The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-β has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-β can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-β directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-β alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-β-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B2) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-β on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-β, in a dose- (0.5–10 ng/ml) and time- (2–24 h) dependent manner, increased mRNA and protein expression of the B2 receptor in cultured human ASM cells. Maximal B2 receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-β treatment and was further confirmed using fluorescence microscopy. TGF-β (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-β-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-β may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B2 receptor expression and signaling.
- airway hyperresponsiveness
- airway remodeling
- intracellular calcium
- isometric force generation
- transforming growth factor-β
asthma is characterized by nonspecific airway hyperresponsiveness (AHR), defined as enhanced contractile response to different agonists that activate G protein-coupled receptors (GPCR). To date, the molecular mechanisms leading to AHR remain uncertain. Recent in vitro studies suggest that enhanced contractile properties of airway smooth muscle (ASM), the primary effector tissue regulating bronchomotor tone (reviewed in Ref. 10), are likely one plausible mechanism promoting the excessive bronchoconstriction that characterizes nonspecific AHR in asthma (54).
Shortening of ASM in response to GPCR stimulation regulates airway caliber and bronchomotor tone by increasing cytosolic Ca2+ concentrations and subsequent actin-myosin cross-bridge cycling via phosphorylation of 20-kDa myosin light chain (8). Studies using cultured ASM cells from human and other animal models demonstrate that abnormal ASM contractile function may result from simultaneous activation of multiple GPCR by different agonists or alternatively may be due to increased expression of specific GPCR and/or by modulation of downstream signaling pathways in ASM such as with the cytokines, TNF-α, or IL-1β (48, 53).
Bradykinin, derived from the kininogen precursor by the action of kallikrein enzymes, is a local-acting blood-borne peptide that mediates its effects through two receptors, namely, bradykinin 1 (B1) and 2 (B2) receptors. Although both B1 and B2 receptors contribute to allergen-induced bronchial hyperresponsiveness in rats (35), in humans, hyperresponsiveness to bradykinin appears to be solely mediated by activation of the B2 receptor and not the B1 receptor (49, 62). Tao et al. (55) demonstrated increased Ca2+ responses to bradykinin in ASM cells derived from hyperresponsive inbred rats. Recent studies have shown that cytokines such as TNF-α and IL-1β increase bradykinin receptor expression and function not only in human lung fibroblasts but also in human bronchial ASM cells (48, 53). Importantly, in subjects with asthma but not in normal subjects, bradykinin induced an excessive airway obstruction (27). Together, these studies raise the hypothesis that the exaggerated bronchial sensitivity to bradykinin in asthmatics may be due in part to modulation of bradykinin receptor signaling pathways in ASM.
Transforming growth factor (TGF)-β, a pleiotropic cytokine, plays a role in the pathophysiology of asthma. In subjects with asthma, TGF-β concentrations are higher in the bronchoalveolar fluid compared with normal subjects (50). Furthermore, studies suggest that TGF-β promotes airway remodeling and irreversible airway obstruction (14, 17, 39, 51). Whether TGF-β regulates AHR by directly modulating ASM contractile function remains unknown. Evidence also suggests that TGF-β potentially reduces ASM relaxation to isoproterenol by decreasing β-adrenergic receptor binding sites in human ASM (45). Based on the observation that TGF-β and bradykinin levels are increased during chronic airway inflammation and in asthmatic airways (12, 58), we hypothesized that TGF-β may directly modulate ASM responsiveness to bradykinin.
In this study, we show that TGF-β enhances bradykinin-induced force generation and intracellular calcium signals in ASM by inducing increased cell surface B2 receptor expression. This is the first report to demonstrate that TGF-β may participate in bradykinin-induced AHR in asthma by directly altering ASM contractile function. Further characterization of the precise pathways by which TGF-β modulates ASM function may provide opportunities for new therapeutic approaches in the regulation of AHR in asthma.
MATERIALS AND METHODS
Measurement of isometric force generation.
Isometric force generation by murine cultured tracheal rings was performed as described previously (19). Briefly, mouse tracheae obtained from 7- to 12-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) were sectioned into 3- to 4-mm-long segments. The tracheal rings were then cultured overnight in Ham’s F-12 medium supplemented with 100 mM HEPES, 1.0 M NaOH, 10% FBS (Hy-Clone, Logan, UT), 0.2 M glutamine, 1.0 M CaCl2, 100 U/ml penicillin, and 100 μg/ml streptomycin. The organ cultures were treated with 12.5 ng/ml TGF-β or diluent alone for 18 h and then washed with Krebs-Henseleit (K-H) solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 11.1 dextrose, 1.2 MgSO4, 2.8 CaCl2, and 25 NaHCO3. Tracheae were mounted on stainless-steel pins, supported by Plexiglas rods in the base of a double-jacketed, glass organ bath maintained at 37°C and bubbled with 95% O2-5% CO2 to maintain a pH of 7.4. Changes in tension of the rings were measured using an FT03 isometric transducer (Astro-Med, West Warwick, RI) and synchronously recorded with an MP 100WS system (BIOPAC Systems, Santa Barbara, CA). All initial tensions of tracheal rings were set at ∼0.5 g. After a 45-min equilibration period, cumulative concentration-isometric force curves were then generated to bradykinin (1 nM to 30 mM). After three washings with 10 ml of K-H buffer, the tracheae were contracted with 100 mM KCl. The institutional review boards of the Wistar Institute and the University of Pennsylvania approved use of all animals.
Human and murine ASM cell culture.
Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings at the University of Pennsylvania. Trachealis muscle was enzymatically dissociated using 640 U/ml collagenase and 10 U/ml elastase in the presence of 1 mg/ml soybean trypsin inhibitor and 0.2 mM CaCl2. The cell suspension obtained was filtered through 105-μm Nytex mesh. After washing the filtrate with equal volumes of cold Ham’s F-12 medium supplemented with 10% FBS (Hyclone Laboratories), cells were plated at a density of 1.0 × 104 cells/cm2 in 75-mm flasks. Human ASM cells in subculture during the second through fifth cell passages were used for all subsequent experiments. These cells have been shown to retain all structural and functional characteristics of ASM (46).
Murine tracheal smooth muscle cells were cultured from explants of excised tracheae using a modification of previously described methods (28, 44). The entire trachea between the larynx and mainstem bronchi was removed and placed in a sterile petri dish containing cold PBS. After additional surrounding tissue and epithelium were removed with the aid of a dissecting microscope, myocytes were dispersed from dissected trachealis using 10 U/ml elastase and 600 U/ml collagenase. Myocytes were seeded and grown on fibronectin-coated plastic culture plates in Ham’s F-12 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml fungizone, 10 μg/ml EGF, and 10 μg/ml PDGF. The explants were incubated at 37°C in a humidified environment of 95% air-5% CO2. Once the monolayer became confluent, the cells were harvested by trypsinization and passaged into 100-mm tissue culture plates at a 1:4 ratio. More than 90% of these cells, as determined by immunohistochemistry, expressed smooth muscle α-actin. All experiments were performed on confluent cells at matched passages 2–4.
Treatment of human ASM with cytokines and/or antagonists.
Confluent human ASM cells were growth arrested by being incubated in serum-free Ham’s F-12 with 0.1% BSA for 48 h before being stimulated with TGF-β (2.5 ng/ml). In some experiments, human ASM were pretreated with indomethacin (2 μM) for 30 min before being treated with TGF-β.
Characterization of bradykinin receptor expression.
RT-PCR analysis was performed as described previously (16). Total cellular RNA was isolated from control and TGF-β-treated human ASM cells using RNeasy mini kit (Qiagen, Valencia, CA) as per the manufacturer’s protocol. Total RNA from control and TGF-β-treated human ASM cells was reverse transcribed using SuperScript II (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The RT reaction was performed at 65°C for 5 min and subsequently at 42°C for 1 h. The first strand of cDNA synthesized by RT was denatured at 94°C for 5 min and used subsequently for amplification by PCR. RT-PCR reactions were performed with the use of B1 receptor (B1R), B2 receptor, and β-actin primers for semiquantitative analysis as described previously (16). The PCR products (B1 receptor, 578 bp; B2 receptor, 447 bp; β-actin, 241 bp) were resolved on 1.5% agarose gel electrophoresis, stained with ethidium bromide, and photographed. Murine RNA was isolated from control and TGF-β-treated murine tracheal rings (3 rings per condition) using RNeasy mini kit. Total murine RNA was reverse transcribed using Moloney murine leukemia virus (Promega, Madison, WI). RT-PCR was performed for 30 cycles at 94°C denaturing, 55°C annealing, and 72°C extension using Taq DNA polymerase (Promega) using specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-GCAGGGGGGAGCCAAAAGGG-3′; reverse: 5′-TGCCAGCCCCAGCGTCAAAG-3′) was used at 200 nM each. B2 receptor (forward: 5′-ATGTTCAACGTCACCACACAAGTC-3′; reverse: 5′-TGGATGGCATTGAGCCAAC-3′) was used at 200 nM each (62). Reaction products were confirmed on 1% agarose (Fisher Biotech, Fair Lawn, NJ) gels with size markers (New England Biolabs, Beverly, MA). Each primer pair produced a specific size product: GAPDH, 565 bp, and B2R, 104 bp.
Western blot analysis was performed as described previously (4). Briefly, human ASM cells were washed with cold PBS and lysed in buffer containing 10 mM Tris·HCl (pH 7.4), 0.5% sodium deoxycholate, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Proteins were analyzed on a 4–12% SDS-PAGE and blotted onto a nitrocellulose membrane. The membranes were blocked with 5% BSA in Tris-buffered saline containing 0.05% Tween 20 and incubated with a mouse monoclonal IgG against the B2R (BD Biosciences, Palo Alto, CA). After being incubated with the appropriate peroxidase-conjugated secondary antibody (Roche Molecular Biochemicals, Minneapolis, MN), the bands were visualized by the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) and autoradiographed. To ensure equal loading, the membranes were stripped and reprobed with β-actin antibodies.
Measurement of intracellular calcium concentration.
Fura-2 AM, a cell-permeant, ratiometric calcium indicator dye was used to determine the intracellular calcium concentration in human ASM cells. Calcium measurements were determined as described previously (21). Briefly, human ASM cells grown on 100-mm dishes were washed with HBSS containing 10 mM HEPES, 11 mM glucose, 2.5 mM CaCl2, and 1.2 mM MgCl2 (pH 7.4) and incubated with 5 μM fura-2 AM for 30 min at 37°C and 5% CO2. Fura-2-loaded cells were trypsinized, and fluorescence was estimated in cell suspensions by excitation at 340- and 380-nm wavelengths. Fluorescence emissions were collected separately for each wavelength at 510 nm using a spectrofluorimeter (Photon Technology International, Lawrenceville, NJ). The ratio of fluorescence intensities at 340- and 380-nm wavelengths was determined and converted to the calcium concentrations using the standard equation (21). The net calcium responses to contractile agonists were calculated by subtracting the basal from that of the peak intracellular calcium concentration. All experiments were performed at 37°C.
Immunolocalization of B2 receptor on human ASM cells.
Human ASM cells were serum starved for 48 h and stimulated with diluent control or TGF-β (2.5 ng/ml) on two-well slides (Nunc, Naperville, IL). Slides were first fixed with 4% formaldehyde and then permeabilized with a mixture of cold acetone/methanol solution (vol/vol) for 10 min. After being washed with PBS, the cells were exposed to 5% BSA in PBS for 60 min at 37°C and incubated with 4 μg/ml anti-B2 receptor antibody (Affinity Bioreagents, Golden, CO) or isotype-matched control (polyclonal rabbit IgG) in 5% BSA in PBS for 60 min at 37°C and washed with PBS followed by incubation with goat anti-rabbit IgG F(ab′)2 Alexa 488 (Molecular Probes). Colocalization of B2 receptor at the cell surface was demonstrated by using the cell surface glycoprotein receptor CD44. Hermes-3, a monoclonal antibody against an extracellular epitope to the cell surface glycoprotein CD44 at a concentration of 4 μg/ml, was used as cell membrane marker (37). Mouse IgG2a antibody at the same concentration was used as the isotype-matched negative control for Hermes-3. Alexa 594-conjugated goat anti-mouse antibody was used as the secondary antibody (1:400 dilution, Molecular Probes). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole. Slides were washed with PBS and mounted with anti-fade agent. Slides were viewed on an Olympus I-70 inverted fluorescent microscope. Fluorescence images were captured using a Hamamatsu 12-bit charge-coupled device camera. Image processing was performed using IMAGE 1 software (Universal Imaging, West Chester, PA).
Tension was calculated as milligram of tension per milligram of tracheal smooth muscle weight (mg/mg). The concentrations of agonists required to produce half-maximal contraction (EC50) were determined, and the EC50 of bradykinin were then converted to log values. Concentration of agonists required to produce a half-maximal relaxation (pD2) was determined with −log values of the EC50. All values were expressed as means ± SE. Comparisons among groups with or without TNF-α were performed by a one-way ANOVA. Student’s unpaired t-test was used to compare the effect of drug treatment. A P value of <0.05 was considered significant.
Tissue culture reagents and primers used for PCR were obtained from Invitrogen (Carlsbad, CA). Recombinant human TGF-β was purchased from R&D Systems (Indianapolis, IN). Carbachol, bradykinin, and fura-2 AM were purchased from Sigma (St. Louis, MO). Hermes-3 antibody was prepared from hybridoma supernatant. Mouse IgG2a antibody was purchased from Jackson ImmunoLabs (West Grove, PA).
TGF-β augments bradykinin-induced isometric force generation in murine cultured tracheal rings.
We have previously shown that cytokines such as TNF-α modulate ASM contractile response to GPCR activation using an organ culture model (19). In this study, we used isolated murine tracheal rings to determine whether TGF-β promotes ASM hyperresponsive to bradykinin. In a concentration-dependent manner, bradykinin evoked isometric force generation in murine tracheal rings, demonstrating that ex vivo tracheal rings retain contractile properties to bradykinin (Fig. 1). Maximal bradykinin-induced force generation represents ∼30% of a maximal methacholine-induced response (data not shown). To determine whether TGF-β modulates agonist-induced force generation, murine tracheal rings were cultured in the presence and absence of 12.5 ng/ml TGF-β for 18 h. In previous experiments, we determined that a minimum of 8–18 h of incubation was needed for cytokine-mediated effects (19). We and others have also shown that concentrations of recombinant human cytokines in the 0.1- to 10-nM range, consistent with the in vitro and ex vivo doses used in this study, are often required when treating murine tracheal rings compared with cultured cell systems (1, 3, 57, 62). This is likely secondary to diffusional barriers in tracheal tissue. In TGF-β-treated murine tracheal rings, there was a significant enhancement of the bradykinin-induced contractile response at concentrations ranging from 10−6 to 10−4 M (P < 0.05) compared with that obtained from diluent-treated rings. In the TGF-β-treated rings, maximal force generation was obtained with bradykinin (3 × 10−5 M), 74 ± 10 mg (n = 6) compared with 34 ± 9 mg (n = 6) in diluent-treated rings (P < 0.05), or a 117% increase in tension. Of note, the effect of TGF-β on bradykinin-evoked maximal force generation was associated with an alteration of the pD2 values (−log EC50), which were 4.73 ± 0.57 and 6.26 ± 0.16 in control and TGF-β-treated rings, respectively (P = 0.02). These data suggest that TGF-β modulates maximal force generation induced by bradykinin by affecting receptor expression or affinity.
TGF-β enhances bradykinin-evoked calcium signals in cultured human ASM.
Because cytokines, such as IL-1β and TNF-α, significantly enhanced carbachol-, bradykinin-, and thrombin-induced calcium responses (2, 33, 61), we postulated that TGF-β may also increase agonist-induced cytosolic calcium levels in fura-2-loaded human ASM cells. As shown in Fig. 2, A and C, we found that TGF-β (2.5 ng/ml for 18 h) significantly increased bradykinin-induced cytosolic free Ca2+ concentration ([Ca2+]i) in human ASM. Baseline [Ca2+]i was similar in both control and TGF-β-treated cells (146.7 ± 20.7, n = 7 vs. 134.3 ± 26.4, n = 11, respectively; P > 0.05). TGF-β pretreatment, however, induced a 50% increase in calcium mobilization with a peak [Ca2+]i of 249.8 ± 34.7 nM in untreated human ASM (n = 3) compared with 376.4 ± 42.6 nM in cells treated with TGF-β for 18 h (n = 5; P < 0.05). Interestingly, this TGF-β-enhanced calcium signal is agonist specific since no difference in 1 or 10 μM carbachol-induced peak [Ca2+]i in untreated (127.2 ± 14.8 nM, n = 4–6) and TGF-β-treated cells (128.5 ± 10.7 nM, n = 4–6) was observed (Fig. 2, A and B).
TGF-β enhances bradykinin-evoked calcium signals in cultured murine ASM.
Peak [Ca2+]i was also measured in cultured murine ASM isolated from BALB/c mice in the presence and absence of TGF-β. TGF-β-treated murine ASM increased peak [Ca2+]i in response to bradykinin, albeit at a higher dose (10 nM) than that observed in human ASM (Fig. 3, A and B). The peak calcium response to 10 nM bradykinin was 78.4 ± 7.6 nM in untreated cells compared with 120 ± 3.3 nM (n = 3, P < 0.05) in TGF-β-treated cells. Even though the responses to 1 nM bradykinin in murine ASM showed a similar trend, the data were not statistically significant (peak calcium values 50.8 ± 8.8 in control vs. 66.5 ± 5.1 in TGF-β-treated cells, n = 3, P > 0.05).
TGF-β effect on bradykinin-induced calcium transient is not inhibited by indomethacin.
Since TGF-β stimulates cyclooxygenase (COX)-2 expression and prostaglandin production and since IL-1β has previously been shown to increase B2 receptor gene transcription via PGE2 synthesis (41), we determined whether prostaglandin production played any role in TGF-β effects on calcium homeostasis. In previous studies, indomethacin at 10 μM blocked IL-1β-induced COX activation and bradykinin-induced IL-6 secretion from ASM (34, 41). As shown in Fig. 4, indomethacin pretreatment (2 μM) had little inhibitory effect on TGF-β-augmented, bradykinin-induced calcium mobilization, suggesting that the effect of TGF-β on agonist-induced [Ca2+]i increase was independent of prostaglandin production. Similarly, indomethacin at 10 μM had little effect on bradykinin-induced [Ca2+]i increases after TGF-β treatment (data not shown).
TGF-β increases B2 mRNA and protein expression in a time-dependent manner.
Because the effect of TGF-β on bradykinin-mediated calcium mobilization was not inhibited by COX inhibitors, we next examined the effect of TGF-β in regulating bradykinin receptor gene expression. As shown in Fig. 5A, TGF-β induced a time-dependent increase in B2 mRNA levels in human ASM. Surprisingly, B1 receptor mRNA levels did not change significantly. At 18 h of treatment of human ASM with 2.5 ng/ml of TGF-β, the relative amount of B2 receptor mRNA was approximately double compared with B2 mRNA from diluent-treated cells when normalized to β-actin. In Fig. 5B, TGF-β treatment for 18 h induced an increase in B2 receptor mRNA levels in murine tracheal rings compared with basal levels of B2 receptor mRNA.
To determine whether B2 receptor protein expression is also increased by TGF-β, human ASM monolayers were treated with TGF-β for varying times and doses. As shown in Fig. 6, A and B, TGF-β markedly increased B2 receptor expression in a dose- and time-dependent manner with maximal B2 receptor levels detected by immunoblot at a concentration of 2 ng/ml. Optimal B2 expression was seen after 17 h of treatment with TGF-β. Collectively, these data suggest that TGF-β enhances expression of bradykinin receptors, promoting ASM calcium mobilization and force generation to bradykinin.
TGF-β increases B2 receptor expression on human ASM cells.
Immunocytochemistry, using an antibody that recognizes a portion of the intracellular COOH terminus, was used to further investigate the protein expression of the B2 receptor on human ASM. As shown in Fig. 7, immunocytochemical analysis revealed that there is a basal expression of B2 receptor that is significantly increased at the cell surface after 24 h of stimulation with TGF-β (2.5 ng/ml). To confirm these results, we localized B2 receptor expression in human ASM cells to the cell surface after TGF-β treatment using double immunofluorescent staining. With the use of the monoclonal antibody Hermes-3, which binds the extracellular epitome of CD44, a receptor that is highly expressed on the cell surface (11), it was possible to demonstrate an increase in colocalization of the B2 receptor at the cell surface after TGF-β (2.5 ng/ml) treatment compared with vehicle control treatment (Fig. 7, C and D). An enhancement of colocalization of B2 receptor and CD44, a cell surface marker highly expressed on ASM (42), was demonstrated by merged imaging. No immunostaining was observed with isotype-matched controls, demonstrating the specificity of the analysis. These results suggest that TGF-β treatment results an in increase in cell surface expression of B2 receptor on human ASM cells.
In this study, we investigated whether TGF-β, a cytokine found to be elevated in asthmatic airways (58), modulates ASM responsiveness to bradykinin, a GPCR agonist known to induce bronchoconstriction in subjects with asthma but not in healthy volunteers. We and others have shown that “proasthmatic” mediators, such as TNF-α, prime ASM cells to become nonspecifically hyperresponsive to different contractile agonists including bradykinin (7, 8, 19, 29, 31, 56). Using isolated murine tracheal rings as an ex vivo model of ASM responsiveness, we now demonstrate that murine tracheal rings treated with TGF-β become hyperresponsive to bradykinin. Furthermore, in cultured human and murine ASM cells, TGF-β increases calcium responses to bradykinin as well as the expression of the B2 receptor at both the RNA and protein level in both a time- and concentration-dependent manner. Using RT-PCR analysis, we also investigated whether B2 receptor expression was altered in our ex vivo model, i.e., TGF-β-treated murine tracheal rings, and found that levels of B2 receptor were significantly increased compared with controls, further supporting the hypothesis that the increased ASM responsiveness to bradykinin induced by TGF-β may involve a modulation of B2 receptor expression. Of note, although both human and murine ASM cells demonstrated an increase in [Ca2+]i to bradykinin, a higher dose concentration of bradykinin (10 nM) was required in murine ASM cells. This may be due to species-dependent differences in bradykinin receptor expression and affinity (32). Differences between murine and human airway responsiveness to bradykinin have been described by others. Bradykinin is a potent bronchoconstrictor of human airways in vivo (27), whereas the effects of bradykinin are negligible in isolated murine airways (62). These results indicate that TGF-β enhances ASM responsiveness to bradykinin, possibly via increased calcium signals in part, at least, due to transcriptional induction of B2 receptor in ASM cells.
The molecular mechanisms promoting AHR, a key feature of asthma, remain unknown. Dysfunction of ASM, the main effector tissue regulating bronchomotor tone, has been considered a plausible mechanism leading to AHR (reviewed in Ref. 6). Data obtained using isolated airway preparations as an ex vivo model support the concept that proasthmatic conditions can contribute to the development of AHR by enhancing ASM responsiveness to contractile agonists (reviewed in Refs. 8, 31, and 43). With the use of cultured murine tracheal rings, previous reports have demonstrated that proasthmatic cytokines, TNF-α, and IL-13 induce nonspecific ASM hyperresponsiveness to different agonists including bradykinin, serotonin, or carbachol (19, 56, 62). Interestingly, we also reported that these cytokines directly modulate ASM cell function via enhancement of GPCR-associated calcium transients, the activating signal that initiates ASM shortening and contraction (7, 38).
We now show that murine tracheal rings treated with TGF-β have significantly increased contractile responses to bradykinin compared with diluent-treated tracheal rings that show modest contractile responses to bradykinin. Whereas TGF-β is an important fibrinogenic and immunomodulatory molecule promoting airway remodeling in asthma (reviewed in Ref. 23), our study is the first to suggest a potential direct role of TGF-β in promoting AHR to bradykinin, a feature described in subjects with asthma (49). The unique finding that TGF-β has little effect on carbachol-evoked calcium responses also contrasts with the nonspecific effects of other cytokines, including TNF-α and IL-13, which enhanced GPCR-associated calcium responses to bradykinin, carbachol, and thrombin (3, 5, 9, 36, 47). The selective effect of TGF-β is confirmed with a suboptimal dose of carbachol as well as at a higher dose. TGF-β appears to have little effect on carbachol-evoked calcium response at 1- or 10-μM doses of carbachol. Although beyond the scope of this study, the selective ability of TGF-β to enhance bradykinin-induced, but not carbachol-induced, calcium response may be regulated by differences in muscarinic receptor expression on ASM.
Previous studies showing that ASM express a family of TGF-β receptors, TβRI, TβRII, and TβRIII (18, 40), suggest that TGF-β alone or in combination with other cytokines can stimulate the expression of a variety of proasthmatic mediators such as connective tissue growth factor (60), vascular endothelial growth factor (59), IL-8 (26), and other structural and extracellular matrix proteins such as collagens (14, 20, 26). Many of these molecules could indirectly mediate TGF-β effects on agonist-induced force generation. IL-1β mediates hyperreactivity of bronchial ASM cells to bradykinin via a rapid increase in the density of B2 receptor. Indomethacin, which blocked IL-1-induced PGE2 production, completely prevented B2 receptor mRNA upregulation, confirming involvement of the prostanoid pathway in the transcriptional induction of B2 receptor (52). Indeed, TGF-β can increase COX-2 expression and PGE2 release in human ASM (25) and pulmonary artery smooth muscle cells (15). Interestingly, we found that in ASM cells, TGF-β increased bradykinin-induced [Ca2+]i via indomethacin-independent pathways. It is, therefore, possible that the enhancing effect of TGF-β on bradykinin-induced contractile responses may be due to a direct effect on ASM cells.
Importantly, using three different complementary techniques [RT-PCR, immunofluorescence with double staining using CD44 as a marker of cell membrane (11, 42), and Western blot analyses], we found that TGF-β enhanced the expression of the B2 receptor at the RNA and protein levels in a time- and concentration-dependent manner (Figs. 5–7). As a means of linking the ex vivo model and the in vitro results, we showed that TGF-β can induce B2 receptor mRNA expression in both murine tracheal rings and human ASM cells. Evidence suggests that other proasthmatic cytokines, including IL-13 or TNF-α, can also increase B2 receptor density in ASM cells or murine tracheal rings (33, 53, 62); however, TGF-β appears to be more agonist selective. Here, we found that in our ex vivo model, i.e., TGF-β-treated murine tracheal rings, levels of B2 receptor were significantly increased compared with controls, further supporting the hypothesis that the increased ASM responsiveness to bradykinin induced by TGF-β may involve a modulation of B2 receptor expression. Other studies showed that TGF-β-induced reduction in β2-adrenoreceptor expression can also serve as a “procontractile” mechanism by decreasing β2-adrenoreceptor function and reducing protein kinase A-mediated ASM relaxation (13, 45). Although our study does not directly address the molecular mechanism behind B2 receptor gene expression, our data support other reports that B2 receptor mRNA levels are enhanced via multiple mechanisms, including increases in the rate of gene transcription and increases in the mRNA stability (52) as well as increases in the rate of B2 receptor synthesis as a consequence of the increase in the steady-state levels of its mRNA. The ability of TGF-β to upregulate the protein as well as the mRNA levels of B2 receptors in ASM cells is shared by other cytokines such as platelet-derived growth factor in arterial smooth muscle (22) and TNF-α in human lung fibroblasts (30). Haddad et al. (30) were able to demonstrate that TNF-α-induced upregulation of B2 receptor in immortalized human embryonic lung fibroblasts is mediated through a posttranscriptional mechanism. Together, our results suggest that transcriptional and posttranscriptional regulation of B2 receptor expression in ASM may represent a general molecular mechanism underlying the cytokine effects of TGF-β on bradykinin-induced calcium signaling and allergic AHR. Further experiments are needed to more fully address the stimulus and cell specificity of the regulation of bradykinin receptor gene transcription.
It should also be noted that TGF-β likely plays a complex regulatory role with some of its effects on ASM being concentration dependent. In Fig. 6, we demonstrate a significant increase in B2 receptor expression on ASM at lower doses of TGF-β, e.g., 1–5 ng/ml, whereas higher concentrations of TGF-β appear to have only a modest effect. This concentration-dependent physiological regulation by TGF-β has been reported in several other cell systems as well as ASM. TGF-β has been shown to release a peak concentration of PGE2 at 1 ng/ml and less release at 10 ng/ml (25). Similar to the biphasic pattern we see with B2 receptor expression in human ASM, cysteinyl leukotriene 1 receptor (CysLT1R) is also induced in a concentration-dependent manner with increasing doses of TGF-β, and at doses >10 ng/ml, CysLT1R significantly decreases (24). TGF-β has been demonstrated to have opposing effects on proliferation at low vs. high concentrations, with higher concentrations inhibiting proliferation in lung fibroblasts (63).
In summary, we show that murine tracheal smooth muscle and human ASM cells treated with TGF-β demonstrate increased ASM responsiveness specifically to bradykinin, an effect that possibly involves increased expression and function of the B2 receptor. Thus the direct effect of TGF-β on ASM responsiveness to bradykinin likely plays a significant role in promoting bronchoconstriction in response to bradykinin seen in asthma and potentially other airway diseases characterized by inflammation.
This work was supported by National Heart, Lung, and Blood Institute Grants T32-HL-007586 (J. H. Kim), HL-067663 (E. Puré), HL-55301 (R. A. Panettieri), and HL-64063 (Y. Amrani). Y. Amrani is a Parker B. Francis Fellow in Pulmonary Research. This project is funded, in part, by a grant from the Pennsylvania Department of Health (E. Puré).
↵* J. H. Kim and D. Jain contributed equally to this work.
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