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J. Biol. Chem., Vol. 281, Issue 11, 6910-6923, March 17, 2006

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The House Dust Mite Allergen Der p 1, Unlike Der p 3, Stimulates the Expression of Interleukin-8 in Human Airway Epithelial Cells via a Proteinase-activated Receptor-2-independent Mechanism^*

Emmanuelle Adam, Kristina K. Hansen, Olaya Fernandez Astudillo, Ludivine Coulon, Françoise Bex^¶, Xavier Duhant^||, Erika Jaumotte, Morley D. Hollenberg, and Alain Jacquet¹

From the Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium, the Canadian Institutes of Health Research Proteinases and Inflammation Network, Departments of Pharmacology and Therapeutics and Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada, ^¶Laboratoire de Microbiologie, Institut CERIA, Université Libre de Bruxelles, 1060 Brussels, Belgium, and the ^||Institute of Interdisciplinary Research, School of Medecine, Department of Immunology, Erasme Hospital, Université Libre de Bruxelles, 1060 Brussels, Belgium

Received for publication, June 30, 2005 , and in revised form, October 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We investigated and compared the mechanisms by which two dust mite proteolytic allergens, Der p 1 and Der p 3, and a peptide agonist of proteinase-activated receptor 2 (PAR₂AP) trigger interleukin (IL)-8 release from human pulmonary epithelial cells (A549). Although all three stimuli tested induced the up-regulation of IL-8 (mRNA and protein), the Der p 1-mediated signaling events did not exactly match those induced by PAR₂AP and Der p 3. First, Der p 1 was less effective in stimulating IL-8 gene transcriptional activity than PAR₂AP and Der p 3. Second, Der p 1-mediated IL-8 expression was mainly dependent on NF-B, whereas Der p 3 and PAR₂AP regulated IL-8 expression through the activation of both NF-B and AP-1. Third, although all three MAP kinases, ERK1/2, p38, and JNK, were activated, Der p 1 induced IL-8 release exclusively via the ERK1/2 signaling pathway, whereas PAR₂AP and Der p 3 also involved the other kinases. Fourth, in HeLa cells, Der p 1 was able to up-regulate IL-8 secretion independent of PAR₂ expression, and in contrast with PAR₂AP and Der p 3, Der p 1 was unable to affect calcium signaling via PAR₂ in PAR₂-expressing KNRK cells. Finally, cleavage by Der p 1 of a synthetic peptide representing the N-terminal activation-cleavage site of PAR₂ did not release a high potency activator of PAR₂ as does Der p 3. We conclude that Der p 1 (but not Der p 3)-induced IL-8 production in A549 epithelial cells is independent of PAR₂ activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
House dust mites are a major source of allergens that contribute to the rising incidence of allergic diseases such as bronchial asthma, perennial rhinitis, and atopic dermatitis (1–3). At least 17 allergen groups have been identified in the two predominant dust mite species, Dermatophagoides pteronyssinus and Dermatophagoides farinae (4). Allergens from group 1 (Der p 1 and Der f 1) display cysteine proteinase activity, whereas group 3, 6, and 9 allergens are serine proteinases that share sequence identity with trypsin, chymotrypsin, and collagenase, respectively. Although the precise pathophysiological role of the proteolytic activity of these house dust mite allergens has not been fully elucidated in vivo, recent data provide evidence that the proteinase activity amplifies allergen-induced bronchial asthma. Indeed, several reports have demonstrated that Der p 1 is capable of cleaving human proteins with potentially immunomodulatory effects, including A1-Pi (1-proteinase inhibitor) (5), CD23 (the human low affinity IgE receptor) (6, 7), CD25 (the subunit of the human IL-2 receptor) (8), and CD40 (9). Moreover, incubation of confluent airway epithelial cells with Der p 1 or the serine proteinases Der p 3, Der p 6, or Der p 9 has been shown to lead to cleavage of the tight junction adhesion proteins, such as ZO-1, causing an increase in the epithelial permeability and access of allergens to dendritic cells (10–13). This event appears to depend on the proteolytic activity of these allergens, since these changes in allergen-induced permeability can be prevented by cysteine or serine proteinase inhibitors. Finally, several studies indicate that Der p 1, as well as Der p 3 and Der p 9, can also induce the release of proinflammatory mediators, such as interleukin (IL)²-8, IL-6, eotaxin, granulocyte macrophage-colony stimulating factor (GM-CSF), or RANTES by airway epithelial cells (14–17). These mediators trigger the accumulation of inflammatory cells, such as eosinophils and neutrophils, to perpetuate the chronic allergic inflammation of the airways. The expression of these cytokines and chemokines has been shown to depend on the enzymatic activity of these dust mite allergens, but the allergen-induced signal transduction mechanism(s) in human epithelial cells has not yet been studied in any depth. The relationship between the proteolytic activity of these allergens and their induction of chemokine production has been strengthened by data pointing to a potential role for proteinase-activated receptors (PARs) 1 and 2 in the action of Der p 1, 3, and 9 (15, 17). PAR₁ and PAR₂ belong to a new subfamily of proteolytically activated G protein-coupled receptors with seven putative transmembrane domains. Activation of PARs is achieved when the extracellular amino terminus of the receptor is cleaved to expose a tethered ligand sequence that binds to the extracellular body of the receptor, leading to the G protein-coupled signal transduction (e.g. activation of phospholipase C, generation of inositol 1,4,5-triphosphate, increased intracellular Ca²⁺, and activation of protein kinase C (reviewed in Ref. 18)). Synthetic peptides based on the sequence of the tethered ligand can bind to and activate the receptor, bypassing the requirement for proteolysis (19).

The chemokine IL-8, which is a major chemoattractant and activator of neutrophils, appears to play a key role in the pathophysiology of asthma (20, 21). Indeed, IL-8 expression is increased in the airway epithelium of patients with asthma (22). Patients with status asthmaticus also exhibit dramatic increases of this cytokine in their airways (23). Moreover, IL-8 has also been found to be an eosinophil chemoattractant relevant to the problem of asthma (21). IL-8 secretion and mRNA synthesis are strongly regulated by more than 100-fold by a plethora of external stimuli (24). A major part of this regulation occurs at the transcriptional level. A relatively small region of the 5'-flanking region of the IL-8 gene (nucleotides –1 to –133) is essential and sufficient for its transcriptional induction by most stimuli (24). This promoter contains an NF-B element that is required for activation in all cell types studied as well as AP-1 and CCAAT enhancer-binding protein (NF-IL6) binding sites. The latter two sites are not essential for induction but are required for maximal gene expression (24). To date, numerous lines of functional evidence indicate that the activity of transcription factors that can bind to NF-B, AP-1, and CCAAT/enhancer-binding protein (NF-IL-6) binding sites in the IL-8 promoter are regulated by the coordinated activation of the MAP kinases ERK, c-Jun NH₂-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK) in response to stimuli that induce IL-8 production (reviewed in Ref. 25). However, the precise molecular mechanisms by which all three types of MAP kinases regulate onset, duration, or extent of IL-8 mRNA synthesis are still not completely elucidated.

In this study, we have investigated and compared the mechanisms whereby Der p 1, Der p 3 and an agonist peptide-activator of PAR₂ (PAR₂AP) induce the expression and the production of IL-8 in human airway epithelial cells. Our data demonstrate that, although Der p 1 and Der p 3 have both been described to trigger cytokine release from respiratory epithelial cells by activation of PAR₂, the signaling events mediated by Der p 1 do not exactly match those induced by either Der p 3 or PAR₂ activation in terms of the transcription factors and MAP kinases mediating IL-8 expression. Moreover, we show that the ability of Der p 1 to up-regulate IL-8 secretion in HeLa cells, in contrast with Der p 3, cannot be attributed to the activation of PAR₂. Finally, we find that cleavage by Der p 1 of a synthetic peptide sequence representing the N-terminal activation-cleavage site of PAR₂ does not release a high potency activator of PAR₂, as does Der p 3. All of our data lead us to conclude that, in contrast with Der p 3, Der p 1-induced IL-8 production in human airway epithelial cells appears to be independent of PAR₂ activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The receptor-selective PAR₂ agonist (PAR₂AP; SLIGRL-NH₂) and the standard PAR₂-inactive control (-PAR₂AP; LRGILS-NH₂) peptide, P20 (corresponding to rat PAR₂ residues 30–45, ³⁰GPNSKGRSLIGRLDTP⁴⁵yggc (sequence of PAR₂ in uppercase letters); yggc added for labeling and coupling) as well as a 27-mer P27 peptide (corresponding to human PAR₂ residues 28–54, Ac-²⁸GTNRSSKGRSLIGKVDGTSHVTGKGVT⁵⁴-NH₂) were synthesized by solid-phase methods (Peptide Synthesis Facility, University of Calgary, Faculty of Medicine). HPLC analysis, mass spectral analysis, and quantitative amino acid analysis confirmed the composition and purity of all peptides. Stock solutions were prepared in 25 mM HEPES buffer, pH 7.4. Trypsin and TNF- were purchased from Sigma and R&D, respectively. The MEK1/2 inhibitor U0126 was obtained from Cell Signaling. The p38 MAP kinase inhibitor, SB203580, the JNK inhibitor, SP 6001125, and actinomycin D were purchased from Calbiochem.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Purification of Der p 1 and Der p 3. A, purified allergens were analyzed by SDS-PAGE, and proteins were detected by Coomassie Blue staining. B, immunoblot analysis with rabbit polyclonal serum raised against recombinant ProDer p 3 and ProDer p 1.

Recombinant ProDer p 1 from CHO spent culture medium was purified to homogeneity as previously described (26). Before use, natural Der p 1 was activated for 15 min at room temperature in PBS, pH 7.3, containing 10 mM L-cysteine. In some experiments, Der p 1 was heat-denatured for 5 min at 100 °C in the presence of 50 mM -mercaptoethanol. The catalytic activity of Der p 1 was determined in a continuous rate (kinetic) assay using the fluorogenic peptide substrate N-tert-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-methylcoumarin (Boc-Gln-Ala-Arg-7-amino-4-methylcoumarin (AMC)). Briefly, Der p 1 was activated for 15 min in 20 mM Hepes buffer, pH 7.4, containing 150 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 10 mM dextrose, 0.25 mM sulfinpyrazone, and 10 mM cysteine. The substrate Boc-Gln-Ala-Arg-AMC (20 µl, 7.5 mM stock in Me₂SO, 75 µM final concentration; Bachem, Torrance, CA) was then added. The rate of the enzymatic activity was measured for 10 min using an AMINCO-Bowman® Series 2 Luminescence spectrometer (Spectronic Unicam, Rochester, NY) with an excitation wavelength of 380 nm and an emission recorded at 460 nm. The amount of substrate cleaved was determined by measuring the fluorescence of known concentrations of AMC (0.75, 1.5, and 2.25 µM; Sigma). The activity of the Der p 1 used for our work (house units/mg) ranged between 200 and 300 units/mg (nmol of Boc-Gln-Ala-Arg-AMC cleaved/min/mg of protein). Sample calculation was as follows: activity = (0.00536697 fluorescence units/s x (2.25 nmol/ml)/(2.41545 fluorescence units) x 2 ml x 60 s/min)/0.0021 mg of Der p 1; activity = 282 nmol of Boc-Gln-Ala-Arg-AMC cleaved/min/mg of protein. A molecular weight of 30,000 was used to calculate the molar concentrations of Der p 1.

The enzymatic activity assay for trypsin (2 nM) and Der p 3 (2 nM) using the same substrate, Boc-Gln-Ala-Arg-AMC (75 µM), in buffer (50 mM Tris, 20 mM CaCl₂, pH 7.4) yielded activities of the two enzymes: 28,700 units/mg (nmol of substrate cleaved/min/mg) for trypsin and 1900 units/mg (nmol of substrate cleaved/min/mg) for Der p 3.

Cell Culture—A549 cells, a human alveolar type II pneumocyte cell line (a kind gift from Prof. J. Pestel, Institut Pasteur, Lille) and HeLa cells (obtained from the European Collection of Cell Culture) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were passed without the use of trypsin by scraping to minimize the proteolytic activation of the PARs. HeLa cells were transiently transfected with human PAR₂ using the pcDNA3 mammalian expression vector, as outlined below.

KNRK rat kidney cells permanently transfected with rat PAR₂ (27) or with the human PAR₂ variant lacking an N-terminal glycosylation site, PAR₂N30A (28), were routinely cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 0.6 mg/ml Geneticin, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin as previously described (27, 28). Cells were subcultured by resuspension in calcium-free isotonic saline/EDTA solution without the use of trypsin.

Activation of A549 and HeLa Cells—A549 and either vector-alone or PAR₂-transfected HeLa cells were cultured to 80% confluence and then incubated in serum-free DMEM for a further 24 h. One hour before stimulation, the growth medium was replaced by fresh serum-free DMEM.

Cells were then incubated with different concentrations of Der p 1 (25–250 nM) or Der p 3 (5–60 nM) for 7 h. In some experiments, cells were then exposed to optimum concentrations of native Der p 1 or Der p 3 for different periods of time in serum-free DMEM. As controls, cells were also incubated with activating peptide (SLIGRL-NH₂) or the standard PAR₂-inactive reverse sequence peptide (PAR₂; LRGILS-NH₂) (final concentration 25 µM), trypsin (final concentration 10 nM), TNF- (final concentration 10 ng/ml), heat-denatured Der p 1, recombinant ProDer p 1 (allergens used at a final concentration of 150 nM), or native Der p 3 treated with the serine proteinase inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride or aprotinin. When appropriate, A549 cells were treated for 1 h at 37°C with 25 µM of the specific MEK inhibitor U0126 (Cell Signaling) or the specific p38 MAP kinase or JNK kinase inhibitors (SB203580 and SP6001125, respectively; Calbiochem) prior to the addition of Der p 1, Der p 3, or positive controls. Negative control cells were preincubated with equivalent amounts of Me²SO. Culture supernatants were collected, centrifuged for 5 min at 10,000 x g, and stored at –80 °C. In another set of experiments, a 1-h activation of A549 cells with Der p 1, Der p 3, or PAR₂AP was followed by incubation with actinomycin D (10 µg/ml) for 2 h at 37 °C.

Measurement of IL-8 Secretion—After stimulation, cell-free supernatants were collected by centrifugation at 400 x g for 5 min and assayed for IL-8 with enzyme-linked immunosorbent assay (ELISA) kits (Pharmingen) according to the manufacturer's protocol. IL-8 concentrations were determined by interpolation from a standard curve performed with purified human IL-8.

Detection of IL-8 mRNA Expression by Reverse Transcription (RT)-PCR—Total RNA was extracted from cultured A549 cells using TriZol reagent (Invitrogen) followed by DNase treatment (RNase-free DNase; Invitrogen). cDNA was prepared with Superscript reverse transcriptase (Invitrogen). Amplification of IL-8 transcripts was accomplished with primers chosen according to the published sequence of human IL-8 cDNA (29). The housekeeping gene fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for verification of equal loading and of RT-PCR efficiency. The sequences of the primers (Invitrogen) were as follows: IL-8, sense 5'-TTGGCAGCCTTCCTGATT-3' and antisense 5'-AACTTCTCCACAACCCTCTG-3' (PCR product, 203 bp); GAPDH, sense 5'-ACCACAGTCCATGCCATCAC-3' and antisense 5'-ATGTCGTTGTCCCACCACCT-3' (PCR product, 405 bp). The PCRs were performed in a DNA thermal cycler (PerkinElmer Life Sciences 9600) with the following conditions: 25 cycles of denaturation (94 °C for 1 min), annealing (55 °C for 1 min), extension (72 °C for 1 min), and final extension for 7 min at 72 °C for GAPDH and IL-8. The intensity of the amplification bands was quantified with the NIH-Image program (version 1.62).

Transient Transfection and Luciferase Assay—The vectors encoding either the wild-type IL-8 promoter (–133/+44)/luciferase reporter, or site mutations of one of the binding sites (AP-1, NF-IL-6, or NF-B) in the IL-8 promoter region were kindly provided by Dr. N. Mukaida (Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Japan). The constructs were described in detail in the publications from Mukaida's laboratory (30). The AP-1-luc construct, driven by 12-O-tetradecanoylphorbol-13-acetate-responsive elements within the 517-bp promoter of the human collagenase gene (MMP-1-luc) (31), and the NF-B-luc reporter plasmid, containing the herpes simplex virus thymidine kinase minimal promoter and four copies of the IL-2 promoter B site, were kindly provided by Dr. P. Angel (German Cancer Research Center, Heidelberg, Germany) and Dr. C. Van Lint (Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, Belgium), respectively. A549 cells were transfected using Fugene (Roche Applied Science) with one of these vectors for 4 h and then cultured in serum-free medium for a further 16 h before the Der p 1, Der p 3, or PAR₂AP treatment for 60 min. The cells were then collected and lysed. Supernatants were evaluated for firefly luciferase activity with a luminometer. Luciferase activities were normalized with respect to protein concentration using the detergent-compatible protein assay (Bio-Rad).

Western Blotting Analysis—Treated A549 cells were washed in cold PBS, lysed with Laemmli sample buffer (60 mM Tris-HCl (pH 7.5), 2% SDS, 5% glycerol, 100 mM dithiothreitol, 0.001% bromphenol blue), and analyzed by SDS-PAGE and Western blotting with polyclonal antibodies specific for both phosphorylated and nonphosphorylated forms of ERK1/2, p38 MAP kinase, or JNK (1:1000 dilution; Cell Signaling) as described previously (41) as well as with a polyclonal antibody to IB (C-21) and p50 NF-B subunits (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The intensities of the bands on the immunoblot were quantified with the NIH-Image program (version 1.62).

Nuclear Extract Preparation—Nuclear extracts from A549 cells were prepared as previously described (32). Briefly, treated A549 cells were scraped and washed in cold phosphate-buffered saline and then resuspended in 500 µl of cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with proteinase inhibitors (Complete; Roche Applied Science). The cells, swollen at 4 °C for 10 min, were lysed in the presence of 0.4% Nonidet P-40. The homogenate was centrifuged for 5 min at 600 x g, and the nuclear pellet was resuspended in 30 µl of cold buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with proteinase inhibitors (Complete; Roche Applied Science) and left for 30 min on ice. After centrifugation (20,000 x g for 15 min at 4 °C), the resulting supernatant was diluted by the addition of 150 µl of cold buffer D (20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with proteinase inhibitors (Complete; Roche Applied Science) and then stored in small aliquots at –80 °C. Protein concentrations were determined by the Bradford method (Bio-Rad protein assay) with bovine serum albumin as a standard.

Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSAs) were performed as previously described (41). Briefly, binding sites for double-stranded oligonucleotides, NF-B(5'-CGTGGAATTTCCTCTG-3'; bp –83 to –68) and AP-1 (5'-GTGATGACTCAGGTT-3'; bp –130 to –116) from the IL-8 promoter were endlabeled with [-³²P]ATP using T4 polynucleotide kinase (Invitrogen) and were used as probes. The binding reactions were performed at room temperature. Nuclear extract (10 µg of protein) was first incubated for 20 min in the absence of probe in a 16-µl reaction mixture containing 10 µg of DNase-free bovine serum albumin (Amersham Biosciences), 1 µg of poly(dI-dC) (Amersham Biosciences) as nonspecific competitor DNA, 1 mM dithiothreitol, 20 mM Tris-HCl (pH 7.5), 60 mM KCl, 1 mM MgCl₂, 0.1 mM EDTA, and 10% (v/v) glycerol. Thirty thousand counts/min of probe was then added to the reaction mixture, and the mixture was incubated for 20 min. Samples were subjected to electrophoresis at room temperature on 6% polyacrylamide gels at 150 V for 2–3 h in 1x TGE buffer (25 mM Tris acetate (pH 8.3), 190 mM glycine, 1 mM EDTA). Gels were dried and autoradiographed at –70 °C. The intensities of the bands on the immunoblot were quantified with the NIH-Image program (version 1.62).

For supershift experiments, antibodies (1 µg/reaction) to p50 (sc-114), p52 (sc-298), p65 (sc-372), c-Rel (sc-70), c-Jun (sc-1694), Jun B (sc-8051), Jun D (sc-74), c-Fos (sc-8047), Fra-1 (sc-605), and Fra-2 (sc-171) (Santa Cruz Biotechnology) were incubated with the extracts at room temperature for 30 min before the addition of the radiolabeled probes.

PCR Cloning of the Human PAR₂ cDNA—Human PAR₂ cDNA was prepared by reverse transcription and PCR amplification of total RNA from A549 cells. PCR primer design was based on the published human PAR₂ sequence (33). The primer sequences were as follows: forward primer, 5'-AAGCTTCCACCATGCGGAGCCCCAGCGCGGCG-3' (containing a HindIII site and Kozak sequence shown in boldface type); reverse primer, 5'-GCGGCCGCTCAATAGGAGGTCTTAACAGTGGT-3' (NotI shown in boldface type). The PCR product was digested with HindIII and NotI, cloned into pcDNA3 vector (Invitrogen), and sequenced.

Transfection of HeLa Cells—HeLa cells were seeded into 6-well plates at a density of 2.5 x 10⁵ cells/well and, on the following day, transfected with 500 ng of DNA using FuGENE^TM-6 (Roche Applied Science) according to the manufacturer's protocol. At 24 h post-transfection, the cells were harvested using nonenzymatic cell dissociation solution and then seeded onto 12-well dishes or glass coverslips. On the next day, the medium of transfected cell monolayers was replaced with serum-free DMEM for 24 h before any treatment.

Immunofluorescence Confocal Microscopy—Transfected HeLa cells were grown on glass coverslips as described above. After treatments, cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The samples were saturated with PBS containing 0.5% gelatin, 0.25% bovine serum albumin, and 125 mM glycine for 1 h and stained for 1 h at room temperature with a 1:200 dilution of the anti-PAR₂ rabbit polyclonal antibody B5 (27). The samples were then washed three times with PBS containing 0.2% gelatin and incubated for 1 h with a 1:300 dilution of the secondary antibody: Alexa 546-coupled goat anti-rabbit immunoglobulin G (Molecular Probes, Inc., Eugene, OR). The samples were then washed three times in PBS with 0.2% gelatin and mounted for analysis on a Zeiss LSM510 laser-scanning confocal microscope.

Calcium Signaling Assay—Changes in intracellular calcium concentration induced by Der p 1, Der p 3, trypsin, or PAR₂AP in rat PAR₂- or human PAR₂N30A-transfected KNRK cells were measured with the intracellular calcium indicator fluo-3 acetoxymethyl ester (Molecular Probes) as previously described (34, 35). Briefly, cells grown to 95% confluence were harvested without the use of proteinase, pelleted by centrifugation, resuspended in DMEM, and loaded with fluo-3 acetoxymethyl ester at a final concentration of 22 µM (25 µg/ml). Indicator uptake was allowed to proceed for 25 min at room temperature in the presence of 0.4 mM sulfinpyrazone, after which the cells were washed two times by centrifugation and resuspended in the following buffer: NaCl (150 mM), KCl (3 mM), CaCl₂ (1.5 mM), HEPES (20 mM), dextrose (10 mM), sulfinpyrazone (0.25 mM), and L-cysteine (10 mM), pH 7.4. Fluorescence measurements, reflecting elevations of intracellular calcium, were conducted at 24 °C using an AMINCO-Bowman® Series 2 Luminescence Spectrometer (Spectronic Unicam, Rochester, NY) with an excitation wavelength of 480 nm and an emission recorded at 530 nm. The fluorescence signals caused by the addition of test agonists (Der p 1, trypsin, or PAR₂AP) were compared with the fluorescence peak height yielded by replicate cell suspensions treated with 2 µM ionophore A23187 [GenBank] (Sigma). This concentration of A23187 [GenBank] was at the plateau of its concentration-response curve for a fluorescence response. To determine if the peptide(s) released from the PAR₂ cleavage/activation sequence (P20) of PAR₂ caused a calcium signal via the activation of PAR₂, a cross-desensitizaton protocol was used (35), wherein a prior desensitization of PAR₂ by exposure of cells twice to a maximally active concentration of the PAR₂-AP (SLIGRL-NH₂, 50 µM) abolishes the calcium signal caused by a subsequent addition of a putative PAR₂-activating agonist but not by an agonist acting via a receptor other than PAR₂. In this cross-desensitization protocol, at least a 5-min time period is allowed between the sequential addition of test compounds to the cell suspension to allow for the refilling of intracellular calcium stores (35).

Proteolysis by Der p 1, Der p 3, and Trypsin of Peptide Sequences Based on the Cleavage/Activation Domain of PAR₂—Der p 1 (700 nM) was first activated as described for the assay of enzyme activity (above) and was then used to hydrolyze (25 min at room temperature) the peptide, P20 (³⁰GPNSKGRSLIGRLDTP⁴⁵yggc; 50 µM), representing the cleavage/activation sequence (boldface type) of rat PAR₂. The P20 hydrolysate was tested for the release of PAR₂-activating sequences in two ways: (a) a PAR₂ cell reporter assay and (b) HPLC and mass spectral analysis. For the PAR₂ cell reporter assay, the P20 hydrolysate was added directly to an equal volume of a test sample of fluo-3-loaded rat PAR₂-expressing KNRK cells in a calcium signaling assay (above). The presence of a calcium signal caused by the hydrolysate provided evidence for the release of a PAR₂-activating peptide from P20. For the chemical analysis, the P20 hydrolysate was subjected to HPLC analysis using a trifluoroacetic acid gradient in acetonitrile, followed by mass spectral analysis of the separated peptides. In another set of experiments, a 27-mer P27 peptide representing the cleavage/activation sequence of human PAR₂ (²⁸GTNRSSKGRSLIGKVDGTSHVTGKGVT⁵⁴-amide) was incubated at room temperature for 25 min with Der p 3 or trypsin (25 nM each) prior to HPLC analysis. Peak HPLC fractions, representing the peptides released from P20 by Der p 1 (50 µM P20, 10 nM enzyme, 10 min at room temperature) or trypsin and from P27 by Der p 3 or trypsin were collected, freeze-dried, and subjected to mass spectral analysis (matrix-assisted laser desorption ionization). Peptide sequences resulting from Der p 1, Der p 3, and trypsin hydrolysis were deconvoluted by analysis of the masses determined for each hydrolysis fragment isolated by HPLC. Since intact P20 alone does not yield a calcium signal via PAR₂ (28) and since in the P20 sequence, only peptides beginning with the SL...motif are capable of activating PAR₂, any signal generated by the hydrolysis of P20 by Der p 1 must come from a cleavage of the Arg³⁶–Ser³⁷ bond.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Der p 1 and Der p 3 stimulation of IL-8 secretion in A549 cells. Serum-starved A549 cells were incubated for 17 h with medium alone or with increasing concentrations of Der p 1 (25–250 nM) or Der p 3 (5–60 nM). An IL-8-specific ELISA was used to quantify levels of IL-8 protein in culture supernatants. All data are expressed as the mean ± S.E. from at least three independent experiments performed in duplicate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Der p 1 and Der p 3 Induce IL-8 Expression in A549 Cells—To determine whether Der p 1 and Der p 3 could enhance the synthesis of IL-8 protein in bronchoalveolar cells, A549 epithelial cells were cultured with various concentrations of Der p 1 and Der p 3 for 7 h, and IL-8 levels were measured in the supernatants by ELISA. Fig. 2 shows that activated Der p 1 and Der p 3 caused IL-8 secretion from A549 cells in a concentration-dependent manner when compared with control medium (p < 0.05). Der p 3 was shown to be more effective than Der p 1 at stimulating IL-8, since maximum responses were observed with 50 nM Der p 3 and 150 nM Der p 1, respectively (p < 0.05). Heat-inactivated Der p 1 and Der p 3 (65 °C for 30 min), proteolytically inactive Der p 1 precursor (ProDer p 1), and Der p 3 preincubated with the irreversible serine proteinase inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride were unable to cause a significant up-regulation of IL-8 production, compared with untreated cells (data not shown), confirming that the allergen-induced IL-8 expression was dependent on the proteolytic activity of Der p 1 and Der p 3.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Time course of Der p 1, Der p 3, and PAR2AP on IL-8 production. A, time course of Der p 1, Der p 3, and PAR2AP on IL-8 mRNA induction. Serum-starved A549 cells were incubated with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), or PAR2AP (25 µM) for various periods of time. Total RNA was isolated, and RT-PCR was performed to determine the IL-8 mRNA expression level. GAPDH mRNA levels were used as an internal control. B, time course of Der p 1, Der p 3, and PAR2AP on IL-8 secretion. Serum-starved A549 cells were incubated with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), or PAR2AP (25 µM) for various periods of time. Released IL-8 in the medium was measured by ELISA. Each data point represents the mean ± S.E. of at least three independent experiments performed in duplicate.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Transcriptional activation of the IL-8 gene by Der p 1, Der p 3, or PAR2AP. A, analysis of IL-8 mRNA stability in A549 cells treated with Der p 1, Der p 3, or PAR2AP. Serum-starved A549 cells were incubated with Der p 1 (150 nM), Der p 3 (50 nM), or PAR2 AP (25 µM) for 1 h before a 2-h treatment with actinomycin D (10 µg/ml) to block RNA synthesis. Total RNA was isolated, and RT-PCR was performed to determine the IL-8 mRNA expression level. GAPDH mRNA levels were used as internal controls. B, culture supernatants were collected after 17 h and assayed for IL-8 levels by ELISA analysis. Each data point represents the mean ± S.E. of at least three independent experiments performed in duplicate.

AP-1 and NF-B Cooperate to Yield Maximal IL-8 Induction by Der p 1 or Der p 3—Many studies have revealed that a sequence spanning nucleotides –1 to –133 within the 5'-flanking region of the IL-8 gene is essential and sufficient for transcriptional regulation of the gene (36–38). This region contains three cis elements important for the induction of IL-8 gene expression: AP-1 (bp –126 to 120), NF-IL6 (bp –94 to –81), and NF-B (bp –80 to –70) binding site (39, 40). To confirm that Der p 1, Der p 3, and PAR₂AP were acting transcriptionally and to identify the transcription binding sites that are important for IL-8 induction, luciferase reporter constructs containing the minimally essential wild-type IL-8 promoter region (bp –133 to +44) or mutated at the level of the binding sites for either AP-1, NF-IL-6, or NF-B, were transfected into A549 cells (Fig. 5A). The point mutations made in each of these cis elements eliminate the ability to bind to their corresponding transcription factors (37). Transfected cells were assayed for luciferase activity in response to Der p 1, Der p 3, PAR₂AP, and trypsin, a well known PAR₂ agonist (33). TNF-, a known potent inducer of IL-8 via the B sites was also used as a positive control of IL-8 induction via the B sites (41).

As shown in Fig. 5B, all of the stimuli tested increased the luciferase activity in cells transfected with the wild-type IL-8 promoter, suggesting that Der p 1 and Der p 3 as well as the enzymatic (trypsin) or the nonenzymatic (PAR₂AP) activator of PAR₂ were acting transcriptionally. Importantly, mutation in the NF-B or AP-1 binding site (NF-B mut or AP-1 mut) resulted in a significant reduction of IL-8 gene induction by all of the tested activators (p < 0.05) (Fig. 5B). In contrast, mutation of NF-IL-6 site (NF-IL-6 mut) failed to reduce the promoter activation by Der p 1, Der p 3, PAR₂AP, trypsin, or TNF-.

These data suggest that the presence of intact B and AP-1 binding sites are both necessary for maximal IL-8 induction by Der p 1 and Der p 3 as well as in response to PAR₂ activation.

Der p 3 and PAR₂AP Increase NF-B and AP-1 Binding Complexes, whereas Der p 1 Induces Mainly the NF-B Complex—To evaluate the effects of Der p 1 and Der p 3 as well as PAR₂AP on AP-1 and NF-B DNA binding activity, nuclear extracts were prepared from A549 cells either untreated or treated with Der p 1, Der p 3, or PAR₂AP for different periods of time (10, 30, 60, and 120 min). A 30-min treatment of A549 cells with TNF-, a known activator of NF-B, was also used as a positive control for NF-B activation in A549 cells. All of these samples were subjected to EMSA analysis using a DNA probe containing either the AP-1 or the B binding site of the human IL-8 promoter (Fig. 6).

As previously reported (42), AP-1 was constitutively expressed in unstimulated A549 cells. Incubation of A549 cells with Der p 1 for various periods of time ranging from 10 to 120 min caused only a marginal increase in the DNA binding activity of AP-1 at 30 and 60 min that persisted at lower levels at 120 min (Fig. 6A). In contrast, Der p 3 rapidly and strongly affected the AP-1 binding activity, which was already increased at 10 min, gradually rose to a maximum at 1 h, and persisted for at least 2 h after cell stimulation (p < 0.05). Interestingly, a similar pattern of increase in AP-1 binding was demonstrated with A549 cells stimulated with the PAR₂AP (Fig. 6A). The specificity of binding of AP-1 was confirmed by supershift assays. The data revealed that the AP-1 complex consists mainly of c-Fos and JunB and, to a lesser extent, JunD, c-Jun, and Fra-2 (Fig. 6B). The AP-1 binding complex was induced slightly by Der p 1 and more significantly by either Der p 3 or PAR₂AP.

The kinetics of NF-B binding was quite different compared with AP-1 in A549 cells. Der p 1, Der p 3, and PAR₂AP caused a comparable and significant NF-B nuclear translocation that was seen only after 30 min post-stimulation, peaked at 60 min, and persisted at lower levels at 120 min (p > 0.05) (Fig. 6A). However, TNF- was consistently a more effective inducer of NF-B binding activity compared with the other three inducers. Supershift experiments using specific antibodies demonstrated that the NF-B complexes were mainly composed of p50/p65 and p52/p65 heterodimers (Fig. 6B). Since the proteolytic degradation of IB subunit precedes the translocation of NF-B to the nucleus, we determined the effect of Der p 1, Der p 3, and PAR₂AP on the protein levels of IB in the cytoplasm of A549 cells treated with each of these three stimuli for times ranging from 10 to 120 min. Western blotting showed that Der p 1, Der p 3, and PAR₂AP induced a slight decrease in the IB level between 30 and 60 min and a complete restoration of the IB levels after 120 min of cell stimulation according to their respective NF-B nuclear levels observed by EMSA analysis (Fig. 6, compare C and A). As expected, a complete degradation of IB was induced by 30 min in response to TNF-.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. The effect of NF-B, AP-1, and NF-IL-6 binding site mutation on the IL-8 promoter activity in response to Der p 1, Der p 3, or PAR2 agonists. A, A549 cells were transiently transfected with luciferase reporter constructs containing the –133 to +44 fragment (WT) or its mutant forms with each of the three transcription factor-binding sites individually destroyed (NF-B mutation (NF-kB-mut), AP-1 mutation (AP-1-mut), and NF-IL-6 mutation (NF-IL6-mut)). B, cells were treated with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), PAR2AP (25 µM), trypsin (20 nM), or TNF- (10 ng/ml) for 6 h, and cellular lysates were tested for luciferase activity. RLU, relative luciferase units. Each data point is expressed as the mean ± S.E. from at least three independent experiments performed in duplicate.

Der p 3 and PAR₂AP Induce NF-B and AP-1 Transcriptional Activity, whereas Der p 1 Induces Mainly NF-B Transcriptional Activity—Nuclear binding of transcription factors provides indirect evidence for their involvement in regulating IL-8 gene activation. Up to this point, we found that the presence of intactB and AP-1 binding sites is necessary for maximum IL-8 induction by Der p 1 and Der p 3 as well as for the response to PAR₂ activation. We next determined whether Der p 1, Der p 3, or PAR₂AP can indeed stimulate AP-1 and NF-B-mediated transcriptional activity in A549 cells. To this end, A549 cells were transfected with AP-1-luc or NF-B-luc luciferase constructs. The NF-B-luc synthetic reporter construct contained four copies of the interleukin 2 promoter B sites in front of the minimal thymidine kinase promoter from the herpes simplex virus (32), whereas the AP-1-luc construct is driven by 12-O-tetradecanoylphorbol-13-acetate-responsive elements present within the 517-bp promoter of the human collagenase gene (31). Transfected cells were assayed for luciferase activity in response to Der p 1, Der p 3, PAR₂AP, and trypsin. TNF- was used as a positive control for NF-B activation.

As shown in Fig. 7, Der p 3, PAR₂AP, and trypsin significantly induced luciferase activity in A549 cells transfected with NF-B-luc or AP-1 luc (p < 0.05). By contrast, Der p 1, like TNF-, was able to induce significantly NF-B-driven luciferase activity. These results corroborated our EMSA data, namely that Der p 3, trypsin, and PAR₂AP increase the transcriptional activities of both NF-B and AP-1, whereas Der p 1 triggers mainly NF-B-dependent gene expression and very slightly AP-1-dependent gene expression.

Taken together, our results suggest that Der p 3 and PAR₂AP regulate the expression of the IL-8 gene through the induction and the activation of both of the transcription factors, NF-B and AP-1, whereas Der p 1-mediated IL-8 expression appears to depend mainly on the activation of NF-B in conjunction with some constitutive AP-1 activity.

Der p 1 and Der p 3 Stimulate ERK1/2, JNK, and p38 MAP Kinases in A549 Cells—Many studies indicate that IL-8 production results mainly from the activation of MAPK signaling cascades, including the ERKs, JNK, and p38 MAP kinases (reviewed in Ref. 25). To understand better the activation of NF-B and AP-1 and the subsequent production of IL-8, we next examined the effect of Der p 1, Der p 3, and PAR₂AP on ERK, JNK, and p38 MAPK activation using a Western blot approach with phosphospecific antibodies that recognize the phosphorylated active forms of these kinases.

As shown in Fig. 8, Der p 1 induced a slightly delayed (not seen at 10 min after stimulation) but marked increase in the phosphorylation of both isoforms of ERK. The Der p 1-mediated activation of ERK1/2 was clearly evident at 30 min, was maximal at 60 min, and began to decline at 120 min. In contrast, treatment of A549 cells with Der p 3, PAR₂AP, or trypsin (data not shown) caused a much more rapid activation of ERK1/2 than did Der p 1 (p < 0.05). ERK1/2 activation was already maximal at 10 min and persisted for up to 120 min (Fig. 8). TNF- also induced a strong phosphorylation of ERK1/2 that was clearly evident as early as 10 min, was maximal at 60 min, and began to decline after 60 min. Pretreatment of cells with the specific MEK inhibitor U0126, which blocks ERK1/2 activation, fully inhibited the increased phosphorylation of ERK1/2 caused by all four agonists (data not shown). To determine whether this induction of MAP kinases was specific to the ERK pathway, all samples were also tested for the activation/phosphorylation of p38 MAPK and JNK. Interestingly, as shown in the bottom panels of Fig. 8, Der p 1, Der p 3, PAR₂AP, and trypsin (data not shown) all induced a transient but very weak increase in the phosphorylation of p38 MAPK and JNK that could be visualized within 30 min of treatment but was no longer detected at 120 min. A much more pronounced activation of p38 MAPK and JNK was observed after TNF- treatment. p38 MAPK and JNK activation induced by TNF- was observed as early as 10 min and persisted up to 60 min (Fig. 8). These data indicated that, whereas Der p 1, Der p 3 and PAR₂AP were able to activate ERK1/2, and to a lesser extent, p38 MAPK and JNK in A549 cells, a more rapid and persistent activation of ERK1/2 was induced by both Der p 3 and PAR₂AP compared with Der p 1.

Inhibition of ERK1/2 Inhibits the Production of IL-8 in Response to Der p 1, Der p 3, and PAR₂AP—Using specific pharmacological inhibitors, we assessed the role of ERK, p38 MAPK, and JNK activation on the production of IL-8 from A549 cells in response to Der p 1, Der p 3, or PAR₂AP. Pretreatment of cells with U0126 (a MEK1/2 inhibitor) for 1 h markedly reduced the Der p 1-, Der p 3-, and PAR₂AP-induced IL-8 production (Fig. 9). On the other hand, the blockade of p38 MAPK or JNK pathways by SB203580 (a p38 MAPK inhibitor) or SP600125 (a JNK inhibitor), respectively, failed to affect the secretion of IL-8 in response to Der p 1 but reduced the up-regulation of IL-8 caused by Der p 3 or PAR₂AP, respectively. Importantly, pretreatment of A549 cells with each of these three inhibitors reduced the IL-8 secretion induced in response to TNF- by more than 50% (data not shown). These data thus indicated that, among the three MAP kinases analyzed, ERK1/2 appears to be the only one involved in the enhanced production of IL-8 in response to Der p 1. However, for Der p 3 and PAR₂AP, in addition to the important contribution of ERK1/2, p38 MAPK (for PAR₂AP) and JNK (for Der p 3) also appear to play a role in the signaling pathways leading to the increased production of IL-8.

View larger version (59K): [in this window] [in a new window]   FIGURE 6. EMSA analysis of NF-B and AP-1 binding activity after Der p 1, Der p 3, or PAR2 stimulation of A549 cells. A, nuclear extracts were prepared from serum-starved A549 cells incubated for various periods of time with either medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), PAR2AP (25 µM), or TNF- (10 ng/ml). An oligonucleotide corresponding to the AP-1 binding site of the human IL-8 promoter or theB binding site of the human IL-6 promoter was used as the probe. Samples were assayed for NF-B and AP-1 DNA binding activity as outlined under "Experimental Procedures." Similar results were obtained in two other independent experiments. B, supershift assays. Nuclear extracts from A549 cells treated for 1 h with Der p 1 (150 nM), Der p 3 (50 nM), or PAR2AP (25 µM) were incubated either with the human IL-8 promoter human AP-1 binding site oligonucleotide probe or with the IL-6 promoter B binding site oligonucleotide probe. Next, antibodies directed against different members of the AP-1 or the NF-B families were added to the binding reaction. C, Western blot analysis of levels of cytoplasmic IB. Cytoplasmic extracts were prepared from serum-starved A549 cells incubated for various periods of time with either medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), PAR2AP (25 µM), or TNF- (10 ng/ml). Equal amounts of proteins were analyzed by Western blotting with anti-IB or anti-p50 NF-B antibodies.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. The effect of Der p 1, Der p 3, and PAR2 stimulation on the activation of NF-B and AP-1 transcription factors. A549 cells were transiently transfected with the indicated luciferase reporter constructs. Cells were treated with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), PAR2AP (25 µM), trypsin (20 nM), or TNF (10 ng/ml) for 6 h, and cellular lysates were tested for luciferase activity. RLU, relative luciferase units. Each data point is expressed as the mean ± S.E. from at least three independent experiments performed in duplicate.

Der p 1 Did Not Induce Changes in Intracellular Calcium in PAR₂-expressing Cells—Previous studies have shown that PAR₂ activation causes an increase in intracellular calcium (35). Thus, monitoring changes in intracellular Ca²⁺ provides a useful method to test the effectiveness and potency of different agonists to activate PAR₂ (27, 28, 35). Reliable PAR₂ calcium signaling assays have been previously developed using stably transfected PAR₂-expressing KNRK cells (27). KNRK cells stably expressing PAR₂ were thus used to monitor changes in calcium signaling upon activation by Der p 1 and Der p 3, in comparison with trypsin and the PAR₂AP (Fig. 11).

View larger version (41K): [in this window] [in a new window]   FIGURE 8. The effect of Der p 1, Der p 3, and PAR2AP on MAPK activation. Serum-starved A549 cells were incubated with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), PAR2AP (25 µM), or TNF- (10 ng/ml) for various periods of time. Equal amounts of proteins were analyzed by Western blotting with the anti-phospho-ERK1/2, anti-phospho-p38, or anti-phospho-JNK antibodies. Blots were reprobed with anti-ERK1/2, anti-p38, or anti-JNK to verify equal loading.

View larger version (18K): [in this window] [in a new window]   FIGURE 9. Requirement of MAP kinases for IL-8 production in response to Der p 1, Der p 3, and PAR2AP. Serum-starved A549 cells were pretreated with either U0126 (25 µM) (ERK1/2 inhibitor), SB203580 (25 µM) (p38 MAPK inhibitor), or SP600125 (25 µM) (JNK inhibitor) for 2 h, followed by treatment with medium alone or with Der p 1 (150 nM), Der p 3 (50 nM), or PAR2AP (25 µM) for 17 h. An IL-8-specific ELISA was used to quantify levels of IL-8 protein in culture supernatants. Each data point is expressed as the mean ± S.E. from at least three independent experiments performed in duplicate.

To ascertain whether Der p 1 has the ability to "disarm" subsequent PAR₂ activation by the proteinases trypsin or Der p 3, cross-desensitization experiments were performed. It was found that pretreatment of PAR₂-expressing KNRK cells with Der p 1 (533 nM) failed to decrease the calcium signal due to a subsequent exposure to trypsin (2 nM) or Der p3 (5 nM) that acted via PAR₂, as compared with cells treated only with trypsin (2 nM) or Der p 3 (5 nM)(n = 3), but without prior exposure to Der p 1 (data not shown). In addition, in two independently conducted experiments, co-incubation of Der p 1 (533 nM) with Der p 3 (5 nM) for 25 min before adding the Der p 1-treated Der p 3 to PAR₂-expressing KNRK cells did not diminish the calcium response due to Der p 3 (5 nM) activation of PAR₂; the signal was virtually the same as that for cells treated with Der p 3 that had not previously been exposed to Der p 1 (5 nM) (data not shown). Thus, not only was Der p 1 incapable of signaling via PAR₂ in these cells, but, in an intact cell system, this cysteine proteinase was not able either to disarm PAR₂ for Der p 3 activation or to inactivate Der p 3 enzymatic ability to trigger PAR₂ signaling in these cells.

To determine whether glycosylation of the N-terminal sequence of PAR₂ just prior to the cleavage/activation site might explain the inability of Der p 1 to activate PAR₂, as previously shown for tryptase (28), calcium mobilization assays were performed using KNRK cells expressing a mutated glycosylation-deficient human PAR₂ receptor, where asparagine 30 at the N-terminal glycosylation site was substituted with alanine (hPAR₂N30A KNRK cells) in order to disrupt the glycosylation sequon. Although both PAR₂AP and trypsin caused a prompt increase in intracellular calcium in the hPAR₂N30A cells, no calcium response was induced by Der p 1, indicating that the absence of PAR₂ glycosylation did not enable Der p 1 to activate PAR₂ (Fig. 11D).

Der p 1 and Der p 3 Cleavage Sites of PAR₂-derived Cleavage/Activation Sequences—Since the calcium signaling experiments with PAR₂-expressing cells failed to demonstrate any appreciable activation of PAR₂ with Der p 1, we wondered if the enzyme was indeed capable of hydrolyzing the N-terminal sequence of PAR₂. Therefore, we used both a biochemical and a functional PAR₂ cell reporter calcium signaling assay to evaluate the ability of Der p 1 to cleave 20 (P20) (GPNSKGRSLIGRLDTPyggc) and 27 (P27) (GTNRSSKGRSLIGKVDGTSHVTGKGVT) amino acid peptides containing the sequence corresponding to the cleavage/activation site of rat and human PAR₂, respectively. Upon enzymatic hydrolysis, the resulting cleavage products were characterized by HPLC and mass spectral analysis and compared with those peptides obtained after trypsin treatment.

Whereas trypsin (10 nM) cleaved >90% of rat P20 (50 µM) during a 10-min incubation period at room temperature, Der p 1 (700 nM) cleaved 60% of P20 upon incubation for 25 min at room temperature. The only detectable hydrolysis product made by trypsin corresponded to SLIGRLDTPYGGC, a sequence that can, on its own, activate PAR₂. This relatively potent PAR₂-activating hydrolysis product was not identified as one of the proteolysis products yielded by Der p 1. However, Der p 1 hydrolysis did yield minor amounts of the shorter peptide SLIGR (Fig. 12A). In previous work, we had determined that the sequence SLIGR is capable of activating PAR₂ (44) but with very low potency. Thus, upon hydrolysis of P20, Der p 1 was not, in contrast with trypsin, capable of producing a peptide that can activate PAR₂ with high potency. Interestingly, the two most abundant hydrolysis products generated by Der p 1 (based on HPLC peak area at 210 nm) corresponded to GPNSKGRSLIGR (rat PAR₂ residues 30–41) and LDTPyggc (rat PAR₂ residues 42–45 plus the added coupling sequence), neither of which would be able to activate PAR₂. Therefore, in the synthetic tethered ligand peptide, Der p 1 preferentially cleaves after PAR₂ arginine residues 36 and 41 in a manner that would disarm the receptor rather than causing activation. Another hydrolysis product observed in small amounts, IGRLDTPYGGC, indicated that Der p1 can also cleave the P20 peptide after leucine residue 38. Thus, the data showed that Der p 1 was able to hydrolyze the rat PAR₂ sequence at its cleavage/activation site less efficiently than trypsin and that it would preferentially disarm the intact receptor, rendering it insensitive to trypsin activation. Of the peptides generated by Der p 1, only SLIGR would be capable of activating PAR₂, albeit with very low potency.

View larger version (40K): [in this window] [in a new window]   FIGURE 10. Comparison of IL-8 release induced by Der p 1, Der p 3, PAR2AP, or trypsin in HeLa cells transfected or not with a human PAR2-expressing vector. A, confocal microscopy was used to analyze the expression of PAR2 in HeLa cells transfected with a human PAR2-expressing vector (A, immunofluorescence; B, phase) or with the empty vector (HeLa) (C, immunofluorescence; D, phase). Indirect immunofluorescence detection was performed with the polyclonal antibody anti-PAR2 (B5). Cells were fixed and stained as described under "Experimental Procedures." B, serum-starved HeLa cells transfected with a PAR2 expression vector (HeLa-PAR2) or with the empty vector (HeLa) were left untreated or were exposed for 7 h to Der p 1 (150 nM), Der p 3 (50 nM); PAR2AP (25 µM), or trypsin (20 nM). Supernatants were harvested, and the concentrations of IL-8 were quantified using ELISA. Data represent the means ± S.E. of at least three independent experiments performed in duplicate.

View larger version (15K): [in this window] [in a new window]   FIGURE 11. Der p 1- and Der p 3-mediated calcium signaling (increased E530 fluorescence) in suspensions of rPAR2-transfected KNRK cells. A, the left-hand side of the trace shows wt-rPAR2 cells treated with Der p 1 (; 233 nM) followed by the PAR2AP SLIGRL-NH2 (; 25µM). A control cell sample treated with SLIGRL-NH2 (; 25µM) is shown on the right-hand side of the trace. B, the left-hand side of the trace shows wt-rPAR2 cells treated with Der p 3 (; 10 nM), followed by the PAR2-activating peptide SLIGRL-NH2 (; 2.5 µM). Control cell samples treated with SLIGRL-NH2 (; 2.5 µM) and trypsin (; 10 nM) are shown in the middle and right traces, respectively. C, wt-rPAR2 cells exposed to receptor-desensitizing concentrations of the PAR2-activating peptide SLIGRL-NH2 (; 50 µM) before treatment with Der p 3 (; 10 nM). D, the left-hand side of the trace shows hPAR2N30A cells treated with Der p 1 (; 350 nM) followed by the PAR2-activating peptide SLIGRL-NH2 (; 25 µM). Control cell samples treated with SLIGRL-NH2 (; 25 µM) and trypsin (; 10 nM) are shown in the middle and right traces, respectively.

View larger version (13K): [in this window] [in a new window]   FIGURE 12. Der p 1 and Der p 3 cleavage sites of PAR2-derived cleavage/activation sequences. A, cleavage of rat PAR2 P20 by trypsin (upper arrow) and Der p 1 (lower arrows). The P20 peptide representing the cleavage/activation sequence of rat PAR2 was subjected to proteolysis by either Der p 1 or trypsin as outlined under "Experimental Procedures," and the resulting peptides were isolated by HPLC and identified by mass spectral analysis. The boldface and dotted arrows indicate the major and minor cleavage sites, respectively, for Der p 1. Only one major hydrolysis site was detected for trypsin under the same conditions (upper arrow). B, the left-hand side of the trace shows the calcium response of wt-rPAR2 cells treated with the hydrolysis products resulting from incubating Der p 1 (350 nM) with a peptide (25 µM) containing residues 30–45 of the rat PAR2 sequence (P20; contains the PAR2 cleavage/activation sequence); the PAR2AP SLIGRL-NH2 (; 1 µM) was then added to the same cell suspension. The right-hand trace shows a control cell sample treated with SLIGRL-NH2 (; 1 µM). C, wt-rPAR2 cells were exposed to receptor-desensitizing concentrations of the PAR2AP SLIGRL-NH2 (; 50 µM) before treatment with the hydrolysis products () resulting from preincubating Der p 1 (350 nM) with P20 (25 µM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the respiratory system, PAR₂ is expressed in the epithelium and is considered to play both a proinflammatory and a bronchodilator role (45, 46). In the human alveolar epithelial A549 cells, activation of PAR₂ has been observed to trigger the secretion of several inflammatory mediators, such as IL-8, IL-6, prostaglandin E₂, matrix metalloproteinase-9, and GM-CSF (48, 49). A comparable effect of PAR₂ activation has also been observed in part in primary bronchial epithelial cells (17). However, the intracellular signaling pathways (other than the G_q/11-mediated activation of phospholipase C) that may regulate PAR₂-induced release of these inflammatory mediators were not evaluated in any detail in those studies. Here, we confirmed that stimulating A549 cells with agonists of PAR₂ induced IL-8 secretion. We demonstrated further that the secretion results from the up-regulation of IL-8 mRNA (in a time-dependent manner) that was mainly dependent on new gene transcription, since the up-regulation of IL-8 mRNA was significantly reduced by actinomycin D. Consistent with this result, transfection studies with a wild-type IL-8 luciferase reporter construct showed that the IL-8 promoter was transcriptionally activated in response to PAR₂ agonists (PAR₂AP, trypsin). Mutational analysis further identified the NF-B and AP-1 binding sites as the essential cis elements involved in IL-8 gene transcription in response to PAR₂ activation in A549 cells. Importantly, our data showed that PAR₂ stimulation led to activation of both NF-B and AP-1 transcription factors, as shown by EMSA and transfection experiments. NF-B binding to the IL-8 promoter peaked at 1 h, which paralleled the time course of degradation of IB. Supershift analysis identified the PAR₂-induced translocated NF-B complexes as p65/p50 and p65/p52 heterodimers, p65 being the major NF-B subunit that regulates IL-8 transcription (24, 49). Interestingly, this time course for PAR₂ induced NF-B activation has also been described in other cell types in response to both trypsin and PAR₂ agonist peptides (50, 51). The kinetics of PAR₂-triggered activation of AP-1 was quite different compared with NF-B. PAR₂ activation (PAR₂AP, trypsin (data not shown)) rapidly and strongly increased the AP-1 binding activity, which was readily detected at 10 min, gradually increased to be maximal at 1 h, and persisted thereafter for at least 2 h. The AP-1 complex induced in response to PAR₂ activation consisted mainly of c-Fos, JunB, and Jun D and, to a lesser extent, c-Jun and Fra-2. Interestingly, c-Fos and Jun D are also the main components of the IL-8 promoter AP-1-DNA binding complex induced by the serine protease coagulation factor VIIa in keratinocytes (52, 53). Thus, in airway epithelial cells, both NF-B and AP-1 transcription factors were activated by PAR₂ stimulation and apparently functioned together to result in IL-8 up-regulation. We believe this is the first report that documents a functional cooperative role for both NF-B and AP-1 transcription factors in IL-8 up-regulation in response to PAR₂ activation in this cell type. Hence, we suggest that the expression of other NF-B and AP-1 target proinflammatory genes (such as IL-6, GM-CSF, COX-2, and iNOS) may also be affected in a similar manner by PAR₂ stimulation.

PAR₂ stimulation has been shown to activate members of the MAP kinase signal transduction family in a way that differs, depending on the cell type affected and the nature of the stimulus (54). In keeping with this diversity of activation of the MAP kinases, we found that PAR₂ agonists induced the phosphorylation/activation of all three MAP kinases (ERK1/2, p38 MAPK, and JNK) in A549 cells. However, the kinetics and extents of activation were quite different for the three kinases. Treatment of A549 cells with the PAR₂AP (or trypsin; data not shown) caused a transient but modest increase in both p38 MAPK and JNK phopsphorylation/activation compared with the persistent and markedly increased levels of phosphorylation/activation of both kinases evoked by TNF-. In contrast, PAR₂-mediated activation of ERK1/2 was strong, rapid in onset (as early as 10 min), and sustained (lasting up to 2 h). The timing of ERK1/2 activation was exactly in step with the appearance of AP-1 binding activity. Using selective MAP kinase inhibitors, we found that inhibition of ERK1/2 and p38 MAPK each decreased IL-8 induction in response to PAR₂ activation, whereas inhibition of JNK appeared to increase the induction of IL-8. The observed requirement of ERK1/2 activation for maximal PAR₂-mediated IL-8 expression may relate to the role of ERK1/2 in AP-1 transactivation. Activation of ERK1/2 signaling has indeed been shown to induce phosphorylation and an increase in the trans-activating activity of the AP-1 transcription factors c-Fos, Jun D, and c-Jun (55, 56). We found that these very same components were part of the PAR₂-induced AP-1-DNA binding complex. Cross-talk between the activation of MAP kinases and NF-B may also occur in a number of settings. For example, in murine fibrosarcoma cells, p38 MAPK and ERK1/2 have both been reported to be required for NF-B p65 transactivation mediated by TNF- (57). JNK has also been described as a potential upstream activator of NF-B (and AP-1) in the regulation of airway epithelial cell IL-8 expression (58). The MAP kinase family is also thought to regulate other steps in the overall process of gene expression, such as chromatin remodeling (59) or in regulating IL-8 mRNA stability (60, 61). Additional studies are thus needed to elucidate the precise mechanism by which ERK1/2, p38, and JNK participate to regulate NF-B and AP-1 transcriptional activity in airway epithelial cells stimulated with PAR₂ agonists.

All of the signaling events described above, as triggered by trypsin and the specific PAR₂AP, were reproduced exactly in timing and extent by treating A549 cells with the mite serine proteinase, Der p 3. As such, our data are entirely consistent with the study of Sun et al. (15), which reported that PAR₂ is one potential target for signaling by Der p 3. However, our study significantly extends these observations by demonstrating that the specific cellular signal transduction pathways linking Der p 3-mediated PAR₂ proteolysis/activation to the up-regulation of IL-8 production involve signaling via the MAP kinases ERK1/2, p38 MAPK, and JNK to activate the transcription factors NF-B and AP-1.

A key question to ask relating to the action of Der p 1, in contrast with Der p 3, is, "Does the mite-derived cysteine proteinase Der p 1 cause the elevation of IL-8 by stimulating PAR₂, a receptor activated by various serine proteinases including Der p 3?" Our data are entirely consistent with previous work showing that Der p 1 triggers the release of IL-8 from A549 cells in a concentration- and time-dependent manner (14). However, in a number of respects, our results raise doubt as to whether Der p 1 acts via PAR₂ activation to increase IL-8 production. First, our data show that although Der p 1 stimulates the transcriptional activity of the IL-8 gene in A549 cells, the magnitude of stimulation by this allergen is much lower than that of either the PAR₂AP or Der p 3 (which we believe does act via PAR₂ cleavage/activation). Further, the duration of the increase in IL-8 mRNA expression resulting from Der p 1 activation, although smaller in magnitude, was much more prolonged, compared with that triggered by the PAR₂AP or Der p 3. Second, although the Der p 1-induced IL-8 mRNA up-regulation relies also primarily on binding sites for NF-B and AP-1, Der p 1 triggers mainly NF-B activation and only to a minor degree on AP-1 activation. Indeed, in agreement with the data reported by Stacey et al. (16), we showed that Der p 1 activates NF-B (consisting of p65/p50 and p65/p52 heterodimers) with a maximum obtained after 60 min. However, in contrast with PAR₂ agonists, Der p 1 induces only a weak delayed recruitment of AP-1 transcription factors (mainly Jun B and Jun D) to the IL-8 promoter. In view of the study realized by Hoffmann et al. (25) on IL-1- and epidermal growth factor-induced IL-8 expression, this result with Der p 1 may explain the low amounts of IL-8 release induced by Der p 1 compared with PAR₂ agonists. Indeed, according to these authors (25), the IL-8 gene behaves like an immediate early gene. In this respect, low, constitutively expressed amounts of AP-1 proteins in conjunction with the stimulus-induced nuclear translocation of p65 NF-B might be responsible for IL-8 transcription within the first 30 min of treatment. Thereafter, de novo synthesis of c-Fos and various Jun proteins might serve to maintain high levels of mRNA synthesis. In the context of Der p 1 treatment, the AP-1 site appears thus to function principally as a basal level enhancer (using the constitutive presence of AP-1 in the nucleus), since Der p 1 induces only a weak delayed recruitment and activation of AP-1 transcription factors (mainly Jun B and Jun D). This situation has already been described for TNF- (review in Ref. 24). In contrast, PAR₂ activation induces a strong and rapid increase in the AP-1 DNA binding complex (in response to PAR₂AP or Der p 3). The complex, composed mainly of c-Fos, Jun D, and Jun B, would contribute to the generation of maximal IL-8 amounts in response to PAR₂ activation. Third, as opposed to the actions of PAR₂AP (or trypsin; data not shown), for Der p 1, ERK1/2 appears to be the principal MAP kinase involved in the up-regulation of IL-8. Further, the time course of ERK1/2 activation (over 30 min) is very distinct from the kinetics of PAR₂-mediated ERK1/2 stimulation (markedly increased after 10 min). Finally, the data obtained with cells (HeLa or KNRK) that did or did not express PAR₂ revealed that Der p 1 was unable to induce either calcium mobilization or IL-8 up-regulation via PAR₂ activation, in clear contrast with the actions of either the PAR₂AP or trypsin. That said, our data did show that in HeLa cells that do not express PAR₂, Der p 1 was nonetheless able to increase IL-8 production (albeit modestly) ostensibly via a non-PAR₂ mechanism that has yet to be elucidated.

The experiments discussed above indicate that Der p 1 can stimulate IL-8 production via a mechanism that does not involve PAR₂. However, we do not exclude the possibility that Der p 1 could cleave PAR₂ in certain circumstances. In this regard, we did find that the polypeptide sequence representing the cleavage/activation site of rat (but not human) PAR₂ can be cleaved by Der p 1 to yield a peptide sequence that in principle might be capable of activating PAR₂ (Fig. 12A), albeit with very low potency. Thus, when present, rat PAR₂ may be subject to a low level of proteolysis by Der p 1. However, when the cells are directly incubated with Der p 1, that low level of activation does not appear to be sufficient to cause an elevation of intracellular calcium. Moreover, it must be pointed out that, although Der p 1 can mainly cleave the synthetic P20 and P27 peptides in vitro, in a manner that in theory would predict a disarming of the receptor rather than causing activation, cross-desensitization experiments using intact PAR₂-expressing KNRK cells indicated that a prior treatment of cells with Der p 1 does not impair the ability of the cells to respond subsequently to Der p 3, trypsin or PAR₂AP. This lack of a disarming action of Der p 1 in intact cells is in keeping with the lack of ability of trypsin to cleave at a potential arginine target residue downstream from the cleavage/activation site (67). It is likely that the Arg⁴¹–Leu⁴² bond in the tethered ligand sequence of rat PAR₂, which is accessible to proteolytic cleavage in the soluble synthetic peptide in vitro, is rendered cryptic in the sequence of the intact receptor, as expressed at the cell surface. Further, it is evident that although Der p 1 can cleave synthetic rat PAR₂-derived peptides at potential activation/disarming sites, this cleavage does not appear to happen when the receptor is present in intact cells.

Receptor glycosylation in the region of the N-terminal cleavage/activation site of the receptor that regulates its activation by tryptase (28) cannot be a factor accounting for the inability of Der p 1 to activate PAR₂, since the receptor construct lacking the N-terminal glycosylation site was as resistant as was the wild type receptor in terms of the lack of ability of Der p 1 to cause a calcium signal in the cells. Trypsin, however was fully active in this regard in the glycosylation-deficient receptor, indicating that if sufficient proteolysis had been caused by Der p 1, a calcium signal should have been generated. Apart from a single previous study pointing to the activation of PAR₂ by Der p 1 (17), very few studies have demonstrated activation of PAR₂ by other cysteine proteinases, except for those like the arginine-specific (trypsin-like) cysteine proteinases (gingipains) produced by P. gingivalis (62, 63). Interestingly, in a study showing that trypsin can activate eosinophils via PAR₂, the effects of papain, a cysteine proteinase archetype that shares a structural motif including the catalytic triad with Der p 1 but is unlike the gingipains, appeared to act independently of PAR₂ (64). Thus, some cysteine proteinases, including Der p 1, appear to be able to activate cells via a PAR₂-independent pathway.

We are not able to explain the differences between our results and those of Asokananthan et al. (17), who provided evidence for an activation of PAR₂ by Der p 1. Unfortunately, the precise specific activity and Western blot analysis of the Der p 1 protein used in that work were not recorded in that paper. A putative contamination of the cysteine protease Der p 1 preparations with serine proteases, such as Der p 3, 6, or 9, could be a contributing factor to explain the discrepancy between our current findings and those of earlier studies (14, 17). Indeed, co-purification of serine proteinase activity with Der p 1 cysteine proteinase by both affinity purification using the monoclonal antibody-coupled column and by active site affinity purification has been reported by several studies dealing with Der p 1 isolated from natural sources (5, 6, 65) as well as for commercially available natural Der p 1 (66).

In our study, we ascertained the purity of our Der p 1 preparations by SDS-PAGE, Western blot analysis, and mass spectrometry in order to verify the absence of contaminating Der p 3 or other proteinases (Fig. 1). In other respects, some of the data previously reported for the action of Der p 1 via PAR₂ (14) appear somewhat contradictory. In one study by those investigators (12), it was shown that Der p 1 and Der p 9 induce the release of GM-CSF, IL-6, and IL-8 in bronchial epithelial cells. However, it was suggested in that study that these two mite allergens act presumably via different receptors (potentially PARs), since the calcium response generated in the BEAS-2B target cells by Der p 1 treatment (Fig. 6 in Ref. 12) did not cross-desensitize a subsequent calcium signal generated in the same cells with Der p 9 (12). It has recently been demonstrated that Der p 9 as well as Der p 3 induce phosphoinositide hydrolysis, transient cytosolic calcium mobilization, and release of GM-CSF and eotaxin in A549 cells by activating PAR₂ (15). Taken together, these two reports (12, 14) would appear to be somewhat self-contradictory, with one study suggesting that Der p 1 causes its effects via a receptor distinct from PAR₂ in BEAS-2B cells (i.e. the Der p 9-activated receptor) (12), whereas the second study suggested that Der p 1 does indeed act via PAR₂ (14). We suggest that the data we report here support the hypothesis that Der p 1 can act via a receptor distinct from PAR₂ to induce an up-regulation of IL-8.

In summary, our study has delineated the transcription factors and the MAP kinases responsible for PAR₂-activation-mediated IL-8 production in airway epithelial cells. We further confirm that the mite allergen Der p 3 induces IL-8 secretion via PAR₂ activation. Finally, we furnish evidence supporting the idea that, relative to trypsin and Der p 3, the cysteine proteinase Der p 1 is not an effective activator of PAR₂ and is unlikely to induce IL-8 release via PAR₂ cleavage. Although the Der p 1 target remains to be defined, we show that the regulation of IL-8 secretion in response to Der p 1 can be mediated at least in part through a PAR₂-independent pathway that triggers the activation of both ERK1/2 MAP kinase and the IL-8 transcription factor, NF-B.

	FOOTNOTES

 
^* This work was supported in part by GlaxoSmithKline (Rixensart, Belgium), by the Walloon Region (Direction Générales des Technologies, de la Recherche, et de l'Energie) of Belgium, by a term grant from the Canadian Institutes of Health Research (to M. D. H.), and by a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (to K. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Applied Genetics, Rue des Professeurs Jeener et Brachet, 12, B-6041 Gosselies, Belgium. Tel.: 32-2-650-99-09; Fax: 32-2-650-99-00; E-mail: alain.jacquet{at}ulb.ac.be.

² The abbreviations used are: IL, interleukin; GM-CSF, granulocyte macrophage-colony stimulating factor; RANTES, regulated on activation normal T cell expressed and secreted; PAR, proteinase-activated receptor; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; PAR₂AP, peptide agonist of proteinase-activated receptor 2; HPLC, high pressure liquid chromatography; TNF, tumor necrosis factor; Boc, t-butoxycarbonyl; AMC, 7-amino-4-methylcoumarin; DMEM, Dulbecco's modified Eagle's medium; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline.

	ACKNOWLEDGMENTS

 
We thank Emmanuelle Kaufman, Lida Garcia, and Mauro Magi for excellent technical assistance. We are most grateful to Drs. Mukaida, Angel, and Van Lint for providing constructs essential for this study.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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