Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells
Abstract
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 fash- ion. 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.
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 nar- rowing in asthmatics is an abnormality of the properties of airway smooth muscle [2,3]. Rapid relief from airway limitation in asth- matic patients by β-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 develop- ment of asthma therapy to understand changes in the contractile signaling of airway smooth muscle cells associated with the disease.
Recently, an importance of RhoA, a monomeric GTP-binding protein, and its downstream Rho-kinases have been demon- strated 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 contrac- tion 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 signal- ing 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 expres- sion 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 has an ability to cause hyperresponsiveness of airway smooth mus- cle [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 IL- 4 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; Cam- brex 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 µg/mL insulin, 2 ng/mL human fibroblast growth factor- basic (hFGF-b), 50 µg/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., Lon- don, 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 vehi- cle (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 µL/well), and used for Western blot analyses.
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 elec- trophoretically transferred to a PVDF membrane. After blocking with 3% skim milk (for RhoA) or 1% BlockAceTM (Dainippon Sum- itomo 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 anti- RhoA (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 Biotech- nology, 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 house- keeping 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 peroxidase- conjugated 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. sta- tistical 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.
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 Sec- tion 2. Our previous study [18] demonstrated that the hBSMCs used expressed IL-4 receptor α (IL4Rα) and IL-13 receptor α chains (IL13Rα1) and STAT6, a major signal transducer activated by IL- 4 [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-
Fig. 1. Effects of interleukin-4 (IL-4) on the expression and phosphorylation of sig- nal 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 immunoblot- tings. (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.
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 time- course 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 sig- nificantly 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 phospho- rylation 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 IL- 4-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 air- way smooth muscle by inflammatory mediators such as cytokines may play a crucial role in the development of airway hyper- responsiveness (AHR) [22], one of the characteristic features of patients with allergic bronchial asthma. In animal models of aller- gic 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],a small GTPase that is involved in the agonist-induced Ca2+ sen- sitization 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.
Fig. 3. Inhibitory effects of an inhibitor of signal transducer and activator of transcription 6 (STAT6), AS1517499, on the interleukin-4 (IL-4)-induced phospho- rylation 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 repre- sents 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 ver- sus 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.
Currently, treatment of the hBSMCs with IL-4 caused a phospho- rylation of STAT6 (Fig. 1). The IL-4-mediated events, including an activation of STAT6, are generated by its binding to the IL-4 recep- tors, the type I IL-4 receptor (a dimer composed of the IL4Rα chain and the common gamma chain of the IL-2 receptor) and/or the type II IL-4 receptor (a dimer consisting of the IL4Rα and IL13Rα1 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 IL4Rα and IL13Rα1 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 µM (1600 nM), and the complex formation of phosphorylated STAT6 with its recognition DNA sequence [30]. The present study sug- gests 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 aller- gic 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 tran- scription 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 IL4Rα–IL13Rα1 receptor complex for its signaling [28].
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.