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Published ahead of print on May 12, 2008, doi:10.1165/rcmb.2007-0410OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 530-535, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0410OC

Chitinase Activates Protease-Activated Receptor-2 in Human Airway Epithelial Cells

Jeong Hee Hong1, Jung Yeon Hong2, Boryung Park1, Syng-Ill Lee1, Jeong Taeg Seo1, Kyu-Earn Kim2, Myung Hyun Sohn2 and Dong Min Shin1

1 Department of Oral Biology, Brain Korea 21 Project, Center for Natural Defense System, Yonsei University College of Dentistry; and 2 Department of Pediatrics, Brain Korea 21 Project, Institute of Allergy, Yonsei University College of Medicine, Seoul, Korea

Correspondence and requests for reprints should be addressed to Dong Min Shin, DDS, PhD, Department of Oral Biology, Brain Korea 21 Project, Center for Natural Defense System, Yonsei University College of Dentistry, Seoul, 120-752, Korea. E-mail: dmshin{at}yuhs.ac or Myung Hyun Sohn, mhsohn{at}yuhs.ac


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mammalian chitinase released by airway epithelia is thought to be an important mediator of disease manifestation in an experimental model of asthma. However, the intracellular signaling mechanisms engaged by exogenous chitinase in human airway epithelial cells are unknown. Here, we investigated the direct effects of exogenous chitinase from Streptomyces griseus on Ca2+ signaling in human airway epithelial cells. Spectrofluorometry was used to measure intracellular Ca2+ concentration ([Ca2+]i) in fura-2-AM–loaded cells. S. griseus chitinase induced dose-dependent [Ca2+]i increases in normal human bronchial epithelial cells and promoted [Ca2+]i oscillations in H292 cells. Chitinase-induced [Ca2+]i oscillations were independent of extracellular Ca2+, suggesting that the observed [Ca2+]i increases were due to Ca2+ release from intracellular stores. Accordingly, after depleting endoplasmic reticulum (ER) Ca2+ with the ER Ca2+ ATPase inhibitor, thapsigargin, chitinase-mediated [Ca2+]i increases were abolished. Treatment with the phospholipase C (PLC) inhibitor U73122 or the 1, 4, 5-trisinositolphosphate (IP3) receptor inhibitor 2-APB attenuated chitinase-induced [Ca2+]i increases. Desensitization of protease-activated receptor-2 (PAR-2) by repetitive agonist stimulation or siRNA-mediated PAR-2 knock-down revealed that chitinase-mediated [Ca2+]i increases were exclusively mediated by PAR-2 activation. Finally, chitinase was found to cleave a model peptide representing the cleavage site of PAR-2 and enhanced IL-8 production. These results indicate that exogenous chitinase is a potent proteolytic activator of PAR-2 that can directly induce PLC/IP3-dependent Ca2+ signaling in human airway epithelial cells.

Key Words: chitinase • protease-activated receptor-2 • calcium signaling • human airway epithelial cells



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Exogenous chitinase is a potent proteolytic activator of protease-activated receptor-2 that can directly induce phospholipase C/inositol 1,4,5-triphosphate–dependent Ca2+ signaling in human airway epithelial cells.

 
Asthma is a chronic inflammatory disorder of the airways characterized by variable airflow obstruction and bronchial hyperresponsiveness (BHR) (1). Although the pathogenesis and etiology of asthma are complex and incompletely understood, it is clear that the respiratory epithelium is important in disease manifestation, as it is the first resident cell encountered by inhaled antigens, and it is capable of releasing mediators and cytokines associated with asthma (2). A variety of antigens, including those from viruses (3), bacteria (4), and fungi (5), have been shown to stimulate cytokine and mediator release from airway epithelium. Several reports have also shown that endogenous or exogenous proteases, including allergens from house dust mites (6, 7), cockroaches (8), or fungi (9), are significant modulators of epithelial function.

Protease-activated receptors (PAR-1, -2, -3, and -4) are a family of proteolytically activated, G protein–coupled receptors (GPCRs) containing seven transmembrane domains (10–13). Activation of PARs involves receptor recognition by a protease and cleavage of an extracellular receptor domain, leading to stimulation of Gq protein-mediated signal transduction. Activated Gq protein stimulates phospholipase C (PLC), which hydrolyzes inositol phosphates to generate inositol 1,4,5-trisphosphate (IP3) and stimulate IP3-receptor (IP3R)-mediated release of Ca2+ from the endoplasmic reticulum (ER) into the cytosol. Ca2+ mobilization plays a pivotal role in many cellular processes, including differentiation, proliferation, and apoptosis (14). PARs are expressed in diverse tissues and are widely distributed throughout the apical surface of airway epithelia (15, 16). Their activity is modulated by endogenous and exogenous airway proteases, which can either activate or disable the receptors. In addition, PARs are positioned at the crossroads between innate immunity and coagulation (17, 18), making those novel pharmacologic targets for the treatment of airway pathologies, such as asthma and chronic obstructive pulmonary disease (15). Thrombin, an activator of PAR-1, is mitogenic and also stimulates the release of the proinflammatory and fibrogenic cytokine, granulocyte-macrophage colony-stimulating factor, in airway smooth muscle cells (17, 19). Moreover, observations from animals overexpressing human PAR-2 and the finding that PAR-2 is up-regulated in the airway epithelium of individuals with asthma suggest that this receptor contributes to allergic inflammation (16, 20–22). Recent reports suggest that, in airway epithelial cells, PAR-2 may be activated by serine/cysteine proteases derived from mite allergens, such as Der p3 and Der p9 (7), or by German cockroach extracts (23). Nevertheless, in many tissues where PAR-2 is found, including airway epithelial cells, the physiologic or pathologic activators of PAR-2 have not been identified.

Chitinases are hydrolysis enzymes that can fragment the chitin polymer. They are present in a wide range of organisms, including bacteria, insects, viruses, plants, and animals. Although chitinase production is important for the life cycle of chitin-containing fungi and parasites, it also plays a key role in regulating innate immune responses to chitin-containing fungi and parasites (24). Zhu and coworkers (25) found that the expression of acidic mammalian chitinase by airway epithelia and pulmonary macrophages is dramatically up-regulated both in a mouse model of asthma and in allergic asthma in humans. In addition, exogenous chitinase allergens, such as Der f 18, Der p 15, and Der p 18 from house dust mites, also have potentially important allergenic activities in humans (26, 27), and chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium were responsible for host penetration through the breakdown of extracellular matrix (28). A recent study has shown that Streptomyces griseus are found in moisture-enriched indoor air and dust, and these bacteria release chitinase extracellularly (29). These findings are consistent with a role for S. griseus–derived chitinase in allergic and nonallergic airway inflammation and asthma as well as in the etiology of other health symptoms associated with indoor air. However, the signaling mechanisms that mediate the effects of exogenous chitinase in human airway epithelial cells remain unknown.

In the present work, we investigated the direct effects of exogenous bacterial chitinase on Ca2+ signaling in human airway epithelial cells and sought to identify the receptors that mediate the effects of chitinase on intracellular Ca2+ signaling and chemokine activation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
S. griseus chitinase (Chi, EC. 3.2.1.14), trypsin, thrombin, 2-aminoethoxydiphenyl borate (2-APB), U73122, and U73343 were purchased from Sigma (St. Louis, MO). The S. griseus chitinase was purified by polymyxin B gel to make endotoxin-free chitinase following the manufacturer's protocol (Sigma). The endotoxin level of polymyxin B–purified chitinase was 0.019 ± 0.00041 E.U./ml. RPMI 1640 media, penicillin, streptomycin, thapsigargin, pluronic acid, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA). Fura-2 acetoxymethyl ester (fura-2-AM) was purchased from Teflabs (Austin, TX). PAR-1 and -2 agonist peptides (AP) were purchased from Tocris (Ellisville, MO). All other chemicals used were of reagent grade. The fluorescence-quenched peptide [5-FAM]-Ser-Lys-Gly-Arg-Ser-Leu-Ile-Gly-Lys(Dabcyl)-Asp (PAR-2) was synthesized by Peptron Co. (Daejeon, South Korea) and purified using a high-performance liquid chromatography (Shimadzu Prominence System; Shimadzu Scientific Instruments, Kyoto, Japan). Peptide structure was confirmed by liquid chromatography/mass spectroscopy (HP 1100 series HPLC System; Agilent Technologies, Santa Clara, CA).

Cell Culture
Normal human bronchial epithelial (NHBE) cells were grown according to the supplier's specifications (Clonetics Corp., Walkersville, MD). The human epithelial carcinoma cell line, H292, was obtained from American Type Culture Collection (ATCC, Manassas, VA) and was cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and streptomycin (Invitrogen). H292 cells were grown in sterile T-75 tissue culture flasks and maintained at 37°C in a humidified 5% CO2 incubator. For experimentation, both cell types were grown in culture dishes on glass coverslips and used at 80% confluence for intracellular Ca2+ concentration ([Ca2+]i) measurements. The medium was replaced every 2 to 3 days.

[Ca2+]i Measurements
[Ca2+]i in NHBE and H292 cells were determined using fura-2-AM dissolved in an extracellular physiologic salt solution (PSS) with the following composition: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose. The solution was titrated to pH 7.4 with NaOH and adjusted 310 mOsm with NaCl. Fura-2–loaded cells were mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan) for imaging. The cells were alternately illuminated at 340 nm and 380 nm, and the fluorescence emitted at 510 nm was captured with a CCD camera and analyzed using the MetaFluor system (Universal Imaging Co., Downington, PA). The 340/380 ratio was taken as a measure of [Ca2+]i, and fluorescence images were obtained at 3-second intervals.

Western Blotting
Cells were lysed with RIPA buffer (10 mM HCl pH 7.8, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) containing 10 µM Na3VO4, 10 µM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml PMSF. Lysates were centrifuged at 12,000 x g for 10 minutes, and supernatants were collected for Western blotting. Protein concentrations were determined using the BCA assay kit (Pierce, Rockford, IL). Equal amounts of protein (50 µg) were separated on 10% SDS-PAGE gels (Bio-Rad, Hercules, CA) and transferred onto nitrocellulose membranes. Membranes were then incubated in 6% skim milk in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl, and 0.1% Tween 20) and sequentially incubated with a PAR-2 polyclonal antibody (Santa Cruz, Santa Cruz, CA) and a horseradish peroxidase–conjugated secondary antibody. Blotted proteins were visualized using enhanced chemiluminescence (IntRON, Sung Nam, South Korea).

RT-PCR
Total RNA was extracted using the TRIZOL reagent (Invitrogen) and reverse transcribed using oligo (dT) primers. PCR reactions were performed as follows: 9 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, 40 seconds at the gene-specific annealing temperature, and 5 minutes at 72°C. RT-PCR reactions were performed according to standard procedures using the following primer sets for PAR-2: forward, 5'-CCCTTTGTATGTCGTGAAGCAGAC-3'; reverse, 5'-TTCCTGGAGTGTTTCTTTGAGGTG-3'. The PCR products were separated on 2% agarose gels and visualized with ethidium bromide. The results were analyzed using the MetaMorph system (Universal Imaging Co.).

Transfection of PAR-2 Small Interfering RNA
Small interfering RNA (siRNA) for PAR-2 (PAR-2 siRNA) was constructed using the siSTRIKE and hMGFP vector systems according to the manufacturer's instructions (Promega, Madison, WI). The hMGFP vector system enabled identification of siRNA-expressing cells based on their GFP fluorescence. The human PAR-2 siRNA target-specific sequences were: forward, 5'-ACCGTCTTCCTTCCAATTGTCTTTCAAGAGAAGACAATTGGAAGGAAGACTTTTTC-3'; reverse, 5'- TGCAGAAAAAGTCTTCC TTCCAATTGTCTTCTCTTGAAAGACAATTGGAAGGAAGA-3'. The human scrambled PAR-2 control sequences were: forward, 5'-ACCGTCTCTTCCAATT CTGTTCTTCAAGAGAGAACAGAATTGGAAGAGACTTTTTC-3'; reverse, 5'-TGCAGAAAAAGTCTCTTCCAATTCTGTTCTCTCTTGAAGAACAGAATTGGAAGAGA-3'. Cells were transfected with PAR-2 siRNA (1.8 µg/ml) and PAR-2 scramble control (2.0 µg/ml) using the Lipofectamine 2000 reagent (Invitrogen), cultured for 4 hours in serum-free media, and maintained in serum-containing media for 48 hours. Transfected cells were selected by culturing with G-418 (Invitrogen). PAR-2 expression levels in siRNA transfectants were measured by RT-PCR and Western blotting and compared with those in scramble control transfectants using the siSTRIKE system in H292 cells. To apply siRNA system in primary cultured NHBE cells, we re-constructed the hMGFP vector system. The siRNA transfected cells were measured by [Ca2+]I using the hMGFP vector system in both cell types.

Enzyme Assays and Measurements of Kinetic Constants
All experiments using chitinase, trypsin, and thrombin were performed in PSS at 37°C. The substrate solution (1 ml) was equilibrated at 37°C for 30 minutes before adding the enzyme solution. Enzyme activity was monitored by continuously measuring emitted fluorescence ({lambda}ex = 490 nm; {lambda}em = 520 nm) using a PMT chamber (Photon Technology International Inc., Birmingham, NJ). Fluorescence of peptide product after exhaustive cleavage by trypsin was found to be proportional to concentration. Accordingly, an increase in fluorescence corresponds to an increase in concentration of the cleaved peptide, allowing the kinetic parameters Km and kcat to be determined from an analysis of initial velocities of product formation obtained at different substrate concentrations.

Measurement of Human IL-8 Production
H292 cells were cultured on 6-well plates were stimulated with chitinase at 37°C by induction times. Human IL-8 levels in the supernatants of medium were determined by a specific ELISA against human IL-8 according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Chitinase Induces Ca2+ Signals in a Dose-Dependent Manner in NHBE and H292 Cells
We hypothesized that chitinase from S. griseus activates Ca2+ signaling in human airway epithelial cells. To investigate Ca2+ signaling evoked by chitinase in NHBE and H292 cells, we measured [Ca2+]i at endotoxin-free chitinase concentrations ranging from 1 to 300 µg/ml. In both cell types, chitinase treatment induced increases in [Ca2+]i within 2 minutes (Figures 1A and 1C). The number of responding cells (i.e., those with an evoked Ca2+ spike/oscillation signal) was dependent on the concentration of chitinase (Figures 1B and 1D). In NHBE cells, the percentage of responding cells was 0% at 10 µg/ml (n = 3), 85.8 ± 8.0% at 30 µg/ml (n = 4), and 100% at concentrations above 100 µg/ml (n = 8). H292 cells were more sensitive to chitinase, with 9.3 ± 11.4% responding at 0.1 µg/ml (n = 3), 22 ± 18.5% at 0.3 µg/ml (n = 3), 31 ± 9.8% at 1 µg/ml (n = 3), 82.2 ± 7.2% at 3 µg/ml (n = 3), 99 ± 0.4% at 10 µg/ml (n > 12), and 100% at 100 µg/ml (n = 3) (Figure 1C). Chitinase induced rapid, transient [Ca2+]i increases at concentrations greater than 30 µg/ml in NHBE cells, and 100 µg/ml in H292 cells (Figures 1A and 1C). These results indicate that chitinase induces Ca2+ signaling in a dose-dependent manner in both cell types.


Figure 1
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  		Figure 1. Chitinase-induced Ca2+ responses in human airway epithelial cells. (A and C) [Ca2+]i increases induced by various concentrations of S. griseus chitinase (Chi) ranging from (A) 10 to 300 µg/ml in normal human bronchial epithelial (NHBE) cells and from (C) 1 to 100 µg/ml in H292 cells. (B and D) Dose-dependent changes in the percentage of responding NHBE and H292 cells. All data are expressed as mean ± SEM.

 
Chitinase Induces Ca2+ Release from Thapsigargin-Sensitive Stores via the PLC/IP3 Pathway
Increases in [Ca2+]i may reflect influx of extracellular Ca2+ or release of Ca2+ from internal stores. To determine the source of the [Ca2+]i increases induced by chitinase, we treated NHBE and H292 cells with 30 or 10 µg/ml chitinase, respectively, in a nominally Ca2+-free medium (n = 4; Figures 2A and 2E). In H292 cells, chitinase-induced [Ca2+]i oscillations persisted in the absence of extracellular Ca2+, suggesting that these oscillations are dependent on the release of Ca2+ from intracellular Ca2+ stores. The major internal Ca2+ store is the ER, which can be selectively depleted by thapsigargin (Tg), a specific inhibitor of the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) pump. Accordingly, NHBE and H292 cells were incubated with 1 µM Tg in a nominally Ca2+-free medium (to prevent Ca2+ influx), and the effects of chitinase on [Ca2+]i were determined (Figures 2B and 2F). Tg treatment elicited an initial increase in [Ca2+]i in the absence of chitinase, reflecting inhibition of the SERCA pump and leakage of Ca2+ from the ER into the cytosol. After depletion of ER Ca2+ with Tg (in the absence of extracellular Ca2+), treatment with chitinase (100 µg/ml) failed to evoke a Ca2+ signal (n = 4). In a complimentary experiment, we found that depletion of internal Ca2+ stores by stimulation with chitinase (in the absence of external Ca2+) prevented Tg-induced [Ca2+]i increases (n = 4). Collectively, the data presented in Figures 2B and 2F indicate that chitinase increases [Ca2+]i by triggering release of Ca2+ from Tg-sensitive ER stores.


Figure 2
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  		Figure 2. Characterization of chitinase-induced Ca2+ signaling. (A and E) Effect of extracellular Ca2+ on chitinase-induced [Ca2+]i increases. (B and F) Effect of 1 µM Tg on chitinase-induced [Ca2+]i increases in the absence of extracellular Ca2+. (C and G) Effect of 5 µM U73122 (dotted line) or 5 µM U73343 (solid line) on chitinase-induced [Ca2+]i increases. (D and H) Effect of 75 µM 2-APB on chitinase-induced [Ca2+]i increases.

 
To determine the role of PLC-dependent IP3 generation in chitinase-induced [Ca2+]i increases, we pretreated NHBE and H292 cells with U73122, a specific blocker of PLC, or its inactive analog, U73343 (Figures 2C and 2G, n = 4) for 5 minutes. U73122 prevented chitinase-induced [Ca2+]i increases, whereas U73343 had no effect. NHBE and H292 cells were also treated with the IP3R blocker, 2-APB (75 µM), to determine whether chitinase-induced [Ca2+]i increases are mediated by IP3Rs. As shown in Figures 2D and 2H (n = 3), treatment with 2-APB during chitinase stimulation inhibited [Ca2+]i increases, suggesting that chitinase increases [Ca2+]i by promoting Ca2+ release from the ER through the PLC/IP3 pathway.

Chitinase-Induced [Ca2+]i Increases Are Exclusively Related to PAR-2 Activation
PARs are highly distributed in human airway epithelia. We hypothesized that chitinase might affect PARs to increase [Ca2+]i. To clarify which type of PAR is activated by chitinase, we used previously described desensitization protocols developed by our laboratory (23). Receptors were desensitized with chitinase (Figure 3A, first and third panels, and 3B, first to third panels), the PAR-1, -3, and -4 agonist thrombin (Figure 3A, second panel) (10–13), or the PAR-2–specific agonist trypsin (Figures 3A and 3B, fourth panels) (12). NHBE and H292 cells were then treated with the Gq-coupled histamine H1 receptor agonist histamine (Figures 3A and 3B, first panel in both), trypsin (Figures 3A and 3B, third panel in both), or chitinase (Figures 3A and 3B, fourth panel in both) (n = 4). Desensitization with chitinase abolished the ability of trypsin to evoke increases in [Ca2+]i, whereas responses to the GPCR receptor agonist histamine were unaffected (Figures 3A and 3B, first and third panels in both). Reversing the order of chitinase and trypsin had the same effect. That is, desensitization with trypsin abolished the Ca2+ signals induced by chitinase (Figures 3A and 3B, fourth panel in both). In H292 cells, desensitization with chitinase also abolished Ca2+ signals induced by PAR-2–activating peptide (PAR-2AP) (Figure 3B, second panel). Although H292 cells do express PAR-1 mRNA, thrombin did not increase [Ca2+]i in these cells (data not shown). Finally, in NHBE cells, desensitization with thrombin, which does not target PAR-2, failed to block chitinase-induced Ca2+ signals (Figure 3A, second panel).


Figure 3
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  		Figure 3. Effects of PAR-2 densensitization/knock-down on chitinase-induced [Ca2+]i increases. (A [NHBE cells, first-fourth panels] and B [H292 cells, first-fourth panels]) Cells were desensitized with the indicated reagent. The arrows indicate the time at which the reagents were added. (C) In H292 cells, RT-PCR and Western blot analysis of PAR-2 in scramble control (SC)- and PAR-2 siRNA-transfected H292 cells. Chitinase-induced [Ca2+]i signals of PAR-2 siRNA-transfected (dotted line) and SC-transfected (solid line) cells. (D) In NHBE cells, chitinase-induced [Ca2+]i signals in PAR-2 siRNA-transfected (dotted line) and SC-transfected (solid line) cells. PAR-2 siRNA-expressing cells were identified based on GFP fluorescence.

 
To confirm the PAR-2 dependence of chitinase-mediated Ca2+ signaling, we used siRNA to disrupt PAR-2 expression. Transient transfection of PAR-2 siRNA suppressed the expression of PAR-2 mRNA and protein by 39.4% and 28.6%, respectively, compared with that of scramble control (Figure 3C, n = 3) in H292 cells. Measurement of [Ca2+]i in siRNA-expressing cells was identified based on GFP fluorescence using hMGFP-siRNA system. PAR-2 knock-down significantly decreased chitinase-induced Ca2+ responses, whereas Ca2+ signals persisted in scramble control-transfected cells (Figures 3C, second panel, and 3D; n = 5). These results indicate that chitinase-induced [Ca2+]i increases are exclusively mediated by PAR-2 in both cell types.

Determination of Kinetic Constants for Chitinase-Mediated PAR-2 Substrate Cleavage
To confirm that chitinase was able to catalyze the cleavage of and activate PAR-2, we synthesized a fluorescence-quenched peptide substrate, [5-FAM]-Ser-Lys-Gly-Arg-Ser-Leu-Ile-Gly-Lys(Dabcyl)-Asp (PAR-2), that was modified to optimize its accuracy as a predicator of receptor cleavage (30). The FAM group fluoresces only when the quenching group Lys(Dabcyl) is released upon proteolytic cleavage. Accordingly, increases in fluorescence are closely correlated with increases in the concentration of cleaved peptide. To test the specificity of this synthetic substrate as a marker of PAR-2 cleavage, we investigated the kinetics of peptide cleavage by serial dose of trypsin and thrombin (Figure 4A) and serial dose of chitinase (Figure 4B). The PAR-2 agonist trypsin bound the peptide with high affinity, as evidenced by its low Km value and cleaved the peptide efficiently, displaying a high kcat/Km value (Table 1). The ability of trypsin, but not that of thrombin, to cleave this synthetic peptide confirm the specificity of this peptide and validate its use in assays for protease activators of PAR-2. To determine whether chitinase is a novel, potentially physiologic, activator of PAR-2, we assessed the ability of chitinase to dose-dependently hydrolyze the synthetic PAR-2 peptide. Chitinase cleaved the synthetic peptide at concentrations as low as 1 µM and with kinetics very similar to those of trypsin (Table 1), indicating that PAR-2 is indeed a substrate of chitinase.


Figure 4
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  		Figure 4. Hydrolysis of synthetic peptide by trypsin or chitinase at different concentrations. (A and B) Dose-dependent increase in fluorescence intensity of synthetic peptide (10 µM) cleaved by (A) trypsin (10–1,000 nM) or thrombin (1 µM) or (B) Chi (1–100 µM).

 

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  		TABLE 1. KINETIC PARAMETERS OF CLEAVAGE OF A SYNTHETIC PEPTIDE OF PAR-2

 
Chitinase Induces IL-8 Production in H292 Cells
Activation of PAR-2 leads to the release of cytokines, including IL-6 and IL-8 in epithelial cells (2–4). Indoor allergens such as cockroach and dust mite induce the release of proinflammatory cytokines such as IL-8 in epithelial cells (6, 7, 32). To check the allergenic effect of chitinase, we measured the level of IL-8 in H292 cells. Chitinase significantly enhanced IL-8 production in a time-dependent manner in H292 cells (Figure 5, n = 4). As a result of 10 µg/ml chitinase treatment, the level of IL-8 production was increased by 175.438 ± 29.187, 414.716 ± 5.537, and 565.217 ± 60.507 pg/ml after 6, 12, and 24 hours, respectively. These results indicate that chitinase-induced IL-8 production are mediated by PAR-2.


Figure 5
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  		Figure 5. Chitinase induces IL-8 production in H292 cells The H292 cells were stimulated with 10 µg/ml chitinase dependent on the times. The concentrations of IL-8 of the supernatants from cultures were measured by ELISA.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Here, we have identified novel actions of exogenous chitinase on Ca2+ signaling in human airway epithelial cells. We found that chitinase triggers [Ca2+]i oscillations and biphasic increases in [Ca2+]i (Figures 1A and 1C). Both cell types increased [Ca2+]i in a dose-dependent manner in response to chitinase stimulation (Figures 1B and 1D). In chitinase-induced Ca2+ signals, intracellular Ca2+ stores were mainly responsible for the generation of Ca2+ signals in both NHBE and H292 cells (Figures 2A and 2E). The Tg-sensitive ER store serves as the source of Ca2+ for chitinase-evoked signals. Chitinase-evoked [Ca2+]i increases were mediated by the PLC/IP3 pathway. Therefore it is possible that chitinase activates PLC/IP3 pathway linked plasma membrane surface receptors.

The most notable finding of this study is that chitinase, a potent protease, activates PAR-2 in human airway epithelial cells. PAR-2 is of particular interest in asthma because it can be activated by mast cell tryptase (31) and aeroallergens, such as house dust mites (2, 7). It has also been implicated in inflammation. Our results show that, after desensitization with trypsin, stimulation with chitinase did not evoke [Ca2+]i increases and that knock-down of PAR-2 also inhibited chitinase-induced [Ca2+]i increases. Collectively, these data indicate that chitinase is a selective activator of PAR-2, with little or no activity at PAR-1, -3, or -4. The activation of PAR-2 by chitinase was not attributable to an up-regulation in PAR-2, as chitinase treatment did not affect PAR-2 expression (data not shown). However, we found that chitinase, at concentrations as low as 1 µM, cleaved the synthetic PAR-2 peptide, indicating that exogenous chitinase is a potent PAR-2 activator. PAR-2 activation by chitinase is on par with trypsin, as seen by their very similar peptide cleavage kinetics.

Recently, we reported that German cockroach extract evokes [Ca2+]i oscillations in airway epithelial cells via the activation of PAR-2 (23), resulting in an intracellular signaling cascade consisting of Ras, mitogen-activated protein kinase, and extracellular-regulated kinase pathways (8, 32). We also showed that these extracts induced cytotoxic inflammatory mediators in eosinophils (33). A recent report also suggests that PAR-2 activation has proinflammatory effects and plays a pathogenic role in the development of BHR and airway inflammation in an experimental model of asthma (34). Accordingly, chitinase-induced Ca2+ signaling via PAR-2 may stimulate related pathways and contribute to pro-inflammatory immune responses. Der f 18 (from D. farinae), which has homology to chitinase, is reported to be a major allergen for humans and dogs that are sensitive to house dust mites (26), and Dermatophagoides pteronyssinus chitinases Der p 15 and Der p 18 were shown to be highly allergenic in human sera (27). Furthermore, serine proteases, including Der p 3 and Der p 9 from house dust mites, were reported to induce [Ca2+]i increases through the IP3/PLCβ pathway (7). Thus, the PAR-2–specific protease activity of exogenous chitinase acting on enhanced IL-8 production described here, may contribute to the pathogenesis of direct allergen exposure.

In summary, chitinase is a potent and selective proteolytic activator of PAR-2 and exogenous bacterial chitinase induces Ca2+ mobilization and enhances IL-8 production through the activation of PAR-2. Our findings may have physiologic and pathologic implications, as they suggest that Ca2+ signaling in response to direct chitinase exposure may mediate intracellular inflammatory reactions including IL-8 in human airway epithelial cells.


   Footnotes
 
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R01-2006-000-10478-0 and R11-2007-040-02003-0).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0410OC on May 12, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 13, 2007

Accepted in final form March 20, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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