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The Journal of Immunology, 1998, 160: 3038-3045.
Copyright © 1998 by The American Association of Immunologists

Role of the Rho GTPase in Bradykinin-Stimulated Nuclear Factor-{kappa}B Activation and IL-1ß Gene Expression in Cultured Human Epithelial Cells1

Zhixing K. Pan*, Richard D. Ye{dagger}, Sandra C. Christiansen*, Mark A. Jagels{dagger}, Gary M. Bokoch{dagger} and Bruce L. Zuraw2,*

Departments of * Molecular and Experimental Medicine and {dagger} Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent evidence suggests a novel role of bradykinin (BK) in stimulating gene transcription. This study examined the effect of BK on nuclear factor {kappa}B (NF-{kappa}B) activation and IL-1ß synthesis in human epithelial cells. Stimulation of A549 cells and primary bronchial epithelial cells with BK rapidly activated NF-{kappa}B. BK also increased the level of secreted immunoreactive IL-1ß in A549 culture supernatants, an effect that was blocked by actinomycin D and the B2 BK receptor antagonist HOE-140. The role of NF-{kappa}B activation in BK-induced IL-1ß synthesis was demonstrated by the ability of BK to stimulate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three {kappa}B enhancers from the IL-1ß gene, while deletion of the {kappa}B enhancer sequences eliminated BK-stimulated CAT activity. C3 transferase exoenzyme, an inhibitor of Rho, abolished BK-induced NF-{kappa}B activation at 10 µg/ml and significantly inhibited BK-stimulated IL-1ß synthesis at 5 µg/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and {kappa}B-dependent manner. Cotransfection of A549 cells with an expression vector encoding a constitutively active form of RhoA (Q63L) along with the IL-1ß promoter-CAT reporter plasmid resulted in a marked increase in NF-{kappa}B activity compared with transfection with the IL-1ß promoter-CAT reporter plasmid alone. These results demonstrate that BK stimulates NF-{kappa}B activation and IL-1ß synthesis in A549 cells, and that RhoA is both necessary and sufficient to mediate this effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bradykinin (BK)3 is a potent mediator of nocieception, increased vascular permeability, eicosanoid synthesis, neuropeptide release, and changes in smooth muscle tone (1, 2). Furthermore, BK has been shown to play a critical role in the development of airway hyper-responsiveness in experimental models of airway inflammation (3, 4, 5). Little is known, however, regarding the mechanism(s) underlying BK’s role in the pathogenesis of airway hyper-responsiveness. Airway inflammation is characterized by bronchial remodeling and increased local cytokine synthesis (6). To elucidate the role of BK in this process, we assessed the effect of BK on cytokine synthesis in human airway epithelial cells.

Recent information suggests that BK has a significant effect on cytokine synthesis. Both BK and its C-terminal arginine-deleted metabolic product, des-Arg-BK, stimulated cytokine release from murine macrophages and spleen cells (7, 8). BK, acting through B2 BK receptors, was shown to stimulate release of multiple cytokines from lung strip explants (9) and synergistically enhance IL-1ß synthesis in human gingival fibroblasts (10). Furthermore, we recently demonstrated that nanomolar concentrations of BK stimulated nuclear factor-{kappa}B (NF-{kappa}B) activation in WI-38 lung fibroblasts, leading to de novo synthesis of IL-1ß, IL-6, and IL-8 (11). BK-induced NF-{kappa}B activation in WI-38 cells was mediated by B2 BK receptors coupled to the Gi/Go class of heterotrimeric G proteins. The ability of BK to activate NF-{kappa}B and thereby stimulate cytokine synthesis represents a novel and potentially important mechanism through which BK may contribute to inflammation.

Several G protein-coupled receptors (GPCR), including the B2 BK receptor, have recently been shown to mediate NF-{kappa}B activation and cytokine gene transcription (11, 12). Little is known, however, regarding the mechanisms of GPCR-mediated NF-{kappa}B activation. BK has been reported to activate the small GTP binding proteins Cdc42 and Rac1, stimulating the formation of peripheral actin microspikes and membrane ruffling in Swiss 3T3 fibroblasts (13). We therefore also investigated the role of the Rho family of small G proteins in BK-induced NF-{kappa}B activation.

We now report that BK, acting through B2 BK receptors, stimulates NF-{kappa}B activation in human airway epithelial cells, and that this is accompanied by increased synthesis of IL-1ß. Furthermore, BK-induced NF-{kappa}B activation and IL-1ß synthesis were inhibited by expression of a dominant-negative RhoA, while a constitutively active RhoA directly stimulated nuclear {kappa}B binding activity. These results show that RhoA is required for BK-stimulated NF-{kappa}B activation and subsequent cytokine synthesis in airway epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The BK agonists, BK and Lys-[des-Arg9]-BK, and the BK antagonists, [des-Arg9,Leu8]-BK and [D-Arg0,Hyp3,Thi5,8,D-Phe7]-BK, were obtained from Peninsula Laboratories (Belmont, CA). The human lung adenocarcinoma cell line, A549, (distal respiratory epithelium like) was obtained from the American Type Culture Collection (Rockville, MD). A549 cells were maintained in Ham’s F12K medium containing 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (50 µg/ml), and 10% FBS at 37°C in a humidified 5% CO2 environment. Immediately before stimulation, epithelial cells were changed into serum-free RPMI 1640 (Irvine Scientific, Santa Ana, CA).

Oligonucleotides and their complementary strands for electrophoretic mobility shift assays (EMSA) were from Promega (Madison, WI) and Santa Cruz Biotechnology (Santa Cruz, CA). The sequences are: murine intronic {kappa}-chain {kappa}B site (underlined), 5'-AGTTGAGGGGACTTTCCCAGGC-3' (NF-{kappa}B) (14), and a mutant {kappa}B site with the G to C substitution (underlined) in the NF-{kappa}B DNA binding motif, 5'-AGTTGAGGCGACTTTCCCAGGC-3'. The NF-{kappa}B oligonucleotide has been shown to represent a consensus {kappa}B sequence (15). Double-stranded oligonucleotide (5 pmol) was 32P-labeled with T4 polynucleotide kinase. [{gamma}-32P]ATP (>5000 Ci/mmol) was from Amersham (Arlington Heights, IL). The plasmid pmTNF-{alpha} (Kravchenko and Nedospasov, manuscript in preparation), was used for preparation of recombinant murine TNF-{alpha} from Escherichia coli. The sp. act. of TNF-{alpha} purified by ion-exchange chromatography was 7 x 107 U/mg protein. Rabbit polyclonal Abs against the subunits of NF-{kappa}B/Rel were purchased from Santa Cruz Biotechnology. They were raised against 1) a peptide corresponding to the basic nuclear localization signal (NLS) sequence and the N-terminal adjacent 11 amino acids of the p105 precursor of the human p50 (anti-p50); 2) a peptide corresponding to amino acids 3 to 19 of the human p65 (anti-p65); 3) a peptide corresponding to carboxyl-terminal 19 amino acids of the human p52 (anti-p52); and 4) a peptide corresponding to amino acids 152 to 176 of the murine c-rel protein (anti-c-Rel). The RhoA dominant-negative (T19N) and constitutively active (Q63L) pCMV plasmids were obtained as previously described (16). Clostridium difficile toxin B was a gift of Dr. K. Aktories (Freiburg, Germany) and Dr. F. Hofmann (Freiburg, Germany) (17). Recombinant Clostridium botulinum C3 transferase exoenzyme was prepared as previously described (18).

Primary bronchial epithelial cells

Human lung samples from unusable organ transplant specimens were obtained through the Organ and Tissue Acquisition Center of Southern California. Preparation of epithelial cell monolayers followed a modification of previously described methods (19). After washing with supplemented culture media, 0.1% Pronase (Sigma, St. Louis, MO) was added and the tissue incubated for up to 2 h at 37°C with 100% humidity and 5% CO2. The cells were dispersed and filtered through sterile 60-µm Nitex mesh (Cellector, E-C Apparatus Corp., Holbrook, NY). After centrifugation, the cells were suspended in complete Bronchial/Tracheal Epithelial Cell Growth Media (BEGM; modified LHC-9 media supplemented with insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), transferrin (10 µg/ml), triiodothyronine (6.5 ng/ml), epinephrine (0.5 µg/ml), hEGF 0.5 ng/ml), gentamicin (50 µg/ml), amphotericin B (50 µg/ml), and 0.4% bovine pituitary extract; Clonetics Corporation, San Diego, CA). Four milliliters of BEGM containing 0.5 to 1.0 x 105 cells were transferred to a T25 tissue culture flask (Falcon, Franklin Lakes, NJ) and incubated in a humidified atmosphere at 37°C with 5% CO2. Medium was changed every 2 days. Epithelial cells in the third passage were utilized in this study. The purity of the cells was confirmed by staining for cytokeratin.

Detection of immunoreactive IL-1ß

A549 cells in six-well plates were stimulated with BK, the conditioned media collected, and secreted IL-1ß measured by ELISA with a commercially available kit (Genzyme Corp., Cambridge, MA) using the manufacturer’s recommended protocol. Quantitation of secreted IL-1ß was accomplished by normalization of the ELISA data with a standard IL-1ß dose curve.

Preparation of nuclear extracts

Nuclear extracts were prepared by a modified method of Dignam, Lebovitz, and Roeder (20). A549 cells were separately plated at a density of 1 x 106 cells in six-well plates, and after stimulation, cells were washed three times with ice-cold PBS, harvested, and resuspended in 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). After 10 min, 23 µl of 10% Nonidet P-40 was added and mixed for 2 s. Nuclei were separated from cytosol by centrifugation at 13,000 x g for 10 s and were resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF). After 30 min at 4°C, lysates were separated by centrifugation (13,000 x g, 30 s) and supernatant containing nuclear proteins were transferred to new vials. The protein concentration of extracts was measured using a protein dye reagent (Bio-Rad, Richmond, CA) with BSA as standard, and samples were diluted to equal concentration in buffer B for use directly or storage at -80°C.

EMSA

EMSAs were performed by incubating 2.5 µg of the nuclear extract in 12 µl of binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl2, 50 mM KCl, 0.5 mM DTT, 0.4 mg/ml poly(dI-dC) (Pharmacia), 0.1 mg/ml sonicated double-stranded salmon sperm DNA, and 10% glycerol) for 10 min at room temperature. Then, approximately 20 to 50 fmol of 32P-labeled oligonucleotide probe (30,000–50,000 cpm) was added and the reaction mixture was incubated for 10 min at room temperature. For reactions involving competitor oligonucleotides, the unlabeled competitor and the labeled probes were premixed before addition to the reaction mixture. For supershift assays, the reaction mixture minus the probe was incubated with 2 µl of specific Abs for 20 min at room temperature. The 32P-labeled oligonucleotide was then added and incubation continued for 15 min. The samples were analyzed on 6% acrylamide gels, which were made in 50 mM Tris-borate buffer containing 1 mM EDTA (TBE) or 50 mM Tris/380 mM glycine/2 mM EDTA (TGE buffer) and were subjected to pre-electrophoresis for 2 h at 12 V/cm. Electrophoresis was conducted at the same voltage for 2 to 2.5 h. Gel contents were transferred to Whatman DE-81 paper (Hillsboro, OR), dried, and exposed for 3 to 5 h at -80°C with an intensifying screen. Using this method, a nonspecific DNA-protein complex of unknown origin is sometimes seen in the autoradiograph.

Chloramphenicol acetyltransferase (CAT) assay

The plasmid pIL-1[-4000]CAT (WT-IL-1ß-CAT) contains three different {kappa}B-like enhancers in a 4-kb fragment from the promoter region of the IL-1ß gene. These enhancers were deleted in the plasmid pIL-1[-133]CAT (MU-IL-1ß-CAT). Both constructs were kindly provided by J. P. Cogswell (Glaxo Research Institute) and were described previously (21). The plasmids p0.2kb[WT]CAT (WT-I{kappa}B-CAT) and p0.2kb(22)CAT (MU-I{kappa}B-CAT), which contain the wild-type and mutant {kappa}B enhancers from the I{kappa}B gene, respectively, were obtained from P. Chiao (The University of Texas, M.D. Anderson Cancer Center) (23). The promoter region of the I{kappa}B{alpha} gene contains one {kappa}B enhancer site (GGAAATTCC), and activation of NF-{kappa}B results in up-regulation of its own inhibitor (23). The plasmid pTri-SVo-CAT was the parent vector for the IL-1ß constructs, and pBLCAT2 was the parent vector for the I{kappa}B constructs. The pSVL-CAT plasmid (Pharmacia, Piscataway, NJ) was used as a positive control for CAT expression. The plasmid pCMVß (Clontech, Palo Alto, CA) was used as a control for monitoring the transfection efficiency by the expression of ß-galactosidase. Plasmid DNA was transiently transfected into A549 cells with the cationic lipid DOTAP (Boehringer Mannheim, Indianapolis, IN), using procedures recommended by the manufacturer. After 6 h, the DNA-containing media was removed, the cells were washed with PBS and incubated with normal medium for 16 h. The cells were then stimulated with agonists for the indicated time and harvested by scraping. Crude cell extracts were prepared for the measurement of CAT activity with the use of [14C]chloramphenicol (Amersham) as substrate and thin layer chromatography (TLC) for the separation of the native from the acetylated forms as described (24). After development, the extent of CAT activity was measured using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BK activates NF-{kappa}B in airway epithelial cells

To assess the ability of BK to activate NF-{kappa}B in A549 cells, subconfluent A549 cells were incubated with BK (20 nM) for varying times, the nuclear extracts prepared, and NF-{kappa}B EMSA performed as described in Materials and Methods. Figure 1GoA shows that BK induced a time-dependent increase in NF-{kappa}B activity. Compared with the media-alone control (lane 1), BK stimulated increased NF-{kappa}B activation within 15 min (lane 4), and the NF-{kappa}B activation peaked at the 60-min time point (lane 6). A549 cells were also incubated with TNF-{alpha} (100 ng/ml for 30 min) as a positive control (lane 2). The specificity of the NF-{kappa}B EMSA was assessed using an excess of unlabeled {kappa}B oligonucleotide to compete for binding to the nuclear translocated NF-{kappa}B. The addition of increasing concentrations of unlabeled {kappa}B oligonucleotide (0.026–2.6 pmol) to the incubation mixture reduced the binding of the 32P-labeled {kappa}B oligonucleotide to the basal level in a dose-dependent manner (Fig. 1GoB, lanes 5-7). In contrast, the addition of 2.6 pmol of an oligonucleotide that differed only in having a mutated nonfunctional {kappa}B site (GCGACTTTCCA) failed to inhibit binding of the labeled probe (Fig. 1GoB, lane 4). The ability of BK to stimulate NF-{kappa}B activation in nontransformed airway epithelial cells was confirmed using primary human bronchial epithelial cells. Figure 2Go shows that BK (lane 3) and TNF-{alpha} (lane 2) but not Lys-[des-Arg9]-BK (lane 4) stimulated NF-{kappa}B activation in bronchial epithelial cells.



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  		FIGURE 1. BK induces NF-{kappa}B activation in A549 cells. A, A549 cells were stimulated with BK (20 nM) for the indicated time. Nuclear extracts were prepared as described in Materials and Methods, and incubated with a 32P-labeled oligonucleotide containing the consensus {kappa}B site (GGGACTTTCC). The EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow.B, Nuclear extracts prepared from BK-stimulated (20 nM, 1 h) A549 cells were incubated with the radiolabeled {kappa}B probe in the absence (lane 3) or presence of identical but unlabeled oligonucleotide (lanes5-7, with concentrations indicated in pmol), or a mutated nonfunctional oligonucleotide (lane4). Nuclear extracts from unstimulated (lane 1) or TNF-{alpha}-stimulated (lane 2) cells were used as negative and positive controls. The samples were subsequently analyzed by EMSA and the autoradiograph is shown.

 


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  		FIGURE 2. BK but not Lys-[des-Arg9]-BK activates NF-{kappa}B in primary human bronchial epithelial cells. Primary human airway epithelial cells were isolated from bronchial tissue as described inMaterials and Methods. Primary epithelial cells were stimulated with either media alone, TNF-{alpha} (100 ng/ml), the B2 BKR agonist BK (100 nM), or the B1 BKR agonist Lys-[des-Arg]-BK (100 nM) for 1 h. Nuclear extracts were then prepared and used to assess NF-{kappa}B activation by EMSA as described in Materials and Methods. The EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow.

 
Additional evidence that the BK-induced DNA binding activity represented NF-{kappa}B was obtained using two known inhibitors of NF-{kappa}B activation: pyrrolidine dithiocarbamate (PDTC), an antioxidant inhibitor of NF-{kappa}B activation (25); and calpain inhibitor I, (N-acetyl-leu-leu-norleucinal), an inhibitor of the ubiquitin-proteasome pathway (26, 27). Figure 3GoA shows that preincubation of the A549 cells with either calpain I (100 µM for 4 h) or PDTC (100 µM for 1 h) completely inhibited subsequent BK-induced NF-{kappa}B activation. These experiments thus demonstrate that BK induces DNA binding activity in A549 cells, and that the BK-induced DNA binding activity is specific for the {kappa}B sequence. To further establish the identity of the DNA binding activity induced by BK, EMSAs were performed using Abs against the p50, p65, c-Rel, and p52 NF-{kappa}B subunits (Fig. 3GoB). Shifted bands were seen with the addition of anti-p50 (lane 4) and anti-p65 (lane 5) Abs. The combination of anti-p50 plus anti-p65 resulted in the appearance of two shifted bands with almost complete loss of the unshifted NF-{kappa}B band (lane 6). These results suggest that BK induces translocation of the p50/p65 NF-{kappa}B heterodimer.



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  		FIGURE 3. Specificity of {kappa}B binding protein. A, Inhibition of BK-induced NF-{kappa}B activation and IL-1ß synthesis by calpain I and PDTC. A549 cells were pre-incubated with media alone (lanes 1-2), calpain inhibitor I (100 µM for 4 h, lane3), or PDTC (100 µM for 1 h, lane4), and then stimulated with BK (20 nM) for 1 h. NF-{kappa}B activation was assessed by EMSA and IL-1ß levels were measured by ELISA as described in Materials and Methods. The EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow. B, Composition of BK-induced {kappa}B binding activity. Nuclear extracts, prepared from BK (20 nM for 1 h)-stimulated A549 cells, were incubated for 20 min at room temperature in the presence of antisera (2 µg/sample), as indicated: lane 1, no antisera;lane 2, anti-c-Rel antisera; lane3, anti-p52 antisera; lane 4, anti-p50 antisera; lane 5, anti-p65 antisera; lane 6, combination of anti-p50 and anti-p65 antisera. 32P-labeled oligonucleotide was then added and EMSA performed as described in Materials and Methods. The unshifted DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow. Shifted DNA-protein bands are seen in lanes 4 to6, with an almost complete loss of unshifted DNA-protein complex in lane 6.

 
Two types of BK receptors have been defined and cloned: the B2 BK receptor binds BK but not Lys-[des-Arg9]-BK; and a B1 BK receptor binds Lys-[des-Arg9]-BK but not BK (28). The receptor specificity of BK-mediated NF-{kappa}B activation in A549 cells was therefore tested using specific BK receptor agonists and antagonists (Fig. 4Go). NF-{kappa}B activation was seen when A549 cells were stimulated with the B2 BK receptor agonist BK (100 nM, lane 4) but not the B1 BK receptor agonist Lys-[Leu8-des-Arg9]-BK (100 nM, lane 3). The addition of an excess (1 µM) of the B2 BK receptor antagonist [D-Arg0,Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE-140) abolished BK-induced NF-{kappa}B activation (lane 10). In contrast, the addition of an excess (1 µM) of the B1 BK receptor antagonist Lys-[Leu8-des-Arg9]-BK had no effect on BK-induced NF-{kappa}B activation (lane 7). Therefore, BK-stimulated NF-{kappa}B activation in A549 cells is mediated primarily through B2 BK receptors.



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  		FIGURE 4. Kinin-stimulated NF-{kappa}B activation is mediated through B2 but not B1 BK receptors. A549 cells were stimulated for 60 min with media alone, TNF-{alpha} (100 ng/ml), the B2 BK receptor agonist (B2 ag.) BK (100 nM), or the B1 BK receptor agonist (B1 ag.) Lys-[des-Arg9]-BK (100 nM) in the presence of absence of the B1 BK receptor antagonist (B1 Antag.) Lys-[Leu8, des-Arg]-BK, or the B2 BK receptor antagonist (B2 Antag.) HOE-140. NF-{kappa}B activation was detected by EMSA as described in Materials and Methods. The EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow.

 
BK stimulates IL-1ß synthesis in A549 cells

NF-{kappa}B is known to stimulate the transcription of many inflammatory genes, including a large number of cytokines (29). We previously showed that BK stimulates IL-1ß, IL-6, and IL-8 synthesis in WI-38 fibroblasts (11). The ability of BK to stimulate IL-1ß synthesis in A549 cells was determined by measuring IL-1ß levels in culture supernatants. Stimulation of A549 cells with BK (100 nM) resulted in a rapid increase of IL-1ß accumulation in the culture supernatant (Table IGo). Pretreatment with the transcription inhibitor actinomycin D (2.5 µg/ml) almost completely abrogated subsequent BK-stimulated IL-1ß synthesis (Table IGo). These results establish that BK also stimulates de novo synthesis of IL-1ß in A549 cells. As shown in Figure 3GoA, the NF-{kappa}B inhibitors calpain I and PDTC each inhibited BK-stimulated IL-1ß synthesis as well as NF-{kappa}B activation.


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  		Table I. BK stimulates IL-1ß synthesis in A549 cells

 
To further define the relationship between BK-stimulated IL-1ß transcription and BK-induced NF-{kappa}B activation, A549 cells were transiently transfected with chimeric reporter plasmids before BK stimulation. Figure 5GoA shows the results in A549 cells transfected with WT-IL-1ß-CAT, containing three {kappa}B-like enhancers. BK (10 nM) and TNF-{alpha} (100 ng/ml) each stimulated increased CAT activity (lanes 6-7) compared with the unstimulated control (lane 5). When the A549 cells were transfected with MU-IL-1ß-CAT (from which the three {kappa}B sites were deleted), neither BK nor TNF-{alpha} stimulated an increase in CAT activity (lanes 3-4 compared with lane 2). Identical results were obtained when A549 cells were transfected with the WT-I{kappa}B-CAT and MU-I{kappa}B-CAT plasmids (see below). We also confirmed that BK-stimulated CAT activity was mediated through B2 BK receptors. BK-stimulated CAT activity in A549 cells transfected with the WT-IL-1ß-CAT plasmid was completely blocked by the B2 BK receptor antagonist HOE-140 (Fig. 5GoB). Taken together with the ELISA data, these results demonstrate that BK stimulates de novo IL-1ß synthesis, and that this is a consequence, at least in part, of NF-{kappa}B activation.



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  		FIGURE 5. NF-{kappa}B regulation of reporter gene expression in BK-stimulated cells. A, A549 cells were cotransfected with 2.5 µg of pSVL-CAT (lane 1, positive control), WT-IL-1ß-CAT (lanes 2-4), or the MU-IL-1ß-CAT (lanes5-7) plasmids, together with 0.5 µg of the pCMVß plasmid as described in Materials and Methods. The transfected cells were stimulated with media alone, BK (10 nM), or TNF-{alpha} (100 ng/ml) for 45 min and then harvested. CAT activity was measured in the crude cell lysates using [14C]chloramphenicol as a substrate, and separated by TLC as described in Materials and Methods. All results were normalized for transfection efficiency using the expression of ß-galactosidase, and reported as relative CAT activity with the pSVL-CAT activity defined as 100%. B, A549 cells were cotransfected with 2.5 µg of the WT-IL-1ß-CAT plasmid together with 0.5 µg of the pCMVß plasmid, then stimulated with media alone, BK (10 nM), or TNF-{alpha} (100 ng/ml) for 45 min in the absence or presence of HOE-140 (1 µM). CAT activity was measured in the crude cell lysates using [14C]chloramphenicol as a substrate, and separated by TLC as described in Materials and Methods. A PhosphoImager screen was exposed, and the autoradiograph is shown.

 
Role of Rho in BK-mediated NF-{kappa}B activation

To assess the role of the Rho family of small GTP-binding proteins in BK-induced NF-{kappa}B activation, we pretreated A549 cells with Clostridium difficile toxin B, an inhibitor of Rho, Rac, and Cdc42 proteins (17). C. difficile toxin B (40 ng/ml for 2 h) completely inhibited BK-induced NF-{kappa}B activation (Fig. 6GoA, lane 4 vs 2). We then tested whether a specific Rho inhibitor would also inhibit BK-induced NF-{kappa}B activation. C3 transferase exoenzyme is an exotoxin produced by Clostridium botulinum that specifically inhibits the Rho small GTP binding proteins (Rho A, B, and C) but does not inhibit Rac or Cdc42 (17). Preincubation of A549 cells with 10 µg/ml of recombinant C3 transferase exoenzyme completely inhibited BK-induced NF-{kappa}B activation (Fig. 6GoB, lane 5 vs 2), but only marginally affected TNF-{alpha}-induced NF-{kappa}B activation (lane 6 vs 3). Preincubation with recombinant C3 transferase exoenzyme also inhibited BK-stimulated IL-1ß synthesis, even when added at a lower dose (Fig. 6GoC, lane 4 vs 3). Consistent with its effects on NF-{kappa}B activation, recombinant C3 transferase exoenzyme did not affect TNF-{alpha}-stimulated IL-1ß synthesis (lane 5 vs 2).



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  		FIGURE 6. Consequences of Rho inhibition on BK-induced NF-{kappa}B activation and IL-1ß synthesis. A, A549 cells were: pre-incubated with media alone or Clostridium difficile toxin B (40 ng/ml) for 2 h, stimulated with media alone or BK (10 nM) for 1 h, and NF-{kappa}B activity determined by EMSA. A PhosphoImager screen was exposed, and the EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow. B, A549 cells were: pre-incubated with media alone or recombinant C3 transferase exoenzyme (rC3 inhibitor; 10 µg/ml overnight); stimulated with media alone, BK (10 nM), or TNF-{alpha} (100 ng/ml) for 45 min; and NF-{kappa}B activity determined by EMSA. A PhosphoImager screen was exposed, and the EMSA autoradiograph is shown. The DNA-protein complex is marked with a bracket, and the unbound probe is indicated by an arrow. C, A549 cells were: pre-incubated with media alone or recombinant C3 transferase exoenzyme (5 µg/ml, overnight); stimulated with media alone, BK (100 nM), or TNF-{alpha} (100 ng/ml) for 2 h; and IL-1ß measured in the culture supernatants by ELISA.

 
To confirm the requirement for Rho in BK-induced NF-{kappa}B activation, A549 cells were cotransfected with an expression vector encoding a dominant-negative form of RhoA (RhoA-T19N) along with chimeric CAT reporter plasmids before BK stimulation. Figure 7GoA shows that the degree of RhoA-T19N-mediated inhibition of BK-stimulated CAT activity progressively lessened as the amount of transfected RhoA-T19N decreased (lanes 3-5). In contrast, the highest concentration of the dominant-negative RhoA plasmid did not affect TNF-{alpha}-stimulated CAT activity (lane 2). Thus, RhoA-T19N blocks BK-induced NF-{kappa}B activation in a dose-dependent manner. Figure 7GoB shows the effect of the dominant-negative RhoA plasmid on BK-stimulated CAT activity in A549 cells transfected with chimeric I{kappa}B-CAT plasmids. BK and TNF-{alpha} each stimulated increased CAT activity in A549 cells transfected with the WT-I{kappa}B-CAT plasmid but not in A549 cells transfected with the MU-I{kappa}B-CAT plasmid (lanes 5-6 vs 2-3). Cotransfection of the dominant-negative RhoA plasmid (2 µg) with the WT-I{kappa}B-CAT plasmid completely blocked BK-stimulated CAT activity (lane 8) but did not affect TNF-{alpha}-stimulated CAT activity (lane 9). These results confirm that RhoA activity is required for BK-induced NF-{kappa}B activation.



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  		FIGURE 7. Effect of a dominant-negative RhoA on NF-{kappa}B regulation of reporter gene expression in BK-stimulated cells. A, A549 cells were cotransfected with 2.5 µg of the WT-IL-1ß-CAT plasmid (lanes 1-6) and the indicated amount of RhoA-T19N plasmid (lanes3-5), together with 0.5 µg of the pCMVß plasmid as described in Materials and Methods. As a positive control, A549 cells were also cotransfected with 2.5 µg pSVL-CAT and 0.5 µg of the pCMVß plasmid (lane6). B, A549 cells were cotransfected with 2.5 µg of the MU-I{kappa}B-CAT (lanes1-3) or WT-I{kappa}B-CAT plasmid and 2.0 µg of either the RhoA-T19N plasmid (lanes7-9) or empty vector (pBLCAT2,lanes 1-6), together with 0.5 µg of the pCMVß plasmid. The pSVL-CAT was again used as a positive control (lane 10). For bothA and B, the transfected cells were stimulated with media alone, BK (20 nM), or TNF-{alpha} (100 ng/ml) for 2 h, and then harvested. CAT activity was measured in the crude cell lysates using [14C]chloramphenicol as a substrate, and separated by TLC as described in Materials and Methods. All results were normalized for transfection efficiency using the expression of ß-galactosidase, and reported as relative CAT activity with the pSVL-CAT activity defined as 100%.

 
The relationship between RhoA activity and subsequent NF-{kappa}B activation was further explored using RhoA-Q63L, a constitutively active form of RhoA. Transfection of A549 cells with RhoA-Q63L resulted in increased CAT activity when the cells were cotransfected with a chimeric CAT reporter plasmid containing functional {kappa}B sites (Fig. 8Go, lanes 2 and 5). When the A549 cells were cotransfected with CAT reporter plasmids containing mutated or absent {kappa}B sites, the constitutively active RhoA did not stimulate CAT activity (Fig. 8Go, lanes 3 and 6). Thus, we have shown that RhoA activity alone is sufficient to generate functional NF-{kappa}B activity. Taken together with the previous results, this strongly suggests that BK-mediated IL-1ß synthesis results from RhoA-dependent NF-{kappa}B activation.



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  		FIGURE 8. Constitutively active RhoA stimulates NF-{kappa}B-mediated reporter gene expression. A549 cells were cotransfected with 2.5 µg of the WT-I{kappa}B-CAT plasmid (lanes1-2), MU-I{kappa}B-CAT (lane3), WT-IL-1ß-CAT (lanes4-5), or MU-IL-1ß-CAT (lane 6), and 2.0 µg of either the RhoA-Q63L plasmid (lanes2-3 and 5-6) or empty vector (pBLCAT2, lane 1 or pTri-SVo-CAT,lane 4), together with 0.5 µg of the pCMVß plasmid. After a 16-h incubation in normal culture media, CAT activity was measured in the crude cell lysates using [14C]chloramphenicol as a substrate, and separated by TLC as described in Materials and Methods. A PhosphoImager screen was exposed, and the autoradiograph is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
BK is recognized to play an important role in asthma. We and others have shown that BK is generated in human airways within minutes of allergen challenge (30, 31). Allergen challenge studies have also established that BK plays a pivotal role in the initiation of chronic airway inflammation (32, 33). B2 BK receptor antagonists effectively block the development of the late phase airway response as well as the development of bronchial hyper-responsiveness following allergen challenge in a number of different experimental animal models (3, 4, 5). Conversely, airway hyper-responsiveness was increased following exposure to BK (34). To explore the mechanism by which BK promotes airway inflammation, we examined the ability of BK to regulate gene transcription and cytokine synthesis in airway epithelial cells.

Chronic asthma is associated with airway remodeling and increased synthesis of cytokines (6). The epithelial cell is now recognized to play a critical role in orchestrating the airway inflammatory response, principally through synthesizing multiple cytokines and chemokines (35). We previously demonstrated that BK stimulates NF-{kappa}B activation and cytokine synthesis in human fibroblasts (11). Using EMSA to detect {kappa}B binding activity in nuclear extracts, we demonstrated that BK also stimulated NF-{kappa}B activation in A549 type II epithelial cells at concentrations in the low nanomolar range. Kinin-stimulated NF-{kappa}B activation was seen only after stimulation with the B2 BK receptor agonist BK and not after stimulation with the B1 BK receptor agonist Lys-[des-Arg9]-BK. Confirming the agonist results, BK-induced NF-{kappa}B activation was totally inhibited by the B2 BK receptor antagonist HOE-140, but was not affected by a B1 BK receptor antagonist.

A549 cells are a relatively differentiated type II alveolar epithelial cell line derived from a human lung adenocarcinoma (36). They were initially developed to study the synthesis of surfactant, but have also been widely used in studies of airway epithelial cytokine and chemokine synthesis (37, 38, 39). In addition, NF-{kappa}B activation in A549 cells has been shown to occur following stimulation with multiple other agents, including: asbestos, ozone, IL-1ß, TNF-{alpha}, IFN-{gamma}, and several types of viruses (40, 41, 42, 43). This is the first report, however, that a GPCR activates NF-{kappa}B in airway epithelial cells, the significance of which is underscored by the large number of different GPCR that have been identified. Since A549 cells are a transformed cell line and may not necessarily reflect the behavior of nontransformed airway epithelial cells, we also showed that BK stimulates NF-{kappa}B activation in primary human bronchial epithelial cells.

BK-induced NF-{kappa}B activation may provide an important link between acute allergic reactions and the development of airway hyper-responsiveness and chronic inflammation. NF-{kappa}B, a heterodimeric member of the rel family, exists in a latent form in the cytoplasm bound to its inhibitor, I{kappa}B. Activation of NF-{kappa}B occurs by phosphorylation of I{kappa}B, causing the complex to dissociate and allowing active NF-{kappa}B to translocate into the nucleus, where it binds to specific {kappa}B sites in the promoter regions of many pro-inflammatory genes and stimulates transcription (44). NF-{kappa}B has been postulated to play a pivotal role in the propagation of asthmatic airway inflammation (29, 45). Cytokine genes expressed in airway epithelial cells and regulated by NF-{kappa}B have been reported to include: IL-1, IL-6, IL-8, TNF-{alpha}, GM-CSF, and IFN-{gamma} (44). We choose to measure IL-1ß synthesis to illustrate the functional consequences of BK-induced NF-{kappa}B activation on cytokine gene expression in A549 cells.

We showed that BK stimulated increased IL-1ß synthesis in A549 cells. The relationship between BK-induced NF-{kappa}B activation and BK-stimulated IL-1ß synthesis was assessed using chimeric CAT reporter plasmids containing either a wild-type (with three {kappa}B-like enhancer sites) or mutant (in which the three {kappa}B sites were deleted) fragment from the promoter region of the IL-1ß gene. BK stimulated increased CAT activity only in the presence of the intact {kappa}B sites, indicating that BK-induced IL-1ß transcription requires intact {kappa}B sites. Additionally, we showed that inhibition of NF-{kappa}B activation and inhibition of IL-1ß synthesis were closely correlated (see below), suggesting a causal relationship between activation of NF-{kappa}B and the subsequent increase in IL-1ß synthesis after stimulation with BK.

Having demonstrated that BK can activate NF-{kappa}B and stimulate cytokine synthesis in airway epithelial cells, we then examined the role of the Rho family of small G proteins in transducing this effect. Several recent studies have implicated involvement of the Rho family in both BK-mediated effects as well as in transcriptional regulation of gene expression (see below). The p21 Ras superfamily of GTP binding proteins often transduces signals initiated through GPCR-mediated activation of heterotrimeric G proteins (46). BK has previously been shown to activate the small G protein Ras (47), and was recently shown to activate the Rho family proteins Cdc42 and Rac (13). BK-mediated voltage-dependent Ca2+ current in NG108–15 neuroblastoma-glioma cells was also shown to be dependent on RacI and possibly Cdc42 activity (48). Rho family proteins have also been shown to participate in GPCR-mediated activation of the serum response element of the c-fos promoter (49), as well as GPCR-mediated transcriptional regulation of the atrial natriuretic factor in rat cardiomyocytes (50).

We demonstrated that Clostridium difficile toxin B inhibited BK-induced NF-{kappa}B activation, suggesting that the Rho family of small GTP-binding proteins was involved. These results were supported by the ability of C3 transferase exoenzyme, an inhibitor of Rho, to abolish BK-induced NF-{kappa}B activation. C3 transferase exoenzyme also significantly inhibited BK-stimulated IL-1ß synthesis, even when used at a lower dose. The role of Rho in BK-induced NF-{kappa}B activation was then confirmed by cotransfecting A549 cells with a dominant-negative RhoA plasmid as well as the chimeric IL-1ß-CAT and I{kappa}B-CAT reporter plasmids. The dominant-negative RhoA plasmid specifically abolished BK-stimulated CAT activity, but had no effect on TNF-{alpha}-stimulated CAT activity. These results suggest that BK-induced NF-{kappa}B activation and IL-1ß synthesis require activation of RhoA.

The mechanism by which BK activates RhoA has not yet been established. The B2 BK receptor is capable of coupling to several heterotrimeric G proteins depending on the cell type, including Gi/Go, Gs, Gq, G11, and G13 (11, 48, 51, 52). After binding of ligand to GPCR, the heterotrimeric G proteins undergo GDP-GTP exchange, whereupon the tightly associated heterotrimeric G protein dissociates into {alpha}- and ß{gamma}-subunits. Constitutively active G{alpha}12 and G{alpha}13 subunits have been shown to stimulate Rho-dependent cytoskeletal changes (53). Similarly, ß{gamma}-subunits have been reported to activate Cdc42, Rac, and Rho (46, 54). We showed, moreover, that RhoA, once activated, is itself a sufficient stimulus to subsequently lead to activation of NF-{kappa}B.

Our results are in agreement with a recent report that constitutively active Rho proteins could activate NF-{kappa}B in NIH3T3 cells (55). However, Perona et al. found that TNF-{alpha}-induced activation of NF-{kappa}B also depended on Cdc42 and RhoA (55), while we found that TNF-{alpha}-induced NF-{kappa}B activation in A549 cells was not affected by inhibition of RhoA. The intracellular signaling pathways linking RhoA and NF-{kappa}B activation need to be further defined. The Rho family of small G proteins has been shown to activate several serine/threonine kinases, which can mediate downstream effects, particularly on the actin cytoskeleton. PAK1 is the primary kinase activated by both Cdc42 and Rac1 but not by RhoA (56). Several closely related serine/threonine kinases appear to be activated by RhoA, including p120 protein kinase N, p160 Rho-associated coiled-coil-containing protein kinase, p164 Rho kinase, and p140 PRK2 kinase (57). Recent studies have suggested that RhoA may also activate protein tyrosine kinases of the focal adhesion family (58). Activated RhoA has also been shown to be involved in phophatidylinositol-3-kinase-mediated activation of protein kinase C-{zeta} (PKC-{zeta}) in a murine T cell line (59). Several studies have implicated PKC-{zeta} in NF-{kappa}B activation (60, 61), and BK has been shown to activate PKC-{zeta} in Chinese hamster ovary cells stably transfected with the human B2 BK receptor (62).

In summary, we have shown that BK, acting through B2 BK receptors, induces activation of the transcription factor NF-{kappa}B and subsequently increases transcription of IL-1ß in human airway epithelial cells. These observations provide a novel possible explanation for the critical role of BK in the transition of acute allergic reactions to chronic airway inflammation. We have further shown that BK-induced NF-{kappa}B activation and IL-1ß synthesis requires activation of RhoA, and that RhoA alone is a sufficient stimulus to activate NF-{kappa}B in A549 cells. Additional experiments are needed to define the signaling steps both upstream and downstream of RhoA that are necessary for BK-induced NF-{kappa}B activation.


   Acknowledgments
 
The authors thank J. P. Cogswell and P. J. Chiao for providing reporter gene constructs.


   Footnotes
 
1 Supported by USPHS Grants AI36220 (to B.L.Z.), AI33503 (to R.D.Y.), and GM37696 (to G.M.B.). The work was done during the tenure of an Established Investigatorship (to R.D.Y.) from the American Heart Association. Oligonucleotide primer design and sequence analysis were performed using the GCRC facility at The Scripps Research Institute supported by National Institutes of Health Grant M01RR00833. This is manuscript no. 10963-MEM from The Scripps Research Institute. Back

2 Address correspondence and reprint requests to Dr. Bruce L. Zuraw, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. E-mail address: Back

3 Abbreviations used in this paper: BK, bradykinin; NF-{kappa}B, nuclear factor-{kappa}B; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidine dithiocarbamate; CAT, chloramphenicol acetyltransferase; GPCR, G protein-coupled receptors; PKC-{zeta}, protein kinase C-{zeta}; TLC, thin layer chromatography. Back

Received for publication August 4, 1997. Accepted for publication November 25, 1997.


   References
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 Materials and Methods
 Results
 Discussion
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