Thrombin stimulates the expression of PDGF in lung epithelial cells

Shino Shimizu1, Esteban C. Gabazza1,2, Tatsuya Hayashi1, Masaru Ido1, Yukihiko Adachi2, and Koji Suzuki1

1 Department of Molecular Pathobiology and 2 Third Department of Internal Medicine, Mie University School of Medicine, Tsu-city, Mie 514-8507, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.

coagulation; lung fibrosis; bronchial asthma; platelet-derived growth factor receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STRUCTURAL REMODELING of the lung tissue is believed to be the consequence of repeated cycles of epithelial injury and repair process associated with chronic pulmonary disease (3). Remodeling of the alveolar compartment of the lung, such as that occurring in pulmonary fibrosis, is characterized by interstitial and intra-alveolar deposition of connective tissue and collapse and distortion of air spaces by scar contraction (5). Tissue remodeling is also a severe complication of airway disorders, particularly of bronchial asthma. It is believed that airway remodeling leading to thickening of the bronchial wall is responsible for the underlying irreversible airway hyperreactivity observed in patients with chronic bronchial asthma (23). The morphological changes of airway remodeling include epithelial sloughing, thickening of the epithelial basement membrane, edema, inflammatory cell infiltration of the submucosa, hyperplasia of mucus glands, accumulation of myofibroblasts, and thickening of medial smooth muscle layers (2, 7, 8). Several growth factors such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta , epidermal growth factor (EGF), and insulin-like growth factor, secreted by inflammatory or resident structural cells, have been implicated in the mechanism of the structural changes associated with lung and airway remodeling (15, 16).

Thrombin is a serine protease that, besides its critical role in thrombosis and hemostasis, has also been described as inducing cellular and molecular events relevant to tissue remodeling. For example, thrombin may induce platelet aggregation and platelet release of thromboxane A2, serotonin, procoagulant, and growth factors. Thrombin may also stimulate chemotaxis of monocytes and lymphocytes, the motility and proliferation of fibroblasts and smooth muscle cells, and the secretion of growth factors and proteases from the vascular endothelium (9, 11, 22). These observations together with previous studies (10, 18, 19) showing increased generation of thrombin in pulmonary diseases suggest that thrombin may also play an important role in the pathogenesis of lung and airway remodeling. We hypothesized that thrombin may also indirectly promote lung and airway remodeling by increasing the production of growth factors from alveolar and bronchial epithelial cells. To demonstrate this possibility, in the present study, we evaluated the effect of thrombin on the secretion of PDGF from alveolar and bronchial epithelial cells and the effect of conditioned medium (CM) of these cells stimulated with thrombin on the proliferation of lung fibroblasts and bronchial smooth muscle cells. It is known that the cellular effects of thrombin depend on stimulation of the protease-activated receptor (PAR)-1, PAR-3, or PAR-4 (6). In the present study, we also evaluated the expression of these receptors in alveolar and bronchial epithelial cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Dulbecco's modified Eagle's medium (DMEM), L-glutamine, vitamin solution, sodium pyruvate, nonessential amino acids, transferrin, bovine pituitary extract, TRIzol reagent, and Superscript preamplification system were purchased from GIBCO BRL (Life Technologies, Grand Island, NY). Fetal bovine serum (FBS) was from BioWhittaker (Walkersville, MD); and penicillin, streptomycin, and dexamethasone were from Nacalai Tesque (Kyoto, Japan). BSA, Ham's F-12 medium, epinephrine, gentamicin, amphotericin B, insulin, triiodothyronine, cholera toxin, endothelial growth supplement, L-leucine, L-lysine, L-methionine, HEPES, and nonspecific protease from Streptomyces griseus were from Sigma (St. Louis, MO). Human EGF was from Higeta Shouyu (Tokyo, Japan). Hydrocortisone and retinoic acid were from Wako (Osaka, Japan). bFGF, cDNA probes of PDGF-A and PDGF-B, goat anti-human PDGF antibody, and Quantikine enzyme immunoassay kits for EGF, bFGF, and PDGF-AB were from R&D Systems (Minneapolis, MN). All other chemicals and reagents used were of the best quality commercially available.

Cell culture. The human lung fibroblast cell line WI-38 was obtained from RIKEN Cell Bank (Ibaraki, Japan). The human lung carcinoma-derived alveolar epithelial cell line A549 and the human bronchial epithelial cell line BEAS-2B were obtained from the American Type Culture Collection (Manassas, VA). Normal human bronchial epithelial (NHBE) cells and human bronchial smooth muscle cells were purchased from Clonetics (Walkersville, MD) as primary culture cells. Both lung fibroblasts and A549 cells were cultured in DMEM containing 10% heat-inactivated FBS, 50 µg/ml of penicillin, 50 µg/ml of streptomycin, 2 mM L-glutamine, 2% vitamin solution, 110 µg/ml of sodium pyruvate, and 0.1 mM nonessential amino acids. NHBE cells were cultured in CCMD161 medium (Clonetics) supplemented with 30 µg/ml of bovine pituitary extract, 0.5 µg/ml of BSA, 0.5 µg/ml of epinephrine, 50 µg/ml of gentamicin, 50 ng/ml of amphotericin B, 0.5 ng/ml of human EGF, 0.5 µg/ml of hydrocortisone, 5 µg/ml of insulin, 7 ng/ml of triiodothyronine, 10 µg/ml of transferrin, and 0.1 ng/ml of retinoic acid. BEAS-2B cells were cultured in serum-free Ham's F-12 medium supplemented with 5 µg/ml of insulin, 5 µg/ml of transferrin, 20 ng/ml of human EGF, 0.1 µM dexamethasone, 20 ng/ml of cholera toxin, 30 µg/ml of bovine pituitary extract, and 1 µM retinoic acid. Nasal epithelial cells were isolated from nasal polyps of patients with chronic sinusitis according to the method previously described (27). Briefly, stroma from polyploid lesions was removed, and the epithelial cells were dissociated by incubation with 0.1% nonspecific protease from Streptomyces griseus in Ham's F-12 medium at 4°C for 20 h. The cell suspension was then filtered through a 60-µm Nitex mesh, centrifuged at 500 g for 10 min, washed, and resuspended in DMEM-Ham's F-12 medium containing 0.5 mM L-leucine, 0.5 mM L-lysine, 0.1 mM L-methionine, 0.3 mM MgCl2, 0.4 mM MgSO4, 1 mM CaCl2, 9 mg/l of phenol red, 14 mM NaHCO3, 7 mM L-glutamine, 50 µg/ml of penicillin, 50 µg/ml of streptomycin, 10 µg/ml of insulin, 0.1 µg/ml of hydrocortisone, 0.1 µg/ml of cholera toxin, 5 µg/ml of transferrin, 25 ng/ml of human EGF, 8 µg/ml of endothelial growth supplement, 50 µg/ml of bovine pituitary extract, and 30 mM HEPES. The human bronchial smooth muscle cells were positive for smooth muscle cell alpha -actin but negative for von Willebrand factor. The cells were cultured with a culture kit (Clonetics) containing a modified MCDB131 medium supplemented with 5% FBS, 50 µg/ml of gentamicin, 50 ng/ml of amphotericin B, 0.5 ng/ml of human EGF, 5 µg/ml of insulin, and 2 ng/ml of human FGF. All cells were cultured in 10-cm dishes in an atmosphere composed of 5% CO2 and 95% air. Confluent cells were harvested by a brief exposure to 0.025% trypsin-0.02% EDTA in HEPES-buffered saline (50 mM HEPES and 150 mM NaCl, pH 7.4) and passaged after 5~7 days.

Preparation of CM. A549, NHBE, and BEAS-2B cells were grown to 80~90% confluence in 10-cm dishes, and human nasal epithelial cells were grown in 12-well tissue culture plates. After incubation for 24 h in basal medium without supplements, the cells were washed and incubated for 48 h in the presence of varying concentrations of thrombin. The CM was harvested, centrifuged at 1,200 g for 15 min, and stored at -80°C until used. To evaluate the participation of the thrombin receptor (PAR-1), after preincubation in FBS- and supplement-free medium for 24 h, NHBE cells were treated with the PAR-1 agonist peptide (SFLLRNP) or its negative control (YFLLRNP) for 48 h, and then the CM was collected and used in the assays.

Proliferation assay. CM was used in the proliferation assays after it was treated with hirudin (250 antithrombin units) at 37°C for 30 min and after confirmation of the absence of thrombin activity in CM with the chromogenic substrate S2238 for thrombin (Chromogenix, Mölndal, Sweden) by amidolytic assay. The proliferative effect of CM on fibroblasts and bronchial smooth muscle cells was assessed by a colorimetric method as previously described (21). Briefly, after the cells were cultured in 96-well plates up to 70~80% of confluence, their growth was arrested by culturing overnight in medium without FBS and growth factors. After the cells were washed, 100 µl of hirudin-treated CM were applied to each well and incubated for 48 h under an atmosphere of 5% CO2 and 95% air. After being appropriately washed, the cells were fixed by treatment with 10% Formalin for 30 min; the wells were then washed, and 1% (wt/vol) methylene blue in 0.01 M borate buffer was added. After the wells were washed with borate buffer, a 1:1 dilution of ethanol in 0.1 M HCl was added, and the absorbance at 650 nm was measured with a microplate reader. This method has been previously validated with a number of characterized growth factors (21). In addition, proliferative assays were also carried out with a cell-counting kit purchased from Dojindo (Kumamoto, Japan); this kit contains a tetrazolium compound, WST-1, a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, which is used for the colorimetric determination of cell number.

In separate experiments to evaluate whether the proliferative effect of CM depends on the presence of PDGF, the CM prepared from cells stimulated with the same concentration of thrombin (250 nM) was treated with varying concentrations of anti-human PDGF antibody at 37°C for 30 min. The anti-PDGF antibody-treated CM was then used in proliferation assays performed as described above.

Enzyme immunoassay. Enzyme immunoassays were carried out to assess the concentration of growth factors in CM. Commercial immunoassay kits (described in Reagents) were used to measure the concentrations of PDGF-AB, bFGF, and EGF.

RNA isolation and RT-PCR. RT-PCR was carried out to evaluate the expression of thrombin receptors (PAR-1, PAR-3, and PAR-4) and PDGF receptors (alpha  and beta ) in alveolar and airway epithelial cells, lung fibroblasts, and bronchial smooth muscle cells. Total RNA was extracted from confluent cells by the guanidine isothiocyanate procedure with the TRIzol Reagent. Two micrograms of total RNA were reverse transcribed with oligo(dT) primer and the Superscript preamplification system kit following the manufacturer's instructions. The primers used in the PCR are listed in Table 1. The PCR mixture contained PCR buffer (100 mM Tris·HCl, pH 8.3, and 200 mM KCl), 25 mM MgCl2, 10 µM deoxyribonucleosides (dATP, dGTP, dCTP, and dTTP), 10 µM each forward and reverse primer, an appropriate amount of diethyl pyrocarbonate-treated distilled water, and 1 U of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The mixture was then divided into aliquots in PCR tubes containing 2 µl of cDNA sample, and the amplification was performed with a PC-800 Programmable Temp control system cycler (Astec, Fukuoka, Japan). The PCR products were then electrophoresed on a 2% agarose gel and stained with 0.5 µg/ml of ethidium bromide. PCR of samples treated without RT was also carried out to ensure the absence of contaminating DNA in total RNA prepared from epithelial cells.

                              
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Table 1.   Primer sequences for RT-PCR

Southern hybridization. Southern hybridization was carried out to assess the effect of thrombin on gene expression of PDGF in airway epithelial cells. Preparation of total RNA and PCR products was performed as described in RNA isolation and RT-PCR. After the PCR products were resolved on a 2% agarose gel, the gel was incubated in a solution containing 1.5 M NaCl and 500 mM NaOH for 30 min at room temperature and then incubated again in a solution containing 1 M ammonium acetate and 20 mM NaOH at room temperature for 30 min. The cDNA was transferred for 24 h by capillary action onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Tokyo, Japan) and fixed to the membrane by ultraviolet irradiation with a Stratalinker (Stratagene, La Jolla, CA). The membrane was then prehybridized in a solution containing 20 mM Tris·HCl, pH 7.5, 750 mM NaCl, 2.5 mM EDTA, 1% SDS, 5× Denhardt's solution, 0.5 µg/ml of denatured salmon sperm DNA, and 50% formamide and then hybridized with cDNA probes of human PDGF-A, PDGF-B, or human glyceraldehyde-3-phosphate dehydrogenase (Cayman Chemical) that were labeled with [alpha -32P]dCTP with a DNA labeling kit (Takara Shuzo, Kyoto, Japan).

Northern hybridization. Northern hybridization was carried out with a digoxigenin Northern starter kit (Boehringer Mannheim). Ten micrograms of total RNA were electrophoretically separated on 1.2% agarose gels and transferred to positively charged Boehringer Mannheim nylon membranes by capillary elution. The membranes were hybridized with digoxigenin-11-UTP-labeled RNA probes. Immunologic detection was then performed following the manufacturer's instructions.

Statistical analysis. All data are expressed as means ± SE unless otherwise specified. The difference between three or more variables was calculated by analysis of variance. Statistical analyses were carried out with the StatView 4.1 package software for the Macintosh (Abacus Concepts, Berkeley, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of CM on proliferation of lung fibroblasts and bronchial smooth muscle cells. To evaluate the role of thrombin in the secretion of PDGF from bronchial and alveolar epithelial cells, the proliferative activity of CM from these cells stimulated with varying concentrations of thrombin on lung fibroblasts and bronchial smooth muscle cells was assessed after complete inhibition of residual thrombin activity in the CM with hirudin. In the methylene blue-based proliferation assays, CM from the primary bronchial epithelial cells (NHBE cells) significantly stimulated the proliferation of fibroblasts and bronchial smooth muscle cells in a dose-dependent manner (Fig. 1A). Similarly, CM from the alveolar epithelial cell line A549 significantly stimulated the proliferation of fibroblasts and bronchial smooth muscle cells in a dose-dependent fashion (Fig. 1B). These findings were in good agreement with the results obtained with the cell-counting kit containing the tetrazolium salt WST-1. For preparation of the control sample, aliquots of culture medium were incubated in the presence of the same concentrations of thrombin but in the absence of epithelial cells; after treatment with hirudin, the control medium was also used in the proliferative assays. These control media did not show proliferative activity on lung fibroblasts (data not shown), further ensuring that hirudin completely inhibited residual thrombin activity in our assays.


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Fig. 1.   Effects of conditioned medium (CM) from normal human bronchial epithelial (NHBE; A) and alveolar epithelial (A549; B) cells treated with indicated concentrations of thrombin for 48 h on proliferation of lung fibroblasts (open bars) and bronchial smooth muscle cells (solid bars). Cell supernatant was used in this assay as CM. Residual thrombin activity in CM was inhibited by hirudin before the assay. Lung fibroblasts and bronchial smooth muscle cells were incubated in each CM for 48 h. CM from NHBE and A549 cells stimulated the proliferation of fibroblasts and bronchial smooth muscle cells. Values are means ± SE, with 100% being the no. of cells that proliferated in the presence of CM from epithelial cells prepared with 0 nM thrombin. Assays were done in triplicate in 5 separate experiments. * P < 0.05. ** P < 0.01. # P < 0.05. ## P < 0.03 [all compared with control samples (0 nM thrombin)].

Effect of anti-PDGF antibody on proliferative activity of CM from airway epithelial cells. To assess whether the proliferative activity of CM depends on the secretion of PDGF from epithelial (NHBE) cells, the inhibitory effect of anti-PDGF antibody that recognizes PDGF-AA, PDGF-BB, and PDGF-AB was evaluated. Compared with control samples (0 µg/ml of anti-PDGF antibody), the anti-PDGF antibody significantly inhibited the proliferative activity of CM prepared with NHBE cells stimulated with 250 nM thrombin in a dose-dependent manner (Fig. 2). This inhibition of fibroblast proliferation was significant above 10 µg/ml of the anti-PDGF antibody.


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Fig. 2.   Effect of anti-platelet-derived growth factor (PDGF) antibody on proliferative activity of CM. CM prepared from NHBE cells stimulated with the same concentration of thrombin (250 nM) was treated with indicated concentrations of anti-human PDGF antibody at 37°C for 30 min before the assay. Fibroblasts were incubated in each medium for 48 h. Values are means ± SE expressed as percentage over basal medium that was taken as 100% of fibroblast proliferation. Assays were done in triplicate in 5 separate experiments. * P < 0.05. ** P < 0.005 [both compared with control sample (0 µg/ml of anti-PDGF antibody)].

Secretion of PDGF and other growth factors by epithelial cells treated with thrombin. To assess whether thrombin stimulation increases the secretion of PDGF and other growth factors from epithelial cells, the concentration of these growth factors in CM was measured. As shown in Fig. 3, thrombin significantly and specifically stimulated the secretion of PDGF-AB but not that of bFGF or EGF from NHBE cells. The increase in the concentration of PDGF-AB in CM stimulated with thrombin was significant from a thrombin concentration of 62.5 nM for BEAS-2B cells (Fig. 4A); in A549 cells (Fig. 4B), thrombin stimulated the secretion of PDGF-AB at a thrombin concentration of 62.5 nM and reached a plateau with 125 nM thrombin. Thrombin also significantly increased the concentration of PDGF-AB in CM prepared with primary cells from bronchial or nasal epithelium; thrombin increased PDGF-AB concentration from a concentration of 62.5 nM for NHBE cells (Fig. 5A) and 125 nM for nasal epithelial cells (Fig. 5B). The secretion of PDGF-AB from thrombin-treated BEAS-2B cells was also time dependent (Fig. 6).


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Fig. 3.   Concentrations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and PDGF-AB in CM from NHBE cells treated with 125 nM thrombin. The cells were grown in 12-well tissue culture plates. They were incubated in the presence (125 nM; solid bars; +) and absence (open bars; -) of thrombin for 48 h, and then the concentration of each growth factor in CM was determined with commercial enzyme immunoassay kits.



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Fig. 4.   Effect of various concentrations of thrombin on PDGF-AB secretion from established epithelial cell lines. BEAS-2B (A) and A549 (B) cells were grown in 10-cm-diameter dishes. The cells were incubated with indicated concentrations of thrombin for 48 h, and then the concentration of PDGF-AB in CM was measured. Values are means ± SE. Assays were done in triplicate in 2 separate experiments. * P < 0.01. ** P < 0.001. # P < 0.0001 [all compared with control samples (0 nM thrombin)].



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Fig. 5.   Effect of indicated concentrations of thrombin on PDGF secretion from primary bronchial (NHBE; A) and nasal epithelial (B) cells. NHBE cells were grown in 10-cm-diameter dishes, and human nasal epithelial cells were grown in 12-well tissue culture plates. The cells were incubated with for 48 h, and then the concentration of PDGF-AB in CM was determined. Values are means ± SE. Assays were done in triplicate in 2 separate experiments. * P < 0.001; ** P < 0.001. # P < 0.005 [both compared with control samples (0 nM thrombin)].



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Fig. 6.   Time course of PDGF-AB secretion from thrombin-treated epithelial cells (BEAS-2B). BEAS-2B cells were grown in 12-well tissue culture plates and incubated in the presence (solid bars) and absence (open bars) of thrombin (125 nM) for indicated times, and then the concentration of PDGF-AB in CM was measured. Values are means ± SE. Assays were done in triplicate in 2 separate experiments.

Effect of thrombin on PDGF gene expression in epithelial cells. The effect of thrombin on the gene expression of PDGF-A and PDGF-B in NHBE cells was evaluated by RT-PCR and Southern blot analysis. As shown in Fig. 7, thrombin significantly increased the expression of the PDGF-B gene but not that of the PDGF-A gene in NHBE cells. Similarly, Northern blot analysis also showed that thrombin stimulates the expression of the PDGF-B gene at a transcriptional level (data not shown).


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Fig. 7.   Effect of thrombin on gene expression of PDGF in epithelial (NHBE) cells. Total RNA was prepared with NHBE cells treated with and without thrombin (125 nM) for 48 h, and RT-PCR and Southern hybridization (top) were performed as described in MATERIALS AND METHODS. The intensities of the bands were densitometrically quantified (bottom), and the results were normalized to the internal standard band glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thrombin increased the expression of PDGF-B gene but not that of PDGF-A gene in NHBE cells.

Effect of PAR-1 agonist peptide on PDGF secretion in epithelial cells. To evaluate the participation of the thrombin receptor on PDGF secretion induced by thrombin, the effect of the PAR-1 agonist peptide on the concentration of PDGF in CM from NHBE cells was assessed. Like thrombin, the PAR-1 agonist peptide also induced PDGF-AB secretion from NHBE cells in a dose-dependent manner (Fig. 8). This effect was significant from a peptide concentration of 25 µM. The negative control peptide did not induce PDGF-AB secretion (data not shown). These results suggest that thrombin stimulation of PDGF-AB secretion from airway epithelial cells is mediated by PAR-1.


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Fig. 8.   Concentration of PDGF-AB in CM from protease-activated receptor (PAR)-1 agonist peptide-treated epithelial cells. NHBE cells were grown in 12-well tissue culture plates and incubated with indicated concentrations of PAR-1 agonist peptide for 48 h, and then the concentration of PDGF-AB in CM was determined. Values are means ± SE. Assays were done in triplicate in 2 separate experiments. Control peptide did not affect PDGF secretion (data not shown). * P < 0.05 compared with control sample (0 µM PAR-1 agonist).

Expression of PARs in epithelial cells. To evaluate the type of thrombin receptors expressed by epithelial cells, mRNAs of PAR-1, PAR-3, and PAR-4 were analyzed by RT-PCR. RT-PCR showed that both alveolar (A549) and airway (NHBE) epithelial cells expressed not only PAR-1 but also PAR-3 and PAR-4; however, the bronchial epithelial cell line BEAS-2B did not express PAR-3 (Fig. 9).


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Fig. 9.   RT-PCR analysis of PAR-1, PAR-3, and PAR-4 in epithelial cells. Total RNA was prepared with cells incubated in basal medium without supplements for 24 h, and RT-PCR was performed as described in MATERIALS AND METHODS. Both alveolar (A549) and bronchial (NHBE) epithelial cells expressed not only PAR-1 (592 bp) but also PAR-3 (513 bp) and PAR-4 (542 bp) thrombin receptors, but BEAS-2B cells did not express PAR-3.

Expression of PDGF receptors in epithelial cells, lung fibroblasts, and bronchial smooth muscle cells. Because it is known that the effect of PDGF depends on the presence of its receptors, alpha  and beta , on target cells, the expression of PDGF receptors was evaluated. RT-PCR showed that both PDGF receptors (alpha  and beta ) are expressed by epithelial cells, lung fibroblasts, and bronchial smooth muscle cells (Fig. 10).


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Fig. 10.   RT-PCR of PDGF receptors in NHBE cells, lung fibroblasts, and bronchial smooth muscle cells (BSMC). Total RNA was prepared with the respective cells incubated in basal medium without supplements for 24 h, and RT-PCR was performed as described in MATERIALS AND METHODS. Both PDGF-alpha (501 bp) and PDGF-beta (216 bp) receptors were expressed by NHBE cells, lung fibroblasts, and BSMC.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study showed that CM from alveolar and bronchial epithelial cells treated with thrombin significantly stimulates the proliferation of lung fibroblasts and bronchial smooth muscle cells compared with that in the medium of untreated cells. Immunoassays and inhibition experiments revealed that the increased growth of these cells depends on the enhanced production and secretion of PDGF-AB induced by thrombin in epithelial cells.

It is well known that increased thrombin generation occurs in the lungs of most patients with inflammatory diseases of the pulmonary parenchyma and airways and that it plays a critical role in tissue remodeling (10, 18, 19). After injury, a tissue factor-activated factor VII complex is formed on lung and airway epithelial and/or endothelial cells, leading to activation of the extrinsic pathway of coagulation, thrombin generation, fibrin formation, and fibrosis (4, 12, 14). In the lung and airways, generated thrombin may modulate the tissue repair response by altering vascular permeability, by stimulating the secretion of proteases and the production or degradation of components of the connective tissue, and by promoting adhesion, spreading, and proliferation of fibroblasts and smooth muscle cells (1). In addition to its direct effects, thrombin may also play a role in lung tissue remodeling by stimulating the secretion of growth factors from several cells. For example, it has been reported that thrombin may induce increased expression of PDGF from lung fibroblasts, vascular smooth muscle cells, endothelial cells, and alveolar macrophages (13, 19, 20, 22, 26). In the present study, we hypothesized that thrombin may also participate in tissue remodeling by stimulating the secretion of growth factors from alveolar and bronchial epithelial cells.

The CM prepared by treating alveolar and bronchial epithelial cells with different concentrations of thrombin was found to stimulate the proliferation of lung fibroblasts and bronchial smooth muscle cells. This CM effect did not depend on the direct proliferative effect of thrombin because its activity in CM was blocked completely by hirudin before use in the proliferative assays. Measurement of growth factors in CM showed that thrombin upregulated the secretion of PDGF-AB from bronchial epithelial cells in dose- and time-dependent manners; RT-PCR analysis showed that this increased secretion of PDGF-AB depends on increased mRNA expression of PDGF-B. It is worth noting that upregulation of PDGF-AB was induced by thrombin not only in transformed epithelial cell lines but also in primary bronchial and nasal epithelial cells, suggesting that this effect is probably also observed in vivo at sites of inflammation. Inhibition assays carried out with anti-PDGF antibody confirmed that PDGF was responsible for the proliferative activity of CM from epithelial cells. Overall, these findings suggest that stimulation of PDGF secretion from epithelial cells may constitute another mechanism of thrombin participation in lung and airway remodeling. The biological relevance of PDGF in the mechanism of tissue remodeling is illustrated by the important role it plays in the pathogenesis of fibroproliferative processes such as wound healing, pulmonary fibrosis, atherosclerosis, myelofibrosis, scleroderma, and inflammatory joint diseases. Properties that implicate PDGF in the mechanism of tissue remodeling are its strong mitogenic and chemotactic activities on fibroblasts and smooth muscle cells and its ability to stimulate angiogenesis and the secretion of cytokines (e.g., transforming growth factor-beta ) that, in turn, may upregulate the expression of extracellular matrix components (17, 24, 25). A previous study (24) has shown that the PDGF-B chain is the polypeptide that is mainly expressed in lung tissues from patients with lung fibrosis (24); the demonstration in the present study that thrombin upregulates the expression of PDGF-B but not that of PDGF-A reinforces the important role that thrombin plays in the process of lung fibrosis.

The cellular effects of thrombin have been found to depend on stimulation of the thrombin receptors PAR-1, PAR-3, and PAR-4 (6). After cellular activation by thrombin, a new NH2-terminal peptide, which functions as a tethered ligand for the receptor itself, is unmasked on PAR-1, PAR-3, or PAR-4. The tethered ligand of PAR-1 has been shown to promote proliferation of fibroblasts and vascular smooth muscle cells, cytokine secretion, and deposition of collagen in the extracellular matrix. To evaluate whether PAR-1 also mediates the effect of thrombin in primary airway epithelial cells (NHBE cells), the effect of the PAR-1 agonist peptide on PDGF-AB secretion was assessed. The PAR-1 agonist peptide induced PDGF-AB secretion from NHBE cells in a similar fashion as thrombin. RT-PCR analysis showed that besides the PAR-1 gene, PAR-3 and PAR-4 genes are also expressed by bronchial (NHBE and BEAS-2B) and alveolar (A549) epithelial cells. These findings suggest that PARs may play an important role in the mechanism of lung and airway remodeling by mediating the mitogenic and secretory activity of thrombin in epithelial cells.

PDGF is known to occur in the form of three dimers (PDGF-AA, PDGF-BB, and PDGF-AB) and to bind to specific high-affinity receptor subunits (alpha alpha , beta beta , and alpha beta ) on the surface of responsive cells (fibroblasts and smooth muscle cells). The effect of PDGF depends on the type of PDGF receptor subunit available on the cell surface. PDGF-BB can bind to any one of these receptor subunits; PDGF-AA can bind only to an alpha alpha -receptor; and PDGF-AB can bind either to an alpha alpha -receptor or to an alpha beta -receptor (24). In the present study, RT-PCR analysis showed that both the alpha - and beta -subunits were equally expressed by lung fibroblasts and bronchial smooth muscle cells. These findings explain the proliferative activity of PDGF-rich CM on fibroblasts and bronchial smooth muscle cells observed in the present study. In addition, airway epithelial cells express both subunit receptors, suggesting that they may also play an important role in epithelial regeneration in the lung and airways.

In conclusion, the results of this study showed for the first time that 1) thrombin regulates PDGF expression and secretion from nasal, bronchial, and alveolar epithelial cells; 2) the cellular effect of thrombin on PDGF secretion by epithelial cells is mediated by its receptor PAR-1; 3) airway epithelial cells express the thrombin receptors PAR-1, PAR-3, and PAR-4; and 4) the thrombin-induced secretion of PDGF is sufficient for stimulating the proliferation of fibroblast and bronchial smooth muscle cells. Overall, these findings suggest that thrombin may play an important role in the process of lung and airway remodeling by stimulating the secretion of PDGF via its PAR-1 receptor from alveolar and bronchial epithelial cells.


    FOOTNOTES

Address for reprint requests and other correspondence: E. C. Gabazza, Third Dept. of Internal Medicine, Mie Univ. School of Medicine, Edobashi 2-174, Tsu-city, Mie 514-8507, Japan (E-mail: gabazza{at}clin.medic.mie-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. §1734 solely to indicate this fact.

Received 8 January 2000; accepted in final form 28 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bar-Shavit, R, Benezra M, Sabbah V, Bode W, and Vlodavsky I. Thrombin as a multifunctional protein: induction of cell adhesion and proliferation. Am J Respir Cell Mol Biol 6: 123-130, 1992[ISI][Medline].

2.   Brewster, CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, and Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 3: 507-511, 1990[ISI][Medline].

3.   Busse, WW, and Parry DE. The biology of asthma. In: Fishman's Pulmonary Diseases and Disorders, edited by Fishman AP.. New York: McGraw-Hill, 1998, p. 721-733.

4.   Chapman, HA, Allen CL, and Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 133: 437-443, 1986[ISI][Medline].

5.   Crouch, E. Pathobiology of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 259: L159-L184, 1990[Abstract/Free Full Text].

6.   Dery, O, Corvera CU, Steinhoff M, and Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am J Physiol Cell Physiol 274: C1429-C1452, 1998[Abstract/Free Full Text].

7.   Djukanovic, R, Roche WR, Wilson JW, Beasley CR, Twetyman OP, Howarth RH, and Holgate ST. Mucosal inflammation in asthma. Am Rev Respir Dis 142: 434-457, 1990[ISI][Medline].

8.   Ebina, M, Takahashi T, Chiba T, and Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 148: 720-726, 1993[ISI][Medline].

9.   Fager, G. Thrombin and proliferation of vascular smooth muscle cells. Circ Res 77: 645-650, 1995[Free Full Text].

10.   Gabazza, EC, Taguchi O, Tamaki S, Takeya H, Kobayashi H, Yasui H, Kobayashi T, Hataji O, Urano H, Zhou H, Suzuki K, and Adachi Y. Thrombin in the airways of asthmatic patients. Lung 177: 253-262, 1999[ISI][Medline].

11.   Glusa, E. Vascular effects of thrombin. Semin Thromb Hemost 18: 296-304, 1992[ISI][Medline].

12.   Gross, TJ, Simon RH, and Sitrin RG. Tissue factor procoagulant expression by rat alveolar epithelial cells. Am J Respir Cell Mol Biol 6: 397-403, 1992[ISI][Medline].

13.   Harlan, JM, Thompson PJ, Ross RR, and Bowen-Pope DF. Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells. J Cell Biol 103: 1129-1133, 1986[Abstract].

14.   Idell, S. Coagulation, fibrinolysis, and fibrin deposition in lung injury and repair. In: Pulmonary Fibrosis, edited by Phan SH, and Thrall RS.. New York: Dekker, 1995, vol. 80, p. 743-776. (Lung Biol Health Dis Ser)

15.   Kelley, J. Cytokines of the lung. Am Rev Respir Dis 141: 765-788, 1990[ISI][Medline].

16.   Kelley, J. Growth factors. In: Asthma, edited by Barnes PJ, Grunstein MM, Leff AR, and Woolcock AJ.. Philadelphia, PA: Lippincott-Raven, 1997, p. 683-694.

17.   Klagsbrun, M, and D'Amore PA. Regulators of angiogenesis. Annu Rev Physiol 53: 217-239, 1991[ISI][Medline].

18.   Kobayashi, H, Gabazza EC, Taguchi O, Wada H, Takeya H, Nishioka J, Yasui H, Kobayashi T, Hataji O, Suzuki K, and Adachi Y. Protein C anticoagulant system in patients with interstitial lung disease. Am J Respir Crit Care Med 157: 1850-1854, 1998[Abstract/Free Full Text].

19.   Ohba, T, McDonald JK, Silver RM, Strange C, LeRoy EC, and Ludwicka A. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor alpha-receptor. Am J Respir Cell Mol Biol 10: 405-412, 1994[Abstract].

20.   Okazaki, H, Majesky MW, Harker LA, and Schwartz SM. Regulation of platelet-derived growth factor ligand and receptor gene expression by alpha-thrombin in vascular smooth muscle cells. Circ Res 71: 1285-1293, 1992[Abstract].

21.   Oliver, MH, and Harrison NK. A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J Cell Sci 92: 513-518, 1989[Abstract].

22.   Papadimitriou, E, Manolopoulos VG, Hayman GT, Maragoudakis ME, Unsworth BR, Fenton JW, II, and Lelkes PI. Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells. Am J Physiol Cell Physiol 272: C1112-C1122, 1997[Abstract/Free Full Text].

23.   Reed, CE. The natural history of asthma in adults: the problem of irreversibility. J Allergy Clin Immunol 103: 539-547, 1999[ISI][Medline].

24.   Ross, R. Platelet-derived growth factor. Lancet 1: 1179-1182, 1989[ISI][Medline].

25.   Ross, R, Raines EW, and Bowen-Pope DF. The biology of platelet-derived growth factor. Cell 46: 155-169, 1986[ISI][Medline].

26.   Tani, K, Ogushi F, Takahashi H, Kawano T, Endo T, and Sone S. Thrombin stimulates platelet-derived growth factor release by alveolar macrophages in rats---significance in bleomycin-induced pulmonary fibrosis. J Med Invest 44: 59-65, 1997[Medline].

27.   Usui, S, Shimizu T, Kishioka C, Fujita K, and Sakakura Y. Secretory cell differentiation and mucus secretion in cultures of human nasal epithelial cells: use of a monoclonal antibody to study human mucin. Ann Otol Rhinol Laryngol 109: 271-277, 2000[ISI][Medline].


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