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Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway

¹Department of Nutrition and Metabolism, Graduate School of Nutrition and Bioscience, and ²Department of Medical Sciences, School of Medicine, University of Tokushima, Tokushima; and ³Department of Clinical Investigation, National Hospital Organization, Kochi National Hospital, Kochi City, Japan

Submitted 2 March 2005 ; accepted in final form 21 September 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20–55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [³H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca²⁺ mobilization in HBF. The HAT-induced increase in Ca²⁺ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts.

mast cell tryptase; mitogen-activated protein kinase

Fibroblast proliferation is involved not only in regeneration and repair of injured or damaged tissues, but also in tissue fibrosis and remodeling (28). Previous studies showed that fibroblasts are involved in fibrosis and remodeling in chronic airway diseases and that fibrogenic mediators released from airway epithelial cells regulate fibroblast proliferation (9, 17, 24, 26, 30). Serine proteases such as thrombin and mast cell tryptase (MCT) have been reported to stimulate fibroblast proliferation, suggesting that they may mediate lung fibrosis and remodeling (1, 6, 15, 27, 28, 33). HAT is a trypsin-like serine protease localized to airway epithelial cells. Therefore, we hypothesized that HAT mediates not only regeneration of airway walls but also remodels airways by stimulating fibroblast proliferation.

Recently, serine proteases such as thrombin, trypsin, and MCT have been shown to regulate biological functions in various kinds of cells via a new class of cell surface receptor, the protease-activated receptor (PAR). Four PARs, PAR-1, PAR-2, PAR-3, and PAR-4 have been identified. These receptors are G protein-coupled receptors that are activated by cleavage of their NH₂-terminal domains by serine proteases (13). PAR-2 is activated by trypsin family proteases such as trypsin and MCT (11, 13, 23). Miki et al. (22) have shown that HAT increased the intracellular Ca²⁺ concentration by activating PAR-2 in primary human bronchial epithelial cells. Recently, we have shown that HAT cleaves a synthetic peptide corresponding to the NH₂ terminus of PAR-2 at the receptor activating site in vitro, as does trypsin (unpublished observations). This result is consistent with HAT activation of PAR-2.

Thus in the present study, we found that HAT stimulates human bronchial and lung fibroblast proliferation in vitro. Our results also indicate that HAT stimulates fibroblast proliferation via PAR-2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents and antibodies. For recombinant HAT (rHAT), 60 U/mg of protein and two kinds of rabbit polyclonal antibody against rHAT and PAR-2 were supplied from Teijin Institute for Bio-Medical Research, Tokyo, Japan. rHAT in PBS containing 0.01% BSA was sterilized by filtration through a Millipore polyvinylidene difluoride membrane (0.22 µm, low-protein binding). Human MCT was purified from human lung tissues by a modification of the method of Smith et al. (29), as previously reported (36). [Methyl-³H]thymidine (sp act 2.9 Ci/mmol) was purchased from Perkin Elmer Life Science (Boston, MA). Bovine insulin and human holotransferrin were purchased from Sigma (St. Louis, MO). PAR-1 activating peptide (PAR-1 AP, TFLLR-NH₂) and PAR-2 activating peptide (PAR-2 AP, SLIGKV-NH₂) were purchased from Bachem (Bubendorf, Switzerland). Human pancreas trypsin was purchased from Athens Research & Technology (Athens, GA). Human thrombin was purchased from MP Biomedicals (Aurora, OH). U0126, a specific inhibitor of MEK, was obtained from Promega (Madison, WI). Anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody and anti-p44/42 MAPK antibody were purchased from Cell Signaling Technology (Beverly, MA). Alexa Fluor 488-labeled goat anti-rabbit IgG was purchased from Molecular Probes (Eugene, OR). Streptavidin-horseradish peroxidase conjugate was purchased from Biosource (Camarillo, CA).

Gel filtration of mucoid sputum extract and measurement of HAT. Gel filtration of mucoid sputum extract on Sephadex G-200 was carried out as previously described (37), using elution buffer (0.05 M Tris, 0.30 M NaCl, pH 7.4). HAT content was measured by ELISA using a rHAt primary antibody, secondary anti-rabbit biotinylated antibody, and streptavidin-peroxidase. Purified rHAT was used as a standard.

Collection of mucoid sputum. Mucoid sputum samples were obtained from 86 patients, 40–70 yr old, who had chronic airway diseases, including bronchial asthma. Samples were collected in a plastic container in an ice bath and kept at –80°C until use.

Cells and cell cultures. The human lung fibroblasts (HLF) were obtained from lung tissues of patients that had undergone resection of localized lung tumors. Before the operation, written informed consent was obtained from patients after a full explanation of the procedures involved. The experimental procedure was approved by the Ethics Committee at the University of Tokushima. For parenchymal lung fibroblasts, tissue was carefully cut into 3- to 5-mm pieces. The pieces were placed into a 6-cm diameter dish (Falcon BD Labware, Lincoln Park, NJ) with 2 ml of DMEM (Sigma) supplemented with 10% FBS and gentamicin (100 mg/ml) and incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. When the tissue had firmly attached to the plastic surface, a further 4 ml of culture medium were slowly added to the dish and incubated, as described above. The medium was changed every 2 days.

For human bronchial fibroblasts (HBF), the bronchial tissues were carefully dissected free of parenchymal connective tissue, and a 4- to 5-mm piece of tissue was cultured, as described for the lung fibroblasts. After 3 wk in culture, fibroblasts had grown out from the explanted tissues. These cells were cultured for at least a further 2 wk until confluent islands of cells were observed. The cultures were then passaged with a split ratio of 1:2 into 75-cm² dishes containing culture medium. Thereafter, confluent cells were passaged with a split ratio of 1:4. Experiments were performed with cells between passages 2 and 6.

Measurement of DNA synthesis. The trypsinized fibroblasts were washed with DMEM twice, suspended in DMEM/10% FBS at a density of 40,000 cells/ml, and cultured in 100 µl of DMEM with 10% FBS in a 96-well plate (Falcon BD Labware) at 37°C in a 5% CO₂ incubator for 48–72 h until 50–70% confluent. Unless otherwise stated, the cells were then rendered quiescent by incubating them in serum-free DMEM containing holotransferrin (1 µg/ml) and BSA (100 µg/ml; DMEM/0.01% BSA) for a further 24 h. The quiescent fibroblasts were incubated for 48 h with a test protease in 100 µl of the same medium in the presence of [³H]thymidine (0.5 µCi/ml). After incubation, 50 µl of 1 N NaOH were added to each well. The lysed cell plates were transferred to a specific glass fiber filter using a harvester (Filtermate 196/micromate 196; Packard, Meriden, CT). Incorporation of [³H]thymidine into the cells was measured using a Matrix 9600 direct beta counter (Packard) and expressed as counts per minute. Six replicate wells were used to test the effects of each growth factor on the fibroblasts and another six-well replicate well without growth factors served as controls.

Cell proliferation assay. HBF were seeded at 4,000 cells/well into a 96-well plate containing 100 µl of DMEM/10% FBS until 70–80% confluent. Then they were cultured in serum-free DMEM/0.1% BSA for 24 h, stimulated with HAT for 48 h in the same medium, harvested from the plate by trypsinization, and counted in a hemocytometer, as described by Tani et al. (33).

RT-PCR analysis of mRNA for PAR-2 in HBF. HBF were seeded into 6-cm cell culture dishes and incubated until confluent. Total cellular RNA was extracted from the cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. RT-PCR procedures were carried out as described previously (22). Sense (CAGTTGGGTCTGAATTGTGTCG) and antisense (TGCACGAGCTTATGCTGCTGAC) primers for PCR of PAR-2 RNA were supplied by the Teijin Institute for Bio-Medical Research. The expected PCR product was a 491-bp fragment for PAR-2. GAPDH was used as an internal control. The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. Semiquantitative measurement of each mRNA was done using VersaDoc (Bio-Rad). The PAR-2 mRNA was normalized to GAPDH mRNA.

Immunofluorescent localization of PAR-2. After activation of PAR-2 on cell surfaces by trypsin, PAR-2 is internalized into early endosomes and lysosomes, indicating that activated PAR-2 was endocytosed and degraded (12–14). To clarify whether intracellular mobilization of the PAR-2 receptor occurs after HAT stimulation, we carried out the following morphological examinations. HBFs were cultured in chambered glass slides (Nalge Nunc, Rochester, NY) in DMEM/10% FBS for 2–3 days until 60–70% confluent. After incubation for 24 h in DMEM/0.01% BSA, the cells were stimulated with test protease or agonist (HAT, trypsin, thrombin, PAR-1 AP, or PAR-2 AP). Cells at 5 min after stimulation were fixed with acetone at –20°C for 20 min. The intracellular mobilization of PAR-2 was observed by indirect immunofluorescence using an anti-PAR-2 antibody according to the method of Iwakiri et al. (18). Briefly, the acetone-fixed cells were pretreated with 10% normal goat serum for 20 min and then incubated overnight at 4°C with a 1:400 dilution of rabbit polyclonal anti-PAR-2. After being washed several times with PBS, the cells were incubated with a 1:200 dilution of Alexa Fluor 488-labeled goat anti-rabbit IgG for 60 min at room temperature in the dark. Nuclear counterstaining was performed using 20 µg/ml ethidium bromide for 30 min at room temperature. Fluorescent images of cell sections excited at 488 and 560 nm were captured using a confocal laser scanning microscope (Leica TCS NT, Heidelberg, Germany) equipped with an argon-krypton laser source. In Fig. 6, PAR-2 is shown in green, and nuclei are shown in red. As a control, the immunostaining procedure was performed with normal rabbit IgG instead of PAR-2 antibody.

View larger version (146K): [in this window] [in a new window]   Fig. 6. Immunofluorescent localization of PAR-2 in HAT-treated HBF cells. HBF were cultured in chambered slides in DMEM/10% FBS for 2–3 days to 60–70% confluence. After incubation for 24 h in DMEM/0.01% BSA, they were incubated with 10 nM HAT, 10 nM trypsin, 200 µM PAR-2 AP, 10 nM thrombin, or 200 µM PAR-1 AP for 5 min at 37°C. Cells were then fixed with cold acetone and immunostained for PAR-2 as described in MATERIALS AND METHODS. A: control (no stimulation). PAR-2 was expressed on the cell surface of HBF. B: cells treated with 10 nM HAT for 5 min show PAR-2 internalization. Both 10 nM trypsin (C) and 200 µM PAR-2 AP (D) treatment for 5 min induced mobilization of PAR-2 in HBF. In contrast, significant mobilization of PAR-2 in HBF was not seen with either 10 nM thrombin (E) or 200 µM PAR-1 AP (F) treatment for 5 min induced. Arrows show PAR-2 localization.

Measurement of intracellular calcium concentration. The concentration of intracellular free Ca²⁺ ([Ca²⁺]_i) was evaluated by microfluorometry using fura-2 AM (Dojindo, Kumamoto, Japan) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm with a specially designed chamber and an ARGUS-50/CA system (Hamamatsu Photonics, Tokyo, Japan). In each experiment, the ratio of fluorescence at 340 nm to that at 380 nm was determined in 10–15 cells selected by the system.

HBF in DMEM/10% FBS were grown to 60–70% confluence on glass coverslips in a 12-well plate. The culture medium was changed to DMEM/0.1% BSA, and the cells were cultured for 24 h. The cells were then washed with HEPES-buffered solution (HBS; 10 mM HEPES, 145 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂, and 10 mM glucose, pH 7.4) containing 0.1% BSA, incubated in HBS with 2 µM fura-2 AM and 0.2% Pluronic (Dojindo) for 20 min at 37°C, and washed twice with dye-free HBS. The coverslips were inserted into the chamber kept at 37°C and perfused with the same solution containing test samples for 15–30 min.

Immunoblotting. HBFs were seeded into 6- or 10-cm dishes and cultured in DMEM/10% FBS until 70–80% confluent. The culture medium was changed to DMEM/0.01% BSA, and the cells were cultured for an additional 24 h. The fibroblasts were stimulated with HAT for 5–120 min. To examine the effects of the agonists on phosphorylation of MAPK, fibroblasts were stimulated with the agonists in 6-cm dishes. After stimulation, cells were washed with ice-cold PBS and incubated in 1 ml of ice-cold cell lysis buffer consisting of 20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na₃VO₄, and 1 µg/ml leupeptin for 5 min. The cells were then scraped off the dishes and transferred to microcentrifuge tubes, sonicated in an ice-bath for 30 s, and microcentrifuged at 14,000 rpm for 5 min at 4°C. Clear supernatants were collected, and protein content was determined using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). Aliquots of cell lysates containing the same amount of protein were adjusted to a volume of 100 µl, mixed with 100 µl of 2x SDS sample buffer, and boiled for 5 min, and 10 µl of the sample were separated by SDS-PAGE on a 7–15% gradient gel (Dai-ichi-Kagaku, Tokyo, Japan). Prestained broad-range protein standards (Promega) were used to estimate molecular mass. The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon, Dai-ichi-Kagaku). For detection of phospho-p44/42 MAPK and p44/42 MAPK, rabbit polyclonal antibodies that detect only phosphorylated MAPK or detect MAPK regardless of its phosphorylation status, respectively, were used. A peroxidase-conjugated anti-rabbit antibody was used as the secondary antibody. Positive reactivity was detected using the ECL chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). Signals were detected on X-ray film.

Assay of trypsin-like activity. Trypsin-like activity was measured by the method of Yasuoka et al. (36).

Statistical analysis. Data are represented as means ± SE. P values were determined by Bonferroni-Duncan multiple-comparison test, and a P < 0.05 value was considered to be statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Gel filtration of mucoid sputum extract. Figure 1 shows the elution pattern of trypsin-like activity during gel filtration on a Sephadex G-200 column loaded with a mucoid sputum extract. Trypsin-like activity was detected only in tubes 44–54 with a peak of activity in tube 48. The peak of purified rHAT was located in tube 48, and the peak of purified lung MCT was located in tube 34 (data not shown). The elution profile of HAT (ng/ml), as measured by the ELISA, was almost identical to that of trypsin-like activity. Similar results were obtained using 15 different mucoid sputum extracts. The HAT concentration in the mucoid sputum samples varied from 50 to 2,700 ng/ml, with a mean value of 408 (±SE of ±54, n = 86).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Sephadex G-200 filtration of mucoid sputum extract from a patient with chronic bronchitis is depicted. Two milliliters of mucoid sputum extract were subjected to gel filtration on Sephadex G-200 (column 2.2 x 65 cm). Elution was carried out at a flow rate of 15 ml/h at 4°C, and 4-ml fractions were collected. Trypsin-like activity was measured by using t-butyloxycarbonyl-L-phenylalanyl-L-seryl-L-arginine-4-methyl-coumaryl-7-amide as a substrate. HAT, human airway trypsin-like protease.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effect of HAT on DNA synthesis in human bronchial fibroblasts (HBF; A) and human lung fibroblasts (HLF; B). Serum-starved cells were stimulated with increasing concentrations of HAT (0.4–400 mU/ml, 0.1–100 nM) for 48 h in 96-well microplates. DNA synthesis was estimated by [3H]thymidine incorporation as described in MATERIALS AND METHODS. Data are presented as means ± SE from 6 wells. *P < 0.05 compared with control. cpm, Counts per minute.

Effect of protease inhibitor on HAT-induced DNA synthesis in HBF. Leupeptin, a serine protease inhibitor, is known to inhibit the peptidolytic activity of HAT (36). Leupeptin at 50 µM almost completely abolished DNA synthesis induced by 50 nM HAT in HBF (Fig. 3). The addition of the inhibitor alone showed no significant effect on the basal level of [³H]thymidine incorporation. Moreover, the inactivation of HAT enzymatic activity by heating at 95°C also abolished its ability to stimulate DNA synthesis (data not shown). These results indicate that the DNA synthesis-stimulating activity of HAT is due to its proteolytic activity.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effect of the protease inhibitor leupeptin on HAT-induced DNA synthesis in HBF. Serum-starved HBF were stimulated with 50 nM HAT for 24 h in the presence or absence of 50 µM leupeptin. Data are presented as means ± SE from 6 wells. *P < 0.05 compared with control.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Analysis of expression of protease-activated receptor (PAR)-2 mRNA in HBF by RT-PCR. HBF were grown in DMEM/10% FBS in a 60-mm dish. Total RNA was extracted from the cells and subjected to RT-PCR for PAR-2 as described in MATERIALS AND METHODS. Some of the products (cDNA) were amplified for 30 cycles with specific primer (denaturation at 85°C for 45 s, annealing at 58°C for 45 s, and elongation at 72°C for 2 min). GAPDH mRNA was used as a control of PAR-2 mRNA. PCR was performed for 30 cycles (denaturation at 85°C for 45 s, annealing at 50°C for 45 s, and elongation at 72°C for 1 min). A: electrophoresis on a 1.5% agarose gel of the PCR products. Molecular markers (100-bp step ladder, Promega) were used to verify the sizes of the RT-PCR products. B: relative levels of mRNA PAR-2/GAPDH.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effect of anti-PAR-2 antibody (Ab) on HAT-induced DNA synthesis in HBF. Experimental procedures were essentially the same as those for Fig. 2. Serum-starved cells were stimulated with HAT (50 nM), PAR-2 activating peptide (AP; 200 µM), thrombin (10 nM), or PAR-1 AP (200 µM) after they had been preincubated in the presence or absence of various concentrations of an anti-PAR-2 antibody (0.1–10 µg/ml) for 1 h. Data are presented as means ± SE from 6 wells. *P < 0.05 compared with response in the absence of antibody. Bars indicate treatment groups being compared.

To estimate the degree of PAR-2 internalization, the change in surface PAR-2 fluorescence intensity after PAR-2 AP stimulation for 0–30 min was analyzed by using Image-Pro software. After stimulation with 50 nM HAT, the fluorescence intensity decreased to 10% of that of the control at 5 min, and the decrease continued for 30 min (Fig. 7). Trypsin at 10 nM and PAR-2 AP at 200 µM also decreased the fluorescence intensity almost to the same extent as 50 nM HAT, whereas 10 nM thrombin and 200 µM PAR-1 AP caused no significant decrease (Fig. 7).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Internalization of PAR-2 in HBF after stimulation with HAT and other substances. Experimental procedures were essentially the same as those described in the Fig. 6. The change in surface PAR-2 fluorescence intensity after agonist stimulation was analyzed by Image-Pro software as described in MATERIALS AND METHODS. Results are expressed as a percentage of the surface PAR-2 immunoreactivity of unstimulated cells, and means ± SE of the fluorescence of 6 cells were expressed as a % of the control.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Effect of HAT on intracellular free Ca2+ concentration ([Ca2+]i) in HBF. HBF grown on 13 x 13 coverslips in a 12-well plate in DMEM/10% FBS to 60–70% confluence were cultured in DMEM/0.1% BSA for 24 h. Cells were loaded with 2 µM fura-2 AM in the presence of 0.2% Pluronic for 20 min in HEPES-buffered solution (HBS) at 37°C. [Ca2+]i was evaluated by microfluorometry as described in MATERIALS AND METHODS and expressed as the ratio of fluorescence intensity at 340 nm to that in 380 nm. The coverslips were inserted into a temperature-controlled chamber kept at 37°C and perfused with the same buffer containing the indicated agonists, trypsin, HAT, or PAR-2 AP, for 15–30 min.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Desensitization of the Ca2+ response to trypsin, PAR-2 AP, or HAT in HBF. The experimental procedures were essentially the same as those for Fig. 8. HBF were exposed to the first agonist in HBS. Then they were exposed to a second agonist 5 min after application of the first agonist, without an intervening wash. The agonists and concentrations were: 50 nM trypsin, 500 µM PAR-2 AP, 100 nM HAT, and 1 µM bradykinin. The first agonists are trypsin (A), PAR-2 AP (B), or HAT (C), and the second agonists are indicated on each graph.

View larger version (11K): [in this window] [in a new window]   Fig. 10. Effect of U0126, an MEK inhibitor, on HAT-induced DNA synthesis in HBF. Unless otherwise stated, experimental procedures were essentially the same as those for Fig. 2. HBF were stimulated with 50 nM HAT in the presence or absence of 10 or 20 nM U0126 for 48 h, and [3H]thymidine incorporation was measured, as described in MATERIALS AND METHODS. Data are presented as means ± SE from 6 wells. *P < 0.05 compared with response in the absence of antibody.

View larger version (17K): [in this window] [in a new window]   Fig. 11. Effect of HAT on phosphorylation of MAPK (p42/44, ERK1/2) in HBF. After HBF were grown to 70–80% confluence in 6-cm dishes in DMEM/10% FBS, they were starved in DMEM/0.01% BSA for 24 h. The cells were then stimulated with HAT for 15 min at 37°C. Phosphorylation of MAPK was analyzed by Western blotting as described in MATERIALS AND METHODS. Phosphorylated MAPK (top) and total MAPK (bottom) were measured using an anti-phosphorylated-MAPK antibody and an anti-MAPK antibody, respectively. A: effect of HAT concentration. Cells incubated with increasing concentrations of HAT showed increased phosphorylation of MAPK (top). The total amounts of MAPK present in the cell lysates are the same (bottom). B: time course of MAPK phosphorylation. Serum-starved HBF were exposed to 50 nM HAT for the indicated time periods, and cell lysates were analyzed as in A. MW, molecular weight.

View larger version (11K): [in this window] [in a new window]   Fig. 12. Effect of mast cell tryptase (MCT) on DNA synthesis in HBF in the presence of HAT. Unless otherwise stated, experimental procedures were essentially the same as those described in Fig. 2. A: HBF were stimulated with various concentrations of MCT. B: HBF were stimulated with MCT in presence of 16 nM HAT. C: HBF were stimulated with insulin in the presence of 16 nM HAT. *P < 0.05 compared with control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, direct cell counting showed that HAT at 10–100 nM stimulated by 53% the proliferation of subconfluent HBF, whereas MCT is known to be a potent mitogen for fibroblasts (1, 8, 15, 27). Our data show that the stimulatory effect of HAT on proliferation of HBF is similar to that of lung MCT but lower than that of thrombin.

The concentration of HAT, as measured by ELISA, in the mucoid sputum samples from patients with chronic airway diseases varies widely from 50 to 2,700 ng/ml, corresponding to a concentration of 2–100 nM. The HAT concentrations on the surfaces of the fibroblasts in bronchial airways are likely to be higher than those measured in airway secretions because HAT released into the airway lumen is diluted by airway secretions. Moreover, HAT seems to act in concert with other fibroblast growth factors. These results strongly suggest that in airway diseases, HAT is released into the airways at concentrations high enough to stimulate fibroblast proliferation.

In the present study, leupeptin almost completely inhibited HAT-induced DNA synthesis in HBF, suggesting that the peptidolytic activity of HAT is markedly inhibited by this serine protease inhibitor (36). The inactivation of HAT enzymatic activity by heating also abolished DNA synthesis. These results indicate that HAT enzymatic activity is required to stimulate DNA synthesis in fibroblasts and suggest that HAT activates some cell surface receptors by limited proteolysis.

It is well known that PARs are localized on the cell surface of various kinds of cells and activated by serine proteases such as thrombin and trypsin family proteases. In the present study, expression of PAR-2 mRNA in HBF as well as HLF was clearly shown by RT-PCR. Akers et al. (1) have also demonstrated expression of PAR-2 mRNA in primary HLF by RT-PCR. Indirect immunofluorescence showed localization of PAR-2 on the cell surface of HBF. PAR-2 is activated by trypsin and MCT, whereas PAR-1, PAR-3, and PAR-4 are activated by thrombin (3, 11, 13, 20, 23). We postulated that HAT might stimulate DNA synthesis via activation of PAR-2 because HAT belongs to the trypsin family of proteases (35, 36).

When PAR-2 on the cell surface is activated by agonists such as trypsin, it is internalized into early endosomes and then lysosomes, and endocytosis is -arrestin dependent (12, 14). Additionally, Iwakiri at el. (18) have reported that HAT induced PAR-2 internalization in a human keratinocyte cell line (HaCat). Therefore, the internalization of PAR-2 is thought to be an important measure of PAR-2 activation by an agonist. In the present immunofluorescence study, we demonstrated that in HBF, internalization of PAR-2 occurs within 5 min after stimulation with 10–100 nM HAT, 10 nM trypsin, or 200 µM PAR-2 AP. No internalization was observed after stimulation with 10 nM thrombin or 200 µM PAR-1 AP. These results support the hypothesis that HAT stimulates DNA synthesis in HBF via activation of PAR-2. Moreover, 10–100 nM HAT rapidly increased intracellular Ca²⁺ in HBF. This HAT-induced increase of intracellular Ca²⁺ was desensitized by pretreatment with trypsin, a known PAR-2 agonist, or by a synthetic PAR-2 AP. Previous investigations have shown that the trypsin-induced increase of intracellular Ca²⁺ concentration is mediated by activation of a heterotrimeric G protein coupled to PAR-2, and Miki et al. (22) also demonstrated that HAT increased intracellular Ca²⁺ in primary human bronchial epithelial cells via PAR-2.

Trypsin cleaves the extracellular NH₂- terminus of PAR-2 at a specific activation site that exposes a "tethered ligand" with the sequence SLIGKV. The newly generated ligand binds to the second extracellular loop and activates the receptor (11, 13). Molino et al. (23) demonstrated that MCT cleaved a synthetic peptide corresponding to the NH₂ terminus of PAR-2 at the activating site in a manner similar to that of trypsin activity. Recently, we found that that 10–100 nM HAT also cleaves a peptide corresponding to the NH₂ terminus of PAR-2 at the activating site (unpublished observation). This result also supports the hypothesis that HAT can activate PAR-2 in HBF and other cells. PAR-2 is coupled to G_q/11 and phospholipase C, leading to hydrolysis of phosphatidylinositol bisphosphate, Ca²⁺ mobilization, and activation of PKC and MAPK (12, 25). In the present study, U0126 inhibited the HAT-induced DNA synthesis in HBF, and HAT induced phosphorylation of MAPK, as shown by Western blot analysis. In addition, U0126 almost completely inhibited HAT-induced phosphorylation of MAPK as detected by Western blot analysis. These results suggest that an MAPK cascade mediates HAT-induced fibroblast proliferation and also support the hypothesis that HAT stimulates DNA synthesis in HBF via PAR-2.

The results shown in Fig. 12, B and C, support the idea that both HAT and MCT stimulate DNA synthesis mainly via PAR-2 and that HAT acts synergistically with other substances, such as insulin, that stimulate fibroblast proliferation via receptors other than PAR-2. Airway fibroblast proliferation may be regulated by a network of various kinds of fibrogenic mediators, which may be classified into two groups: nonenzymatic substances and serine proteases, including HAT, MCT, and thrombin. Ammit et al. (2) have reported that mast cell number is nearly the same in nonsensitized and sensitized human bronchi, including the epithelium, lamina propria, and adventitia, but is higher in the smooth muscle of sensitized bronchi. These findings indicate that MCT is released into not only the subepithelial layer but also into the epithelial layer of airways. However, the immunohistochemical study using anti-MCT antibody did not detect MCT in extracellular spaces, probably because MCT concentration is lower in the extracellular space compared with the concentration in mast cells (2). Like in the case of MCT, immunohistochemical examination using anti-HAT antibody also did not detect HAT in the extracellular spaces of airways (31). However, HAT is thought to be released from epithelial cells not only into the epithelial layer but also into the subepithelial layer during inflammation and tissue damage. Nakamura et al. (24) showed that bronchial epithelial cells regulate fibroblast proliferation by releasing both growth-stimulating factors and growth-inhibiting factors. Cambrey et al. (9) showed that primary human airway epithelial cells produce insulin-like growth factor I, which stimulates fibroblast proliferation. These reports indicate that the mediators released from bronchial epithelial cells may be related to peribronchial fibrosis. The results of the present study strongly suggest that HAT is one of the fibrogenic mediators released from airway epithelial cells.

Even in healthy individuals, the mucous membranes of airways are frequently injured by various kinds of inhaled toxic substances and infectious agents. In patients with chronic airway diseases, the mucous membranes of airways are more often injured than in healthy subjects due to the chronic inflammation associated with immunological responses and infection. Under these circumstances, fibroblasts proliferate and participate in the regeneration of tissues both in the normal and disease states, as well as in the progression of tissue fibrosis and remodeling in chronic inflammation (17, 19, 26, 28). The pathophysiological roles of fibroblasts may differ depending on the circumstances. For instance, fibroblasts are involved in such remodeling events as the thickening of the subepithelial layer following the deposition of interstitial collagen and fibronectin in chronic bronchial asthma (7, 17) and in the regeneration of tissue during acute inflammation of bronchial airways. The results of the present study strongly suggest that HAT may function to regulate fibroblast proliferation and that it might be involved in certain airway processes during chronic or acute airway diseases.

Recently, we found that HAT stimulates synthesis of IL-8 in primary bronchial epithelial cells via PAR-2 activation (21, 22). Therefore, HAT is thought to be involved in regulation of the biological function of airway epithelial cells via PAR-2. Chokki et al. (10) reported that HAT stimulates mucus production in NCI-H292 cells. Together with previous reports, the present results lead us to propose that HAT may be involved primarily in the defense of airways in healthy as well as diseased individuals and that it may lead to an amplification of the pathological state in chronic airway diseases. Further study will be necessary to clarify whether HAT is involved in the amplification of the subepithelial fibrosis associated with bronchial asthma.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Hiroshi Eguchi and his colleagues at Teijin Institute for Bio-Medical Research, Tokyo, Japan, for the kind gifts of human airway trypsin-like protease, its polyclonal antibodies, and a polyclonal antibody to PAR-2.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Matsushima, Dept. of Nutrition and Metabolism, Graduate School of Nutrition and Bioscience, Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, Japan 770-8503 (e-mail: rie{at}nutr.med.tokushima-u.ac.jp)

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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