help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dulon, S.
Right arrow Articles by Pidard, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dulon, S.
Right arrow Articles by Pidard, D.
American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 339-346, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4908

Proteinase-Activated Receptor-2 and Human Lung Epithelial Cells

Disarming by Neutrophil Serine Proteinases

Sophie Dulon, Céline Candé, Nigel W. Bunnett, Morley D. Hollenberg, Michel Chignard and Dominique Pidard

Unité de Défense Innée et Inflammation, Unité Associée IP/Inserm 485, Institut Pasteur, Paris, France; Departments of Surgery and Physiology, University of California, San Francisco, California; and Department of Pharmacology and Therapeutics and Department of Medicine, The University of Calgary, Calgary, Alberta, Canada

Address correspondence to: Michel Chignard, Unité de Défense Innée et Inflammation, Unité Associée IP/Inserm 485, Institut Pasteur, 25, rue du Dr. Roux, 75015 Paris, France. E-mail: chignard{at}pasteur.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH2 leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH2. Preincubation of a PAR-2–transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH2–binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2–mediated lung inflammation.

Abbreviations: bovine serum albumin, BSA • PMSF-inactivated cathepsin G, CG-PMSF • Dulbecco's modified Eagle's medium, DMEM • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • human bronchial epithelial cells, HBEC • Hank's balanced salt solution, HBSS • PMSF-inactivated elastase, HLE-PMSF • interleukin, IL • Kirsten murine sarcoma virus-transformed rat kidney, KNRK • proteinase-activated receptor, PAR • polymerase chain reaction, PCR • prostaglandin, PG • soybean trypsin inhibitor, SBTI


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proteinase-activated receptors (PAR) are a subfamily of G-protein–coupled, seven-transmembrane domain receptors, which are cleaved within the aminoterminal exodomain by certain serine proteinases at a specific peptide bond (1, 2). After cleavage, the new aminoterminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor. Until their discoveries, mechanisms by which serine proteinases modulate cellular responses were largely unknown (1, 2). The first PAR to be characterized was the thrombin receptor or PAR-1 (3, 4). Since then, three other receptors have been identified, PAR-2 (5, 6), PAR-3 (7), and PAR-4 (8). PAR-1 and PAR-3 are both preferentially cleaved by thrombin, PAR-2 by trypsin and tryptase, whereas PAR-4 is activated by thrombin, cathepsin G, and trypsin. All PAR, except PAR-3, can be selectively activated by the short synthetic peptide that corresponds to their tethered ligand (1, 2).

Experimental studies have shown that trypsin cleaves the aminoterminal extracellular domain of human PAR-2 at SKGR36{downarrow}37SLIGKV (where {downarrow} designates the trypsin cleavage site), unmasking the aminoterminal intramolecular tethered ligand SLIGKV (1, 2). Accordingly, the synthetic peptide corresponding to this sequence activates PAR-2 without the need for receptor cleavage (5, 6). PAR-2 is highly expressed in the gastrointestinal tract (9), indicating a possible function in this system where it may be exposed to physiologic concentrations of pancreatic trypsin (10). PAR-2 is also expressed in tissues like prostate, kidney, liver, heart, vascular endothelium, skin, pancreas, and lung (9). Except for the pancreas, it is of note that under normal physiologic conditions, these different organs or tissues are not exposed to pancreatic trypsin; and although trypsinogens are widely expressed, including in the lung (11), a role for extrapancreatic trypsin in PAR-2 activation remains to be established, thus indicating that the physiologic activator(s) of PAR-2 remains mostly unknown (1, 2). One potential source of PAR-2 activation is the mast cell, located in many tissues, which may degranulate and release tryptase that has been reported to activate PAR-2 in isolated cell preparations (1, 11). However, a role for tryptase in activating PAR-2 in vivo is open to question (12).

PAR-2 activation by trypsin or the synthetic peptide SLIGKV leads to the production of inositol 1,4,5-trisphosphate and diacylglycerol, which messengers result in an increase of cytosolic calcium concentration, and an activation of protein kinase C (2). Activation of PAR-2 has been observed to cause a variety of responses, including the triggering of prostaglandin (PG) E2 and F2{alpha} secretion from enterocytes (10), production of interleukin (IL)-8 by keratinocytes (13), a mitogenic response in primary human cells in culture, including vascular endothelial cells, lung fibroblasts, and airway smooth muscle cells (2), as well as a secretion of inflammatory neuropeptides from rat primary spinal afferent neurons (14). In vivo, activation of PAR-2 leads to effects such as bronchoconstriction of airways in guinea pigs (15), neutrophil extravasation in rats (16), and hyperalgesia in rats and in mice (1). These results indicate that PAR-2 may have a dual proinflammatory/anti-inflammatory role, in keeping with its bronchoprotector role in mouse airways (17) and anti-inflammatory activity in a mouse model of colitis (18), but to be contrasted with its inflammatory effect in a rat paw edema bioassay (19) and with the delayed onset of inflammation observed in PAR-2–deficient mice (20).

Recent studies have shown that a functional PAR-2 is expressed in the respiratory epithelium (17, 21) as well as in many human respiratory cell lines (6, 22–25). Indeed, these cells can be activated through PAR-2 to release matrix metalloproteinase-9 (MMP-9) (23), granulocyte monocyte-colony stimulating factor (22, 24), and eotaxin (24), as well as PGE2, IL-6, and IL-8 (25), suggesting that PAR-2 may well participate in some aspects of lung inflammation. On the other hand, it is well recognized that during such an inflammatory response, polymorphonuclear neutrophils migrate into the lung tissue and release their serine proteinases, including elastase and cathepsin G, in the airway environment. A growing body of evidence suggests that neutrophil-derived proteinases play an important role in the onset of acute lung inflammatory diseases, as well as in the perpetuation of chronic respiratory inflammation such as in cystic fibrosis (26, 27). For example, elastase has been identified as a major inducer of IL-8 expression in bronchial epithelial cells (27). However, the ability of inflammatory proteinases to regulate PAR-2 on airway epithelial cells has not been examined.

In the present study, we first show that PAR-2 is expressed by human alveolar and bronchial epithelial cell lines and that it is activated by trypsin and by the human PAR-2–activating peptide SLIGKV-NH2. Moreover, we demonstrate that human leucocyte elastase and cathepsin G do not activate, but rather disarm PAR-2, thereby rendering the receptor refractory to trypsin activation, most likely by a proteolysis of the large extracellular aminoterminal sequence downstream of the trypsin cleavage/activation site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
A549 cells (ATCC CCL-185) are type II alveolar epithelial cells from a human adenocarcinoma, whereas 16HBE14o- cells, hereafter designated 16HBE, are human SV40-transformed bronchial epithelial cells and are a gift from Dr D. Gruenert (University of Vermont, Colchester, VT) (28). A permanent human PAR-2–expressing cell line (KNRK/PAR-2) was prepared essentially as previously described (6) using a pcDNA3 expression vector and the Kirsten murine sarcoma virus-transformed rat kidney cell line, KNRK. The expression and functionality of PAR-2 in these cells have been previously documented (6). Complementary DNA (cDNA) prepared from a total extract of primary human bronchial epithelial cells (HBEC) RNA was a gift from Dr P. S. Hiemstra (Leiden University, Leiden, the Netherlands). The PAR-2 antiserum B5 was raised in rabbits against a peptide fragment of rat PAR-2 (30GPNSKGR{downarrow}SLIGRLDTP45YGGC, where {downarrow} designates the trypsin cleavage site, with the sequence YGGC added for derivitization) and cross-reacts with both rat and human PAR-2 (10, 12). Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibodies were obtained from Rockland (Gilbertsville, PA).

Hanks' balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), F12/DMEM, and F12-K media (all from Gibco BRL, Paysley, UK) were used for cultures of KNRK/PAR-2, 16HBE, and A549 cells, respectively. L-glutamine, penicillin, streptomycin, fungizone, hygromycin B, and trypsin-EDTA solutions were obtained from Gibco BRL. Fetal calf serum (FCS) was from D. Dutscher S.A. (Brumath, France). FURA2/AM was from Calbiochem (La Jolla, CA). Bovine serum albumin (BSA) was from Euromedex (Strasbourg, France). Trypsin type XI from bovine pancreas (catalog number T1005, specific activity 6000–9000 BAEE U/mg protein), as well as soybean trypsin inhibitor type I-S (SBTI), came from Sigma Chemical Co (St. Louis, MO). Trypsin was prepared as a 10 µM stock solution in NaCl 0.9% and stored at -20°C. The human PAR-2–activating peptide SLIGKV-NH2, corresponding to the tethered ligand exposed after trypsin cleavage of the receptor and its reverse form VKGILS-NH2, were synthesized by Neosystem Laboratories (Strasbourg, France) and were resuspended in deionized water at 2 mM and stored at –20°C. Oligonucleotide primers for human PAR-2 and ß-actin were purchased from Genset (Les Ullis, France). Eglin C was a gift from Dr H. P. Schnebli (Novartis, Basel, Switzerland).

Purification of Neutrophil Proteinases
Human leukocyte elastase and cathepsin G were purified as previously described (29). Briefly, neutrophils were isolated free of erythrocytes from human blood, and neutrophil granules were obtained through disruption of cells by nitrogen cavitation followed by ultracentrifugation. After mechanical disruption of isolated granules and ultracentrifugation, serine proteinase-containing supernatants underwent a two-step separation procedure on aprotinin-Sepharose and CM-Trisacryl columns for affinity and ion exchange chromatography, respectively. Protein fractions corresponding to either elastase or cathepsin G at a concentration of 200 µM in 10 mM sodium acetate, 20 mM NaCl, 200 mM sucrose, pH 6.0, were stored at -80°C. The purity of the neutrophil proteinases was assessed by SDS-PAGE, and their enzymatic activity determined spectrophotometrically by monitoring the hydrolysis of N-succinyl-Ala-Ala-Ala-p-nitroanilide or of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, respectively (both substrates from Sigma). Each proteinase preparation was shown to be free from the other proteinase activity.

To block their catalytic site, the purified proteinases were incubated for 60 min at 25°C with 1.25 mM PMSF, and the mixture was subsequently dialyzed to remove the free inhibitor. PMSF-treated proteinases (HLE-PMSF and CG-PMSF) were shown to be proteolytically inactive by testing the lack of hydrolysis of their respective specific synthetic substrates.

Cell Cultures
A549 and 16HBE cells were grown in their respective medium supplemented with 10% (v/v) FCS, 0.3 mg/ml L-glutamine and 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml fungizone. KNRK/PAR-2 cells were grown in DMEM containing hygromycin B (0.15 mg/ml) supplemented with 10% FCS and penicillin, streptomycin and fungizone as above. For weakly passage, KNRK/PAR-2 cells were detached from culture flasks using the Versene medium of the following composition (mM) : NaCl 126, KCl 5, EDTA 1, Hepes 50, pH 7.4. All cell lines were cultured at 37°C in a 5% CO2, water-saturated atmosphere.

Reverse Transcriptase–Polymerase Chain Reaction Analysis
Total RNA was extracted from 16HBE, A549, or KNRK/PAR-2 cells according to the method described by Chomczynski and Sacchi (30). RNA (5 µg for each cell preparation) was reverse-transcribed with random hexamers. Polymerase chain reaction (PCR) was performed with 5 U of Q-Bio Taq polymerase (Quantum, Montreal, PQ, Canada) on 2 µl of template cDNA with the following human PAR-2 oligonucleotide primers: forward 5'-CTTGAA GATTGCCTATCACATACA-3' and reverse 5'-TCTTAATCA GAAAATAATGC ACCA-3', which were chosen to amplify a 560-bp fragment. PCR was achieved with 1.25 mM dNTP (Amersham Pharmacia Biotech, Piscataway, NJ) and Q-BioTaq buffer, as follows: denaturation at 95°C for 4 min, 40 cycles of replication at 95°C for 45 s, 56°C for 40 s, 72°C for 40 s and a final elongation step at 72°C for 5 min. PCR products were analyzed by electrophoresis at 100 V for 1 h on a 1.5% agarose gel containing ethidium bromide. The PCR amplification of PAR-2 cDNA was quantified using the ImageQuant software on a Storm device (Molecular Dynamics, Sunnyvale, CA) and compared with PCR amplification of human ß-actin cDNA obtained with the following oligonucleotide primers: forward 5'-AAGGAGAAGCTGTGCTACGTCGC-3' and reverse 5'-AGACAGCACTGT GTTGGCGTACA-3'.

Flow Cytometry Analysis
Adherent KNRK/PAR-2, 16HBE, and A549 cells were detached from culture flasks using the Versene medium. Cells were pelleted by centrifugation and resuspended in HBSS-BSA 0.25% (wt/vol) at a final concentration of 3 x 106 cells/ml. Before immunostaining, cells were pretreated or not with either 12.5 to 50 nM trypsin, 62.5–500 nM elastase, 125–500 nM cathepsin G, 500 nM HLE-PMSF or CG-PMSF, or the combination of 50 nM trypsin and 3 µM of its inhibitor SBTI, for 10 min at 37°C. After one wash, cell immunolabeling was achieved using the PAR-2 rabbit B5 antiserum (31) or rabbit pre-immune serum, both at 1:500 dilution, for a 1-h incubation at 4°C. After one wash, immunofluorescence staining was performed with FITC-conjugated goat anti-rabbit antibodies (20 µg/ml) incubated at 4°C for 1 h. Then, cells were washed, resuspended in a medium containing 0.1 µg/ml propidium iodide (Sigma) and fluorescence emission was recorded using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ). Propidium iodide-positive dead cells were excluded from analysis which was performed using the CellQuest software (v3.3; Becton Dickinson) and binding of the B5 antiserum immunoglobulins to the specific epitope on PAR-2 at the surface of viable cells was expressed as the geometric mean of fluorescence intensity, to which the background binding measured with the rabbit pre-immune serum was substracted.

Measurement of Intracellular Ca2+ Mobilization
Adherent KNRK/PAR-2, 16HBE, and A549 cells were detached from culture flasks using the Versene medium. After centrifugation, pulmonary epithelial cells were resuspended in their respective medium at a final concentration of 3 x 106 cells/ml, whereas KNRK/PAR-2 cells were resuspended at a final concentration of 1.5 x 106 cells/ml. Cells were incubated with the calcium probe FURA 2/AM (5 µM for epithelial cells and 2 µM for KNRK/PAR-2) for 30 min at room temperature. Cells were then washed twice in the assay buffer HBSS-BSA 0.25% (wt/vol) and resuspended at the same final concentration in the assay buffer containing 1 mM CaCl2 and 1 mM MgCl2. Fractions (1 ml) of cell suspensions were equilibrated at 37°C for 3 min for 16HBE and A549 or 5 min for KNRK/PAR-2 under stirring in a cuvette placed in a spectrofluorimeter. Cells were then exposed to various concentrations of either trypsin, the activating peptide SLIGKV-NH2 or the reverse peptide VKGILS-NH2, added in a maximal volume of 150 µl, and the changes in fluorescence were continously recorded for time periods of 1–4 min. In some sets of experiments, cells were preincubated for 3 min in the spectrofluorimeter cuvette with various concentrations of either purified elastase or cathepsin G, or HLE-PMSF or CG-PMSF before addition of trypsin or peptides. Fluorescence was measured at wavelengths of 340 nm excitation and 510 nm emission. Cytosolic calcium ion concentrations were calculated as described previously (32), and data are expressed as the maximal variation of intracellular concentration of calcium elicited by the agonist over the basal concentration ({Delta}[Ca2+]i). EC50 values for the activation of PAR-2 by trypsin or SLIGKV-NH2 in the various cell lines were estimated from the concentration response curves, when possible, using the GraphPad Prism 2.0 software (GraphPad Software, Inc., San Diego, CA) and fitting the data points for a one-phase exponential binding site mode.

Statistics
Results are expressed as mean ± SEM for the indicated number of independently performed experiments. Statistical significance between the different values was analyzed by the Student's t test for unpaired data with a threshold of P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PAR-2 mRNA and Protein Expression by Human Pulmonary Epithelial Cells
Total RNA extracted from KNRK/PAR-2, 16HBE, and A549 cells were reverse transcribed, and the obtained cDNA, as well as cDNA from subcultures of primary HBEC, were then processed for PCR amplification of specific messengers for PAR-2 or ß-actin taken as a constitutive control mRNA. As shown in Figure 1A, PAR-2 messengers were detected in the four cell types. The highest relative expression compared with ß-actin expression was observed for the transfected KNRK/PAR-2 cells and for the primary HBEC, whereas both 16HBE and A549 cells showed an ~ 2-fold lower expression (Figure 1A).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. Expression of PAR-2 by human pulmonary epithelial cells. (A) Total cell RNA were isolated from KNRK/PAR-2, 16HBE, and A549 cells, reverse-transcribed into cDNA, then amplified by the PCR technique in parallel with cDNA from subcultures of primary human bronchial epithelial cells (HBEC). Upper panel: electrophoresed and ethidium bromide-stained PAR-2 cDNA at 560 bp. Lower panel: specific PAR-2 and ß-actin PCR products obtained from each cell type were quantified by densitometry analysis of ethidium bromide-stained agarose gels, and ratios of PAR-2 to ß-actin calculated. (B) Cell monolayers were dispersed and incubated at 3 x 106 cells/ml with the B5 PAR-2 antiserum (b) or with a nonimmune rabbit serum (a) at a 1:500 dilution. Following washes, cells were incubated with FITC-coupled goat anti-rabbit antibodies at 20 µg/ml. Binding of antibodies was analyzed by flow cytometry as described in MATERIALS AND METHODS. Illustrated are fluorescence intensity histograms representative of three distinct experiments.

 
The expression of the protein at the cell surface was analyzed by flow cytometry using the B5 antiserum, which is directed to a PAR-2–specific domain spanning the trypsin cleavage site (10, 31). Membrane expression of PAR-2 was observed for both 16HBE and A549 cells, whereas KNRK/PAR-2 cells, used as control positive cells, showed a much higher expression (Figure 1B).

Functionality of PAR-2 Expressed by Human Pulmonary Epithelial Cells
KNRK/PAR-2, 16HBE, and A549 cells loaded with the calcium probe FURA-2 were challenged with either 50 nM trypsin or 100 µM of the activating peptide SLIGKV-NH2. As shown in Figure 2A, a cell response was observed in each case as a rapid and transient rise of cytosolic Ca2+. It can be noted that the kinetics of signals induced by either trypsin or SLIGKV-NH2 are comparable for a given cell line, which is in agreement with the fact that the two agonists activate the same receptor, and consequently trigger the same intracellular signaling pathway. By contrast, these kinetics are markedly different between cell lines. The main difference is observed for the kinetics of the decrease of the signals, which decrease is very fast for KNRK/PAR-2, slower for 16HBE, and almost absent within the time frame of measurement for A549 cells. Results obtained with KNRK/PAR-2 and A549 cells are in agreement with those previously reported for the same cell lines (6, 25). When considering the variations of calcium concentrations between basal and peak levels, calcium mobilization was greater for KNRK/PAR-2 cells than for the two pulmonary epithelial cells. This result is illustrated in Figure 2B, when testing a wide range of concentrations of both trypsin and peptide. Determination of the EC50 values for trypsin activation of KNRK/PAR-2, 16HBE, and A549 cells yielded mean values within the same range, i.e., 40 nM, 18 nM, and 13 nM, respectively. Again, these values are quite similar to those previously reported for KNRK/PAR-2 and A549 cells (6). For activation by SLIGKV-NH2, the EC50 has a mean value of 117 µM for KNRK/PAR-2 cells, whereas it could not be accurately calculated for 16HBE and A549 cells because the peptide SLIGKV-NH2 cannot be used at concentrations beyond 300 µM due to limited solubilization. It is of note that the reverse peptide VKGILS-NH2 used at 300 µM did not induce any calcium mobilization (data not shown), as previously reported (6, 10, 24, 25).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Activation of KNRK/PAR-2, 16HBE, and A549 cells by trypsin or the PAR-2 agonist peptide SLIGKV-NH2. FURA 2-loaded KNRK/PAR-2 (1.5 x 106 cells/ml), 16HBE (3 x 106 cells/ml), or A549 (3 x 106 cells/ml) cells were challenged with purified bovine trypsin or the synthetic SLIGKV-NH2. (A) Cells were activated with 50 nM trypsin or 100 µM SLIGKV-NH2 peptide and variations in fluorescence reflecting changes of cytosolic Ca2+ concentrations were recorded as a function of time. In the upper part of each tracing are indicated the value (in nM) of the maximal variation of intracellular concentration of Ca2+ elicited by the agonist over the basal concentration ({Delta}[Ca2+]i). (B) Cells were activated with increasing concentrations of trypsin or SLIGKV-NH2. Each point represents the {Delta}[Ca2+]i and is the mean ± SEM of at least three experiments. Fitted curves (filled squares, KNRK/PAR-2 cells; filled circles, 16HBE cells; and filled triangles, A549 cells) were produced by calculations based on a one-phase exponential binding site model.

 
Effects of the Neutrophil Serine Proteinases on PAR-2 Functions
To first determine if neutrophil elastase and cathepsin G might be potential activators of PAR-2, KNRK/PAR-2 cells were challenged with increasing concentrations of these two serine proteinases up to 3 µM. In no case was an intracellular calcium mobilization observed, indicating an absence of PAR-2 activation by these proteinases (data not shown), in agreement with previous reports (33, 34). Nonetheless, when trypsin was added 4.5 min after the exposure to elastase or cathepsin G at 0.5 µM, the trypsin-induced calcium mobilization was reduced by 81 ± 9% and 74 ± 13%, respectively, compared with that observed in untreated cells activated by the same concentration of trypsin (Figure 3). A similar pattern of inhibition was observed when elastase- or cathepsin G–treated 16HBE and A549 cells were activated by trypsin, with increases in cytosolic calcium concentration reduced by 73 ± 2 and 72 ± 11%, respectively, for 16HBE cells, and by 60 ± 6 and 55 ± 15% for A549 (Figure 3). Inhibition was due to the enzymatic activity of the proteinases, because pretreatment of KNRK/PAR-2 cells with the inactivated proteinases HLE-PMSF or CG-PMSF failed to reduce the calcium mobilization responses to trypsin (Figure 4).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. Effects of elastase and cathepsin G on trypsin- or SLIGKV-NH2–induced KNRK/PAR-2, 16HBE, and A549 cell activation. FURA 2-loaded cells (1.5 x 106 cells/ml for KNRK/PAR-2 cells and 3 x 106 cells/ml for 16HBE and A549) were challenged for 3 min with 0.5 µM human leukocyte elastase (HLE) or cathepsin G (CG). The proteolytic activity of the leukocyte proteinases was then blocked with 1.2 µM eglin C, and trypsin (50 nM) or SLIGKV-NH2 (100 µM) was added 90 s later. Variations in fluorescence reflecting changes of cytosolic Ca2+ concentrations were recorded over a total period of 10 min. As controls, batches of cell suspensions were processed exactly as above except that HLE and CG were replaced by HBSS-BSA 0.25%. Histograms (black bars, trypsin; and hatched bars, SLIGKV-NH2) represent the means ± SEM of three experiments with values expressed as the percentage of {Delta}[Ca2+]i measured in trypsin- or SLIGKV-NH2–treated control cells.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Effects of PMSF-inactivated elastase and cathepsin G on trypsin-induced KNRK/ PAR-2 cell activation. FURA 2–loaded KNRK/ PAR-2 cells (1.5 x 106 cells/ml) were challenged for 4.5 min with 0.5 µM PMSF-inactivated human leukocyte elastase (HLE-PMSF) or cathepsin G (CG-PMSF). Next, trypsin (50 nM) was added. Variations in fluorescence reflecting changes of cytosolic Ca2+ concentrations were recorded over a total period of 10 min. As controls, batches of cell suspensions were processed exactly as above except that HLE- and CG-PMSF were replaced by HBSS-BSA 0.25%. Histograms represent the means ± SEM of three experiments with values expressed as the percentage of {Delta}[Ca2+]i measured in trypsin-treated control cells. Histograms obtained for HLE- or CG-treated KNRK/PAR-2 cells subsequently exposed to trypsin and corresponding to data shown in Figure 3, are also represented.

 
In parallel experiments, KNRK/PAR-2 cells pretreated with elastase or cathepsin G at 0.5 µM were challenged with the activating peptide SLIGKV-NH2 instead of trypsin. In this case, cytosolic calcium mobilizations were comparable with those observed in untreated control cells, with maximum concentrations reaching 512 ± 130, 564 ± 186, and 575 ± 169 nM for untreated, elastase-pretreated, and cathepsin G–pretreated KNRK/PAR-2 cells, respectively (Figure 3). Increasing the leukocyte proteinase concentrations up to 3 µM left the reactivity to SLIGKV-NH2 unchanged. Again, the reverse peptide VKGILS-NH2 did not induce cell activation (data not shown). Similar results were obtained with the 16HBE and A549 cell lines (Figure 3).

Effects of Neutrophil Serine Proteinases on PAR-2 Expression
Taken together, the above data pointed to a cleavage of PAR-2 by leukocyte elastase and cathepsin G that would be downstream of the trypsin cleavage/activation site within the aminoterminal extracellular extension of the receptor, and should leave functional the SLIGKV-NH2 binding site, thus abrogating responses to trypsin but sparing those to the activating peptide, as we observed previously for PAR-1 (32). This assumption was supported by flow cytometry analysis of cells exposed to the proteinases using the antiserum B5 raised against a peptide encompassing the trypsin cleavage/activation site (10, 31). Indeed, treatment of KNRK/PAR-2 cells with 62.5–500 nM of elastase or cathepsin G for 10 min significantly reduced in a concentration-dependent manner, the detection of the targeted epitope by the B5 antiserum to an extent similar to that observed with 12.5–50 nM trypsin. As compared with control untreated cells, a reduction of 94 ± 3, 99 ± 2, and 80 ± 13% of the detected fluorescence was noted for 50 nM trypsin, 500 nM elastase, and 500 nM cathepsin G, respectively (Figure 5). Similar results were obtained with the 16HBE and A549 cell lines (data not shown). When cells were exposed to the highest concentrations of inactivated proteinases (HLE-PMSF, CG-PMSF, or SBTI-treated trypsin), no reduction in the B5 epitope expression was observed (Figure 5).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. B5 antibody binding at the surface of trypsin-, elastase- or cathepsin G–treated KNRK/PAR-2 cells. Cells (3 x 106 cells/ml) were incubated without (untreated) or with either 12.5–50 nM trypsin, 62.5–500 nM HLE, or 125–500 nM CG for 10 min at 37°C before enzymatic activities were blocked with either 3 µM SBTI for trypsin or 1.2 µM eglin C for the neutrophil proteinases. Alternatively, cells were incubated with either 50 nM SBTI-inhibited trypsin, or 500 nM HLE-PMSF or CG-PMSF for 10 min at 37°C. Cells were then incubated with the B5 PAR-2 antiserum or control rabbit serum and processed for flow cytometry analysis as described in the legend of Figure 1. Curves (open squares, trypsin; open triangles, HLE; open circles, CG) and single points (filled squares, trypsin-SBTI; filled triangles, HLE-PMSF; filled circles, CG-PMSF) represent the means ± SEM of three experiments, with the fluorescence of proteinase-treated cells expressed as the percentage of that of untreated cells.

 
Of note is the observation that treatment of cell lines with either trypsin, elastase, or cathepsin G did not increase the number of propidium iodide–positive dead cells, as compared with untreated cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that PAR-2 is expressed at the mRNA, protein, and functional levels by the bronchial epithelial cell line 16HBE, while confirming similar results in the alveolar cell line A549 in agreement with previous reports (6, 25). Expression of PAR-2 in these cell lines would appear to reflect a physiologic situation, because specific messengers for this receptor are also present in primary human bronchial epithelial cells, confirming previous observations (25). In fact, the present results, as well as previous data based on immunohistochemistry or RT-PCR analysis of human bronchial smooth muscle and epithelial cells (9, 21, 25), indicate that PAR-2 is expressed at various cellular levels in human lungs. We further confirm that this receptor is activated upon challenge of the cells either with trypsin or with the synthetic peptide SLIGKV-NH2, corresponding to the PAR-2 tethered ligand. This result is in keeping with the presence of active PAR-2 in murine (17) and guinea-pig (15) airways, in which it modulates contractility, and in human pulmonary epithelial cells in culture, in which it induces the release of various mediators of the inflammatory response (22–25).

As mentioned in the introduction, the physiologic or pathologic proteinase(s) for activating PAR-2 in vivo remains poorly defined. Trypsin and mast cell tryptase, and more recently, the activated coagulation factors VIIa and Xa, have been identified as serine proteinases able to activate mammalian PAR-2 (11, 35). If within the small intestine, a physiologic role for pancreatic trypsin can be suggested (10), within the lung the role of tryptase is only speculative, especially because PAR-2 is expressed at the apical side of epithelial cells (11) and because the activation of PAR-2 by tryptase is restricted by receptor sialylation (12). Another potential source of PAR-2–activating enzymes is represented by the proteinases secreted by pathogens, particularly at mucosal surfaces, such as in the airways. Thus, it is of note that two major dust mite antigens, Der p3 and Der p9, endowed with a serine proteinase activity, have been recently shown to activate a lung epithelial cell line in part through PAR-2 (24). Similarly, an Arg-specific cysteine proteinase produced by Porphyromonas gingivalis has been shown to activate PAR-2 in an oral epithelial cell line (36). Finally, leukocyte serine proteinases such as elastase and cathepsin G, which can be released in the epithelial environment by neutrophils present in the airspaces during lung inflammation (26), might also act as PAR-2 regulating enzymes. This hypothesis was supported by two observations: (i) leukocyte elastase can activate human bronchial epithelial cells, leading to IL-8 expression (27); and (ii) cathepsin G has been shown to be an activating proteinase for PAR-4 (37).

Actually, our data show that elastase and cathepsin G do not activate PAR-2 in the A549 and 16HBE human lung cell lines, as well as in the PAR-2–transfected cell line. This is in agreement with previous reports showing that cathepsin G at 100 nM does not induce calcium mobilization in A549 cells and HBEC (34), and that elastase in the 100–250 nM range does not activate a PAR-2–expressing human microvascular endothelium cell line (33). Although a recent report indicates that 100 nM elastase can induce a slight calcium mobilization in primary rat brain microvascular endothelial cells, it was not demonstrated that this activation proceeds through PAR-2 (38). On the contrary, our data establish that neutrophil serine proteinases disarm PAR-2 expressed in the three cell lines used in this study. Indeed, preincubation of cells with these serine proteinases prevents activation by trypsin. This disarming of the receptor is accompanied by the disappearance of the epitope for the antibody B5, which maps over the trypsin cleavage/activation site. This extends to lung epithelial cells, previous observations made on PAR-2–expressing microvascular endothelial cells, and showing that exposure to elastase in the 100–250 nM range reduces a subsequent trypsin-induced calcium mobilization by 50–80% (33, 38). However, these studies did not consider the effect of elastase on the PAR-2–specific activating peptide, and were performed on cells expressing several PAR subtypes, thus not allowing conclusion of an effect specifically targeted at PAR-2.

Possible interpretations of our data include the following: (i) PAR-2 disappears from the plasma membrane through an endocytotic process triggered by leucocyte proteinases; (ii) leucocyte proteinases bind to PAR-2 at its cleavage site, thus preventing proteolysis by trypsin and masking the epitope recognized by B5; and (iii) elastase and cathepsin G cleave the aminoterminal exodomain of PAR-2 downstream of the trypsin cleavage/activation site. Because it was observed that the PAR-2–activating peptide SLIGKV-NH2 remained fully active upon prior exposure of cells to neutrophil serine proteinases, it can be inferred that PAR-2 remains located at the surface of cells exposed to these proteinases. The second hypothesis can be discarded because pretreatment of cells with inactivated neutrophil proteinases did not prevent the intracellular calcium increase induced by trypsin. Based on these data and on our previous study describing the effects of the same enzymes on PAR-1 (32), we thus conclude that elastase and cathepsin G cleave the aminoterminal exodomain of PAR-2 downstream of the trypsin cleavage/activation site. This assumption is further supported by a recent report investigating the proteolysis of a recombinant exodomain of PAR-2 (33), which predicts that the receptor should be inactivated by both elastase and cathepsin G, through cleavages at peptide bonds carboxyterminal to the Arg36-Ser37 activation site, including six sites for elastase between Val42 and Phe77 and three for cathepsin G between Leu38 and Ser65. Our data clearly suggest that such cleavages observed with a recombinant polypeptide may occur in situ on the native cellular receptor. Interestingly, it has been recently reported that the bacterial metalloproteinase thermolysin has the capacity to abrogate the response of A549 cells and HBEC to 50 nM trypsin, while leaving the response to 100 µM SLIGKV-NH2 unchanged (34), in a process resembling that described here for neutrophil elastase and cathepsin G. It thus appears that, in an infectious and inflammatory context, both endogenous and exogenous proteinases have the capacity to downregulate PAR-2 activity.

As already indicated, PAR-2 is believed to be involved in inflammation. This role for PAR-2 implies that elastase and cathepsin G would paradoxically display an anti-inflammatory property by disarming PAR-2. An anti-inflammatory role for these two enzymes has been suggested previously by the observations that the same enzymes can downregulate the biological activity of receptors participating in the inflammatory process such as the thrombin PAR-1 receptor in platelets and endothelial cells (32), the LPS-binding molecule CD14 in monocytes (39), and a number of cytokine and chemokine receptors (40). As far as the lung is concerned, it has been shown that the activation of PAR-2 expressed by pulmonary epithelial cells leads to the induction of expression and release of metalloproteinases, prostaglandins, and cytokines (22–25), all important mediators of lung inflammation. There is also evidence that activation of murine PAR-2 within bronchi induces the formation of PGE2 and PGF2{alpha}, which are also potent inflammatory molecules, although it is of note that these molecules also protect from bronchoconstriction (17). The latter observation has to be balanced, however, by data obtained in guinea pigs, in which in vivo administration of PAR-2 agonists causes bronchoconstriction (15). Despite such strong arguments for considering PAR-2 as a proinflammatory receptor, it has been recently suggested that PAR-2 could also be involved in a physiologic process of cytoprotection, and in so doing could be linked to anti-inflammatory pathways (11). This anti-inflammatory role is supported by the observation that an intranasal administration of the PAR-2 activating peptide to mice is unable to trigger inflammation, but on the contrary reduces LPS-induced neutrophil influx (41). If such an anti-inflammatory property is confirmed, this would indicate that by disarming PAR-2, elastase and cathepsin G actually could also participate in the enhancing inflammation. As far as human lung is concerned, a recent report demonstrates that PAR-2 agonists induce contraction of isolated intact bronchi (42), whereas in another study which compares PAR-2 expression in the central airways of smokers and nonsmokers, a possible protective role for PAR-2 in the development of chronic airflow limitation is suggested (21). It thus appears that PAR-2 activation clearly affects airways, although the effects depend strongly on the species and the model used. The exact role of PAR-2 in lungs has to be clarified, but nonetheless, neutrophil serine proteinases may represent major regulators of its activity.


   Acknowledgments
 
The authors thank Dr. Agustin Valenzuela-Fernandez (Pasteur Institute, Paris, France) for assistance in data analysis of calcium mobilization measurements. This study was supported by the nonprofit association "Vaincre la Mucoviscidose" (S.D.), by NIH grants DK43207, DK 57480, DK 39957 (N.W.B.), and by the Centre National de la Recherche Scientifique (D.P.).

Received in original form June 7, 2002

Received in final form August 9, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vergnolle, N., J. L. Wallace, N. W. Bunnett, and M. D. Hollenberg. 2001. Protease-activated receptors in inflammation, neuronal signaling and pain. Trends Pharmacol. Sci. 22:146–152.[CrossRef][Medline]
  2. Macfarlane, S. R., M. J. Seatter, T. Kanke, G. D. Hunter, and R. Plevin. 2001. Proteinase-activated receptors. Pharmacol. Rev. 53:245–282.[Abstract/Free Full Text]
  3. Vu, T. K. D., D. T. Hung, V. I. Wheaton, and S. R. Coughlin. 1991. Molecular cloning of a functional receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057–1068.[CrossRef][Medline]
  4. Rasmussen, U. B., V. Vouret-Craviari, S. Jallat, Y. Schlessinger, G. Pagers, A. Pavirani, J. P. Lecocq, J. Pouyssegur, and E. Van Obberghen-Schilling. 1991. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization. FEBS Lett. 288:1323–1328.
  5. Nystedt, S., K. Emilsson, A. K. Larsson, B. Strombeck, and J. Sundelin. 1995. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur. J. Biochem. 232:84–89.[Medline]
  6. Böhm, S. K., W. Kong, D. Brömme, S. P. Smeekens, D. C. Anderson, A. Connolly, M. Kahn, N. A. Nelken, S. R. Coughlin, D. G. Payan, and N. W. Bunnett. 1996. Molecular cloning, expression and potential function of the human proteinase-activated receptor-2. Biochem. J. 314:1009–1016.
  7. Ishihara, H., A. J. Connolly, D. Zeng, M. L. Kahn, Y. W. Zheng, C. Timmons, T. Tram, and S. R. Coughglin. 1997. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386:502–506.[CrossRef][Medline]
  8. Xu, W.-F., H. Anderson, T. E. Whitmore, S. R. Presnell, D. P. Yee, A. Ching, T. Gilbert, E. W. Davie, and D. C. Foster. 1998. Cloning and characterization of human protease-activated receptor 4. Proc. Natl. Acad. Sci. USA 95:6642–6646.[Abstract/Free Full Text]
  9. D'Andrea, M. R., C. K. Derian, D. Leturcq, S. M. Baker, A. Brunmark, P. Ling, A. L. Darrow, R. J. Santulli, L. F. Brass, and P. Andrade-Gordon. 1998. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J. Histochem. Cytochem. 46:157–164.[Abstract/Free Full Text]
  10. Kong, W., K. McConalogue, L. M. Khitin, M. D. Hollenberg, D. G. Payan, S. K. Böhm, and N. W. Bunnett. 1997. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2. Proc. Natl. Acad. Sci. USA 94:8884–8889.[Abstract/Free Full Text]
  11. Cocks, T. M., and J. D. Moffatt. 2001. Protease-activated receptor-2 (PAR2) in the airways. Pulm. Pharmacol. Ther. 14:183–191.[CrossRef][Medline]
  12. Compton, S. J., B. Renaux, S. J. Wijesuriya, and M. D. Hollenberg. 2001. Glycosylation and the activation of proteinase-activated receptor 2 (PAR2) by human mast cell tryptase. Br. J. Pharmacol. 134:705–718.[CrossRef][Medline]
  13. Hou, L., S. Kapas, A. T. Cruchley, M. G. Macey, P. Harriot, C. Chinni, S. R. Stone, and G. L. Howells. 1998. Immunolocalization of proteinase-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro. Immunology 94:356–362.[CrossRef][Medline]
  14. Steinhoff, M., N. Vergnolle, S. H. Young, M. Tognetto, S. Amadesi, H. S. Ennes, M. Trevisani, M. D. Hollenberg, J. L. Wallace, G. H. Caughey, S. E. Mitchell, L. M. Williams, P. Geppetti, E. A. Mayer, and N. W. Bunnett. 2000. Agonists of proteinase-activated receptor 2 induce inflammation by neurogenic mechanism. Nat. Med. 6:151–158.[CrossRef][Medline]
  15. Ricciardolo, F. L., M. Steinhoff, S. Amadesi, R. Guerrini, M. Tognetto, M. Trevisani, C. Creminon, C. Bertrand, N. W. Bunnett, L. M. Fabbri, S. Salvadori, and P. Geppetti. 2000. Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways. Am. J. Respir. Crit. Care Med. 161:1672–1680.[Abstract/Free Full Text]
  16. Vergnolle, N. 1999. Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo. J. Immunol. 163:5064–5069.[Abstract/Free Full Text]
  17. Cocks, T. M., B. Fong, J. M. Chow, G. P. Anderson, A. G. Frauman, R. G. Goldie, P. J. Henry, M. J. Carr, J. R. Hamilton, and J. D. Moffatt. 1999. A protective role for protease-activated receptors in the airways. Nature 398:156–160.[CrossRef][Medline]
  18. Fiorucci, S., A. Mencarelli, B. Palazzetti, E. Distrutti, N. Vergnolle, M. D. Hollenberg, J. L. Wallace, A. Morelli, and G. Cirino. 2001. Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis. Proc. Natl. Acad. Sci. USA 98:13936–13941.[Abstract/Free Full Text]
  19. Vergnolle, N., M. D. Hollenberg, K. A. Sharkey, and J. L. Wallace. 1999. Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw. Br. J. Pharmacol. 127:1083–1090.[CrossRef][Medline]
  20. Lindner, J. R., M. L. Kahn, S. R. Coughlin, G. R. Sambrano, E. Schauble, D. Bernstein, D. Foy, A. Hafezi-Moghadam, and K. Ley. 2000. Delayed onset of inflammation in proteinase-activated receptor-2-deficient mice. J. Immunol. 165:6504–6510.[Abstract/Free Full Text]
  21. Miotto, D., M. D. Hollenberg, N. W. Bunnett, A. Papi, F. Braccioni, P. Boschetto, F. Rea, A. Zuin, P. Geppetti, M. Saetta, P. Maestrelli, L. M. Fabbri, and C. E. Mapp. 2002. Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers. Thorax 57:146–151.[Abstract/Free Full Text]
  22. Vliagoftis, H., D. Befus, M. D. Hollenberg, and R. Moqbel. 2001. Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2. J. Allergy Clin. Immunol. 107:679–685.[CrossRef][Medline]
  23. Vliagoftis, H., A. Schwingshackl, C. D. Milne, M. Duszyk, M. D. Hollenberg, J. L. Wallace, D. Befus, and R. Moqbel. 2000. Proteinase-activated receptor-2-mediated matrix metalloproteinase-9 release from airway epithelial cells. J. Allergy Clin. Immunol. 106:537–545.[CrossRef][Medline]
  24. Sun, G., M. A. Stacey, M. Schmidt, L. Mori, and S. Mattoli. 2001. Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells. J. Immunol. 167:1014–1021.[Abstract/Free Full Text]
  25. Asokananthan, N., P. T. Graham, J. Fink, D. A. Knight, A. J. Bakker, A. S. McWilliam, P. J. Thompson, and G. A. Stewart. 2002. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E(2) release from human respiratory epithelial cells. J. Immunol. 168:3577–3585.[Abstract/Free Full Text]
  26. Lucey, E. C., P. J. Stone, and G. L. Snider. 1997. Consequences of proteolytic injury. In The lung. R. G. Crystal, J. B. West, P. F. Barnes, and E. Weibel, editors. Lippincott-Raven, New York. 2237–2250.
  27. Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal. 1992. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J. Clin. Invest. 89:1478–1484.
  28. Cozens, A. L., M. J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W. E. Finkbeiner, J. H. Widdicombe, and D. C. Gruenert. 1994. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 10:38–47.[Abstract]
  29. Renesto, P., V. Balloy, T. Kamimura, K. Masuda, A. Imaizumi, and M. Chignard. 1993. Inhibition by recombinant SLPI and half-SLPI (Asn55-Ala107) of elastase and cathepsin G activities: consequences for neutrophil-platelet cooperation. Br. J. Pharmacol. 108:1100–1106.[Medline]
  30. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.[Medline]
  31. Al-Ani, B., M. Saifeddine, A. Kawabata, B. Renaux, S. Mokashi, and M. D. Hollenberg. 1999. Proteinase-activated receptor 2 (PAR(2)): development of a ligand-binding assay correlating with activation of PAR(2) by PAR(1)- and PAR(2)-derived peptide ligands. J. Pharmacol. Exp. Ther. 290:753–760.[Abstract/Free Full Text]
  32. Renesto, P., M. Si-Tahar, M. Moniatte, V. Balloy, A. Van Dorsselaer, D. Pidard, and M. Chignard. 1997. Specific inhibition of thrombin-induced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor. Blood 89:1944–1953.[Abstract/Free Full Text]
  33. Loew, D., C. Perrault, M. Morales, S. Moog, C. Ravanat, S. Schuhler, R. Arcone, C. Pietropaolo, J. P. Cazenave, A. Van Dorsselaer, and F. Lanza. 2000. Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. Biochemistry 39:10812–10822.[CrossRef][Medline]
  34. Ubl, J. J., Z. V. Grishina, T. K. Sukhomlin, T. Welte, F. Sedehizade, and G. Reiser. 2002. Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin. Am. J. Physiol. Lung Cell. Mol. Physiol. 282:L1339–L1348.[Abstract/Free Full Text]
  35. Camerer, E., W. Huang, and S. R. Coughlin. 2000. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc. Natl. Acad. Sci. USA 97:5255–5260.[Abstract/Free Full Text]
  36. Lourbakos, A., J. Potempa, J. Travis, M. R. D'Andrea, P. Andrade-Gordon, R. Santulli, E. J. Mackie, and R. N. Pike. 2001. Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion. Infect. Immun. 69:5121–5130.[Abstract/Free Full Text]
  37. Sambrano, G. R., W. Huang, T. Faruqi, S. Mahrus, C. Craik, and S. R. Coughlin. 2000. Cathepsin G activates protease-activated receptor-4 in human platelets. J. Biol. Chem. 275:6819–6823.[Abstract/Free Full Text]
  38. Dömötör, E., K. Bartha, R. Machovitch, and V. Adam-Vizi. 2002. Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase. J. Neurochem. 80:746–754.[CrossRef][Medline]
  39. Le-Barillec, K., D. Pidard, V. Balloy, and M. Chignard. 2000. Human neutrophil cathepsin G down-regulates LPS-mediated monocyte activation through CD14 proteolysis. J. Leukoc. Biol. 68:209–215.[Abstract/Free Full Text]
  40. Bank, U., and S. Ansorge. 2001. More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J. Leukoc. Biol. 69:197–205.[Abstract/Free Full Text]
  41. Moffat, J. D., K. L. Jeffrey, and T. M. Cocks. 2002. Proteinase-activated receptor-2 activating peptide SLIGRL inhibits bacterial lipopolysaccharide-induced recruitement of polymorphonuclear leukocytes into the airways of mice. Am. J. Respir. Cell Mol. Biol. 26:680–684.[Abstract/Free Full Text]
  42. Schmidlin, F., S. Amadesi, R. Vidil, M. Trevisani, N. Martinet, G. Caughey, M. Tognetto, G. Cavallesco, C. Mapp, P. Geppetti, and N. W. Bunnett. 2001. Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle. Am. J. Respir. Crit. Care Med. 164:1276–1281.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. C. Chin, W. Y. Lee, A. Nusrat, N. Vergnolle, and C. A. Parkos
Neutrophil-mediated Activation of Epithelial Protease-Activated Receptors-1 and -2 Regulates Barrier Function and Transepithelial Migration
J. Immunol., October 15, 2008; 181(8): 5702 - 5710.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. J. Ramsay, Y. Dong, M. L. Hunt, M. Linn, H. Samaratunga, J. A. Clements, and J. D. Hooper
Kallikrein-related Peptidase 4 (KLK4) Initiates Intracellular Signaling via Protease-activated Receptors (PARs): KLK4 AND PAR-2 ARE CO-EXPRESSED DURING PROSTATE CANCER PROGRESSION
J. Biol. Chem., May 2, 2008; 283(18): 12293 - 12304.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. J. Moraes, R. Martin, J. D. Plumb, E. Vachon, C. M. Cameron, A. Danesh, D. J. Kelvin, W. Ruf, and G. P. Downey
Role of PAR2 in murine pulmonary pseudomonal infection
Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L368 - L377.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
I. Kuwahara, E. P. Lillehoj, T. Koga, Y. Isohama, T. Miyata, and K. C. Kim
The Signaling Pathway Involved in Neutrophil Elastase Stimulated MUC1 Transcription
Am. J. Respir. Cell Mol. Biol., December 1, 2007; 37(6): 691 - 698.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Geraghty, M. P. Rogan, C. M. Greene, R. M. M. Boxio, T. Poiriert, M. O'Mahony, A. Belaaouaj, S. J. O'Neill, C. C. Taggart, and N. G. McElvaney
Neutrophil Elastase Up-Regulates Cathepsin B and Matrix Metalloprotease-2 Expression
J. Immunol., May 1, 2007; 178(9): 5871 - 5878.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Ramachandran, L. R. Sadofsky, Y. Xiao, A. Botham, M. Cowen, A. H. Morice, and S. J Compton
Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4
Am J Physiol Lung Cell Mol Physiol, March 1, 2007; 292(3): L788 - L798.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Oikonomopoulou, K. K. Hansen, M. Saifeddine, I. Tea, M. Blaber, S. I. Blaber, I. Scarisbrick, P. Andrade-Gordon, G. S. Cottrell, N. W. Bunnett, et al.
Proteinase-activated Receptors, Targets for Kallikrein Signaling
J. Biol. Chem., October 27, 2006; 281(43): 32095 - 32112.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
I. Kuwahara, E. P. Lillehoj, W. Lu, I. S. Singh, Y. Isohama, T. Miyata, and K. C. Kim
Neutrophil elastase induces IL-8 gene transcription and protein release through p38/NF-{kappa}B activation via EGFR transactivation in a lung epithelial cell line
Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L407 - L416.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. Csernok, M. Ai, W. L. Gross, D. Wicklein, A. Petersen, B. Lindner, P. Lamprecht, J. U. Holle, and B. Hellmich
Wegener autoantigen induces maturation of dendritic cells and licenses them for Th1 priming via the protease-activated receptor-2 pathway
Blood, June 1, 2006; 107(11): 4440 - 4448.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. Chignard and D. Pidard
Neutrophil and Pathogen Proteinases versus Proteinase-Activated Receptor-2 Lung Epithelial Cells: More Terminators than Activators.
Am. J. Respir. Cell Mol. Biol., April 1, 2006; 34(4): 394 - 398.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
I. Kuwahara, E. P. Lillehoj, A. Hisatsune, W. Lu, Y. Isohama, T. Miyata, and K. C. Kim
Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter
Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L355 - L362.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
C. C. Taggart, C. M. Greene, T. P. Carroll, S. J. O'Neill, and N. G. McElvaney
Elastolytic Proteases: Inflammation Resolution and Dysregulation in Chronic Infective Lung Disease
Am. J. Respir. Crit. Care Med., May 15, 2005; 171(10): 1070 - 1076.
[Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. Dulon, D. Leduc, G. S. Cottrell, J. D'Alayer, K. K. Hansen, N. W. Bunnett, M. D. Hollenberg, D. Pidard, and M. Chignard
Pseudomonas aeruginosa Elastase Disables Proteinase-Activated Receptor 2 in Respiratory Epithelial Cells
Am. J. Respir. Cell Mol. Biol., May 1, 2005; 32(5): 411 - 419.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. Sokolova, Z. Grishina, F. Buhling, T. Welte, and G. Reiser
Protease-activated receptor-1 in human lung fibroblasts mediates a negative feedback downregulation via prostaglandin E2
Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L793 - L802.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
V. S. OSSOVSKAYA and N. W. BUNNETT
Protease-Activated Receptors: Contribution to Physiology and Disease
Physiol Rev, April 1, 2004; 84(2): 579 - 621.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
P. J. O'Brien, H. Koi, S. Parry, L. F. Brass, J. F. Strauss III, L.-P. Wang, J. E. Tomaszewski, and L. K. Christenson
Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation: Receptor Activation Mediates Extravillous Trophoblast Invasion in Vitro
Am. J. Pathol., October 1, 2003; 163(4): 1245 - 1254.