < 0.001 versus the group that were treated with IL-4 (100 ng/mL) in the absence of AS1517499 (the second column from the left, respectively) by Bonferroni/Dunn’s test.
patients with allergic bronchial asthma. In animal models of allergic bronchial asthma, an increased contractility of isolated BSM to contractile agonists has been found in rats [23] and mice [24]. The augmented BSM contraction induced by antigen challenge has reportedly been associated with an upregulation of RhoA [9–11],
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a small GTPase that is involved in the agonist-induced Ca2+ sensitization of smooth muscle contraction [25,26]. An importance of RhoA and its downstream Rho-kinases was also demonstrated in contraction of human BSM [8]. Here, we show that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. Currently, treatment of the hBSMCs with IL-4 caused a phosphorylation of STAT6 (Fig. 1). The IL-4-mediated events, including an activation of STAT6, are generated by its binding to the IL-4 receptors, the type I IL-4 receptor (a dimer composed of the IL4R␣ chain and the common gamma chain of the IL-2 receptor) and/or the type II IL-4 receptor (a dimer consisting of the IL4R␣ and IL13R␣1 chains). In nonhematopoietic cells including the airway smooth muscle cells, the type II IL-4 receptor is suggested as the main receptor for IL-4 [27,28]. Our previous study also demonstrated the expression of IL4R␣ and IL13R␣1 chains in the cultured hBSMCs [18]. It is thus possible that IL-4 can act on the hBSMCs directly and activate its signal transduction, the Janus kinases (JAKs)/STAT6 pathway [19–21]. In the present study, AS1517499 was used as an inhibitor of STAT6 activation. AS1517499 is a novel selective STAT6 inhibitor synthesized on the basis of the structure of a reported STAT6 inhibitor, TMC-264, discovered from the fungus Phoma [29,30]. Nagashima et al. [31] synthesized a series of 2-{[2(4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities using a STAT6 reporter assay in cells stably transfected with an IL-4-responsive luciferase reporter plasmid. Among these compounds, AS1517499 showed a potent STAT6 inhibition with a 50% inhibitory concentration (IC50 ) of 21 nM [31]. AS1517499 also inhibited the IL-4-induced Th2 cell differentiation of mouse spleen T cells with an IC50 value of 2.3 nM without influencing the IL-12-induced Th1 cell differentiation [31]. Although the exact mechanism of inhibition of STAT6 by AS1517499 is unclear now, the parent compound TMC-264 is known to inhibit both tyrosine phosphorylation of STAT6, with an IC50 value of 1.6 M (1600 nM), and the complex formation of phosphorylated STAT6 with its recognition DNA sequence [30]. The present study suggests that mode of action of AS1517499 is at least the inhibition of phosphorylation of STAT6 (Fig. 3A). One of the important findings of the current study is that IL-4 is capable of inducing an upregulation of RhoA protein (Fig. 2). There is increasing evidence that RhoA is involved in the contraction of airway smooth muscle, and that an augmented contraction due to the upregulated RhoA protein is a cause of AHR [8–10,26]. Although the mechanism of upregulation of RhoA in the diseased bronchial smooth muscle is not fully understood to date, the observation indicates that IL-4, which is upregulated in the airways of allergic bronchial asthmatics [14,32,33], is one of the candidate factors responsible for the event. Indeed, neutralizing IL-4 with anti-IL-4 antibodies prevented the development of antigen-induced AHR in mice [34]. In addition, mice in which targeted deletion of IL-4 was performed fail to develop the AHR [35]. The current observation that the IL-4-induced upregulation of RhoA protein was inhibited by the co-incubation with AS1517499 suggests that this event is mediated by an activation of STAT6 in the cultured hBSMCs. Although the exact mechanism of RhoA transcription in BSMCs is not fully understood, the upstream genomic DNA sequence of human RhoA contains several STATs binding sites, e.g., −77 (from the transcription start) to −69 (score 85.6), −270 to −261 (score 86.5), −417 to −409 (score 78.8) and −518 to −510 (score 84.6), when analyzed using the TFSEARCH program (http://mbs.cbrc.jp/research/db/TFSEARCH.html). In addition, a STAT6-dependent upregulation of RhoA was reported in hBSMCs that were stimulated with IL-13 [18], and IL-13 can also utilize IL4R␣–IL13R␣1 receptor complex for its signaling [28].
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In conclusion, the current findings indicate that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics [14,32,33] might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. Conflict of interest statement None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgements We thank Yuka Narushima and Tomoko Minemura for their technical assistance. This work was partly supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 2006;355:2226–35. [2] Seow CY, Schellenberg RR, Pare PD. Structural and functional changes in the airway smooth muscle of asthmatic subjects. Am J Respir Crit Care Med 1998;158:S179–86. [3] Martin JG, Duguet A, Eidelman DH. The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease. Eur Respir J 2000;16:349–54. [4] Sears MR. Asthma treatment: inhaled beta-agonists. Can Respir J 1998;5(Suppl. A):54A–9A. [5] Rodrigo GJ. Inhaled therapy for acute adult asthma. Curr Opin Allergy Clin Immunol 2003;3:169–75. [6] O’Byrne PM. Acute asthma intervention: insights from the STAY study. J Allergy Clin Immunol 2007;119:1332–6. [7] Papi A, Caramori G, Adcock IM, Barnes PJ. Rescue treatment in asthma. More than as-needed bronchodilation. Chest 2009;135:1628–33. [8] Yoshii A, Iizuka K, Dobashi K, Horie T, Harada T, Nakazawa T, et al. Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization. Am J Respir Cell Mol Biol 1999;20:1190–200. [9] Chiba Y, Takada Y, Miyamoto S, Mitsui-Saito M, Karaki H, Misawa M. Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol 1999;127:597–600. [10] Chiba Y, Ueno A, Shinozaki K, Takeyama H, Nakazawa S, Sakai H, et al. Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice. Respir Res 2005;6 [Art. No. 4]. [11] Chiba Y, Sakai H, Wachi H, Sugitani H, Seyama Y, Misawa M. Upregulation of rhoA mRNA in bronchial smooth muscle of antigen-induced airway hyperresponsive rats. J Smooth Muscle Res 2003;39:221–8. [12] Gosens R, Schaafsma D, Nelemans SA, Halayko AJ. Rho-kinase as a drug target for the treatment of airway hyperresponsiveness in asthma. Mini Rev Med Chem 2006;6:339–48. [13] Schaafsma D, Gosens R, Zaagsma J, Halayko AJ, Meurs H. Rho kinase inhibitors: a novel therapeutical intervention in asthma? Eur J Pharmacol 2008;585:398–406. [14] Batra V, Musani AI, Hastie AT, Khurana S, Carpenter KA, Zangrilli JG, et al. Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts. Clin Exp Allergy 2004;34: 437–44. [15] Steinke JW, Borish L. Th2 cytokines and asthma. Interleukin-4: its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin4 receptor antagonists. Respir Res 2001;2:66–70. [16] Dabbagh K, Takeyama K, Lee HM, Ueki IF, Lausier JA, Nadel JA. IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo. J Immunol 1999;162:6233–7. [17] Bryborn M, Adner M, Cardell LO. Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogen-activated protein kinase pathways. Clin Exp Allergy 2004;34:1291–8. [18] Chiba Y, Nakazawa S, Todoroki M, Shinozaki K, Sakai H, Misawa M. Interleukin13 augments bronchial smooth muscle contractility with an up-regulation of RhoA protein. Am J Respir Cell Mol Biol 2009;40:159–67. [19] Chatila TA. Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends Mol Med 2004;10:493–9.
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