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Published ahead of print on January 30, 2004, doi:10.1165/rcmb.2003-0223OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 886-892, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0223OC

Protease-Activated Receptor-2–Mediated Inhibition for Ca2+ Response to Lipopolysaccharide in Guinea Pig Tracheal Epithelial Cells

Akihiro Oshiro, Hitomi Otani, Yasuhiro Yagi, Shirou Fukuhara and Chiyoko Inagaki

Department of Pharmacology and Department of Internal Medicine 1, Kansai Medical University, Osaka, Japan

Address correspondence to: Chiyoko Inagaki, M.D., Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan. E-mail: inagaki{at}takii.kmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The protease-activated receptor-2 (PAR-2) has been implicated in airway inflammation. Here, we examined the interaction between PAR-2 and lipopolysaccharide (LPS), a major proinflammatory factor, using cultured guinea pig tracheal epithelial cells. In fura2-loaded cells, LPS (1 µg/ml) transiently increased intracellular Ca2+ concentrations ([Ca2+]i), this effect being abolished by a Ca2+ channel blocker, verapamil, and Ca2+ removal. Prestimulation of PAR-2 with trypsin (0.1–1 U/ml) or an agonist peptide (SLIGRL-NH2, 1 µM) for 60 min inhibited the LPS-induced [Ca2+]i increase. Such an inhibitory effect of trypsin was abolished by inhibitors of protein kinase C (PKC), chelerythrine and staurosporine. A PKC activator, phorbol 12,13-dibutylate, also reduced the LPS response. Trypsin also inhibited a transient increase in [Ca2+]i caused by a Ca2+ channel opener, Bay K 8644. When the trypsin-pretreated cells were incubated in normal buffer for 10–60 min before LPS exposure, the effect of trypsin on the Ca2+ response to LPS diminished in a time-dependent manner. Such a recovery was slowed by incubation with a protein phosphatase inhibitor, okadaic acid. Further, trypsin induced sustained activations of PKC{alpha} and -{epsilon}. Thus, PAR-2 stimulation reduced the epithelial cell response to LPS, probably through the inactivation of Ca2+ channels via PKC-mediated phosphorylation.

Abbreviations: 1,4-Dihydro-2, 6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester, (±)-Bay K 8644 • fetal calf serum, FCS • intracellular Ca2+ concentration, [Ca2+]i • Joklik's modified Eagle's medium, JMEM • lipopolysaccharide, LPS • protease-activated receptor, PAR • phorbol 12, 13-dibutylate, PDBu • cAMP-dependent protein kinase, PKA • protein kinase C, PKC • cGMP-dependent protein kinase, PKG • soybean trypsin inhibitor, SBTI


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The respiratory epithelium plays an active role in the defense and inflammatory reactions of airway exposed to endogenous and exogenous stimuli such as allergens, mast cell products, and air particles (1, 2). These stimulants activate epithelial cells to release various proinflammatory mediators like cytokines, oxygen radicals, and extracellular proteases, thereby altering ciliary motility, mucus secretion, and inflammatory cell infiltration (1–5).

Recently, the ability of some serine proteases to regulate inflammation through protease-activated receptors (PARs) has received attention (6, 7). The PARs are G-protein–coupled receptors that are activated by proteolytic cleavage of their extracellular amino-terminus domain to expose a new N-terminus that acts as tethered ligands. To date, four PARs (PAR-1 to PAR-4) have been cloned, and airway epithelial cells express at least two types, a thrombin-activated receptor (PAR-1) and a thrombin-independent type, PAR-2 (6–8). The PAR-2 activated by trypsin and mast cell tryptase triggers phosphoinositide breakdown-derived signal transduction (6, 7). Indeed, recent reports including our own have suggested that inositol trisphosphate-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) (9) and activation of protein kinase C (PKC) (10) participate in the signaling cascade. There is now substantial evidence that this receptor is involved in the regulation of epithelial cell functions. For example, activation of PAR-2 by trypsin, mast cell tryptase or a PAR-2 synthetic agonist peptide has been demonstrated to induce a release of epithelium-derived relaxing factors (8) and production of interleukin-6, interleukin-8 (11), and matrix metaloproteinase-9 (12). Furthermore, increased PAR-2 expression has been reported in bronchial epithelium from patients with asthma (13). However, the pathophysiologic role of PAR-2 in pulmonary disorders such as asthma is not yet fully understood.

Lipopolysaccharide (LPS), an endotoxin found in the cell wall of gram-negative bacteria, is ubiquitously present in air dust and cigarette smoke. In the airway, exposure to LPS stimulates many types of cells to secrete proinflammatory cytokines such as interleukin(s) and tumor necrosis factor-{alpha} (14, 15), which have been recognized to induce immediate and strong cellular inflammatory responses and pulmonary injury. In vivo and in vitro studies have demonstrated that administration of LPS induces apoptotic cell death in broncheal epithelium (16) and infiltration of neutrophils (17). Further, in several types of cells, LPS is reported to increase [Ca2+]i mainly by promoting the Ca2+ influx through voltage-dependent Ca2+ channel (18). However, little is known about the functional interaction between PAR-2 and LPS in respiratory systems. In general, Ca2+ channel activity is known to be regulated by protein kinases including PKC. Therefore, we hypothesized that PAR-2 stimulation may interfere with LPS response through PKC-dependent signaling pathways. In the present study, we examined the effects of PAR-2 stimulation on tracheal epithelial response to LPS using [Ca2+]i increase as a parameter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Tracheal Epithelial Cells
All animals were handled in accordance with "Rules of the Animal Experimentation Committee, Kansai Medical University."

Hartley guinea pigs (400–450 g) were anesthetized with an intraperitoneal injection of pentobarbital (80–100 mg/kg). Primary cell culture was achieved using a slight modification of procedures described previously (19). Briefly, freshly excised trachea was washed two times with phosphate-buffered solution, and its laryngeal end was cannulated with polyethylene tube (2.3 mm diameter). The lumen of the excised trachea was first washed with cold Joklik's modified Eagle's medium (JMEM) and then flushed with JMEM containing 0.1% pronase (type 14) using a syringe fitted with an 18-gauge needle. After the tightening of the distal end of the trachea, pronase-containing JMEM was added until the trachea was fully expanded, and then the proximal end was bent and closed. The entire tissue was immersed in ice-cold JMEM and incubated at 4°C for ~ 16 h. Following protease digestion, the epithelial cells were flushed out with cold JMEM containing 10% fetal calf serum (FCS) and filtered through a 60-µm nylon mesh before being centrifuged at 150 x g for 10 min. The resulting pellets were dispersed with cold JMEM containing 10% FCS and centrifuged again. The second cell pellets were resuspended in Ham's F12 medium supplemented with 10% FCS, recombinant human epidermal growth factor (25 ng/ml), 1% HL-1, retinal acetate (0.1 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (0.75 µg/ml), and gentamicin (5 µg/ml), and were then plated on collagen (cellmatrix type 1-C, 0.03%)-coated circular glass coverslips placed inside the plastic dishes at a density of 40,000/dish. The viability of epithelial cells after isolation was checked by trypan-blue exclusion and was found to be > 98%. Cultures were incubated in humidified 95% air–5% CO2 at 37°C. Confluence was usually achieved in ~ 7 d. Cells were immunohistochemically characterized using antibodies against keratin and vimentin as respective markers for epithelial cells and fibroblasts according to procedures described previously (20). The majority of cells cultured on the collagen-coated coverslips were epithelial cells with a squamous shape. Beating cilia were occasionally observed. The contamination of fibroblasts was very low (< 2%). Between Days 7 and 10, the cells were subjected to experiments.

Measurement of [Ca2+]i
[Ca2+]i was measured using a calcium-sensitive fluorescent dye, fura2-AM. Epithelial cells grown on circular glass coverslips were washed with Krebs-HEPES buffer that contained (in mM): NaCl, 120; KCl, 5.4; CaCl2, 1.5; MgSO4, 1.2; NaH2PO4, 1.0; HEPES, 20; and glucose, 10 (pH 7.2), and were loaded with 5 µM fura2-AM for 40 min at 37°C. Coverslips were mounted on a Rose chamber (Ikemoto, Tokyo, Japan), which was then filled with 2 ml of fresh Krebs-HEPES solution and transferred to the stage of an inverted microscope (IX70; Olympus, Tokyo, Japan). The temperature was kept at 37°C by a ring heater surrounding the dish. Fura2-loaded epithelial cells were excited with light at a wavelength of 340 or 380 nm produced by a xenon lamp and fluorescence at 510 nm emission was detected with a cooled CCD camera (model C6790–81; Hamamatsu Photonics, Shizuoka, Japan). Ca2+ fluorescence images obtained at 10-s intervals were stored on the hard disk of a computer, and then the fluorescence ratio of 340–380 nm was measured to calculate the [Ca2+]i. All analyses were performed using a computer-based fluorescence image analysis system (Argus Hisca Imaging System; Hamamatsu Photonics). Autofluorescence from the cells was negligible throughout the experiment. Samples were used for one protocol only.

In situ calibration of the fura2 fluorescence signal was performed by the use of the fura2 fluorescence-pCa calibration curve. The calibration medium contained (mmol/liter): KCl, 122.5; NaCl, 5.4; MgSO4, 1.1; glucose, 10; HEPES, 20; EGTA, 2; and CaCl2, 1.5 to achieve the desired pCa (5–8), and the pH was adjusted to 7.4 at 37°C with NaOH. This calibration was done at the end of each experiment and the fluorescence-pCa calibration curve was almost linear in the range of pCa 5–8.

Western Blot Analysis of PKC Translocation
Epithelial cells were stimulated with trypsin or PDBu for the indicated durations followed by harvest. Preparation of membranes and cytosol was performed at 4°C.

The cells were homogenized in a buffer (pH 7.4) containing 20 mM Tris, 1 mM EDTA, 10 mM EGTA, 20 µg/ml leupeptin, 200 U/ml aprotinin, and 0.4 mM phenylmethylsulfonyl fluoride and then centrifuged at 100 x g for 5 min to remove cell debris. The upper fraction was centrifuged again at 100,000 x g for 30 min. The resulting pellet was resuspended in the homogenization buffer, the supernatant being concentrated using centrifugal filtration (ultrafree MC; Milipore, Bedford, MA), and they were saved as membrane and cytosol fractions, respectively. Protein concentrations of the samples were measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The membrane and cytosol fractions were mixed with a sodium dodecyl sulfate–Laemmli sample buffer and heated at 100°C for 2 min. Proteins (25 µg) were separated by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to an immobilon P membrane (Milipore) using electroblotting apparatus. The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.5) overnight at 4°C. After washing with TBS containing 0.1% Tween 20, the membrane was incubated with specific antibodies for 7 different PKC isoforms for 2 h at room temperature. The membrane was then washed and incubated for 1 h with peroxidase-conjugated goat anti-mouse or -rabbit immunoglobulin. Reactive bands were visualized using enhanced chemiluminescence method. Equal loading was verified by reprobing the blots with anti-ß-actin (Sigma, St. Louis, MO). The signal intensities were determined with a densitometer (DMU-33C; Advantec Digital Densitorol, Tokyo, Japan).

Materials
Trypsin, phorbol ester, verapamil and okadaic acid were purchased from Sigma Chemical Co. Thrombin was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Synthetic peptides corresponding to the tethered ligand sequences of rat/mouse PAR-2 (SLIGRL-NH2) were synthesized by the Peptide Institute, Inc. (Osaka, Japan). Fura2-AM was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). (±)-Bay K 8644, KT 5720, KT 5823, and staurosporine were from Calbiochem (La Jolla, CA). Ham F-12 medium, penicillin, streptomycin, and amphotericin B were purchased from ICN Biomedicals, Inc. (Aurora, OH). JMEM and FCS were from Gibco BRL Life Technologies (Grand Island, NY). HL-1 was from BioWhittaker A Cambrex Co. (Walkersville, MD). Recombinant human epidermal growth factor (rhEGF) was from R&D Systems, Inc. (Minneapolis, MN). Cellmatrix type 1-C was purchased from Nitta Gelatin Biochemical Institute, Inc. (Osaka, Japan). Polyclonal rabbit antibodies against human keratin and monoclonal mouse antibody against human vimentin were purchased from Dako Corporation (Carpinteria, CA). Anti-{alpha}, -{delta}, and -{epsilon} PKC monoclonal antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA) and anti-ß, -{gamma}, -{zeta}, and -{eta} PKC polyclonal antibodies were purchased from Oxford Biomedical Research (Oxford, MI). Rodamine red–conjugated goat anti-mouse IgG was from Molecular Probes (Eugene, OR). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, peroxidase-conjugated goat anti-mouse and -rabbit IgGs were from Cappel, ICN Pharmaceutical (Aurora, OH). PermaFluor aqueous mounting medium was purchased from Shandon/Lipshaw Co. (Pittsburgh, PA). Enhanced chemiluminescence was from NEN Life Science Products (Boston, MA). All other chemicals including LPS (Escherichia coli O111: B4) and soybean trypsin inhibitor (SBTI) were from Wako Pure Chemical (Osaka, Japan) and were of the highest purity available. (±)-Bay K 8644, KT 5720, KT 5823, staurosporine, and okadaic acid were prepared as 1,000 x stocks in dimethyl sulfoxide.

Statistical Analysis
Student's t test was used for statistical analysis. Differences between mean values with P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PAR-2 Agonists on LPS-Induced Changes in [Ca2+]i
Figure 1 shows typical changes in [Ca2+]i in ciliated epithelial cells during exposure to 1 µg/ml LPS. The basal [Ca2+]i was estimated to be 45.5 ± 2.1 nM (mean ± SEM, n = 34) in normal Krebs-HEPES solution. After addition of LPS in the presence of 1% FCS as a source of LPS-binding protein, the [Ca2+]i transiently increased to a peak of 208.9 ± 14.9 nM (n = 34) within 1 min. It then gradually decreased to near the basal level 5 min after stimulation (thick line). In contrast, when the cells were pretreated with trypsin (1 U/ml) for 60 min followed by a washout with normal Krebs-HEPES buffer, the LPS-induced increase in [Ca2+]i was obviously attenuated (thin line). Although trypsin itself produced an immediate and transient increase in [Ca2+]i, the concentration subsequently returned to near the basal level, being stable thereafter. This Ca2+ response was reduced by pretreatment with a PKC inhibitor, chelerythrine (1 µM, 10 min) to ~ 40% of the control value, suggesting the contribution of PKC to trypsin action. As shown in our previous report (9), this trypsin-induced transient increase in [Ca2+]i did not induce a depletion of stored [Ca2+]i, because histamine (5 µM) applied 5 min later without an intervening wash was able to produce an increase in [Ca2+]i, with almost the same magnitude as that observed without trypsin pretreatment. SBTI (100 nM) abolished the inhibitory effect of trypsin on the LPS response (dotted line), indicating its requirement for the enzymatic activity. SBTI did not affect basal [Ca2+]i.



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  		Figure 1. Representative traces of [Ca2+]i changes during exposure to LPS with or without trypsin pretreatment in single guinea pig tracheal epithelial cells. Cells loaded with fura2-AM were pretreated for 60 min with (thin or dotted line) or without (thick line) test reagents; 1 U/ml trypsin (thin line) or 1 U/ml trypsin plus soybean trypsin inhibitor (SBTI, 100 nM, dotted line) and then washed with normal Krebs-HEPES buffer. After 5 min, they were exposed to 1 µg/ml LPS. [Ca2+]i was measured fluorometrically as described in MATERIALS AND METHODS.

 
Figure 2 shows the dose- and time-dependent effects of trypsin on the LPS-induced Ca2+ response. Compared with LPS alone (control), pretreatment with trypsin (0.1 and 1 U/ml) for 60 min reduced the LPS (1 µg/ml)-induced increase in [Ca2+]i in a dose-dependent manner, and this trypsin action was abolished by co-incubation with SBTI (Figure 2A). Further, such a downregulation by trypsin was dependent on the duration of treatment, complete inhibition being attained in 60 min (Figure 2B). In subsequent experiments, therefore, the duration of pretreatment was set at 60 min.



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  		Figure 2. Dose- and time-dependent effects of trypsin on [Ca2+]i changes in tracheal epithelial cells stimulated with LPS. (A) Epithelial cells were pretreated with 0.1, 1.0 U/ml trypsin (Try), or 1.0 U/ml trypsin plus 100 nM SBTI for 60 min, and then exposed to 1 µg/ml LPS. (B) Cells were pretreated with 1 U/ml trypsin for the indicated periods before exposure to 1 µg/ml LPS. Changes in [Ca2+]i were determined as net increase ({Delta}; peak value minus basal value) after the addition of LPS. Each bar represents the mean ± SEM for 5–10 preparations. *P < 0.05, **P < 0.01 compared with LPS alone.

 
Because our recent work has shown that trypsin induces a Ca2+ response in tracheal epithelial cells through the activation of PAR-2 (9), experiments were performed using a synthetic peptide corresponding to the tethered ligand of rat/mouse PAR-2 (SLIGRL-NH2). Figure 3 shows typical changes in [Ca2+]i in ciliated epithelial cells during pretreatment with agonist peptide and subsequent stimulation with LPS. After pretreatment with peptide or vehicle for 60 min, the cells were washed with normal Krebs-HEPES buffer and stimulated with 1 µg/ml LPS 5 min later. SLIGRL-NH2 (1 µM) also produced an immediate and transient increase in [Ca2+]i followed by a gradual return to the baseline level. Pretreatment with this peptide for 60 min significantly inhibited the LPS-induced increase in [Ca2+]i. The Figure 3 inset shows the pooled data obtained from 8–10 cultures derived from four different tracheal tissues. The net increase in [Ca2+]i caused by LPS was obviously reduced from 163.3 ± 13.6 nM to 15.8 ± 1.6 nM (P < 0.01) by pretreatment with SLIGRL-NH2.



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  		Figure 3. Effects of PAR-2 activating peptide on LPS-induced changes in [Ca2+]i in tracheal epithelial cells. Cells loaded with fura2-AM were pretreated for 60 min with (thin line) or without (thick line) SLIGRL-NH2 (1 µM) and then exposed to 1 µg/ml LPS. (Inset) Summaries of the net increase ({Delta}) in [Ca2+]i induced by 1 µg/ml LPS with (filled column) or without (open column) SLIGRL-NH2 pretreatment. Each bar represents the mean ± SEM for 5–10 preparations. **P < 0.01 compared with LPS alone.

 
Effects of PKC Inhibitors on the Inhibitory Actions of PAR-2 Agonists
Because trypsin has been demonstrated to induce the hydrolysis of phosphoinositide through PAR-2 stimulation in a variety of cell types including tracheal epithelial cells (7, 9, 10), the activation of PKC may be responsible for the present trypsin action. To test this possibility, the effects of PKC inhibitors on PAR-2–mediated inhibitory action were examined (Figure 4). Epithelial cells were pretreated with PKC inhibitor, either 10 nM staurosporine or 1 µM chelerythrine, for 10 min, then treated with 1 U/ml trypsin for an additional 60 min, and finally washed with Krebs-HEPES solution before being exposed to 1 µg/ml LPS. Both PKC inhibitors abolished the inhibitory effects of trypsin on the LPS-induced increase in [Ca2+]i. However, other kinase inhibitors, KT 5,720 (5 µM, for cAMP-dependent protein kinase; PKA) and KT 5,823 (5 µM, for cGMP-dependent protein kinase; PKG) did not have any significant effect on the LPS response. In addition, pretreatment with a PKC activator, phorbol ester (phorbol 12, 13-dibutylate; PDBu, 100 nM) for 60 min also produced significant inhibitory effects on the LPS-induced increase in [Ca2+]i (Figure 4, dashed column). These findings suggest that PKC stimulation may play a role in the PAR-2–mediated inhibitory process against the LPS response.



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  		Figure 4. Effects of various kinase inhibitors on the inhibitory effects of trypsin on LPS-induced [Ca2+]i changes in tracheal epithelial cells. Tracheal epithelial cells were pretreated for 10 min with or without (control) various kinase inhibitors (10 nM staurosporine [stauro] and 1 µM chelerythrine [chele] for PKC, 5 µM KT 5720 for PKA, 5 µM KT 5823 for PKG) before the trypsin (1 U/ml) stimulation. In the groups depicted by hatched bar, cells were treated with PDBu (100 nM). After 60 min, cells were washed and exposed to 1 µg/ml LPS, 5 min later. Basal [Ca2+]i did not change with these kinase inhibitors or PDBu. Changes in [Ca2+]i were determined as net increase ({Delta}). Each bar represents the mean ± SEM for 5–10 preparations. **P < 0.01 compared with control.

 
Involvement of Voltage-Dependent Ca2+ Channels in LPS-Induced Transient [Ca2+]i Increase
To elucidate the role of PKC in the mechanism of PAR-2–mediated inhibition for the response to LPS, we first estimated the source of Ca2+ contributing to the [Ca2+]i response induced by LPS using a voltage-dependent Ca2+ channel blocker, verapamil and Ca2+-free medium. As shown in Figure 5, the LPS-induced transient increase in [Ca2+]i was significantly attenuated in the presence of verapamil (10 µM). A similar inhibition of the LPS response was observed in the cells incubated in Ca2+-free Krebs-HEPES buffer containing 5 mM EGTA (n = 5). In addition, LPS-induced increase in [Ca2+]i was significantly inhibited by 5 min pretreatment with chelerythrine (1 µM) ({Delta}increase in [Ca2+ ]i by LPS with or without chelerythrine pretreatment; **52 ± 11 nM or 152 ± 12 nM, respectively, **P < 0.01 versus without chelerythrine, n = 6). These results imply that the influx of Ca2+ through PKC-dependent opening of verapamil-sensitive (probably voltage-dependent) Ca2+ channels, rather than intracellular Ca2+ mobilization, is responsible for the LPS-induced increase in [Ca2+]i and further raised a possibility that trypsin inhibits the response to LPS by reducing Ca2+ influx. To test this possibility, we examined the effect of trypsin on the Ca2+ increase induced by the direct opening of this channel.



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  		Figure 5. Effects of Ca2+ removal and verapamil on LPS-induced Ca2+ response in tracheal epithelial cells. Fura2-loaded epithelial cells were pretreated for 10 min with 10 µM verapamil or incubated in Ca2+-free Krebs-HEPES solution, and then stimulated with 1 µg/ml LPS. The removal of extracellular Ca2+, but not verapamil, reduced the basal [Ca2+]i to ~ 20 nM. Changes in [Ca2+]i were determined as net increase ({Delta}) after the addition of LPS. Each bar represents the mean ± SEM for 5–10 preparations. **P < 0.01 compared with LPS alone.

 
PAR-2–Mediated Inhibition of Bay K 8644 Response
Figure 6A shows typical changes in [Ca2+]i in ciliated epithelial cells exposed to Bay K 8644, an active Ca2+ channel agonist. A dihydropyridine agonist, Bay K 8644 is known to induce an increase in L-type Ca2+ current density via a direct effect on channel gating (21), and is therefore used as an excellent tool for examining the functions of this channel. After pretreatment with trypsin or vehicle for 60 min, the cells were washed with normal Krebs-HEPES buffer and 5 min later exposed to 10 µM Bay K 8644. In the control (vehicle) experiment, Bay K 8644 transiently increased [Ca2+]i to a peak of 162.8 ± 17.4 nM (n = 5) within 1 min. This increase was clearly reduced by pretreatment with trypsin (1 U/ml) for 60 min. As shown in Figure 6B, the Bay K 8644-induced net increase in [Ca2+]i was significantly reduced in the trypsin-pretreated group. A similar inhibition was observed on the activation of PKC by 100 nM PDBu, indicating that the PKC-mediated reduction of Ca2+ channel activity may be involved in the inhibitory effect of trypsin on the LPS response.



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  		Figure 6. Effect of trypsin and PDBu on Bay K 8644-induced changes in [Ca2+]i in tracheal epithelial cells. Cells loaded with fura 2-AM were pretreated with trypsin (1 U/ml) or PDBu (100 nM) 5 min later. After 60 min, cells were washed and then exposed to 10 µM Bay K 8644. (A) Representative traces of changes in [Ca2+]i. (B) Summaries of net increase ({Delta}) in [Ca2+]i induced by Bay K 8644 in the presence or absence (Bay K alone) of pretreatment. Each bar represents the mean ± SEM for 5–10 preparations. **P < 0.01 compared with Bay K 8644 alone.

 
Effects of a Phosphatase Inhibitor on the Trypsin/PDBu-Induced Inhibition and the Time-Dependent Loss of Trypsin Action
To substantiate whether or not PKC-mediated phosphorylation of a target protein (probably Ca2+ channel) plays a key role in the inhibitory effect of trypsin, we examined the effects of okadaic acid on the action of trypsin or PDBu. Okadaic acid inhibits the serine/threonine phosphatases PP2A (Ki: ~ 0.1 nM), PP1 (Ki: ~ 10 nM), and also PP2B (calcineurin; Ki: ~ 5 µM) with much less potency (22), and is therefore thought to potentiate or prolong the phosphorylation state. In epithelial cells pretreated with trypsin (1 U/ml) or PDBu (100 nM) for 30 min in the presence or absence of okadaic acid (0.1 µM), okadaic acid potentiated the inhibitory effect of trypsin or PDBu on the LPS-induced increase in [Ca2+]i, compared with each control (Figure 7A).



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  		Figure 7. Effect of okadaic acid on the inhibitory action of trypsin or PDBu in LPS-induced [Ca2+]i changes in tracheal epithelial cells. (A) Epithelial cells were pretreated with trypsin (1 U/ml) or PDBu (100 nM) for 30 min in the presence (hatched column) or absence (0.1% DMSO; filled column) of okadaic acid (OKA, 100 nM), and then exposed to 1 µg/ml LPS. (B) Cells pretreated with 1 U/ml trypsin for 60 min were washed and then treated with vehicle (0.1% DMSO; filled column) or okadaic acid (OKA, 100 nM; hatched column) for 10, 30, or 60 min before exposure to 1 µM LPS. Open column shows the changes in [Ca2+]i induced by 1 µM LPS alone. Changes in [Ca2+]i were determined as net increase ({Delta}). Each bar represents the mean ± SEM for 5–10 preparations. *P < 0.05 between the okadaic acid–treated and untreated groups.

 
Next, after being pretreated with trypsin for 60 min, the epithelial cells were washed and incubated for 10, 30, or 60 min in normal Krebs-HEPES buffer with or without 100 nM okadaic acid and exposed to 1 µg/ml LPS (Figure 7B). In the cells incubated without okadaic acid, inhibition of the LPS-induced increase in [Ca2+]i diminished in a time-dependent manner. Such a recovery was much slower in the okadaic acid–treated group, compared with the untreated group. These results suggest that this time-dependent loss of trypsin action was due to okadaic acid–inhibitable phosphatase activity.

Determination of Activations of PKC Isoforms in Response to Trypsin
In the last experiment, to verify that PKC activation is involved in PAR-2–mediated inhibitory effect on LPS response, we performed Western blot analyses using seven PKC isoform (Ca2+-dependent types; {alpha}, ß, {gamma}; Ca2+-independent types; {delta}, {epsilon}, {eta}, {zeta})–specific antibodies. When activated by extracellular stimuli, these kinases are translocated from the cytosol to plasma membrane to exert kinase activities. Among these isoforms, exposure to trypsin (1 U/ml, 5–30 min) increased the amounts of immunoreactive PKC{alpha} (molecular mass, 78 kD) and -{epsilon} (97 kD) in the membrane (particulate:P) fractions associated with concomitant decrease in the cytosol (C) fractions, compared with control (Ct: no treatment) (Figure 8A). Stimulation with PDBu (100 nM), as a positive control, for 5 min also induced typical translocations of PKC{alpha} and -{epsilon}. This reagent unaffected the immunoreactivities on other PKC isoforms (data not shown). The Figure 8B shows the pooled data obtained from four to five cultures derived from four different tracheal tissues. As shown by the densitometric analyses, redistributions of these isoforms were maintained at least for 30 min in the presence of trypsin. These findings suggest that PAR-2 stimulation resulted in the sustained activations of PKC{alpha} and -{epsilon}.



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  		Figure 8. Western blot analyses of PKC{alpha} and -{epsilon} activations in epithelial cells. (A) Representative time courses of translocation of PKC{alpha} and -{epsilon} induced by trypsin. Epithelial cells were incubated with trypsin (1 U/ml) or PDBu (100 nM) for the times indicated. Cytosol (C) and membrane (particulate; P) fractions were prepared, and immnoblotted with anti-PKC{alpha} and -{epsilon} antibodies as described in MATERIALS AND METHODS. Ct: control (no treatment). (B) Effects of trypsin on the subsellular distributions of PKC{alpha} (a, c) and -{epsilon} (b, d). Results are based on densitometric scanning of immunoblot data of cytosol (a, b) and membrane (c, d) fractions. Data are expressed as percentages of control value (no treatment). Each bar represents the mean ± SEM for 4–5 preparations. **P < 0.01, *P < 0.05 compared with the control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, pre-stimulation of PAR-2 with trypsin and SLIGRL-NH2 inhibited the LPS-induced increase in [Ca2+]i. This is the first report to demonstrate that LPS induces a rapid increase in [Ca2+]i in tracheal epithelial cells. Based on the fact that the PAR-2–mediated inhibitory effect on this LPS response was attenuated by specific PKC inhibitors (Figure 4) and was mimicked by a PKC activator, a PKC-regulated mechanism is thought to underlie the inhibition process. In addition, we have shown for the first time that in airway epithelial cells, PKC{alpha} and -{epsilon} isoforms were activated under PAR-2 stimulation (Figure 8). This evidence supports our hypothesis.

LPS has been demonstrated to produce various inflammatory responses associated with an increase in [Ca2+]i. For example, in alveolar and hepatic macrophages, LPS reportedly induces superoxide or tumor necrosis factor-{alpha} production through a Ca2+-requiring process (18, 23). Regarding the mechanisms of this increase in [Ca2+]i, there are two possibilities, i.e., release from an internal Ca2+ store and stimulation of voltage-dependent Ca2+ channel. In this study, the PKC-dependent latter mechanism appears to be involved because the LPS-induced increase in [Ca2+]i was almost completely or significantly supressed by verapamil, Ca2+ removal (Figure 5), or chelerythrine, respectively. The presence of voltage-dependent Ca2+ channel in lower airway epithelial cells has not been reported, but it has been suggested in nasal epithelial cells, i.e., substance P–induced increase in ciliary beat frequency depends on Ca2+ influx via this type of Ca2+ channel (24).

In general, the voltage-gated Ca2+ channels are known to be regulated via protein phosphorylations by various protein kinases, i.e., PKC, PKA, and PKG (25). Among these kinases, the cAMP/PKA and cGMP/PKG pathways have been shown to positively and negatively regulate this channel activity, respectively (25). In contrast, activation of PKC has resulted in variable effects on this channel activity. In cardiac and smooth muscle preparations, neurohormones (for example, endothelin) linked to PKC have been reported to induce various effects on the Ca2+ current, i.e., increase, decrease, or no change (25, 26). However, there are reports demonstrating that direct stimulation of PKC by phorbol ester inhibits the activity of L-type Ca2+ channels (26, 27). Thus, the PKC-mediated regulation may vary depending on the cell type, the isoform(s) of PKC activated by a particular signaling pathway, and chemicals used to modify PKC activity, as described in a previous study (26). In the present study, both trypsin and PDBu inhibited the increases in [Ca2+]i induced by Bay K 8644 as well as by LPS (Figure 6). Therefore, PAR-2 in the airway epithelia appears to negatively regulate the LPS response by reducing the Ca2+ channel activity through the activation of PKC. Such interference by PKC has also been observed in the LPS-induced Ca2+ response in hepatic macrophages (18). The phosphorylation of verapamil-sensitive (i.e., voltage-dependent) Ca2+ channels might be responsible for this desensitization to LPS, as we propose in this study.

To clarify the functional relevance of channel phosphorylation to the trypsin–LPS interaction, we examined the effects of okadaic acid on PAR-2/PKC-mediated action (Figure 7). Okadaic acid at 100 nM, a concentration sufficient to inhibit PP1/2A, potentiated the inhibitory effects of trypsin or PDBu on Ca2+ response to LPS. Further, this reagent significantly slowed the time-dependent loss of the inhibitory effect of trypsin, implying the prolongation of the phosphorylated state of Ca2+ channels. Therefore, it may be reasonable to conclude that PAR-2 activation resulted in the inhibition of the LPS response through the inactivation of Ca2+ channels via PKC-mediated phosphorylation. Although we did not provide direct evidence of PAR-2–mediated phosphorylation of Ca2+ channels, our idea is reinforced by previous reports showing (i) the phosphorylation of {alpha}1 and ß2 subunits as well as reduction of the channel activity under PKC activation (27, 28) and (ii) the association of protein phosphatase 2A with L-type Ca2+ channels (22). Further, the phosphorylation of threonines at amino acid 27 and 31 on the {alpha}1 subunits has been demonstrated to be responsible for PKC-dependent inhibition of Ca2+ current (27).

Besides airways, interactions between PAR-2 and LPS have also been examined in some PAR-2–mediated responses in vivo. For example, in animals treated with LPS for 24 h, saliva secretion by the PAR-2 agonist was reduced probably through the functional desensitization of PAR-2 (29). However, in LPS-treated rats, trypsin-induced hypotension was augmented in conjunction with increased levels of PAR-2 mRNA (30). Thus, the mode of interaction between PAR-2 and LPS differs depending on the tissue type or experimental design (application order or treatment period).

From the present data, we propose that the short (30–60 min)-term effect of PAR-2 on the airway epithelia is protective for allergic reactions to proinflammatory stimuli such as LPS, although many studies suggest that long (> 24 h)-term stimulation of this receptor conversely aggravates allergic reactions by increasing the production of various cytokines in the epithelium (6, 12). Although the exact reason for the requirement of 60 min to exert maximal effect of trypsin (Figure 2) is unclear, one possible explanation is the time-dependent balance between protein kinase activity and phosphatase activity. A recent report (22) describes that protein phosphatases localizing at or near the Ca2+ channel play roles in the regulations of channel function. Accumulation of phosphorylated state of Ca2+ channel or other signaling protein is thought to be required to exert inhibitory action, and this idea is supported by the evidence that the effects of trypsin or PDBu was potentiated in the presence of okadaic acid (Figure 7A). Furthermore, PAR-2–mediated inhibitory action on LPS response was transient (< 60 min) (Figure 7B). Possible physiologic effects of this inhibition may include the modification of some Ca2+-dependent kinase activities, e.g., PKC-mediated regulation of inflammatory cytokine production and apoptotic signal (31, 32). Thus, PAR-2–mediated inhibition may attenuate LPS-induced inflammatory responses at least partly by reducing the Ca2+-dependent activities of signaling molecules.

As for the protective role of PAR-2 in the airway, Cocks and coworkers have reported that PAR-2 activation causes the relaxation of broncheal smooth muscle, and explained that this response was due to PGE2 production by epithelium (8). In addition to this information, our results point to a new cytoprotective mechanism mediated by PAR-2. Further study is required to identify the type of LPS-mediated pathophysiologic response that can be downregulated by PAR-2. Recruitment of neutrophils into the targeting tissues, being a characteristic features of the inflammation, is a likely candidate (17).

In conclusion, in airway epithelial cells, PAR-2 stimulation reduces the response to LPS by inhibiting the Ca2+ influx probably through the phosphorylation of Ca2+ channels via activation of PKC{alpha} and -{epsilon}. Epithelial PAR-2 stimulation may be a new strategy to protect the airway from injury induced by LPS derived from enviromental pollutants such as organic dust and cigarette smoke.


   Acknowledgments
 
This study was supported by grants from Kansai Medical University and Japanese Private School Promotion Foundation.

Received in original form June 15, 2003

Received in final form December 24, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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