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Tumor Necrosis Factor–α (TNF-α) Induces Upregulation of RhoA via NF-κB Activation in Cultured Human Bronchial Smooth Muscle Cells
Journal of Pharmacological Sciences
J Pharmacol Sci 110, 437 – 444 (2009)4
©2009 The Japanese Pharmacological Society
Full Paper
Tumor Necrosis Factor–α (TNF-α) Induces Upregulation of RhoA via NF-κB Activation in Cultured Human Bronchial Smooth Muscle Cells Kumiko Goto1, Yoshihiko Chiba1,*, Hiroyasu Sakai1, and Miwa Misawa1 1
Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
Received March 13, 2009; Accepted June 1, 2009
Abstract. RhoA plays an important role in Ca2+ sensitization of bronchial smooth muscle in antigen-induced airway hyperresponsiveness (AHR). Tumor necrosis factor–α (TNF-α), a major proinflammatory cytokine, is capable of inducing AHR, but the mechanisms for this are still unknown. In the present study, the effect of TNF-α on RhoA protein expression was examined in cultured human bronchial smooth muscle cells (hBSMCs). To investigate the role of NF-κB in the TNF-α–induced upregulation of RhoA, the effects of an inhibitor of IκB kinase (IKK), BMS-345541, were also determined. Both immunoblot and immunocytochemical analyses revealed that incubation of the hBSMCs with TNF-α caused an activation of NF-κB (determined by a translocation of p65 proteins to nuclei): the peak response was observed when cells were incubated with 10 ng/mL of TNF-α for 30 min. An upregulation of RhoA protein was also observed at 12 – 24 h after the incubation with TNF-α (10 ng/mL). Both the activation of NFκB and upregulation of RhoA were concentration-dependently inhibited by the co-incubation with BMS-345541. These results suggest that TNF-α–induced upregulation of RhoA might be mediated by an activation of NF-κB in hBSMCs. Keywords: RhoA, NF-κB, human bronchial smooth muscle cell (hBSMC) TNF-α receptor 1 (TNFR1). The nuclear transcription factor NF-κB, typical of the p50 and p65 heterodimer, regulates a large number of genes involved in immune and inflammatory responses. Recent evidence suggests an essential role of NF-κB overactivation in increased expression of many inflammatory genes and in airway inflammation in asthma (10, 11). In the normal condition, NF-κB activity is suppressed by binding to an intrinsic NF-κB inhibitor called IκBα. Activated IκB kinase (IKK)β by TNF-α is essential for activation of the classical NF-κB pathway through phosphorylation of serine residues 32 and 36 of IκBα, resulting in the ubiquitination and subsequent degradation of IκBα by the 26S proteasome. The degradation of IκBα exposes a nuclear translocation sequence facilitating translocation of NF-κB to the nucleus. AHR associated with heightened airway resistance and inflammation is an asthmatic characteristic feature (12). The importance of AHR in the pathogenesis of asthma has been suggested by its relevance to the severity of the disease (13). It has been demonstrated that bronchial smooth muscle (BSM) responsiveness to contractile agonists is significantly increased in the
Introduction Tumor necrosis factor–α (TNF-α), one of the proinflammatory cytokines released from inflammatory cells, is directly linked to airway inflammation and hyperresponsiveness (1). TNF-α is elevated in the sputa and bronchoalveolar lavage fluids (BALFs) of patients with bronchial asthma (2, 3). Increased levels of TNF-α have also been detected in the BALFs of sensitized animals after challenge with antigen in mouse and guinea-pig models of lung inflammation (4, 5). In addition, incubation of airway smooth muscle tissues with TNF-α augments contractile response to agonists in mice, guinea pigs, and rats (6 – 8). TNF-α blockade with anti–TNF-α antibody (2 μg, i.p.) resulted in a significant inhibition of airway hyperresponsiveness (AHR) without affecting airway eosinophilia and inflammation in mice (9). TNF-α is known to activate NF-κB via binding to *Corresponding author. [email protected] Published online in J-STAGE on July 14, 2009 (in advance) doi: 10.1254 / jphs.09081FP
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AHR rats and mice (14 – 16). Smooth muscle contraction is mainly regulated by an increase in cytosolic Ca2+ concentration in myocytes. Recently, an additional mechanism, termed Ca2+ sensitization, has also been suggested in the agonist-induced contraction of smooth muscle including airway regions (14, 15, 17 – 21). Our previous studies demonstrated that the agonist-induced, RhoA-mediated Ca2+ sensitization of contraction was augmented in BSMs of rats and mice with allergic bronchial asthma. A marked upregulation of RhoA in BSMs has also been demonstrated in these animal models of allergic bronchial asthma (14, 15). Although the detailed mechanism(s) of RhoA upregulation is not fully understood, TNF-α might be one of the causes of the phenomenon (8, 22). We show here that TNF-α has an ability to upregulate RhoA directly in cultured human BSM cells (hBSMCs). To determine the role of NF-κB in the TNF-α–induced RhoA upregulation, the effect of an IKK inhibitor, BMS345541, on the phenomenon was also investigated. Materials and Methods Chemicals All biochemicals were of analytical grade and were purchased from commercial suppliers: recombinant human TNF-α (rhTNF-α; Peprotech, Paris, France) and BMS-345541 (Sigma-Aldrich, St. Louis, MO, USA). Cell culture hBSMCs (Cambrex, MD, USA) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum (FBS), 2 ng/mL hFGF-B, 0.5 ng/mL hEGF, 5 μg/ mL insulin, and 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 6 – 9) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and 8-well chamber slides (Nage Nunc International, Naperville, IL, USA) at a density of 3,500 cells/cm2, and when 80 – 85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human TNF-α. The culture technique was used in experiments involving stimulation with 10 ng/mL TNF-α or sterile phosphate-buffered saline (PBS) as its vehicle control for 30 min or 24 h. BMS-345541, an IKK inhibitor, was coincubated with TNF-α. Reverse transcription–polymerase chain reactions (RTPCR) The expression of TNFR1 mRNA was examined
by RT-PCR. Briefly, total RNA was extracted from hBSMCs with a one-step guanidium–phenol–chloroform extraction procedure using TRI Reagent (SigmaAldrich). cDNAs were prepared from the total RNA (1.0 μg) by using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Inc., Hanover, MD, USA) in a total volume of 20 μL reaction buffer containing 50 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mM dNTP mixture, 1 U/μL RNase inhibitor, 10 ng/μL random 6-mers, and 10 U /μL M-MuLV reverse transcriptase. The reaction mixture (1 μL) was subjected to PCR (0.1 μM forward and reverse primers, 0.025 U /L Taq DNA polymerase, 2 mM MgCl2, 0.2 mM dNTPs) in a final volume of 10 μL. The PCR primer sets used were as follows: 5'-ACCAAGTGCCACAAAGGAAC3' (sense) and 5'-CTGCAATTGAAGCACTGGAA-3' (antisense) for TNFR1, which were designed from published sequences (Accession No. NM_001065). The thermal cycle profile used was 1) denaturing for 30 s at 95°C, 2) annealing primers for 30 s at 60°C, and 3) extending the primers for 60 s at 72°C. The PCR amplification was performed at 30 cycles according to the preliminary cycle-dependence experiment. The PCR products were subjected to electrophoresis on 1.2% agarose gel and visualized by ethidium bromide staining. Nuclear protein extraction Cells were washed in PBS and lysed in 15 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.6), 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.1% (v/ v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 μg/mL leupeptin, and 5 μg/ mL aprotinin for 10 min on ice. Nuclei were pelletted by centrifugation at 14,000 × g for 5 min at 4°C. Proteins were extracted from the nuclei by incubation at 4°C with vigorous vortexing in buffer [420 mM NaCl, 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF, 2.5 μg/mL leupeptin, and 5 μg/mL aprotinin]. Nuclear debris was pelletted by centrifugation at 13,000 × g for 30 min at 4°C, and the supernatant extract was collected as a nuclear protein and stored at −85°C until use. Total protein extraction Cells were washed in PBS and lysed in SDS sample buffer containing 0.0625 M Tris-HCl, 2% SDS, 0.005% bromophenol blue, 5% 2-mercaptoethanol, and 8% glycerol. Extracted proteins were collected and stored at −85°C until use. Immunoblotting analyses The nuclear and total protein samples (10 μg of
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total protein per lane) were subjected to 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane that was incubated with polyclonal rabbit anti–NF-κB p65 (1:1,000 dilution; Biolegend, San Diego, CA, USA) or polyclonal rabbit anti-RhoA (1:2,500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight. Then the membrane was incubated with horseradish peroxidase– conjugated donkey anti–rabbit IgG (1:2,500 dilution; Amersham Biosciences, Co., Piscataway, NJ, USA), detected by an enhanced chemiluminescent system (Amersham Biosciences), and analyzed by a densitometry system. Detection of house-keeping gene was also performed on the same membrane by using polyclonal rabbit anti–histone H1 (1:1,000 dilution, Santa Cruz) for nuclear samples or monoclonal mouse anti–β-actin (1:5,000 dilution, Santa Cruz) for total protein samples to confirm the same amount of proteins loaded. Real-time RT-PCR The quantitative analyses of mRNA levels of RhoA were examined by real-time RT-PCR. Briefly, cDNAs were prepared from the total RNA (1.0 μg) by using QuantiTect Reverse Transcriptase (Qiagen, Germany) after incubation with genomic DNA wipeout buffer at 42°C for 3 min to remove genomic DNA contamination. The reaction mixture (2 μL) was subjected to PCR (50 nM forward and reverse primers, iQ SYBR Green Supermix; BIO RAD, CA, USA) in a final volume of 20 μL. The PCR primer sets used were as follows: Hs_RHOA_1_SG QuantiiTect Primer Assay (Qiagen) for RhoA, 5'-GGAGCCAAAAGGGTCATCATCTC-3' (sense) and 5'-AGGGATGATGTTCTGGAGAGCC-3' (antisense) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were designed from published sequences (Accesion No. NM_001664 and NM_145071, respectively). The thermal cycle profile used was 1) denaturing for 30 s at 95°C and 2) annealing primers for 30 s at 60°C. The PCR amplification was performed at 40 cycles. Data are expressed as the relative expression to GAPDH mRNA as a house-keeping gene using the 2−ΔΔCT method. Immunohistochemistory Immunostaining was performed to detect translocation of NF-κB to nuclei induced by TNF-α in hBSMCs cultured on 8 chamber slides. After the TNF-α stimulation, cells were fixed and permeabilized, and nonspecific binding was blocked by 5% skim milk for 1 h, followed by incubation with control or primary polyclonal rabbit anti–NF-κB p65 (1:100 dilution) for 1 h. After washing,
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cells were incubated with biotinylated affinity-purified anti–rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 h. Then the slides were incubated with Vectastain® ABC reagent for 30 min. After washing, immune complexes were detected using 3,3'-diaminobenzidine (DAB) staining. Counter staining was carried out by hematoxylin after the immunostaining. For negative control experiments, the primary antibody was omitted. Statistical analyses All the data were expressed as the mean with S.E.M. Statistical significance of difference was determined by one-way analysis of variance (ANOVA) with the post hoc Bonferroni /Dunn test (StatView for Macintosh ver. 5.0; SAS Institute, Inc., Cary, NC, USA). A value of P<0.05 was considered as significant. Results NF-κB activation by TNF-α in hBSMCs As shown in Fig. 1A, the RT-PCR analysis revealed an expression of TNFR1 in the cultured hBSMC, indicating that TNF-α is capable of activating signal transduction in BSM cells directly. Indeed, incubation of the cells with TNF-α caused concentration-dependent activation of NF-κB, as assessed by nuclear translocation of p65 (Fig. 1B). A significant increase in the nuclear p65 was observed when the hBSMCs were incubated with 10 or 100 ng/ mL of TNF-α for 30 min (Fig. 1B). Figure 2 shows the time-course changes in the levels of nuclear p65 induced by TNF-α. A peak response was observed when cells were stimulated with 10 ng/mL of TNF-α for 30 min (Fig. 2). RhoA upregulation induced by TNF-α in hBSMCs Figures 3 and 4 show the time-course changes in the expression levels of RhoA mRNA (Fig. 3) and protein (Fig. 4) after application of TNF-α. Consistent with our previous rat study (22), TNF-α caused an upregulation of RhoA in hBSMCs. A significant increase in the level of RhoA mRNA was observed when the cells were stimulated with 10 ng/mL of TNF-α for 6, 12, and 24 h (Fig. 3); and protein was observed when the cells were stimulated with 10 ng/mL of TNF-α for 12 and 24 h (Fig. 4). Involvement of NF-κB activation in the TNF-α–induced upregulation of RhoA To determine the role of NF-κB signaling in the TNF-α–induced upregulation of RhoA, the cells were also treated with the IKK inhibitor BMS-345541. As shown in Fig. 5, the increase in nuclear p65 induced
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K Goto et al Fig. 1. Expression of TNF-α receptor 1 (TNFR1, A) and concentration-dependent translocation of p65 to nuclei induced by TNF-α (B) in cultured human bronchial smooth muscle cells (hBSMCs). A: The expression of mRNA encoding TNFR1 was evaluated by RT-PCR. A molecular weight standard is shown in the left lane. A clear band of TNFR1 was observed in hBSMC. B: Nuclear proteins of hBSMCs treated with TNF-α (1, 10, or 100 ng / mL) or its vehicle (Cont) were assayed for p65, subunit of NF-κB, by Western blotting analyses. Typical blots for p65 and histone H1 (upper photos). The expression levels of p65 are summarized in the lower panel. Each column represents the means with S.E.M. from 3 independent experiments. ***P<0.001 vs. Cont by one-way ANOVA with the post hoc Bonferroni / Dunn test.
Fig. 2. Time-course changes in the translocation of p65 to nuclei induced by TNF-α in cultured human bronchial smooth muscle cells (hBSMCs). Nuclear proteins of hBSMCs extracted after treatment with TNF-α (10 ng / mL) or its vehicle (Cont) were assayed for p65 by Western blotting analyses. Typical blots for p65 and histone H1 (upper photos). The expression levels of p65 are summarized in the lower panel. Each column represents the mean with S.E.M. from 3 independent experiments. **P<0.01, ***P<0.001 vs. Cont by oneway ANOVA with the post hoc Bonferroni / Dunn’s test.
by treatment with 10 ng/mL of TNF-α for 30 min was significantly inhibited by the co-incubation with BMS345541 in a concentration-dependent manner. Immunohistochemical examinations also revealed that the nuclear translocation of p65 induced by 10 ng/mL of TNF-α was inhibited by co-incubation with 0.3 μM of BMS-345541 (Fig. 6). These observations indicate that the TNF-α–mediated activation of NF-κB at 30 min after the treatment with TNF-α is surely inhibited by
Fig. 3. Time-course changes in the upregulation of RhoA mRNA induced by TNF-α in human bronchial smooth muscle cells (hBSMCs). Data are expressed as the relative expression to GAPDH mRNA as a house-keeping gene using the 2−ΔΔCT method. Each column represents the mean with S.E.M. from 6 independent experiments. The cDNA samples, synthesized by the reverse transcription reaction using total RNAs, were analyzed by real-time PCR. *P<0.05 vs. Cont by the Bonferroni /Dunn test.
BMS-345541. Under these conditions, the TNF-α– induced increase in RhoA protein at 24 h after the treatment with TNF-α was also inhibited by the coincubation with BMS-345541 in a concentrationdependent manner (Fig. 7). Both the nuclear translocation of p65 (Figs. 5 and 6) and the upregulation of RhoA (Fig. 7) induced by TNF-α were almost completely inhibited when 0.3 μM BMS-345541 was used. Discussion TNF-α induces several different signaling pathways, leading to inflammatory gene transcription. Transcriptional regulation in various cells by TNF-α is mediated by the activation of NF-κB and AP-1 (23, 24). Both
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Fig. 4. Time-course changes in the upregulation of RhoA protein induced by TNF-α in human bronchial smooth muscle cells (hBSMCs). Total proteins of hBSMCs treated with TNF-α (10 ng / mL) or its vehicle (Cont) were assayed for RhoA by immunoblotting (upper photos). Typical blots for RhoA and β-actin. The expression levels of RhoA are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. *P<0.05 vs. Cont by one-way ANOVA with the post hoc Bonferroni / Dunn test.
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Fig. 5. Inhibitory effects of BMS-345541, an IKK inhibitor, on translocation of p65 to nuclei induced by treatment with TNF-α (10 ng /mL) for 30 min in cultured human bronchial smooth muscle cells (hBSMCs). Nuclear proteins of hBSMCs were assayed for p65 by immunoblotting (upper photos). Typical blots for p65 and histone H1. The expression levels of p65 are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. **P<0.01 vs. Cont, #P<0.05, ##P<0.01 vs. TNF-α by two-way ANOVA with the post hoc Bonferroni / Dunn test.
Fig. 6. Immunocytochemical localization of p65 protein in human bronchial smooth muscle cells (hBSMCs). The hBSMCs were treated with vehicle (A and B), TNF-α (10 ng /mL, C) or BMS-345541 (0.3 μM) + TNF-α (D) for 30 min. Counter staining was carried out by hematoxylin after the immunostaining. A: negative control (no 1st antibody). The immunoreactive p65 was located in the cytoplasm in naive cells (B). In the cells treated with TNF-α, immunoreactive p65 was mainly detected in nuclei (C). The TNF-α–induced translocation of p65 to nuclei was inhibited by the co-incubation with BMS-345541 (D).
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Fig. 7. Effects of BMS-345541, an IKK inhibitor, on upregulation of RhoA induced by treatment with TNF-α (10 ng / mL) for 24 h in cultured human bronchial smooth muscle cells (hBSMCs). Total proteins of hBSMCs were assayed for RhoA by immunoblotting (upper photos). Typical blots for RhoA and β-actin. The expression levels of RhoA are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. **P<0.01 vs. Cont, #P<0.05 vs. TNF-α by two-way ANOVA with the post hoc Bonferroni /Dunn test.
NF-κB and AP-1 are activated in the airways of asthmatic patients and animals (25, 26). NF-κB is rapidly activated as a result of the binding with TNFR1, a TNF-α receptor, leading to transcriptional upregulation of adhesion molecules and chemokines (27), because it is likely that NF-κB is the main transcription factor activated by TNF-α in airway smooth muscle (28, 29). In the present study, translocation of p65 was induced by TNF-α in hBSMCs (Fig. 1B). The NF-κB family of transcription factors is crucial to the immune responses. It is composed of five different members, p65, c-Rel, RelB, p50, and p52, which are maintained as homo- and heterodimeric complexes in the cytoplasm. p65, c-Rel, and RelB contain transactivation domains, which are required for gene transcription, whereas p50 and p52 do not contain transactivation domains (30). When cells were stimulated with TNF-α, heterodimers of p65 and p50 are free to translocate into the nucleus (31, 32). A translocation of p65 to nuclei is widely used as an index of activation of NF-κB (33). In the present study, a peak of p65 translocation was observed when cells were stimulated with 10 ng/mL of TNF-α for 30 min (Fig. 2), indicating an activation of NF-κB by the TNF-α stimulation in hBSMCs. In the present study, TNF-α also induced an upregulation of RhoA in hBSMCs (Figs. 3 and 4). The Rho family of small GTPases, consisting of Rho, Rac, and
Cdc42, is a group of 20 – 40-kDa monomeric G proteins that can regulate a number of cellular biologic functions, including actin stress fiber formation, focal adhesion, motility, aggregation, proliferation, and transcription (34, 35). A small GTPase, RhoA, is a key protein participating in the agonist-induced Ca2+ sensitization of smooth muscle contraction including airway smooth muscle (14). The importance of RhoA and its downstream Rho-kinases in contraction of human bronchial smooth muscle was also suggested (20), and the RhoA /Rho-kinase pathway has now been proposed as a new target for the treatment of AHR in asthma (36). Glucocorticoids are the most effective therapy currently available for the treatment of allergic asthma. Our previous study revealed that prednisolone, a glucocorticoid, inhibited RhoA upregulation in bronchial smooth muscle of AHR rats (37). There are many reports that glucocorticoids inhibited activation of NF-κB in human airway smooth muscle (38 – 40). In addition, treatment of hBSMCs with prednisolone abolished TNF-α–induced activation of NF-κB and upregulation of RhoA (data not shown). Although the exact RhoA promoter and/or enhancer regions have not yet been identified, the DNA sequence analysis for the directly upstream region of the rat RhoA transcriptional start site (from −1 to −1191) using the TFSEARCH program (http:/ /www.cbrc.jp/research/ db /TFSEARCH.html) revealed 2 putative binding sites for NF-κB. In addition, we have previously reported that the RhoA upregulation in bronchial smooth muscle observed in a rat model of experimental asthma was inhibited by prednisolone (37), which is known to inhibit the activation of NF-κB (36 – 38). So next, the effect of BMS-345541, an IKK inhibitor that also inhibits NF-κB activation indirectly, on the TNF-α induced upregulation of RhoA was determined to identify the role of NF-κB in the phenomenon. BMS-345541 (4(2'-aminoethyl) amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKKβ IC50 = 0.3 μM, IKKα IC50 = 4 μM; data based on the assays using recombinant proteins) as binding an allosteric site of IKK and blocked NF-κB– dependent transcription (41, 42). As shown in Figs. 5 and 6, nuclear translocation of p65 induced by TNF-α was inhibited by the co-incubation with BMS-345541, suggesting that NF-κB activation induced by TNF-α could be inhibited by BMS-345541. Interestingly, the RhoA upregulation induced by TNF-α was also inhibited by BMS-345541 (Fig. 7). These findings indicate that the RhoA upregulation induced by TNF-α is mediated by an activation of NF-κB in hBSMCs. In conclusion, the current study clearly showed that TNF-α upregulates RhoA protein via an activation of
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NF-κB in hBSMCs. Selective inhibition of the TNFα/NF-κB signaling in airway smooth muscles might be useful for treatment of the AHR in asthmatics. Acknowledgments We thank Mr. Kunihiko Orui and Ms. Satomi Aso for their technical assistance.
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CD38 expression by tumor necrosis factor-alpha in human airway smooth muscle cells: Role of NF-kappaB and sensitivity to glucocorticoids. FASEB J. 2006;20:1000–1002. Tirumurugaan KG, Kang BN, Panettieri RA, Foster DN, Walseth TF, Kannan MS. Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-alpha and dexamethasone. Respir Res. 2008;9:26. Zhu YM, Bradbury DA, Pang L, Knox AJ. Transcriptional regulation of interleukin (IL)-8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF)-IL-6 and prostanoid-independent activation of NF-kappaB. J Biol Chem. 2003;27:29366–29375. Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappaB-dependent transcription in mice. J Biol Chem. 2003;278:1450–1456. McIntyre KW, Shuster DJ, Gillooly KM, Dambach DM, Pattoli MA, Lu P, et al. A highly selective inhibitor of I kappa B kinase, BMS-345541, blocks both joint inflammation and destruction in collagen-induced arthritis in mice. Arthritis Rheum. 2003;48: 2652–2659.
J Pharmacol Sci 110, 437 – 444 (2009)4
©2009 The Japanese Pharmacological Society
Full Paper
Tumor Necrosis Factor–α (TNF-α) Induces Upregulation of RhoA via NF-κB Activation in Cultured Human Bronchial Smooth Muscle Cells Kumiko Goto1, Yoshihiko Chiba1,*, Hiroyasu Sakai1, and Miwa Misawa1 1
Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
Received March 13, 2009; Accepted June 1, 2009
Abstract. RhoA plays an important role in Ca2+ sensitization of bronchial smooth muscle in antigen-induced airway hyperresponsiveness (AHR). Tumor necrosis factor–α (TNF-α), a major proinflammatory cytokine, is capable of inducing AHR, but the mechanisms for this are still unknown. In the present study, the effect of TNF-α on RhoA protein expression was examined in cultured human bronchial smooth muscle cells (hBSMCs). To investigate the role of NF-κB in the TNF-α–induced upregulation of RhoA, the effects of an inhibitor of IκB kinase (IKK), BMS-345541, were also determined. Both immunoblot and immunocytochemical analyses revealed that incubation of the hBSMCs with TNF-α caused an activation of NF-κB (determined by a translocation of p65 proteins to nuclei): the peak response was observed when cells were incubated with 10 ng/mL of TNF-α for 30 min. An upregulation of RhoA protein was also observed at 12 – 24 h after the incubation with TNF-α (10 ng/mL). Both the activation of NFκB and upregulation of RhoA were concentration-dependently inhibited by the co-incubation with BMS-345541. These results suggest that TNF-α–induced upregulation of RhoA might be mediated by an activation of NF-κB in hBSMCs. Keywords: RhoA, NF-κB, human bronchial smooth muscle cell (hBSMC) TNF-α receptor 1 (TNFR1). The nuclear transcription factor NF-κB, typical of the p50 and p65 heterodimer, regulates a large number of genes involved in immune and inflammatory responses. Recent evidence suggests an essential role of NF-κB overactivation in increased expression of many inflammatory genes and in airway inflammation in asthma (10, 11). In the normal condition, NF-κB activity is suppressed by binding to an intrinsic NF-κB inhibitor called IκBα. Activated IκB kinase (IKK)β by TNF-α is essential for activation of the classical NF-κB pathway through phosphorylation of serine residues 32 and 36 of IκBα, resulting in the ubiquitination and subsequent degradation of IκBα by the 26S proteasome. The degradation of IκBα exposes a nuclear translocation sequence facilitating translocation of NF-κB to the nucleus. AHR associated with heightened airway resistance and inflammation is an asthmatic characteristic feature (12). The importance of AHR in the pathogenesis of asthma has been suggested by its relevance to the severity of the disease (13). It has been demonstrated that bronchial smooth muscle (BSM) responsiveness to contractile agonists is significantly increased in the
Introduction Tumor necrosis factor–α (TNF-α), one of the proinflammatory cytokines released from inflammatory cells, is directly linked to airway inflammation and hyperresponsiveness (1). TNF-α is elevated in the sputa and bronchoalveolar lavage fluids (BALFs) of patients with bronchial asthma (2, 3). Increased levels of TNF-α have also been detected in the BALFs of sensitized animals after challenge with antigen in mouse and guinea-pig models of lung inflammation (4, 5). In addition, incubation of airway smooth muscle tissues with TNF-α augments contractile response to agonists in mice, guinea pigs, and rats (6 – 8). TNF-α blockade with anti–TNF-α antibody (2 μg, i.p.) resulted in a significant inhibition of airway hyperresponsiveness (AHR) without affecting airway eosinophilia and inflammation in mice (9). TNF-α is known to activate NF-κB via binding to *Corresponding author. [email protected] Published online in J-STAGE on July 14, 2009 (in advance) doi: 10.1254 / jphs.09081FP
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AHR rats and mice (14 – 16). Smooth muscle contraction is mainly regulated by an increase in cytosolic Ca2+ concentration in myocytes. Recently, an additional mechanism, termed Ca2+ sensitization, has also been suggested in the agonist-induced contraction of smooth muscle including airway regions (14, 15, 17 – 21). Our previous studies demonstrated that the agonist-induced, RhoA-mediated Ca2+ sensitization of contraction was augmented in BSMs of rats and mice with allergic bronchial asthma. A marked upregulation of RhoA in BSMs has also been demonstrated in these animal models of allergic bronchial asthma (14, 15). Although the detailed mechanism(s) of RhoA upregulation is not fully understood, TNF-α might be one of the causes of the phenomenon (8, 22). We show here that TNF-α has an ability to upregulate RhoA directly in cultured human BSM cells (hBSMCs). To determine the role of NF-κB in the TNF-α–induced RhoA upregulation, the effect of an IKK inhibitor, BMS345541, on the phenomenon was also investigated. Materials and Methods Chemicals All biochemicals were of analytical grade and were purchased from commercial suppliers: recombinant human TNF-α (rhTNF-α; Peprotech, Paris, France) and BMS-345541 (Sigma-Aldrich, St. Louis, MO, USA). Cell culture hBSMCs (Cambrex, MD, USA) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum (FBS), 2 ng/mL hFGF-B, 0.5 ng/mL hEGF, 5 μg/ mL insulin, and 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 6 – 9) were seeded in 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and 8-well chamber slides (Nage Nunc International, Naperville, IL, USA) at a density of 3,500 cells/cm2, and when 80 – 85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human TNF-α. The culture technique was used in experiments involving stimulation with 10 ng/mL TNF-α or sterile phosphate-buffered saline (PBS) as its vehicle control for 30 min or 24 h. BMS-345541, an IKK inhibitor, was coincubated with TNF-α. Reverse transcription–polymerase chain reactions (RTPCR) The expression of TNFR1 mRNA was examined
by RT-PCR. Briefly, total RNA was extracted from hBSMCs with a one-step guanidium–phenol–chloroform extraction procedure using TRI Reagent (SigmaAldrich). cDNAs were prepared from the total RNA (1.0 μg) by using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Inc., Hanover, MD, USA) in a total volume of 20 μL reaction buffer containing 50 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mM dNTP mixture, 1 U/μL RNase inhibitor, 10 ng/μL random 6-mers, and 10 U /μL M-MuLV reverse transcriptase. The reaction mixture (1 μL) was subjected to PCR (0.1 μM forward and reverse primers, 0.025 U /L Taq DNA polymerase, 2 mM MgCl2, 0.2 mM dNTPs) in a final volume of 10 μL. The PCR primer sets used were as follows: 5'-ACCAAGTGCCACAAAGGAAC3' (sense) and 5'-CTGCAATTGAAGCACTGGAA-3' (antisense) for TNFR1, which were designed from published sequences (Accession No. NM_001065). The thermal cycle profile used was 1) denaturing for 30 s at 95°C, 2) annealing primers for 30 s at 60°C, and 3) extending the primers for 60 s at 72°C. The PCR amplification was performed at 30 cycles according to the preliminary cycle-dependence experiment. The PCR products were subjected to electrophoresis on 1.2% agarose gel and visualized by ethidium bromide staining. Nuclear protein extraction Cells were washed in PBS and lysed in 15 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.6), 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.1% (v/ v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 μg/mL leupeptin, and 5 μg/ mL aprotinin for 10 min on ice. Nuclei were pelletted by centrifugation at 14,000 × g for 5 min at 4°C. Proteins were extracted from the nuclei by incubation at 4°C with vigorous vortexing in buffer [420 mM NaCl, 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF, 2.5 μg/mL leupeptin, and 5 μg/mL aprotinin]. Nuclear debris was pelletted by centrifugation at 13,000 × g for 30 min at 4°C, and the supernatant extract was collected as a nuclear protein and stored at −85°C until use. Total protein extraction Cells were washed in PBS and lysed in SDS sample buffer containing 0.0625 M Tris-HCl, 2% SDS, 0.005% bromophenol blue, 5% 2-mercaptoethanol, and 8% glycerol. Extracted proteins were collected and stored at −85°C until use. Immunoblotting analyses The nuclear and total protein samples (10 μg of
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total protein per lane) were subjected to 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane that was incubated with polyclonal rabbit anti–NF-κB p65 (1:1,000 dilution; Biolegend, San Diego, CA, USA) or polyclonal rabbit anti-RhoA (1:2,500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight. Then the membrane was incubated with horseradish peroxidase– conjugated donkey anti–rabbit IgG (1:2,500 dilution; Amersham Biosciences, Co., Piscataway, NJ, USA), detected by an enhanced chemiluminescent system (Amersham Biosciences), and analyzed by a densitometry system. Detection of house-keeping gene was also performed on the same membrane by using polyclonal rabbit anti–histone H1 (1:1,000 dilution, Santa Cruz) for nuclear samples or monoclonal mouse anti–β-actin (1:5,000 dilution, Santa Cruz) for total protein samples to confirm the same amount of proteins loaded. Real-time RT-PCR The quantitative analyses of mRNA levels of RhoA were examined by real-time RT-PCR. Briefly, cDNAs were prepared from the total RNA (1.0 μg) by using QuantiTect Reverse Transcriptase (Qiagen, Germany) after incubation with genomic DNA wipeout buffer at 42°C for 3 min to remove genomic DNA contamination. The reaction mixture (2 μL) was subjected to PCR (50 nM forward and reverse primers, iQ SYBR Green Supermix; BIO RAD, CA, USA) in a final volume of 20 μL. The PCR primer sets used were as follows: Hs_RHOA_1_SG QuantiiTect Primer Assay (Qiagen) for RhoA, 5'-GGAGCCAAAAGGGTCATCATCTC-3' (sense) and 5'-AGGGATGATGTTCTGGAGAGCC-3' (antisense) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which were designed from published sequences (Accesion No. NM_001664 and NM_145071, respectively). The thermal cycle profile used was 1) denaturing for 30 s at 95°C and 2) annealing primers for 30 s at 60°C. The PCR amplification was performed at 40 cycles. Data are expressed as the relative expression to GAPDH mRNA as a house-keeping gene using the 2−ΔΔCT method. Immunohistochemistory Immunostaining was performed to detect translocation of NF-κB to nuclei induced by TNF-α in hBSMCs cultured on 8 chamber slides. After the TNF-α stimulation, cells were fixed and permeabilized, and nonspecific binding was blocked by 5% skim milk for 1 h, followed by incubation with control or primary polyclonal rabbit anti–NF-κB p65 (1:100 dilution) for 1 h. After washing,
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cells were incubated with biotinylated affinity-purified anti–rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) for 1 h. Then the slides were incubated with Vectastain® ABC reagent for 30 min. After washing, immune complexes were detected using 3,3'-diaminobenzidine (DAB) staining. Counter staining was carried out by hematoxylin after the immunostaining. For negative control experiments, the primary antibody was omitted. Statistical analyses All the data were expressed as the mean with S.E.M. Statistical significance of difference was determined by one-way analysis of variance (ANOVA) with the post hoc Bonferroni /Dunn test (StatView for Macintosh ver. 5.0; SAS Institute, Inc., Cary, NC, USA). A value of P<0.05 was considered as significant. Results NF-κB activation by TNF-α in hBSMCs As shown in Fig. 1A, the RT-PCR analysis revealed an expression of TNFR1 in the cultured hBSMC, indicating that TNF-α is capable of activating signal transduction in BSM cells directly. Indeed, incubation of the cells with TNF-α caused concentration-dependent activation of NF-κB, as assessed by nuclear translocation of p65 (Fig. 1B). A significant increase in the nuclear p65 was observed when the hBSMCs were incubated with 10 or 100 ng/ mL of TNF-α for 30 min (Fig. 1B). Figure 2 shows the time-course changes in the levels of nuclear p65 induced by TNF-α. A peak response was observed when cells were stimulated with 10 ng/mL of TNF-α for 30 min (Fig. 2). RhoA upregulation induced by TNF-α in hBSMCs Figures 3 and 4 show the time-course changes in the expression levels of RhoA mRNA (Fig. 3) and protein (Fig. 4) after application of TNF-α. Consistent with our previous rat study (22), TNF-α caused an upregulation of RhoA in hBSMCs. A significant increase in the level of RhoA mRNA was observed when the cells were stimulated with 10 ng/mL of TNF-α for 6, 12, and 24 h (Fig. 3); and protein was observed when the cells were stimulated with 10 ng/mL of TNF-α for 12 and 24 h (Fig. 4). Involvement of NF-κB activation in the TNF-α–induced upregulation of RhoA To determine the role of NF-κB signaling in the TNF-α–induced upregulation of RhoA, the cells were also treated with the IKK inhibitor BMS-345541. As shown in Fig. 5, the increase in nuclear p65 induced
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K Goto et al Fig. 1. Expression of TNF-α receptor 1 (TNFR1, A) and concentration-dependent translocation of p65 to nuclei induced by TNF-α (B) in cultured human bronchial smooth muscle cells (hBSMCs). A: The expression of mRNA encoding TNFR1 was evaluated by RT-PCR. A molecular weight standard is shown in the left lane. A clear band of TNFR1 was observed in hBSMC. B: Nuclear proteins of hBSMCs treated with TNF-α (1, 10, or 100 ng / mL) or its vehicle (Cont) were assayed for p65, subunit of NF-κB, by Western blotting analyses. Typical blots for p65 and histone H1 (upper photos). The expression levels of p65 are summarized in the lower panel. Each column represents the means with S.E.M. from 3 independent experiments. ***P<0.001 vs. Cont by one-way ANOVA with the post hoc Bonferroni / Dunn test.
Fig. 2. Time-course changes in the translocation of p65 to nuclei induced by TNF-α in cultured human bronchial smooth muscle cells (hBSMCs). Nuclear proteins of hBSMCs extracted after treatment with TNF-α (10 ng / mL) or its vehicle (Cont) were assayed for p65 by Western blotting analyses. Typical blots for p65 and histone H1 (upper photos). The expression levels of p65 are summarized in the lower panel. Each column represents the mean with S.E.M. from 3 independent experiments. **P<0.01, ***P<0.001 vs. Cont by oneway ANOVA with the post hoc Bonferroni / Dunn’s test.
by treatment with 10 ng/mL of TNF-α for 30 min was significantly inhibited by the co-incubation with BMS345541 in a concentration-dependent manner. Immunohistochemical examinations also revealed that the nuclear translocation of p65 induced by 10 ng/mL of TNF-α was inhibited by co-incubation with 0.3 μM of BMS-345541 (Fig. 6). These observations indicate that the TNF-α–mediated activation of NF-κB at 30 min after the treatment with TNF-α is surely inhibited by
Fig. 3. Time-course changes in the upregulation of RhoA mRNA induced by TNF-α in human bronchial smooth muscle cells (hBSMCs). Data are expressed as the relative expression to GAPDH mRNA as a house-keeping gene using the 2−ΔΔCT method. Each column represents the mean with S.E.M. from 6 independent experiments. The cDNA samples, synthesized by the reverse transcription reaction using total RNAs, were analyzed by real-time PCR. *P<0.05 vs. Cont by the Bonferroni /Dunn test.
BMS-345541. Under these conditions, the TNF-α– induced increase in RhoA protein at 24 h after the treatment with TNF-α was also inhibited by the coincubation with BMS-345541 in a concentrationdependent manner (Fig. 7). Both the nuclear translocation of p65 (Figs. 5 and 6) and the upregulation of RhoA (Fig. 7) induced by TNF-α were almost completely inhibited when 0.3 μM BMS-345541 was used. Discussion TNF-α induces several different signaling pathways, leading to inflammatory gene transcription. Transcriptional regulation in various cells by TNF-α is mediated by the activation of NF-κB and AP-1 (23, 24). Both
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Fig. 4. Time-course changes in the upregulation of RhoA protein induced by TNF-α in human bronchial smooth muscle cells (hBSMCs). Total proteins of hBSMCs treated with TNF-α (10 ng / mL) or its vehicle (Cont) were assayed for RhoA by immunoblotting (upper photos). Typical blots for RhoA and β-actin. The expression levels of RhoA are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. *P<0.05 vs. Cont by one-way ANOVA with the post hoc Bonferroni / Dunn test.
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Fig. 5. Inhibitory effects of BMS-345541, an IKK inhibitor, on translocation of p65 to nuclei induced by treatment with TNF-α (10 ng /mL) for 30 min in cultured human bronchial smooth muscle cells (hBSMCs). Nuclear proteins of hBSMCs were assayed for p65 by immunoblotting (upper photos). Typical blots for p65 and histone H1. The expression levels of p65 are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. **P<0.01 vs. Cont, #P<0.05, ##P<0.01 vs. TNF-α by two-way ANOVA with the post hoc Bonferroni / Dunn test.
Fig. 6. Immunocytochemical localization of p65 protein in human bronchial smooth muscle cells (hBSMCs). The hBSMCs were treated with vehicle (A and B), TNF-α (10 ng /mL, C) or BMS-345541 (0.3 μM) + TNF-α (D) for 30 min. Counter staining was carried out by hematoxylin after the immunostaining. A: negative control (no 1st antibody). The immunoreactive p65 was located in the cytoplasm in naive cells (B). In the cells treated with TNF-α, immunoreactive p65 was mainly detected in nuclei (C). The TNF-α–induced translocation of p65 to nuclei was inhibited by the co-incubation with BMS-345541 (D).
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Fig. 7. Effects of BMS-345541, an IKK inhibitor, on upregulation of RhoA induced by treatment with TNF-α (10 ng / mL) for 24 h in cultured human bronchial smooth muscle cells (hBSMCs). Total proteins of hBSMCs were assayed for RhoA by immunoblotting (upper photos). Typical blots for RhoA and β-actin. The expression levels of RhoA are summarized in the lower panel. Each value is the mean with S.E.M. from 3 independent experiments. **P<0.01 vs. Cont, #P<0.05 vs. TNF-α by two-way ANOVA with the post hoc Bonferroni /Dunn test.
NF-κB and AP-1 are activated in the airways of asthmatic patients and animals (25, 26). NF-κB is rapidly activated as a result of the binding with TNFR1, a TNF-α receptor, leading to transcriptional upregulation of adhesion molecules and chemokines (27), because it is likely that NF-κB is the main transcription factor activated by TNF-α in airway smooth muscle (28, 29). In the present study, translocation of p65 was induced by TNF-α in hBSMCs (Fig. 1B). The NF-κB family of transcription factors is crucial to the immune responses. It is composed of five different members, p65, c-Rel, RelB, p50, and p52, which are maintained as homo- and heterodimeric complexes in the cytoplasm. p65, c-Rel, and RelB contain transactivation domains, which are required for gene transcription, whereas p50 and p52 do not contain transactivation domains (30). When cells were stimulated with TNF-α, heterodimers of p65 and p50 are free to translocate into the nucleus (31, 32). A translocation of p65 to nuclei is widely used as an index of activation of NF-κB (33). In the present study, a peak of p65 translocation was observed when cells were stimulated with 10 ng/mL of TNF-α for 30 min (Fig. 2), indicating an activation of NF-κB by the TNF-α stimulation in hBSMCs. In the present study, TNF-α also induced an upregulation of RhoA in hBSMCs (Figs. 3 and 4). The Rho family of small GTPases, consisting of Rho, Rac, and
Cdc42, is a group of 20 – 40-kDa monomeric G proteins that can regulate a number of cellular biologic functions, including actin stress fiber formation, focal adhesion, motility, aggregation, proliferation, and transcription (34, 35). A small GTPase, RhoA, is a key protein participating in the agonist-induced Ca2+ sensitization of smooth muscle contraction including airway smooth muscle (14). The importance of RhoA and its downstream Rho-kinases in contraction of human bronchial smooth muscle was also suggested (20), and the RhoA /Rho-kinase pathway has now been proposed as a new target for the treatment of AHR in asthma (36). Glucocorticoids are the most effective therapy currently available for the treatment of allergic asthma. Our previous study revealed that prednisolone, a glucocorticoid, inhibited RhoA upregulation in bronchial smooth muscle of AHR rats (37). There are many reports that glucocorticoids inhibited activation of NF-κB in human airway smooth muscle (38 – 40). In addition, treatment of hBSMCs with prednisolone abolished TNF-α–induced activation of NF-κB and upregulation of RhoA (data not shown). Although the exact RhoA promoter and/or enhancer regions have not yet been identified, the DNA sequence analysis for the directly upstream region of the rat RhoA transcriptional start site (from −1 to −1191) using the TFSEARCH program (http:/ /www.cbrc.jp/research/ db /TFSEARCH.html) revealed 2 putative binding sites for NF-κB. In addition, we have previously reported that the RhoA upregulation in bronchial smooth muscle observed in a rat model of experimental asthma was inhibited by prednisolone (37), which is known to inhibit the activation of NF-κB (36 – 38). So next, the effect of BMS-345541, an IKK inhibitor that also inhibits NF-κB activation indirectly, on the TNF-α induced upregulation of RhoA was determined to identify the role of NF-κB in the phenomenon. BMS-345541 (4(2'-aminoethyl) amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKKβ IC50 = 0.3 μM, IKKα IC50 = 4 μM; data based on the assays using recombinant proteins) as binding an allosteric site of IKK and blocked NF-κB– dependent transcription (41, 42). As shown in Figs. 5 and 6, nuclear translocation of p65 induced by TNF-α was inhibited by the co-incubation with BMS-345541, suggesting that NF-κB activation induced by TNF-α could be inhibited by BMS-345541. Interestingly, the RhoA upregulation induced by TNF-α was also inhibited by BMS-345541 (Fig. 7). These findings indicate that the RhoA upregulation induced by TNF-α is mediated by an activation of NF-κB in hBSMCs. In conclusion, the current study clearly showed that TNF-α upregulates RhoA protein via an activation of
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NF-κB in hBSMCs. Selective inhibition of the TNFα/NF-κB signaling in airway smooth muscles might be useful for treatment of the AHR in asthmatics. Acknowledgments We thank Mr. Kunihiko Orui and Ms. Satomi Aso for their technical assistance.
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