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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0048OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 92-100, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0048OC

Lung Fibroblast Proteoglycan Production Induced by Serum Is Inhibited by Budesonide and Formoterol

Lizbet Todorova, Eylem Gürcan, Anna Miller-Larsson and Gunilla Westergren-Thorsson

Department of Cell and Molecular Biology, Lund University; and AstraZeneca R&D Lund, Lund, Sweden

Correspondence and requests for reprints should be addressed to Lizbet Todorova, Division of Vascular and Airway Research, Department of Experimental Medical Science, C13, BMC, Lund University, S-221 84 Lund, Sweden. E-mail: lizbet.todorova{at}med.lu.se


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Proteoglycans contribute to extracellular matrix remodeling in asthmatic airways. We investigated the effects of budesonide, a glucocorticoid, and formoterol, a long-acting {beta}2-adrenergic agonist, on serum-induced proteoglycan production by human lung fibroblasts. In 10% serum, total proteoglycan production was increased 1.5-fold (P < 0.01) compared with basal production in 0.4% serum. Budesonide (10–8 M) reduced this increase by 44% (P < 0.01) and, whereas formoterol (10–10–10–8 M) had no inhibitory effects, the drug combination abolished the increase (P < 0.01) without affecting fibroblast proliferation. This synergistic effect required functional glucocorticoid and {beta}-adrenergic receptors. The production of the proteoglycans decorin, biglycan, perlecan, and versican was increased 2.5- to 5-fold (P < 0.01) in 10% serum. Combination treatment with budesonide (10–8 M) and formoterol (10–10 M) abolished this increase to a significantly greater extent than either drug alone. In 10% serum, only versican mRNA was increased 1.4-fold (P < 0.05), whereas decorin mRNA was reduced to 39% (P < 0.01) of basal expression. These serum effects were counteracted by the drug combination, but there were no significant differences between the combination and either drug alone. Thus, the budesonide and formoterol combination seems to synergistically control serum-induced proteoglycan production, primarily at the post-transcriptional level. In conclusion, the proteoglycan upregulation characteristic of asthmatic airways may be limited by combination therapy with budesonide and formoterol.

Key Words: lung fibroblasts • airway remodeling • proteoglycans • glucocorticoids • {beta}2-agonists


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lung fibroblasts are the principal source of collagens, glycoproteins, and proteoglycans, molecules that constitute airway extracellular matrix (ECM) and levels of which are elevated in the subepithelial tissue of patients with asthma (1–3). The increased deposition of ECM components in asthmatic airways is associated with an increase in the number of activated fibroblasts, known as myofibroblasts (4, 5). It has been shown that an allergen challenge leads to a rapid increase in fibroblast activation and myofibroblast formation (6, 7) with upregulated synthesis of ECM components, contributing to the structural remodeling in the airways of patients with asthma (6). Such increased deposition of ECM molecules in asthmatic airways is probably driven by ongoing inflammatory processes, and is boosted by acute asthma episodes. In turn, an altered ECM may perpetuate the inflammatory process (8). One fundamental feature of inflammation is an increase in plasma exudation, a process in which plasma leaks out of blood vessels, thereby exposing the extravascular tissue to various plasma components. This may provide an important innate stimulus for rapid inflammatory responses to allergens, irritants, and infections (9, 10). Indeed, elevated concentrations of various serum proteins were observed in bronchoalveolar lavage fluid and induced sputum from patients with asthma exposed to allergens or other stimuli (11–13), and an increased albumin level in bronchoalveolar lavage fluid was found to be the best predictor of prolonged bronchial inflammation (11). Moreover, it was shown that the concentration of albumin in induced sputum correlates with asthma symptoms (14) and asthma severity (15), as well as several other hallmarks of asthmatic airway inflammation (14, 15). In addition, various in vitro studies have shown that serum is a potent cell stimulator that increases fibroblast proliferation and ECM synthesis (16–18), and may thereby promote airway ECM remodeling.

Several clinical studies have revealed that, in patients with asthma, treatment with an inhaled glucocorticoid (GC) and a long-acting {beta}2-agonist (LABA) provides better asthma control, including fewer disease exacerbations, than with a GC alone (19–21). It is conceivable that the reduction in asthma exacerbations is a consequence of enhanced antiinflammatory efficacy (22), together with greater relaxation and lesser proliferation of bronchial smooth muscle cells (23, 24). Less is known about the potential of combined GC and LABA treatment to regulate the ECM remodeling and fibrotic processes in asthma that is relatively resistant to therapy with inhaled GC (25, 26). ECM remodeling is a crucial target for asthma pharmacotherapy because abnormal ECM structure may contribute to pathologic alterations in the mechanics, reactivity, and function of asthmatic airways. Accordingly, bronchial subepithelial fibrosis was shown to be positively correlated with airway hyperresponsiveness and asthma severity (27, 28).

Essential elements for airway and lung ECM homeostasis are the proteoglycans—a family of molecules covalently linked with one or more glycosaminoglycans. They have diverse functions as a result of their structural heterogeneity, such as different sulfation patterns of the glycosaminoglycan chains, in addition to differences in the amino acid sequence of the core protein. Proteoglycans have both structural and regulatory properties that affect not only fluid balance and tissue mechanics, but also activities of cytokines and growth factors, as well as various cellular functions (29). Proteoglycans are known to participate in a range of biological processes, including inflammation, wound healing, and fibrosis. Several studies have revealed that versican, biglycan, perlecan, and decorin are hallmarks of early and/or late progression of the ECM remodeling seen in asthma (1–3, 30). A positive correlation has been found between the deposition of versican and biglycan in the subepithelial layer of the airway wall and airway hyperresponsiveness in patients with asthma (3). Similarly, in vitro production of perlecan and biglycan by bronchial fibroblasts isolated from the airways of patients with asthma was positively correlated with the donors' airway hyperresponsiveness (30).

Given the importance of increased proteoglycan deposition in the ECM remodeling seen in asthmatic airways, we undertook the present study to examine (1) whether proteoglycan production by activated lung fibroblasts is affected by treatment with a combination of GC and LABA, and (2) whether combination therapy has the potential to provide better control over proteoglycan upregulation than either drug alone. We studied the effects of budesonide (BUD), a GC, and formoterol (FORM), a LABA, alone and in combination, on both total and specific proteoglycan production at protein and mRNA levels in serum-stimulated human lung fibroblasts.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human fetal lung fibroblasts (cell line HFL-1, passage 12; CCL-153; American Type Culture Collection, Rockville, MD) between passages 14 and 20 were used. The morphology of HFL-1 is fibroblast-like, and the cells have features that resemble those of lung biopsy–derived fibroblasts (31).

Study Design
Experiments were performed on lung fibroblasts cultured in 96, 24, or 6-well plates in Earle's minimal essential medium (Sigma-Aldrich, Irvine, UK), containing 10% (vol/vol) donor calf serum, 1% glutamine, and 1% penicillin-streptomycin, at 37°C in a humidified 5% CO2 atmosphere.

Total proteoglycan production was measured as [35S]-sulfate incorporation into the proteoglycan glycosaminoglycan chains after incubation for 24 h with BUD (10–12–10–6 M) and FORM (10–12–10–6 M), separately or in combination (BUD 10–8 M with FORM 10–10–10–8 M), in sulfate-deprived Dulbecco's modified Eagle's medium supplemented with 10% serum. Cell medium with 0.4% serum was used as a control of basal activity. Drug control consisted of cell medium with 10% serum and 0.1% ethanol. Measured proteoglycan production in the medium was related to the total amount of protein in the corresponding cell layer. In a limited number of experiments, the effect of the drugs on proteoglycan production was also investigated by measuring [3H]-leucine incorporation into the proteoglycan core protein.

To determine whether the effects of BUD and FORM on the total proteoglycan production were mediated via the GC and {beta}-adrenergic receptor, respectively, the antagonists mifepristone (10–6 M) and propranolol (10–7 M) were used as cotreatments with drugs.

The effects of BUD (10–8 M) and FORM (10–10 M), separately and in combination, on the production of various specific proteoglycans were further investigated at both protein and, in separate experiments, mRNA levels. To examine whether these drugs inhibited the proteoglycan production directly or by affecting the cell proliferation, the effects of BUD and FORM on fibroblast proliferation were further investigated in the presence of 10% serum. All experiments were performed with duplicate or triplicate samples, and were repeated at least three times.

Labeling and Extraction of Proteoglycans
After incubating fibroblasts for 2 h in cell culture medium with or without drugs, 50 µCi/ml [35S]-sulfate or 25 µCi/ml [3H]-leucine (Perkin-Elmer Life Science, Boston, MA) was added to the cell cultures. After a total incubation time of 24 h, the cell media were decanted and supplemented with 0.1% phenylmethylsulfonyl fluoride (Sigma-Aldrich Co., St Louis, MO) and 2 mM EDTA, together with 0.1 mg/ml chondroitin sulfate-6 and 0.4 mg/ml dextran as carriers. The cell layers were extracted with RIPA-PBS lysis buffer (0.1% SDS, 0.5% deoxycolic acid, and 0.5% Triton X-100).

Proteoglycan Isolation
Collected cell media were applied to columns of diethylaminoethyl (DEAE) cellulose (Whatman, Maidstone, UK), pre-equilibrated with 6 M urea, 50 mM acetic acid, 5 mM N-ethylmaleimide, 1 mM EDTA, 5 µg/ml ovalbumin, pH 5.8. Unincorporated radioactive precursors were washed out with 140 vols of equilibration buffer, followed by 6 vols of 6 M urea, 0.6 M sodium acetate, 5 mM N-ethylmaleimide, 1 mM EDTA, 5 µg/ml ovalbumin, pH 5.8. Finally, proteoglycans were eluted with 2 x 1.5 ml of 4 M guanidine hydrochloride (ICN Biomedicals, Inc., Aurora, OH), 50 mM sodium acetate, 5 µg/ml ovalbumin, pH 5.8. Column eluates were analyzed for [35S]-sulfate activity using liquid scintillation counting.

Proteoglycan Identification
After the isolation process, equal volumes of total proteoglycan fractions obtained were precipitated with 3 vols of 95% ethanol with 0.4% acetic acid. Pellets were dried and dissolved in 0.1 M Tris-acetate, pH 7.3, followed by separation using SDS-PAGE. Electrophoresis was performed on 3–12% gradient gel using a 3% stacking gel. The gels were fixed in acetate:methanol:water (ratio, 7:50:43), dried in a Speed Gel SG 200 (Savant Instruments Inc., Farmingdale, NY) and exposed to Fuji imaging plates (Fujicolor Sverige AB, Skärholmen, Sweden) for 24 h. The intensity of the different bands containing versican, perlecan, biglycan, or decorin was visualized, further analyzed on Fuji FLA 3000 image analyzer (Seikagaku Kogyo, Tokyo, Japan), and related to the protein amount in the respective cell layers. The separation pattern of these proteoglycans has been identified in earlier studies by Western blot (30) and mass spectrometry, MALDI-TOF (32).

Protein Determination
The production of proteoglycans was related to the corresponding cell layer's total amount of protein, which was determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Bovine serum albumin was used as a standard.

RNA Isolation and cDNA Synthesis
Total RNA was extracted from cells incubated with combinations of BUD and FORM, as described previously here, using RNeasy kit (Quagen GmbH, Hilden, Germany), according to the manufacturer's instructions. Quantity and purity of total isolated RNA was measured spectrophotometrically using a NanoDrop ND-1000 (NanoDrop Technologies, Delaware, MD). To produce first-strand cDNA, 100 ng of RNA was used for RT-PCR using the first-strand cDNA synthesis kit for RT-PCR (Roche Applied Science, Indianapolis, IN). Briefly, cDNA transcription mixture was prepared using 1x reaction buffer, 5 mM MgCl2, 1.0 mM deoxynucleotide mix, 0.2 mM random primer, 50 U RNAse inhibitor, and 20 U avian myeloblastosis virus (AMV) reverse transcriptase in 20 µl total volume. For primer annealing and cDNA synthesis, reaction mixture was incubated at 25°C for 10 min, and then at 42°C for 60 min, followed by additional incubation at 99°C for 5 min and then at 4°C for 5 min.

Real-Time PCR
A total of 2 µl RT product, diluted 1:50, was added to 18 µl of SYBR Green I PCR Master Mix (Roche Diagnostics Scandinavia AB, Bromma, Sweden) containing 5 µM of each primer. The thermal profile was as follows: 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 5 s, and amplification at 72°C for 10 s. A melting curve was performed to determine the melting temperature of the amplicons, and consequently the specificity of the PCRs. The results were analyzed using the Lightcycler software (Roche Applied Science, Indianapolis, IN). After normalization to the housekeeping gene 18S, relative mRNA expression was calculated from differences in threshold cycle at which the fluorescence curve reaches its second derivate maximum. All real time PCRs were performed in duplicate or triplicate. As a negative control for the cDNA, mRNA samples were used that had not undergone reverse transcription.

Oligonucleotide Primers for Real-Time PCR
All primers used were generated using the oligonucleotide design program Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), except for versican (33), and obtained from A/S DNA Technology (Aarhus C, Denmark). The product size of all primers ranged between 100–160 base pair. Biglycan, forward 5'-gga ctc tgt cac acc cac ct -3' and reverse 5'-agc tcg gag atg tcg ttg tt -3'; decorin, forward 5'-tgg caa caa aatbcag cag ag -3' and reverse 5'-gcc att gtc aac agc aga ga -3'; perlecan, forward 5'-ggc ata cga tgg ctt gtc tc -3'and reverse 5'-agc cag cat gtc ctc atc at -3'; 18S, forward 5'-cga acg tct gcc cta tca ac -3' and reverse 5'-tgc ctt cct tgg atg tgg ta -3'.

Cell Proliferation
HFL-1 cells were allowed to grow for 5 h in a 96-well plate (5,000 cells/well) in Earle's minimal essential medium (Sigma-Aldrich, Irvine, UK) containing 10% serum. Cells were serum-starved overnight to synchronize cell cycle stage. They were then incubated in 10% serum–supplemented cell medium with BUD 10–10 or 10–8 M, FORM 10–10 or 10–8 M, or combinations, for 24, 48, or 72 h. Drug control consisted of cell medium containing 10% serum and 0.1% ethanol. As a basal proliferation control, cells were incubated in cell medium with 0.4% serum. Using crystal violet dye, cell proliferation was determined spectrophotometrically at 595 nm (34).

For additional control, fibroblast proliferation (for cells grown in 24- or 6-well plates) was also assayed in separate experiments, in which DNA was quantified using a modified fluorometric assay (35) with Hoechst 33,258 dye (Riedel-de Haën, Hannover, Germany). DNA was determined by excitation at 356 nm and emission at 458 nm, and the amount was then calculated using a known standard for bovine DNA.

Statistical Analysis
Data are expressed as percent of background control (where 100% corresponds to background conditions [i.e., at 0.4% serum]) and presented as means ± SEM. Statistical analysis was performed with Astute software 1.5 (DDU Software, Leeds, UK) using one-way analysis of variance (ANOVA), and included predetermined pair-wise comparisons of the effects of drug combination versus the effects of either drug alone (two comparisons for each variable). The issue of multiple comparisons was addressed by looking at the significance of overall P values from the ANOVA (these were all significant, except when specifically indicated in the text). Drug effects were analyzed as percent inhibition and calculated according to the following formula: % inhibition = 100 – (100 x [D–B]/[S-B]), where B, S, and D correspond to background control, 10% serum with drug vehicle, and 10% serum with drug, respectively. Comparisons with positive control (S – B) and background control (B) were performed by analysis of 95% and 99% confidence intervals obtained from ANOVA, i.e., analyses of whether zero value (test versus positive control) and 100 value (test versus background control) are included in these intervals. Differences were considered statistically significant at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of BUD and FORM on Total Proteoglycan Production
The total proteoglycan production by human lung fibroblasts, measured by [35S]-sulfate incorporation, was increased 1.5-fold (P < 0.01) in the presence of 10% serum compared with basal conditions (0.4% serum). BUD at 10–8 M reduced this increase by 44% (P < 0.01), whereas lower concentrations had no effect (Figure 1A). Higher concentration of BUD (10–7 M) resulted in a 23% reduction, but did not reach statistical significance. Similarly, BUD at 10–6 M (complementary experiment, n = 2; data not shown), showed no greater inhibition than BUD at 10–8–10–7 M.




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  		Figure 1. The effects of budesonide (BUD) and formoterol (FORM), either alone or in combination, on 10% serum–induced total proteoglycan production by lung fibroblasts (A), and the effects of receptor antagonists (B). Proteoglycan production was measured as [35S]-sulfate incorporation into the glycosaminoglycan side chains (or [3H]-leucine incorporation into the core protein [insert in A]) after 24 h of treatment with 10% serum with/without drugs: BUD 10–12–10–7 M (B12–B7), FORM 10–12–10–7 M (F12–F7), and their combinations, in the absence (A) or presence (B) of receptor antagonists: glucocorticoid (GC) receptor antagonist mifepristone 10–6 M (M6) and the {beta}-adrenergic receptor antagonist propranolol 10–7 M (P7). Open bars represent 10% serum without drug treatment (with drug vehicle). Data are expressed as percent of control (0.4% serum with drug vehicle) and presented as mean ± SEM. ++P < 0.01 versus 0.4% serum; *P < 0.05, **P < 0.01 versus positive control (10% serum minus 0.4% serum); §P < 0.05, §§P < 0.01 versus BUD alone; ##P < 0.01 versus FORM alone.

 
FORM exerted rather inconsistent effects on serum-induced proteoglycan production. When using 10–9 M, FORM increased proteoglycan production by 51% (P < 0.05), and a slight but not significant increase was also observed at 10–8 M. At 10–12–10–10 M and 10–7 M, there was an overall but not significant reduction of 7–30% (Figure 1A). Even at 10–6 M (complementary experiment, n = 2; data not shown), the reducing effect of FORM was not greater than that seen at 10–7 M.

To examine the effects of BUD and FORM in combination, we combined BUD 10–8 M with equal and lower concentrations of FORM (10–10–10–8 M). All combinations inhibited the 10% serum–induced total proteoglycan production down to the level seen under low-serum conditions (P < 0.01) (no significant difference versus 0.4% serum level; Figure 1A). The inhibitory effects of the combinations were generally significantly greater than the effects of the same concentrations of either drug alone. Thus, although FORM had no inhibitory effects when administered alone, it synergistically increased the effect of BUD ~ two-fold, resulting in complete inhibition of 10% serum–induced proteoglycan production.

The GC receptor antagonist, mifepristone (10–6 M), significantly decreased total proteoglycan production; however, administered together with BUD 10–8 M, it abolished the inhibitory effect of BUD (Figures 1A and 1B). Similarly, mifepristone abolished the inhibitory effects of the combination of BUD 10–8 M and FORM 10–10 M. The {beta}–adrenergic receptor antagonist, propranolol (10–7 M), had no effect on its own, but reduced the inhibitory effect of the combination of BUD and FORM to the level of BUD alone (Figure 1B). These results indicate that the synergistic effect of FORM on BUD-mediated inhibition of proteoglycan production, as shown in Figure 1A, depends on the presence of functional GC and {beta}-adrenergic receptors.

In addition, the effects of the drugs were also examined on basal proteoglycan production at 0.4% serum. Although BUD 10–8 M tended to decrease proteoglycan production, and FORM 10–10 M to increase it, these effects were not statistically significant. The same drug concentrations in combination resulted in a modest (13%) but significant decrease (P < 0.05; data not shown).

In a separate series of experiments, we examined the effects of BUD and FORM on total proteoglycan production measured as [3H]-leucine incorporation into proteoglycan core proteins. The data obtained (insert in Figure 1A) were similar to those described previously here using [35S]-sulfate incorporation in proteoglycan glycosaminoglycan chains.

Effects of BUD and FORM on Individual Proteoglycan Production
To examine whether BUD and FORM differentially affected specific proteoglycans, a part of the fraction containing total proteoglycan amount was subjected to SDS-PAGE and separated into versican, perlecan, biglycan, and decorin (Figure 2A). The production of these proteoglycans was significantly increased in the presence of 10% serum compared with the production under low-serum conditions (Figure 2B); versican was increased 5.2-fold (P < 0.01), whereas perlecan, biglycan, and decorin were increased 2.6- to 3.0-fold (P < 0.01 for all). BUD 10–8 M significantly decreased the production of versican, perlecan, and biglycan by 62% (P < 0.01), 35% (P < 0.05), and 50% (P < 0.01), respectively, but not of decorin production, where the 28% reduction was not statistically significant (Figure 2B). FORM 10–10 M significantly decreased the decorin production by 35% (P < 0.05), and reduced the production of the other proteoglycans to a similar extent as BUD. The combination of BUD and FORM significantly decreased the production of versican by 98% and decorin by 85%, and completely inhibited perlecan and biglycan (P < 0.01 for all). Statistically, all drug combinations reduced proteoglycan production to levels that were not significantly different from the basal levels (i.e., those seen under low-serum conditions). The inhibitory effects of the combination treatment were approximately additive for the serum-induced production of versican and biglycan, and synergistic for perlecan and decorin (Figure 2B). For all proteoglycans studied, the effect of the drug combination was significantly greater than the effects of either drug alone.




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  		Figure 2. Representative SDS-PAGE gel (A) for separation of the [35S]-sulfate–labeled proteoglycans produced by fibroblasts incubated for 24 h with 0.4% serum, or 10% serum with or without drugs: BUD 10–8 M (B8), FORM 10–10 M (F10), and their combination (B8 + F10). Equal volumes of all the total proteoglycan pools were first precipitated and then separated on 3–12% SDS-PAGE as: versican (molecular weight > 400 kD), perlecan (~ 300 kD), biglycan (~ 200 kD), and decorin (~ 100 kD). The intensity of each band was measured, and the percentage of each individual proteoglycan in each pool was calculated and related to the protein concentration in the respective cell layers (B). BUD 10–8 M (dark gray bars); FORM 10–10 M (light gray bars); their combination (hatched bars). Open bars represent 10% serum without drug treatment (with drug vehicle). Data are expressed as percent of control (0.4% serum with drug vehicle) and shown as mean ± SEM. ++P < 0.01 versus 0.4% serum; *P < 0.05, **P < 0.01 versus positive control (10% serum minus 0.4% serum); §P < 0.05, §§P < 0.01 versus BUD alone; #P < 0.05 versus FORM alone.

 
Effects of BUD and FORM on Individual Proteoglycan mRNA Expression
Real-time PCR experiments were performed to determine whether the changes in proteoglycan synthesis seen after treatment with BUD and FORM involved effects on mRNA expression. Incubation of fibroblasts with 10% serum affected the expression of the individual proteoglycan mRNA transcripts in different ways. Versican mRNA expression was induced 1.4-fold (P < 0.05) and this increase was completely or almost completely abolished by BUD 10–8 M (P < 0.01), FORM 10–10 M (P < 0.05), and the combination of both drugs (P < 0.05); there was no significant difference between the combination and either drug alone (Figure 3). Similarly, biglycan mRNA tended to increase 1.3-fold (P < 0.1); this increase was completely prevented by the combination of BUD 10–8 M and FORM 10–10 M (P < 0.05), but the 48% and 28% reductions seen with the single respective doses were not statistically significant. The 10% serum induction of perlecan mRNA expression was not statistically significant, and drug treatments had no effect. The overall P values from ANOVA were not significant for versican, biglycan, or perlecan. In contrast with other proteoglycans studied, decorin mRNA expression in 10% serum was reduced to 39% of the basal expression in 0.4% serum (P < 0.01; Figure 3). This effect was partly counteracted by BUD 10–8 M alone (P < 0.05), and in combination with FORM 10–10 M (P < 0.01). FORM alone had no effect, and there was no significant difference between the effects of BUD alone and in combination with FORM.



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  		Figure 3. The effects of BUD and FORM, either alone, or in combination, on mRNA expression of versican, perlecan, biglycan, and decorin. Lung fibroblasts were stimulated for 24 h with 0.4% serum, or 10% serum in the absence or presence of BUD 10–8 M (dark gray bars), FORM 10–10 M (light gray bars), and their combination (hatched bars). Individual proteoglycan mRNA expression was determined by real-time PCR as described in MATERIALS AND METHODS. Open bars represent 10% serum without drug treatment (with drug vehicle). Data are expressed as percent of control (0.4% serum with drug vehicle) and shown as mean ± SEM. +P < 0.05; ++P < 0.01 versus 0.4% serum; *P < 0.05; **P < 0.01 versus positive control (10% serum minus 0.4% serum); ##P < 0.01 versus FORM alone.

 
Effects of BUD and FORM on Fibroblast Proliferation
After 24-h incubation in 10% serum, fibroblast proliferation increased 31% (P < 0.01; Figure 4). BUD or FORM alone (both at 10–8 or 10–10 M) did not affect fibroblast proliferation after 24-h incubation. The combination of BUD 10–8 M with FORM 10–8 or 10–10 M resulted in 12–27% reduction of the fibroblast proliferation; however, these effects did not achieve statistical significance (Figure 4). Very similar drug effects were obtained with 48- and 72-h incubations (data not shown). Using light microscopy, no morphologic differences were observed between untreated and drug-treated cells.



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  		Figure 4. The effects of BUD and FORM on serum-induced fibroblast proliferation. Serum-starved cells at a density of 5,000 cells/well were incubated for 24 h in 10% serum with BUD 10–10 M (B10) or BUD 10–8 M (B8), FORM 10–10 M (F10), or10–8 M (F8), or their combinations. Cell proliferation was assessed spectrophotometrically using crystal violet dye (absorbance at 595 nm). Open bar represents 10% serum without drug treatment (with drug vehicle). Data are expressed as percent of control (0.4% serum with drug vehicle) and shown as mean ± SEM. ++P < 0.01 versus 0.4% serum.

 
Thus, the inhibitory effects of BUD and FORM on serum-induced proteoglycan production were not exerted through effects on cell proliferation.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have demonstrated that proteoglycan production in cultured human lung fibroblasts, when serum-induced, is inhibited by treatment with BUD and FORM in combination. Whereas BUD alone only modestly decreased total proteoglycan production, and FORM had no inhibitory effects, when combined, FORM significantly enhanced the effect of BUD—a synergistic effect that was shown to be dependent on functional GC and {beta}-adrenergic receptors. Regarding the individual proteoglycans studied— versican, perlecan, biglycan, and decorin—both BUD and FORM alone had similar inhibitory effects, but an enhanced and complete inhibition was seen with the drugs in combination. Furthermore, we have shown that BUD and FORM, either alone or in combination, decreased proteoglycan production per se, and this was achieved predominantly on the post-transcriptional level, and not by affecting fibroblast proliferation.

We measured total proteoglycan production by [35S]-sulfate incorporation into the proteoglycan glycosaminoglycan chains. All proteoglycans consist of a core protein to which different glycosaminoglycan chains of heparan sulfate or chondroitin/dermatan sulfate are covalently attached. Labeling glycosaminoglycan with [35S]-sulfate is considered to be a reliable method for the measurement of proteoglycan levels (36), as the glycosaminoglycan chains of proteoglycans are heavily sulfated. To address the question of whether the inhibitory drug effects were confined to the inhibition of glycosaminoglycan chains, we also investigated the production of proteoglycans by measuring the incorporation of [3H]-leucine into the proteoglycan core protein. We found similar effects with both methods, implying that the combination of BUD and FORM similarly affected both the glycosaminoglycans and the core proteins of the proteoglycans.

The range of drug concentrations used in this study is considered to reflect concentrations achieved in the airway and lung tissue 24 h after inhalation of GC (37–39) and LABA. Although airway/lung tissue concentrations of inhaled LABA have not yet been reported, maximal plasma concentrations after inhalation of a very high dose of FORM have been shown not to exceed 10–9 M (40). Studies involving inhaled GC have suggested that plasma concentrations achieved after inhalation of lipophilic drugs are 10- to 100-times lower than concentrations in airway and lung tissue (37, 38). This indicates that 10–8–10–7 M is the maximal concentration that can be achieved in airway and lung tissue after inhalation of FORM. The high (10–6 M) concentration of BUD and FORM examined in our complementary study is probably not reached in the airway and lung tissue even after inhalation of high clinical doses. If, however, this concentration is reached, it declines rapidly with time (39), and is interesting only as the top of the concentration–response curve. To examine the effects of BUD and FORM in combination, we used the lowest BUD concentration (10–8 M) that significantly inhibited total proteoglycan production, together with lower or equal concentrations of FORM (10–10–10–8 M). This reflects the fact that, in asthma therapy, LABA doses are typically 10- to 100-times lower than GC doses, but also that multiple inhalations of short-acting {beta}2-agonists for symptom relief during asthma exacerbations may result in equal doses of {beta}2-agonists and GC.

In our study, fibroblasts were stimulated with 10% donor calf serum, which contains high concentrations of various active components, including growth factors and hormones (hence its use as a cell activator in various cell systems). Stimulation with serum resulted in an enhancement of both fibroblast proliferation and proteoglycan production. After normalization to protein content, the increase in the individual proteoglycans examined was greater than that in the total proteoglycan production. Aside from experimental variation between separate series of experiments, this difference may be explained by the fact that fibroblasts produce and secrete several proteoglycans, not all of which are increased upon stimulation (41)—some may even be decreased (42). In fact, at the mRNA level, we have observed differential regulation of the proteoglycans, where serum stimulation resulted in a slight increase of versican and biglycan, whereas decorin mRNA was reduced. A similar pattern, as in our study, was previously reported in TGF-{beta}–stimulated lung fibroblasts, where induction of proteoglycan mRNA was lower than the induction of proteoglycans, and where an upregulation of decorin was accompanied by a downregulation of its mRNA (43). The slight increase of the proteoglycans' mRNA by serum, accompanied by the several-fold increase of proteoglycans measured, suggests that serum increased proteoglycan production predominantly at the post-transcriptional level.

Regarding the effects of drug treatments, BUD and FORM inhibited serum-induced total and individual proteoglycan production in an additive and synergistic manner. BUD partially decreased both the total and the individual proteoglycan production. FORM, on the other hand, had no significant inhibitory effects on total production, but it decreased the individual proteoglycans to a degree similar to that obtained by the use of BUD. This inconsistency in FORM effect suggests that there might be a pool of proteoglycans that are not inhibited by FORM, and may even be increased. However, at the mRNA level, FORM had no effects on the individual proteoglycans, except for a decrease of versican expression. Interestingly, BUD alone and in combination with FORM significantly counteracted both serum-induced increase of versican mRNA and the decrease of decorin mRNA. Similar action was earlier shown for dexamethasone, which reduced the TGF-{beta}–induced increase of biglycan mRNA and prevented a decrease of decorin mRNA in human skin fibroblasts (44). When comparing effects of the drugs at the protein (Figure 2B) and mRNA levels (Figure 3), it seems that BUD and FORM regulate serum-induced proteoglycan production primarily at the post-transcriptional level, for example, by increased proteolytic degradation.

In our study, the synergistic inhibition of serum-induced proteoglycan production by the BUD and FORM combination was due to the inhibition of fibroblast proteoglycan production and not to any effects on cell proliferation. The BUD and FORM combination tended to decrease fibroblast proliferation, although this effect was not statistically significant. A decrease in fibroblast proliferation has been observed by another combination of the GC, fluticasone propionate, and the LABA, salmeterol. (45).

There have been several reports on the cooperating inhibitory effects of GC and LABA on various activation markers of cultured human lung fibroblasts (45, 46). Most recently, it was demonstrated that salmeterol enhanced the inhibitory effect of fluticasone propionate on the hyaluronan production induced in fibroblasts by IL-1{beta} and TNF-{alpha} (47). Similarly, preliminary results from ongoing experiments in our laboratory show enhanced inhibition by the combination of BUD and FORM on TGF-{beta}1–induced proteoglycan production in lung fibroblasts (48). Additive and synergistic effects of GC and LABA have also been described in airway smooth muscle cells (22, 23).

The mechanisms of the interactions between GC and LABA are not completely elucidated, and may be specific to cell, stimulus, and/or response studied. However, there is an increasing body of evidence to suggest that the effects of both GC and LABA may be mutually enhanced via interactions at the receptor level and interwoven signal transduction pathways. For example, GC increase the expression of {beta}2-adrenergic receptors and promote their activation (49), whereas {beta}2-agonists activate the GC receptor in various cells (23, 50)—an effect that was also demonstrated very recently in a human study (51). On the other hand, many of the effects of LABA, in particular, but also of GC, have been reported to be independent of the {beta}-adrenergic receptor (52) and classic, nuclear GC receptor (53), respectively. Therefore, we investigated the effects of receptor antagonists, and showed that the inhibitory effects of BUD were mediated via the GC receptor, whereas the synergistic effects of BUD and FORM in combination were dependent on both the GC and {beta}-adrenergic receptors. Paradoxically, mifepristone—which blocked the effects of both BUD alone and BUD and FORM in combination—had its own inhibitory effect when applied alone; it is known, however, that mifepristone may sometimes act in vitro as a receptor agonist. The inhibitory effect of FORM on the individual proteoglycan production remains to be elucidated. However, it has been suggested that the antifibrotic effects of {beta}-agonists in serum-stimulated lung fibroblasts are mediated via the cyclic adenosine monophosphate pathway (18).

The specific proteoglycans investigated in this study have been reported to be involved in the early or late processes of ECM remodeling in asthmatic airways (1–3, 30, 54), as well as in lung fibrosis and other diseases characterized by fibrotic tissue remodeling (54). Versican, a large chondroitin sulfate proteoglycan, forms large aggregates with hyaluronic acid, and therefore has an impact on fluid balance and tissue mechanics. Perlecan, a heparan sulfate, is a basal lamina proteoglycan that regulates stability and permeability of basement membrane, and binds and activates the basic fibroblast growth factor that promotes angiogenesis (55). Versican, and the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, directly and indirectly affect cell responses to growth factors and cytokines, in addition to cell phenotype, proliferation, and migration (56). Versican and biglycan are implicated early in airway remodeling, as increased levels of these components are observed in the airways of patients with mild asthma (3). In contrast, decorin, although structurally related to biglycan, appears to be involved to a greater extent in a later phase of the airway remodeling process (1, 54). Interestingly, decorin has been reported to have an antifibrotic effect under certain conditions in vivo (57). On the other hand, decorin is associated with the increased deposition of collagens type I and III (58), and is colocalized with TGF-{beta} in asthmatic airways (59). Recent studies have shown that cells treated with decorin differentiate into myofibroblasts with increased production of {alpha}–smooth muscle actin, proteoglycans, and collagen type I (60, 61). These data suggest that decorin may be involved in generating a more fibrotic ECM type. Our in vitro findings on the inhibition of decorin production by the combination of BUD and FORM were recently confirmed in vivo in a mouse model of asthma, in which inhaled allergen induced a 10-fold increase of decorin in the epithelial basement membrane and submucosa of bronchi and bronchioles (62).

In summary, we have demonstrated that total proteoglycan production in cultured human lung fibroblasts is modestly inhibited by BUD, and that this effect is synergistically enhanced by the addition of FORM. We have also shown that the combination of BUD and FORM is more efficient than either drug alone in the inhibition of versican, biglycan, and perlecan (proteoglycans known as markers of an early-inflammatory ECM), as well as decorin (which is usually upregulated in the more fibrotic ECM). The drug effects were exerted primarily at the post-transcriptional level. We conclude that increased early and late deposition of proteoglycans in asthmatic airways may be limited by treatment with a combination of BUD and FORM. Clinical studies are warranted to confirm these conclusions.


   Acknowledgments
 
The authors thank Dr. Ralph Brattsand for initiating this study and for his scientific input into the article, and Dr. Ian Naya for his valuable comments. They also greatly appreciate the technical assistance of Marie Wildt.


   Footnotes
 
This work was supported by the Medical Faculty of Lund University, the Swedish Medical Research Council (11550), and AstraZeneca.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0048OC on September 15, 2005

Conflict of Interest Statement: L.T. received educational research grants from AstraZeneca in the amount of $47,000 in 2002 and $63,000 in 2003. E.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.M.-L. is a permanent employee of AstraZeneca. G.W.-T. received $14,000 in research grants from AstraZeneca in 2002.

Received in original form February 1, 2005

Accepted in final form September 9, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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