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Am J Physiol Lung Cell Mol Physiol 290: L259-L269, 2006. First published September 9, 2005; doi:10.1152/ajplung.00215.2005
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Tryptase activates phosphatidylinositol 3-kinases proteolytically independently from proteinase-activated receptor-2 in cultured dog airway smooth muscle cells

James K. Brown,1,2 Morley D. Hollenberg,3 and Cary A. Jones1

1Pulmonary and Critical Care Medicine Section, Medical Service, Department of Veterans Affairs Medical Center, San Francisco; 2Cardiovascular Research Institute and Department of Medicine, University of California San Francisco, San Francisco, California; and 3Departments of Pharmacology, Therapeutics, and Medicine, University of Calgary Faculty of Medicine, Calgary, Canada

Submitted 17 May 2005 ; accepted in final form 2 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mast cell tryptase is a potent mitogen for many cells in the airways and lung, but the cellular mechanisms for its growth stimulatory effects are poorly understood. Our major goal was to determine whether tryptase activates phosphatidylinositol 3-kinases (PI 3-kinases) in cultured dog tracheal smooth muscle cells to induce its mitogenic effects. After exposure to tryptase, cells were lysed. Immunocomplexes prepared from the lysates using an antibody to the p85 subunit of PI 3-kinase, but not using anti-phosphotyrosine antibodies, possessed increased capacity to phosphorylate inositol on its D3 hydroxyl group. Tryptase also increased phosphorylation of Akt, a downstream target of PI 3-kinases. This effect was abolished by one PI 3-kinase inhibitor, wortmannin, and attenuated by another, LY-294004, which also blocked tryptase's mitogenic effects. Treatment of tryptase with p-amidino phenylmethanesulfonyl fluoride, to abolish its proteolytic activity irreversibly, inhibited its stimulatory effects on Akt phosphorylation. Proteinase-activated receptor-2 (PAR-2)-activating peptides failed to increase Akt phosphorylation in cultured dog tracheal smooth muscle cells, but the PAR-2-activating peptides did induce brisk increases in Akt phosphorylation in Madin-Darby canine kidney cells. We concluded that tryptase activates PI 3-kinases in cultured dog tracheal smooth muscle cells to induce its potent mitogenic effects. These effects of tryptase on PI 3-kinases appear to occur via novel proteolytic mechanisms independent from PAR-2. Also, tryptase, although comparable in mitogenic potency to platelet-derived growth factor (PDGF), induces considerably less tyrosine phosphorylation on proteins than occur in response to PDGF.

mast cell; airway smooth muscle hyperplasia; asthma

HYPERPLASIA OF AIRWAY SMOOTH MUSCLE is one of several relatively irreversible, structural changes, collectively referred to as airway remodeling, that occur in the airways of asthmatic patients (16). Such structural alterations may contribute to bronchial hyperreactivity by thickening and stiffening the airway wall such that it narrows to a greater degree than does a normal airway in response to a given concentration of contractile agonist (42). Remodeling of the airways also may account for the progressive decline in spirometric values that occurs in some patients with asthma (58). Smooth muscle hyperplasia in the airways of asthmatic patients first was recognized in autopsy studies of patients dying from asthma (30). However, morphometric analyses of endobronchial biopsies now have established its presence in living patients with even mild asthma (78).

Recent investigation into the mechanisms of airway smooth muscle hyperplasia has focused on defining the intracellular signaling cascades that lead to mitogenesis in cultured airway smooth muscle cells after their exposure to the broad array of extracellular factors that can induce them to proliferate (for review, see Ref. 81). One important cascade involves activation of the extracellular regulated kinases (ERKs or p44/42), members of the MAP kinase superfamily (50). Use of several approaches, including cell-permeable inhibitors (14), intracellular injection of neutralizing antibodies (3), and overexpression of dominant negative mutant proteins (54), has suggested that activation of this pathway is required for mitogenesis in cultured airway smooth muscle cells. More recent evidence has indicated that activation of phosphatidylinositol 3-kinases (PI 3-kinases) (33) also is important (49). Thus cell-permeable inhibitors of PI 3-kinases, such as wortmannin and LY-294002 (4, 76), markedly attenuated mitogenesis induced by platelet-derived growth factor (PDGF), epidermal growth factor, and thrombin in cultured airway smooth muscle cells (49). Also, overexpression of the catalytic subunit of PI 3-kinase in these cells, by transient transfection, resulted in activation of cyclin D1, a critical regulator of G1 progression in the same cells (55). PI 3-kinases promote phosphorylation on the D3 position of the inositol ring of membrane-associated phosphatidylinositol lipids (33), resulting in the production of phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate, that serve as second messengers leading to the activation of specific intracellular serine/threonine kinases including Akt (19). To date, most evidence suggests that the ERK and PI 3-kinase pathways are separate and parallel, rather than interconnecting, pathways that mediate proliferative responses to extracellular stimuli in airway smooth muscle cells (49, 55).

Among mitogens of potential relevance to the induction of airway smooth muscle hyperplasia in asthma, mast cell tryptases have been of special interest (7, 13–15, 18, 25, 63) for several reasons (68). In bronchial mucosal biopsies, mast cell numbers in the smooth muscle layer were far greater in biopsies obtained from asthmatic patients than in those obtained from normal controls and or from patients with eosinophilic bronchitis (10–12). The finding supports the possibility that mast cell mediators contribute to the induction of smooth muscle hyperplasia in the airways of asthmatic patients. Human tryptases are an expanding family of trypsin-like serine proteinases that are encoded by a cluster of genes on chromosome 16p13.3 (56). Tryptases accumulate in abundance in mast cell granules, making up 25% of the total intracellular protein in human lung mast cells (65). Once released during degranulation, tryptases are relatively unique among serine proteinases in their resistance to degradation by endogenous proteinase inhibitors and therefore have the potential to remain catalytically active in the extracellular space for long periods. When purified from human lung tissue, tryptases consist predominantly of their {beta}-isoenzymes (39), whose amino acid sequences are 98–99% identical to one another (56), and therefore they often are referred to as "tryptase" (68). We have found that human lung tryptase is a potent mitogen for cultured human and dog airway smooth muscle cells with growth stimulatory effects that occur over the nanomolar range of concentrations and with maximal effects on mitogenesis that far exceed those induced by thrombin (or other mitogenic serine proteinases of the coagulation cascade), trypsin, or other mast cell-associated serine proteinases we have tested (13–15). Other investigators also have described potent mitogenic effects of human lung tryptase in cultured airway smooth muscle cells (7, 18, 25).

Considerable uncertainty remains regarding the mechanisms by which tryptase induces mitogenesis in cultured airway smooth muscle cells, both in terms of the relevant intracellular signaling cascades as well as the earliest changes, presumably at or near the cell membrane, that tryptase induces to activate these pathways. We reported previously that activation of the p44/p42 ERK class of MAP kinases was required for tryptase-induced mitogenesis in these cells (14), but whether tryptase also activates PI 3-kinases has not been examined in airway smooth muscle cells, to our knowledge. Also, of great interest was the demonstration that tryptase has the capacity to cleave and activate proteinase-activated receptor-2 (PAR-2) (52), one of the four known members of the G protein-coupled family of PARs (27). This observation has spawned a series of studies that have implicated PAR-2 activation as one potential mechanism for tryptase's effects on cells and tissues (for review, see Ref. 26). Several investigators have found that human lung fibroblasts and airway smooth muscle cells express PAR-2 (1, 7, 40). Also, PAR-2-activating peptides, which stimulate PAR-2 nonenzymatically, can induce mitogenesis in these cells (1, 7). However, largely due to a lack, to date, of suitable PAR-2 antagonists, it has not yet been proved unequivocally that tryptase induces its mitogenic effects in cultured lung fibroblast and airway smooth muscle cells via PAR-2 activation. Clearly, in some cells expressing PAR-2, tryptase cannot activate PAR-2 because of the receptor's glycosylation status (21, 23). In one study using cultured guinea pig airway smooth muscle cells, the authors found that tryptase stimulated growth via a proteolytic mechanism independent of PAR-2 activation, even though PAR-2 clearly was expressed in the cells (25).

The major goals of this study were to determine whether tryptase stimulates PI 3-kinases and Akt phosphorylation in cultured airway smooth muscle cells and whether this signaling pathway contributes to the induction of tryptase's mitogenic effects. We also have sought to determine whether tryptase's effects on this signaling cascade are mediated via its proteolytic actions and activation of PAR-2. For these experiments, we have employed cultured dog tracheal smooth muscle cells, a preparation in which we have demonstrated previously that tryptase induces its mitogenic effects largely via proteolytic actions (13), because of our interest in examining the potential role of PAR-2 activation in mediating stimulation of PI 3-kinase by tryptase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. We purchased porcine heparin (4–6 kDa), N-p-tosyl-Gly-L-Pro-L-Lys p-nitroanilide (GPK), p-amidino phenylmethanesulfonyl fluoride (p-APMSF), silica gel thin-layer chromatography (TLC) plates, myelin basic protein (MBP), and Triton X-100 from Sigma Chemical (St. Louis, MO). Purified human lung tryptase, LY-294002, wortmannin, and PD-098059 were obtained from Calbiochem (La Jolla, CA). Other reagents and their sources were as follows: M199 culture media and Cellgro COMPLETE serum-free medium were from Mediatech (Herndon, VA); recombinant human PDGF-BB homodimer was from Peprotech (Rocky Hill, NJ); PAR-2-activating peptides SLIGRL-NH2 and SLIGKV-NH2 were from Bachem (Torrance, CA); polyclonal anti-phospho-Akt (Ser473) antibody, phospho-Erk antibody, and anti-Akt antibody were from Cell Signaling Technology (Beverly, MA); monoclonal anti-Erk1/2 antibody and Histostain-SP kit were from Zymed Laboratories (South San Francisco, CA); polyclonal anti-PI 3-kinase (p85 subunit, 06-195) and monoclonal 4G10 antibodies were from Upstate Biotechnology (Lake Placid, NY); phosphatidylinositol (bovine liver) and phosphatidylinositol 4-phosphate (PI-3P) were from Avanti Polar Lipids (Alabaster, AL); enhanced chemiluminescence reagents were from Pierce (Rockford, IL); and Western Re-Probe was from Geno Technology (St. Louis, MO). For immunolocalization of PAR-2, we employed anti-PAR-2 A5 antibody, a polyclonal antibody raised in rabbits that is similar to anti-PAR-2 B5 antibody described previously (48). The B5 antibody has been demonstrated in a previous study to react with PAR-2 in canine cells (53). The immunizing peptide used for generation of A5 was GPNSKGRSLIGRLDTPyggc, corresponding to a region encompassing the cleavage/activation site of the receptor. The peptide we used for immunoadsorption, GPNSKGRSLIGRLDTP, was prepared by the University of Calgary Peptide Synthesis Service (Peplab@ucalgary.ca).

Cell culture. Primary cultures of dog tracheal smooth muscle cells were established and maintained as we have described previously (72). Cells were fed on alternate days and passaged when they approached confluence. As a positive control for our experiments with PAR-2-activating peptides, we employed the Madin-Darby canine kidney (MDCK) cell line, a cell line from the same species in which other investigators showed that PAR-2 was present and susceptible to activation with PAR-2-activating peptides (60). MDCK cells were obtained from American Type Culture Collection (Manassas, VA), cultured in MEM containing 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% FBS, and incubated in 5% CO2.

Measurement and inhibition of serine proteinase activity. Catalytic activity of tryptase was measured by assessing its ability to cleave GPK. Tryptase was added to 50 mM Tris buffer, pH 7.7, containing 120 mM NaCl, 20 µg/ml heparin, and 100 µM GPK (final volume 100 µl). Rates of GPK hydrolysis were determined at room temperature for 3 min by following the change in absorbance at 405 nm using a Beckman spectrophotometer. The concentration of tryptase was calculated from the amount of cleaved substrate (molar extinction coefficient = 8,800) assuming a molecular weight of 134,000 for tetrameric tryptase (66).

To inhibit its catalytic activity irreversibly, tryptase (25.5–48 nM) was incubated with p-APMSF (10 µM) for 100–120 min at 4°C in 10 mM Bis-Tris buffer, pH 6.1, as we have described previously (14). For a control, tryptase was incubated with the p-APMSF diluent alone (1:1 acetonitrile:dimethylformamide), diluted 1:5 in serum-free Cellgro COMPLETE medium, pH 7.4, and placed on ice for 3–5 h before application to cells in 96-well plates. To test for possible cytotoxic effects, p-APMSF that had been incubated alone was added to wells containing starvation media, and the effects on DNA synthesis were determined. Before application on cells, tryptase that had been treated with p-APMSF was assayed for completeness of catalytic inhibition using the GPK assay, as described above.

Western blot analysis. Cells were rendered quiescent before treatments and then lysed with buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 10 mM {beta}-glycerophosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF, and 1 mM sodium vanadate. Lysates were run on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with specific antibodies that were then detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. When appropriate, blots were stripped using Re-Probe, according to the manufacturer's instructions, and reprobed using another primary antibody. Autoradiograms of blots were scanned, and densitometric analysis was performed using NIH Image software 1.62.

Measurement of PI 3-kinase activity. After being exposed to mitogens, cells grown in six-well culture plates were washed and lysed with 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM sodium vanadate, 1 mM PMSF, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. Equal amounts of cell lysates were incubated overnight at 4°C with either monoclonal 4G10 antiphosphotyrosine or polyclonal anti-p85 PI 3-kinase antibodies for immunoprecipitation. Protein A/G agarose was added to the samples for an additional 2-h incubation. The agarose-antibody-antigen complex was isolated by centrifugation and sequentially washed twice with PBS/1% Nonidet P-40/1 mM DTT/1 mM vanadate, 100 mM Tris (pH 7.4)/500 mM LiCl, 10 mM Tris (pH 7.4)/100 mM NaCl, and 25 mM MOPS (pH 7.0)/10 mM MgCl2/1 mM EGTA. The final pellet was resuspended in 30 µl of 20 mM HEPES assay buffer before addition of 50 µM MgATP, 200 µM adenosine (to inhibit PI 4-kinases) (33), and sonicated phosphoinositol. Reactions were initiated by the addition of [{gamma}-32P]ATP (10 µCi/sample) and continued for 15 min at 37°C. Reactions were terminated by the addition of 4 N HCl, and lipids in the reaction mix were extracted with ice-cold chloroform/MeOH (1:1). The lipid phase was washed once with MeOH/HCl, spotted on a silica TLC plate (treated with sodium oxalate), and eluted with H2O:glacial acetic acid:methanol:acetone:chloroform (14:24:26:30:80). 32P-labeled PI-3P were visualized by autoradiography and identified by their ability to comigrate with phosphatidylinositol 4-phosphate standard stained with iodine vapor. Autoradiograms were quantitated by densitometry using NIH Image software 1.62.

Measurement of ERK MAP kinase activity. These assays were carried out as we have described previously (14). In brief, cellular lysates were incubated overnight at 4°C with polyclonal anti-Erk2 antibody followed by a 2-h incubation with Protein G agarose. Erk2 immunocomplexes were assayed for kinase activity in 25 mM Tris, pH 7.0, 0.7 mM EGTA, and 0.33 mg/ml MBP. Reactions were initiated by the addition of 35 mM magnesium acetate and 35 µM ATP (1 µCi [{gamma}-32P]ATP). After a 30-min incubation at 30°C, the assay was terminated by pipetting the reaction mixture onto circles of phosphocellulose paper that were then immersed in 0.5% phosphoric acid. Papers were washed four times with phosphoric acid and once with acetone, dried, and then counted using liquid scintillation spectrophotometry.

Measurement of DNA synthesis. To quantify DNA synthesis in the cells, we measured the incorporation of bromodeoxyuridine (BrdU) into cellular DNA using an ELISA as we described previously (13). Cultured smooth muscle cells, at passages 1–5, were seeded at a density of 10,000 cells/well in a 96-well format. After a 24-h incubation in M199 media containing 10% FCS, the cells in each well were washed once with PBS and then starved for 24 h in 100 µl of Cellgro COMPLETE serum-free media before the addition of mitogens. BrdU (10 µM) was added 24 h after mitogens.

Immunolocalization of PAR-2. Subconfluent dog tracheal smooth muscle and MDCK cells were grown on coverslips, washed with PBS, and fixed for 10 min in cold methanol. After rehydration, coverslips were incubated for 2 h at 37°C in a humidified chamber with PBS alone (no primary antibody), anti-rat PAR-2 antibody (A5, 1:500 dilution), or anti-rat PAR-2 antibody (A5, 1:500 dilution) that had been preincubated with 20 µg/ml of the immunoadsorption peptide (GPNSKGRSLIGRLDTP) for 24 h at 4°C. Coverslips were processed using a Histostain-SP kit with diaminobenzidine as chromogen, and cell nuclei were counterstained with hematoxylin.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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DISCUSSION
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Tryptase-induced increases in PI 3-kinase activity are detectable in anti-p85 PI 3-kinase, but not in anti-phosphotyrosine, immunocomplexes. As an initial approach to testing for increases in PI 3-kinase activity in tryptase-treated cells, we exposed cells to tryptase (or PDGF as a positive control), lysed the cells, and tested for increases in kinase activity in anti-phosphotyrosine immunocomplexes. Preliminary immunoblot analysis, using the anti-p85 PI 3-kinase antibody, indicated that treatment of the cells with PDGF increased the amounts of p85 regulatory subunit of PI 3-kinase in anti-phosphotyrosine immunocomplexes (Fig. 1A). Thus the small amounts of p85, detected in these immunocomplexes isolated from unstimulated cells, were markedly increased in immunocomplexes isolated from cells exposed to PDGF (Fig. 1A). These anti-phosphotyrosine immunocomplexes prepared from PDGF-stimulated cells also contained augmented PI 3-kinase activity, as assessed by thin-layer chromatographic demonstration of increases in PI-3P (Fig. 1B). Of note is that in these anti-phosphotyrosine immunocomplexes, PI-3P was barely detectable when the immunocomplexes were isolated from unstimulated cells, presumably reflecting very low basal PI 3-kinase activity. PDGF treatment of the cells induced rapid increases that were present 2 min after PDGF exposure and declined thereafter (Fig. 1B). However, when the same approach was employed using tryptase, rather than PDGF, as the mitogen, we were unable to detect increases in p85 in anti-phosphotyrosine immunocomplexes (data not shown). Also, PI 3-kinase activity did not increase significantly above control in anti-phosphotyrosine immunocomplexes isolated from cells treated with tryptase (Fig. 1C).



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  		Fig. 1. Mitogen exposure in airway smooth muscle cells increases phosphatidylinositol 3-kinase (PI 3-kinase) activity in immunocomplexes. Confluent growth-arrested cells were treated with either 100 ng/ml PDGF-BB (A–C) or tryptase (4.2–9.6 nM) (C–E) and lysed, and immunoprecipitates (IP) from cellular lysates were prepared using either a monoclonal anti-phosphotyrosine antibody (A–C, left) or anti-p85 (D and E, right), as described in MATERIALS AND METHODS. A: SDS-PAGE immunoblot analysis using polyclonal anti-p85 in anti-phosphotyrosine immunocomplexes. B and C: PI 3-kinase activity in anti-phosphotyrosine immunocomplexes, assayed using inositol as substrate with reaction products separated by thin-layer chromatography. 32P-labeled phosphatidylinositol 3-phosphate (PI-3P) is shown by arrows. Cells were exposed to PDGF, tryptase, or diluent alone (cont) for the indicated times. D: immunoblot analysis of anti-p85 immunocomplexes using anti-p85 for detection. E: PI 3-kinase activity of anti-p85 immunocomplexes in lysates isolated from cells treated with tryptase or PDGF. F: combined effects of tryptase on PI 3-kinase activity, expressed as fold increase in PI-3P over control, quantified by densitometry (n = 5 experiments, means ± SE). *Significantly different from control values (P < 0.05, paired t-test).

 
We began these experiments using anti-phosphotyrosine antibodies for immunoprecipitation because this approach had been used by others for measurement of PI 3-kinase activity in cultured airway smooth muscle cells (49, 55). As an alternative approach in our own experiments, we used the anti-p85 PI 3-kinase antibody for the initial immunoprecipitation (74). Treatment of the cells with tryptase resulted in clear-cut increases in the PI 3-kinase activity in these anti-p85 immunocomplexes (Fig. 1E). Data from five experiments are summarized in Fig. 1F and demonstrate that tryptase-induced increases in PI 3-kinase activity reached a maximum at 10–30 min and then began to decline. Of interest was that, in the control state, the amount of PI 3-kinase product (i.e., amounts of PI-3P) recovered in anti-p85 immunocomplexes (Fig. 1E) was noticeably greater than in anti-phosphotyrosine immunocomplexes (Fig. 1B).

Tryptase increases Akt phosphorylation via PI 3-kinase activation and proteolytic actions. Because Akt phosphorylation is a well-known downstream effect of PI 3-kinase activation (19) and because well-characterized anti-phospho-Akt antibodies are available (9), in our next series of experiments we tested the ability of tryptase to increase Akt phosphorylation using immunoblot analysis and an antibody to Akt phosphorylated at Ser473 (9). Once again, PDGF was used as a positive control in these experiments. Treatment of cells with either PDGF or tryptase increased Akt phosphorylation. Intensity of phospho-Akt signals was greater in response to PDGF than to tryptase (Fig. 2A). Also, time-course experiments showed a relatively rapid response to PDGF, peaking at 15 min (Fig. 2B). Responses to tryptase were slower in onset and more sustained (Fig. 2B).



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  		Fig. 2. Tryptase increases Akt phosphorylation. Airway smooth muscle cells were growth arrested in media containing 0.5% FBS for 24 h and then exposed to tryptase (4.2–8.2 nM) or, as a positive control, to PDGF-BB (100 ng/ml), for the indicated times. A: cellular lysates were analyzed by SDS-PAGE and immunoblotting using anti-phospho-Akt (p-Akt) antibody. To control for protein loading, the blot was stripped and reprobed with antibody to Akt (Akt). B: time course of tryptase-induced Akt phosphorylation. Cells were treated with either tryptase or PDGF for the indicated times, and lysates were prepared for immunoanalysis. Densitometric quantification of phospho-Akt signals was performed on autoradiograms of anti-phospho-Akt immunoblots. Data are expressed as a percent of the maximum (max) signal detected in the presence of tryptase or PDGF (means ± SE, n = 5 experiments).

 
Akt phosphorylation was abolished in tryptase-exposed cells that were preincubated with one PI 3-K inhibitor, wortmannin (100 nM), and attenuated by another, LY-294002 (10 µM; Fig. 3). These inhibitors also blocked PDGF-induced Akt phosphorylation (data not shown). In the same experiments, we tested the effects of PD-098059 (50 µM), an inhibitor of MAP kinase kinases 1 and 2 (2), the major upstream activators of ERKs. Inhibition of MEK 1 and 2 with PD-098059, using concentrations we showed previously inhibited tryptase-induced activation of ERK in these cells (14), did not alter tryptase-induced Akt phosphorylation significantly (Fig. 3B).



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  		Fig. 3. PI 3-kinase inhibitors attenuate tryptase-induced Akt phosphorylation. Confluent airway smooth muscle cells were growth arrested for 24 h in M199 media containing 0.5% FBS. Cells were then preincubated in the presence of 10 µM LY-294002 or 100 nM wortmannin (PI 3-kinase inhibitors). Effects of MEK1/2 inhibition by 50 µM PD-098059 also were tested in these experiments. After a 45-min exposure to one of these inhibitors, tryptase (5.0–8.2 nM) was applied for 60 min. Cellular lysates were analyzed with SDS-PAGE and immunoblotted using anti-phospho-Akt (p-Akt) antibody. A: immunoblot shown is representative of results from 4 experiments. The same pattern of inhibition was observed in cells stimulated with tryptase for 15 or 30 min. B: combined results of 4 immunoblots were analyzed by densitometry and expressed as percent of responses to tryptase alone. In the presence of wortmannin, responses to tryptase were not detectable (100% inhibition) in any experiment (P < 0.05), and therefore this experimental condition is not shown on the histogram. LY-294002 reduced the response to tryptase alone by 48 ± 20.1 (means ± SE, n = 4 experiments), although the degree of inhibition did not achieve statistical significance (P = 0.09, paired t-test). MEK1/2 inhibition with PD-098059 had no significant effects on responses to tryptase in these 4 experiments.

 
In other experiments, we examined the importance of tryptase's properties as a proteinase on its ability to induce Akt phosphorylation. When tryptase was preincubated with p-APMSF (final concentration 10 µM), its catalytic activity, measured in the GPK assay, decreased to <1% of control. The capacity of tryptase to induce Akt phosphorylation at 15, 30, and 60 min also was greatly reduced when the tryptase was pretreated with p-APMSF (Fig. 4A). These decreases in tryptase-induced Akt phosphorylation ranged from 73.9% to 88.3% (Fig. 4B) and were statistically significant (P < 0.05).



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  		Fig. 4. Inhibiting tryptase's proteolytic activity decreases its ability to promote Akt phosphorylation. Cells were growth arrested in M199 media with 0.5% FBS for 24 h and then exposed for 15, 30, or 60 min to tryptase (5.1–9.6 nM), the irreversible serine inhibitor p-amidino phenylmethanesulfonyl fluoride (p-APMSF) alone, or to tryptase that had been preincubated with p-APMSF. A: representative immunoblots using anti-phospho-Akt and anti-Akt antibodies. B: densitometric quantification of phospho-Akt signals is expressed as a percent of the signal produced in the presence of tryptase alone (control) for 60 min. Results are means ± SE from 5 experiments. *Akt phosphorylation signals significantly less using tryptase treated with p-APMSF compared with tryptase alone (P < 0.05, paired t-test).

 
Inhibiting PI 3-kinases blocks tryptase-induced mitogenesis without altering its stimulatory effects on ERK MAP kinases. In other experiments, we measured mitogenic responses to tryptase and PDGF using a BrdU ELISA and tested the effects of PI 3-kinase inhibition. A 45-min preincubation of the cells with LY-294002 completely abolished the ability of both mitogens to increase BrdU uptake (Fig. 5).



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  		Fig. 5. PI 3-kinase inhibition abolishes tryptase-induced mitogenesis. Cells were growth arrested in serum-free medium and then preincubated for 45 min in the absence (control) or presence of 50 µM LY-294002 before exposure to PDGF (100 ng/ml) or tryptase (3.3–14 nM). DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation using an ELISA and expressed as percent response to 10% FBS (%max). Results are expressed as means ± SE, n = 8 or 4 experiments with PDGF or tryptase, respectively. *Significant effects compared with control responses (P < 0.05, paired t-test).

 
In a previous paper, we showed that tryptase-induced mitogenesis in cultured airway smooth muscle cells required activation of the ERK (p44/p42) cascade (14). Most evidence suggests that the ERK and PI 3-kinase pathways are separate and parallel pathways that mediate proliferative responses to extracellular stimuli in airway smooth muscle cells (49, 55), although interconnections between the two pathways have been described (24). Therefore, in other experiments, we wished to be certain that LY-294002 did not alter tryptase's ability to stimulate this alternative pathway. Cells were preincubated with LY-294002 for 45 min before exposure to tryptase or PDGF-BB. Treatment of cells with LY-294002 had no significant effect on the ability of immunoprecipitated Erk2 to phosphorylate MBP (14) in response to either mitogen (Fig. 6).



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  		Fig. 6. LY-294002 does not alter mitogen-induced increases in ERK activity. Cells were growth arrested for 24 h in 0.5% FCS and then preincubated for 60 min with 10 µM LY-294002 or with DMSO diluent (control) before stimulation for 10 min with either tryptase (4 nM) or PDGF-BB (100 ng/ml). Cell lysates were prepared, Erk2 was immunoprecipitated, and Erk2 activity was measured, as described in MATERIALS AND METHODS. Results are expressed as a fold of the activity measured in lysates from cells not treated with mitogen or inhibitor (basal). Data are expressed as means ± SE, n = 3 experiments for both tryptase and PDGF.

 
PAR-2-activating peptides do not increase Akt phosphorylation in cultured dog tracheal smooth muscle cells but do so in MDCK cells. Our findings indicated that the major portion of tryptase's stimulatory effects on Akt phosphorylation in these cells was via tryptase's proteolytic actions (Fig. 3). Therefore, we were interested to explore the possibility that these effects of tryptase were mediated by tryptase-induced activation of PAR-2. For most members of this family of G protein-coupled PARs, proteolytic cleavage of the NH2 terminus of the receptor results in cellular activation via a tethered ligand such that the truncated, but still attached, NH2 terminus interacts with an extracellular domain in the receptor to initiate signaling (27). Oligopeptides with an identical sequence to the attached, truncated NH2 terminus generally have the capacity to activate the receptor via binding to the same extracellular domain and therefore serve as receptor-specific activating peptides (27). Therefore, in our experiments, we tested for the ability of PAR-2-activating peptides to stimulate Akt phosphorylation in the cultured airway smooth muscle cells. We exposed these cells to either 100 µM or 1 mM concentrations of mouse (SLIGRL-NH2) or human (SLIGKV-NH2) PAR-2-activating peptides, as well as to tryptase, and tested for increases in Akt phosphorylation. In each of four experiments, no increases in phospho-Akt above control were detected in lysates from the smooth muscle cells treated with 1 mM concentrations of the PAR-2-activating peptides, even though tryptase caused brisk increases in the same signal (Fig. 7A). The same pattern was observed in response to a lower concentration of the peptides (100 µM). In parallel experiments, we repeated the same experiments using MDCK cells. In MDCK cells, the opposite pattern of responses was observed in that the PAR-2-activating peptides increased Akt phosphorylation but tryptase did not (Fig. 7B). Approximately equal amounts of cellular protein from the two different cell types were employed for the immunoblot analyses in these experiments.



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  		Fig. 7. Proteinase-activated receptor (PAR)-2-activating peptides do not increase Akt phosphorylation in cultured dog tracheal smooth muscle cells but do so in Madin-Darby canine kidney (MDCK) cells. A: dog tracheal smooth muscle cells were growth arrested for 24 h and then exposed for the indicated times to vehicle alone (control) or to 1 mM concentrations of mouse (SLIGRL) or human (SLIGKV) PAR-2-activating peptides or to 4 nM tryptase. Proteins in cellular lysates were separated on 10% SDS-PAGE gels and immunoblotted with anti-phospho-Akt antibody. B: MDCK cells were treated and analyzed as in A. Results shown are representative of those obtained from 4 separate experiments.

 
PAR-2 is present in both dog tracheal smooth muscle and MDCK cells. Using A5 anti-PAR-2 antibody, we observed immunoreactivity over both tracheal smooth muscle and MDCK cells (Fig. 8, A and B). Immunoreactivity was markedly reduced both by preincubation of the primary antibody with the immunoadsorption peptide (Fig. 8, C and D) and by omission of the primary antibody (Fig. 8, E and F).



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  		Fig. 8. PAR-2 is expressed in cultured dog tracheal smooth muscle and MDCK cells. PAR-2 immunostaining was present over both tracheal smooth muscle (A) and MDCK (B) cells when the anti-PAR-2 A5 antibody was used (1:500 dilution) as primary antibody. Preincubation of A5 antibody with the immunoadsorption peptide (C and D) or omission of the primary antibody (E and F) markedly diminished immunostaining in both the tracheal smooth muscle and MDCK cells. Photographs were taken at x400 magnification.

 

   DISCUSSION
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In eukaryotic cell membranes, phosphatidylinositol, although present in relatively small amounts compared with other phospholipids, is unique in that its inositol ring is susceptible to phosphorylation on five different sites (33). PI 3-kinases catalyze phosphorylation on the D3 hydroxyl group resulting in the generation of the phosphoinositides phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (33) and in the induction of downstream effects, such as activation of Akt (19) and 70-kDa ribosomal S6 kinase (2), that mediate diverse cellular effects including mitogenesis (19). Many mitogens activate PI 3-kinases (33), but there also are examples of growth stimulation by extracellular factors that do not involve this signaling cascade (61). In cultured airway smooth muscle cells, the mitogens PDGF, epidermal growth factor, and thrombin each has been shown to activate PI 3-kinases (49, 55, 67, 77). The major findings of our work are that mast cell tryptase, a well-recognized cellular mitogen (7, 13–15, 18, 25, 63), activates PI 3-kinases in cultured dog airway smooth muscle cells and that this event can be linked to its capacity to induce mitogenesis. In the cultured dog airway smooth muscle cells employed in our current experiments, the activating effects of tryptase on PI 3-kinases and Akt phosphorylation were mediated largely via tryptase's proteolytic effects (Fig. 4). This finding is consistent with our prior observation that tryptase's mitogenic effects in these cells also depended largely on proteolytic mechanisms (13, 14). Although PAR-2 is a recognized proteolytic target for activation by tryptase in at least some cells (26, 52), our experiments suggested that tryptase-induced Akt phosphorylation, and presumably PI 3-kinase stimulation, were not mediated via PAR-2 in these cells (Fig. 7).

Although PI 3-kinases are a growing family of structurally diverse enzymes, in eukaryotic cells the class IA enzymes are primarily responsible for the production of D3 phosphoinositides in response to growth factors (33). Class IA PI 3-kinases are heterodimers, each consisting of one catalytic (p110{alpha}, {beta}, or {delta}) and one adaptor/regulatory subunit (p85{alpha}, {beta}, or {delta}, each encoded by its own specific gene, as well as p55{alpha} or p50{alpha} produced by alternate splicing of the p85{alpha} gene) (33). The catalytic subunit possesses the lipid kinase domain, and the adaptor/regulatory subunits mediate its activation by one of several mechanisms, including direct interaction of src homology 2 domains in the p85 regulatory subunits with phosphotyrosine residues on activated growth factor receptor tyrosine kinases (17). Such interactions also result in translocation of the PI 3-kinase dimer to the plasma membrane where its potential substrates are located (17). Wortmannin irreversibly inhibits the lipid kinase activity of p110 subunits with IC50 values in the 1- to 10-nM range (4), and LY-294002 is a reversible inhibitor with an IC50 of ~1 µM (76). An important concern related to the use of these PI 3-kinase inhibitors is emerging evidence that they have other effects besides PI 3-kinase inhibition (5, 29, 32, 71). In our experiments, we feel it is likely that wortmannin and LY-294002 attenuated tryptase-induced mitogenesis via their inhibitory effects on PI 3-kinases, rather than via other actions, in part because tryptase's stimulatory effects on ERK MAP kinases were not altered by these agents (Fig. 6) and in part because the concentrations used in our experiments were below those causing some of the reported nonspecific effects (29). In addition, our overall conclusion that tryptase mediates its mitogenic effects in part via PI 3-kinase activation is supported by some of our other findings, including the observation that tryptase increased phosphorylation of D3 phosphoinositides (Fig. 1, E and F) and Akt (Fig. 2) in our cells, as well as by the demonstration by others that activation by PI 3-kinases contributes importantly to mitogenic responses in cultured airway smooth muscle cells (49, 55).

In our experiments, we detected tryptase-induced increases in PI 3-kinase activity in immunocomplexes obtained using an anti-p85, but not an anti-phosphotyrosine, antibody (Fig. 1). Other investigators found that, when cultured airway smooth muscle cells were treated with PDGF, mitogen-induced increases in PI 3-kinase activity clearly were detectable in anti-phosphotyrosine immunocomplexes (55), and we have confirmed this finding (Fig. 1B). The ability to detect such increases presumably reflected the presence of newly tyrosine-phosphorylated domains in one or more sites, including PDGF receptors, p85 subunits of PI 3-kinases (45), or other proteins contained within the anti-phosphotyrosine immunocomplexes (17). Our failure to detect tryptase-induced increases in PI 3-kinase activity in anti-phosphotyrosine immunocomplexes may have several explanations. First, tryptase likely does not induce its mitogenic effects via activation of PDGF receptors or other receptor tyrosine kinases, because we showed previously that, in lysates prepared from tryptase-treated airway smooth muscle cells, anti-phosphotyrosine immunoblots lacked large-molecular-weight bands corresponding to known receptor tyrosine kinases (14). Second, tryptase may activate PI 3-kinases via one of the many well-recognized pathways for PI 3-kinase activation that may not require extensive new tyrosine phosphorylation (33). These include activation of small GTPase Ras proteins that in turn bind and stimulate p110 PI 3-kinase subunits (79), activation of Src family kinases (44), or Rho, Rac, or Cdc42 proteins (80) that in turn activate p85 subunits of PI 3-kinases and activation of heterotrimeric G proteins whose {beta}{gamma}-subunits stimulate p110{delta}-subunits of PI 3-kinases (70). However, previous work by other investigators demonstrated that cultured airway smooth muscle cells did not express p110{delta} (49) and therefore activation of PI 3-kinases via G protein {beta}{gamma}-subunits is a less likely mechanism than the other alternatives. It is also important to point out that in other cells, mitogen-induced activation of PI 3-kinases was associated with tyrosine phosphorylation of several motifs on p85 PI 3-kinase subunits, but the amounts and patterns of p85 tyrosine phosphorylation varied considerably depending on the specific mitogen (45). Therefore, in our experiments, it is possible that tryptase-induced activation of PI 3-kinases in airway smooth muscle cells was associated with tyrosine phosphorylation on relatively few sites on p85 such that the amounts were insufficient for, or inaccessible to, immunoprecipitating effects of the anti-phosphotyrosine antibodies.

In our experiments, tryptase and PAR-2-activating peptides had discrepant effects on Akt phosphorylation in cultured dog tracheal smooth muscle and MDCK cells. In the smooth muscle cells, tryptase, but not PAR-2-activating peptides, induced brisk increases in Akt phosphorylation (Fig. 7A), whereas in MDCK cells, PAR-2-activating peptides, but not tryptase, increased Akt phosphorylation (Fig. 7B). Although to our knowledge, information about canine PAR-2 DNA and protein sequences is not yet available, other investigators found, by functional and immunohistochemical criteria, that PAR-2 was expressed in MDCK and other canine cells and could be activated by the human and murine PAR-2-activating peptides (53, 60). Our own experiments confirmed PAR-2 expression in MDCK cells using an anti-PAR-2 antibody comparable to the one used to localize PAR-2 in canine pancreatic duct cells (53) (Fig. 8B). Our findings regarding the effects of the PAR-2-activating peptides in MDCK cells require cautious interpretation because of reports that PAR-2-activating peptides on occasion can activate cells via mechanisms independent from their capacity to activate classic PAR-2, albeit at concentrations much higher than those used in our study (69, 75). Thus, in our experiments, it is highly likely that the PAR-2-activating peptides promoted Akt phosphorylation via PAR-2 activation in the MDCK cells. Hence, our findings indicate that PAR-2 receptors were coupled to activation of the PI 3-kinase signaling cascade in these cells. In this context, tryptase's lack of ability to activate PAR-2 in the same cells might be explained by several factors, including N-linked glycosylation of PAR-2 in MDCK cells, an effect shown in other cells to restrict tryptase's capacity to activate PAR-2 (22).

In the cultured dog tracheal smooth muscle cells, PAR-2-activating peptides failed to induce Akt phosphorylation (Fig. 7A) despite abundant PAR-2 expression in the same cells (Fig. 8A). The likely explanation of these findings is that PAR-2 simply was not coupled to PI 3-kinase and Akt activation in these cells. The fact that tryptase induced brisk increases in Akt phosphorylation in the same dog tracheal smooth muscle cells (Fig. 7A) suggested quite strongly that tryptase-induced Akt phosphorylation in these cells did not proceed via PAR-2 activation. Other investigators have reported somewhat similar observations. For example, in cultured guinea pig tracheal smooth muscle cells, tryptase induced mitogenic effects via a proteolytic mechanism independent from PAR-2, even though PAR-2 clearly was expressed in these cells (25). Also, in cultured human dermal fibroblasts, tryptase induced potent mitogenic effects via a proteolytic mechanism independent from PAR-2, although in these cells, PAR-2 expression was relatively sparse (1). An interesting and somewhat parallel observation was that PAR-1 did not mediate thrombin's mitogenic effects in cultured human airway smooth muscle cells, even though 1) thrombin's mitogenic effects were attenuated by proteinase inhibitors in those cells, 2) the cells clearly expressed functional PAR-1 (73), and 3) thrombin has a well-known capacity to cleave and activate PAR-1 in other cells (27).

We can only speculate as to other proteolytic mechanisms, besides PAR-2 activation, that tryptase may be employing to activate PI 3-kinases in our cells. Among the four known members of the PAR family, PAR-4, the most recently described (43), is, like PAR-2, susceptible to activation by tryptic enzymes (43). Therefore, if the cultured airway smooth muscle cells employed in our experiments express PAR-4, tryptase may have been activating this receptor, rather than PAR-2, to stimulate PI 3-kinases. Of interest in our experiments was our observation that tryptase activated Akt phosphorylation with much slower kinetics of onset than did PDGF (Fig. 2B). We reported previously that tryptase's stimulatory effects on ERK MAP kinases were also slow in onset compared with PDGF’s (14), so delayed onset kinetics may be a characteristic of cellular responses to tryptase. The finding points to a possible involvement of PAR-4 activation in tryptase's effects, because slow onset kinetics is also characteristic of PAR-4's cellular actions (64). In addition, there is already evidence that the PAR-activating peptides, as well as the serine proteinase trypsin, can activate proteolytically sensitive receptors other than the four cloned PARs that have been characterized to date (75). Thus tryptase may activate as yet undiscovered PAR family members to stimulate PI 3-kinases in our cells. Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes with important roles in growth promotion in many cells (51). MMPs exist in latent proforms that often require proteolytic cleavage for activation. Of interest is a prior observation that tryptase cleaves pro-MMP-3 to yield active MMP-3, also known as stromelysin, in synovial cells (37), and cultured airway smooth muscle cells have been shown to express MMP-3 (31). Thus MMP activation by tryptase could account for the PI 3-kinase stimulation observed in our experiments.

In our experiments, we employed p-APMSF to inhibit tryptase's proteolytic activity and showed that treating tryptase with p-APMSF substantially reduced its capacity to phosphorylate Akt (Fig. 4). Advantages of using p-APMSF, compared with other proteinase inhibitors, include p-APMSF's irreversible inhibitory effects, its lack of cytotoxicity, and its nearly complete (>99%) inhibition of tryptase's proteolytic activity (14, 15). That p-APMSF inhibits tryptase's activation of Akt provides fairly strong evidence that this effect of tryptase is mediated by its active catalytic site (59). The finding parallels our previous demonstration that treating tryptase with p-APMSF substantially reduces tryptase-induced mitogenesis in these preparations of cultured dog tracheal smooth muscle cells (15). Interestingly, in cultured human airway smooth muscle cells, we have found that p-APMSF treatment of tryptase does not alter the capacity of tryptase to induce mitogenesis in those cells (15). Thus, in cultured human airway smooth muscle cells, a nonproteolytic mechanism likely accounts for tryptase's potent mitogenic effects. The nonproteolytic mechanisms for tryptase's effects are not known. However, it is increasingly clear that a number of proteinases, including thrombin and tissue plasminogen activator, can activate cells via nonproteolytic mechanisms, often involving noncatalytic sequences on the surface of the proteinases that serve as ligands for specific plasma membrane-associated binding sites (6, 36, 41, 57).

Besides those observed in cultured airway smooth muscle cells, tryptase has mitogenic effects in several other cell types and may have important roles in diverse pathological disorders involving fibroproliferation and remodeling, especially in tissues where mast cells are abundant. Tryptase's capacity to stimulate mitogenesis was first demonstrated by Ruoss et al. (63) in Chinese hamster lung and Rat-1 fibroblasts. These observations have been extended to include primary cultures of human lung fibroblasts (1, 34). The latter findings are of particular interest because lung tissue from patients with fibrotic lung diseases contains increased numbers of mast cells, many in close apposition to proliferating fibroblasts (46). Tryptase is also a mitogen for human renal fibroblast cell lines (47) and hepatic stellate cells (35), suggesting that tryptase may play a part in the induction of renal interstitial and hepatic fibrosis. In support of tryptase's potential role as an angiogenic factor, mast cells accumulate near sites of new capillary sprouting (62), and tryptase is a mitogen for human dermal microvascular endothelial cells (8). Related observations in a murine model of squamous epithelial carcinogenesis have suggested that tryptase participates in the induction of stromal architectural reorganization at the tumor margin, including inducing dermal fibroblast proliferation and activation of angiogenesis (28). Importantly, tryptase's mitogenic effects are cell type specific and do not extend to vascular smooth muscle cells (38). Tryptase also has nonmitogenic cellular effects, such as increasing expression of adhesion molecules and cytokines (20), that add to its potential importance in remodeling and fibroproliferation.

Because of its diverse roles as a potent mitogen, more information is needed regarding the mechanisms through which tryptase stimulates cells to grow. The findings of our study indicate that tryptase activates PI 3-kinases in cultured dog airway smooth muscle cells and that this effect likely is important in the induction of its mitogenic effects in these cells. Our findings also suggest that this effect of tryptase involves a proteolytic action that is independent of PAR-2 activation.


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This work was supported by the Research Service of the Department of Veterans Affairs, by the University of California San Francisco’s Research Evaluation and Allocation Committee and Academic Senate Committee on Research (J. K. Brown), and by a Canadian Institutes of Health Operating grant and Proteinases and Inflammation Network group grant (M. D. Hollenberg).


   ACKNOWLEDGMENTS
 
The authors thank George H. Caughey and W. Michael Kavanaugh for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: James K. Brown, Pulmonary and Critical Care Medicine Section, Dept. of Veterans Affairs Medical Center (111-D), 4150 Clement St., San Francisco, CA 94121 (e-mail: jkbrown{at}itsa.ucsf.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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