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Published ahead of print on July 3, 2003, doi:10.1165/rcmb.2003-0005OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 91-100, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0005OC

Role of Cytosolic Phospholipase A2 in Prostaglandin E2 Production by Lung Fibroblasts

Moumita Ghosh, Allison Stewart, Dawn E. Tucker, Joseph V. Bonventre, Robert C. Murphy and Christina C. Leslie

Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver; Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, Colorado; Department of Medicine, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts; and Astra Zeneca R&D Charnwood, Safety Assessment, Loughborough, United Kingdom

Address correspondence to: Christina C. Leslie, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. E-mail: lesliec{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin (PG)E2 acts in an autocrine fashion to suppress proliferation of lung fibroblasts and production of collagen, and may negatively regulate pulmonary fibrosis. The role of Group IVA cytosolic phospholipase A2 {alpha} (cPLA2{alpha}) in PGE2 production was investigated by comparing lung fibroblasts from wild-type and cPLA2{alpha}-deficient mice. Arachidonic acid release from wild-type mouse lung fibroblasts (MLF+/+) was stimulated by serum, A23187 plus phorbol-myristate acetate (PMA), and lysophosphatidic acid (LPA) plus platelet-derived growth factor, but was >= 80% lower from cPLA2{alpha}-deficient MLF (MLF-/-). Transforming growth factor-ß increased cyclooxygenase-2 (COX2) expression to similar levels in MLF+/+ and MLF-/-, but MLF+/+ produced an order of magnitude more PGE2 than MLF-/- in response to A23187/PMA or platelet-derived growth factor/LPA. MLF+/+ synthesized less collagen than MLF-/-, supporting a role for PGE2 in suppressing collagen production. An SV40 immortalized line developed from MLF+/+ released arachidonic acid and expressed COX2 to levels similar to those of primary fibroblasts but produced 30-fold lower amounts of PGE2. Unlike primary fibroblasts, immortalized cells were deficient in microsomal PGE synthase (mPGES) but expressed slightly higher levels of cytosolic PGES. The results demonstrate a primary role for cPLA2{alpha} in providing arachidonic acid for PGE2 production in mouse lung fibroblasts and support a role for this pathway in regulating collagen production.

Abbreviations: 5-lipoxygenase, 5-LO • cyclooxygenase-2, COX2 • cytosolic PGES, cPGES • cytosolic PLA2-{alpha}, cPLA2{alpha} • Dulbecco's modified Eagle's medium, DMEM • extracellular matrix, ECM • fetal bovine serum, FBS • interleukin, IL • immortalized MLF, IMLF • lysophosphatidic acid, LPA • mitogen-activated protein kinases, MAPK • mouse lung fibroblasts, MLF • microsomal prostaglandin E synthase, mPGES • polymerase chain reaction, PCR • platelet-derived growth factor, PDGF • prostaglandin, PG • protein kinase C, PKC • phospholipase A2, PLA2 • phorbol-myristate acetate, PMA • sodium dodecyl sulfate, SDS • transforming growth factor-ß, TGF-ß • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fibroblasts play an important role in tissue repair following injury. However, during chronic diseases excessive fibroblast responses can lead to scarring and compromised organ function, as occurs in fibrotic diseases of the lung (1). The pathologic consequences of pulmonary fibrosis are in part due to the excessive proliferation of fibroblasts and deposition of extracellular matrix (ECM) proteins such as collagen. The extent of lung injury may be determined by the balance between the levels of pro- and antifibrotic mediators (2, 3). Certain cytokines and growth factors have been proposed to contribute to fibroproliferative diseases of the lung and include platelet-derived growth factor (PDGF), transforming growth factor-ß (TGF-ß) and tumor necrosis factor-{alpha} (TNF-{alpha}). Proliferating epithelial cells, fibroblasts- and inflammatory cells are sources of these mitogens and ECM-inducing proteins. In addition to increased production of profibrotic agents, a decrease in the production of antifibrotic mediators may also contribute to the extent of lung disease. Agents that inhibit fibroblast responses have been described and include interferon-{gamma}, glucocorticoids, prostaglandin (PG)E2, and epidermal growth factor (2, 4).

There is evidence that oxygenated metabolites of arachidonic acid can play opposing roles in the development of fibrosis. Arachidonic acid is converted to PGs through the cyclooxygenase (COX) pathway by the constitutive enzyme COX1 or by the inducible COX2. Arachidonic acid is also converted to leukotrienes by 5-lipoxygenase (5-LO), an enzyme primarily found in inflammatory cells (5). Lung fibroblasts produce PGE2, which acts in an autocrine fashion to inhibit proliferation and production of collagen (6, 7). TGF-ß can exert both pro- and antiproliferative effects on lung fibroblasts, and the antiproliferative effects are blocked when COX upregulation and PGE2 production are prevented with indomethacin, suggesting that PGE2 suppresses growth (4). Fibroblasts from fibrotic lungs have been shown to produce less PGE2 both basally and in response to various agonists (8, 9). This was attributed to a diminished ability of the fibrotic fibroblasts to upregulate COX2. In addition, COX2-deficient mice exhibit enhanced susceptibility to pulmonary fibrosis (8, 10). Collectively, these results suggest that PGE2 negatively regulates fibroblast proliferation, deposition of ECM proteins, and the pathogenesis of fibrosis. In contrast to the protective role of PGE2, proinflammatory eicosanoids derived from the 5-LO pathway, leukotriene C4, and leukotriene B4 are implicated in the induction of pulmonary fibrosis. Leukotrienes are increased in tissues from patients with idiopathic pulmonary fibrosis, and 5-LO–deficient mice are protected from bleomycin-induced pulmonary fibrosis (11, 12).

The production of eicosanoids is controlled by the availability of free arachidonic acid, which is released from membrane phospholipid by the action of phospholipase A2 (PLA2). There are three main types of PLA2s in mammalian cells: the Group IV enzymes, which includes the well studied Group IVA 85-kD cytosolic PLA2 {alpha} (cPLA2{alpha}); the Group VI calcium-independent PLA2s; and the secretory PLA2s, of which there are 10 different genes in mammalian cells (13). There are data suggesting that members from each of these families of PLA2 can contribute to the release of arachidonic acid (13). However, cPLA2{alpha} is the only PLA2 that exhibits specificity for arachidonic acid, and its role in mediating agonist-induced arachidonic acid release for eicosanoid production in a variety of cell models is well established (14). A role for cPLA2{alpha} in mediating acute lung injury induced by sepsis or acid aspiration, and in mediating bronchial reactivity associated with anaphylaxis, has been demonstrated in studies comparing cPLA2{alpha}-deficient and wild-type mice (15, 16). It has recently been demonstrated that pulmonary fibrosis is attenuated in cPLA2{alpha}-deficient mice treated with bleomycin, which correlated with a decrease in influx of inflammatory cells and a decrease in production of the proinflammatory eicosanoids, thromboxane A2, and leukotrienes (17). These data demonstrate an important role for cPLA2 in mediating arachidonic acid release for production of profibrotic eicosanoids by inflammatory cells. However, the identity of the PLA2 that mediates arachidonic acid release for production of PGE2 by primary lung fibroblasts from adult tissue has not been established. By comparing lung fibroblasts isolated from cPLA2+/+ and cPLA2-/- mice, we have found that cPLA2{alpha} plays a primary role in mediating agonist-induced arachidonic acid release and PGE2 production. In addition, cPLA2{alpha}-deficient fibroblasts were found to synthesize increased amounts of collagen compared with wild-type fibroblasts, supporting a role for PGE2 in regulating levels of ECM protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and Culture of Mouse Lung Fibroblasts
Mice in which the Group IVA 85-kD cPLA2{alpha} gene had been disrupted were generated using 129 embryonic stem cells in a C57BL/6 strain background (18). Lungs from these and strain-matched wild-type littermates were removed and used for fibroblast isolation. Lungs were minced in sterile phosphate-buffered saline and the washed tissue pieces placed in 100-mm tissue culture dishes containing Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 1% penicillin/streptomycin/glutamine, nonessential amino acids, and sodium pyruvate (supplemented DMEM). The medium was replenished approximately every 3 d. After 14 d, the fibroblast growing out of the explants were trypsinized and plated in supplemented DMEM. Lung fibroblasts were also isolated by a technique involving enzymatic digestion of mouse lungs (19). Lungs from 3–4 cPLA2{alpha}+/+ and cPLA2{alpha}-/- mice were minced into 1–2 mm3 pieces and incubated in calcium and magnesium-free Hanks' balanced salt solution containing 0.3 mg/ml type IV collagenase and 0.5 mg/ml trypsin for 60 min at 37°C with shaking as previously described (19). The dissociated cells were centrifuged and plated in supplemented DMEM. After a 1-h incubation, the adherent fibroblasts were rinsed with Hanks' balanced salt solution and cultured in supplemented DMEM. The primary lung fibroblasts (MLF) were used between passages 3 and 8. To compare the growth rate of MLF from cPLA2{alpha}+/+ (MLF+/+) and cPLA2{alpha}-/- (MLF-/-) mice, cells were plated in 12-well tissue culture plates at a density of 1.25 x 104 cells/well in supplemented DMEM. Cells were trypsinized and cell numbers determined 6 h after plating and every 24 h for 4 d.

Immortalization of MLF
For immortalization, MLF+/+ and MLF-/- (second passage) were trypsinized and suspended in supplemented DMEM. The cells (2 x 106) were incubated with SV40 (kindly provided by Dr. James Cook, University of Illinois, Chicago) at a multiplicity of infection of 30 for 1 h at 37°C with occasional mixing. The fibroblasts were then plated at 5 x 105 cells in 100-mm dishes in supplemented DMEM. After 3–5 d the culture medium was replaced with DMEM containing 2% FBS to favor growth of transformed cells. Well-defined colonies (visible by 14 d) were isolated with cloning cylinders and expanded. SV40 transformation was confirmed by Western blotting using a monoclonal antibody against the large T antigen.

Arachidonic Acid Release
Fibroblasts were plated in 24-well tissue culture plates at a density of 2.5 x 104 cells/well in supplemented DMEM for the primary fibroblasts and supplemented DMEM with FBS decreased to 2% for the SV40 immortalized fibroblasts and incubated for 6 h at 37°C with 5% CO2. Cells were washed twice and incubated overnight in serum-free DMEM containing 0.1% bovine serum albumin and 0.2 µCi [3H]arachidonic acid/well. The cells were washed three times and then incubated in serum-free DMEM containing 0.1% bovine serum albumin and stimulated with agonists for the times indicated. The culture medium was removed and centrifuged at 15,000 rpm for 15 min. Cells were solubilized with 0.1% Triton-X-100. The level of radioactivity in the culture supernatant and the cells was measured, and the amount released was calculated as a percentage of the total radioactivity. The [3H]label released into the medium was confirmed to be [3H]arachidonic acid by extracting the medium according to Bligh and Dyer and separating the labeled lipids by thin-layer chromatography using hexane/diethyl ether/acetic acid, 80:20:1.

Western Blotting
Fibroblasts were washed with phosphate-buffered saline and then scraped into ice-cold lysis buffer: 50 mM Hepes, pH 7.4, 150 mM sodium chloride, 1.5 mM magnesium chloride, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 200 µM sodium vanadate, 10 mM terasodium pyrophosphate, 100 mM sodium fluoride, 300 nM p-nitrophenyl phosphate, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. After incubation on ice for 30 min, lysates were centrifuged at 15,000 rpm for 15 min and protein concentration in the supernatant determined by the bicinchoninic acid method. Lysates were boiled for 5 min after addition of Laemmli electrophoresis sample buffer (5x), and then proteins (15–25 µg total protein) were separated on 10% or 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels for analysis of cPLA2{alpha} and COX2, respectively. For Western blot analysis of mPGES and cytosolic PGES (cPGES), 10–20% gradient SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) were used. After electrophoresis, samples were transferred to nitrocellulose membrane, blocked with 5% nonfat milk for 1 h, and then incubated overnight at 4°C with polyclonal antibody against mPGES or cPGES (Cayman Chemical, Ann Arbor, MI) at 1:500, with anti-cPLA2{alpha} polyclonal antibody at 1:5,000 dilution, or with anti-COX2 and COX1 polyclonal antibody (Cayman Chemical) at 1:1,000 dilution, in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween containing 5% nonfat milk. The membranes were then incubated with anti-rabbit IgG horseradish peroxidase antibody (1:5,000 dilution) for 30 min at room temperature. The immunoreactive proteins were detected using the Amersham ECL system (Piscataway, NJ).

Collagen Synthesis Assay
Deposition of collagen into ECM by MLF+/+ and MLF-/- was assayed by measuring the amount of L-2,3 [3H]proline (NEN life-Sciences, Boston, MA) incorporated into collagen as a proportion of total deposited ECM protein as previously described (20). Briefly, MLF+/+ and MLF-/- were plated in a 12 well plate (5 x 104 cells/well) in supplemented DMEM. After reaching ~ 90% confluence, the cells were incubated in fresh supplemented DMEM containing 0.5 µCi/ml of [3H]proline and 10 µg/ml ascorbic acid for 48 h. The culture medium was removed, the cells washed with TCP buffer (50 mM Tris-HCl, pH 7.5, 1 mM CaCl2 and 1 mM proline) and then lysed with 25 mM NH4OH for 10 min at room temperature. The deposited ECM was ethanol fixed (70% ethanol, two times for 15 min at room temperature), washed twice with TCP buffer and then incubated for 4 h at 37°C in 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2 and 62.5 mM N-ethyl maleimide with or without 30 U/ml collagenase (from Clostridium histolyticum; Sigma, St. Louis, MO). Supernatants containing the degraded collagen were removed after 4 h and residual deposited ECM on the dish was solubilized by overnight incubation in 0.3 M NaOH containing 1% SDS. The amount of [3H]proline in the supernatants and solubilized residual ECM was determined by liquid scintillation counting. The amount of collagen in ECM was calculated by comparing the difference in counts in collagenase-digested and collagenase free samples as previously described (20).

PGE2 Assays
PGE2 levels in the culture medium were quantitated by enzyme-linked immunosorbent assay (Cayman Chemical) or by mass spectrometry for samples containing serum. For analysis of samples by mass spectrometry, culture medium (2 ml) from MLF+/+ and MLF-/- grown in DMEM containing 10% FBS was mixed with 2 parts methanol. Stable isotope internal standard D4-PGE2 (Cayman Chemical) was added. After centrifugation, the supernatant was diluted to 20% methanol with water and applied to a 100 mg STRATA C18 solid phase extractor cartidge (Phenomenex, Torrance, CA) that was prewashed with 2 ml ethanol followed by 3 ml water. The loaded cartridge was washed with 3 ml water followed by 2.5 ml 20% acidified methanol, and the sample eluted with 1 ml methanol. Analysis was performed by reverse phase high-performance liquid chromatography on a Columbus C18, 150 mm x 1 mm column (Phenomenex,Torrance, CA) coupled to an Applied Biosystems (Framingham, MA) API 3,000 LC/MS/MS system operated in the negative ion MRM mode. Specific MRM transitions for PGE2 (351.2/271.2) and for the internal standard D4-PGE2 (355.2/275.2) were monitored. Peak areas of the analytes were compared with the internal standards, and these ratios were compared with a calibration curve to determine the amounts of PGE2.

Polymerase Chain Reaction Analysis of mPGES
cDNA was generated (Advantage RT PCR kit; Clontech, Palo alto, CA) from 1 µg of total RNA isolated (SV Total RNA Isolation Kit; Promega, Madison, WI) from the lung fibroblasts. Polymerase chain reaction (PCR) was performed using 10 µl of cDNA for mPGES and 5 µl of cDNA for glyceraldehyde phosphate dehydrogenase (control). Specific primers for mouse mPGES were 5'-GAGTTGAAGTCCAGGCCGGCTAGCCG-3' and 5'-GCTGAGGAGGCTTCAGCTGCTGGTCAC-3'. After an initial denaturation at 95°C for 2 min, 30 cycles of PCR were performed (94°C for 1 min, 55°C for 2 min and 72°C for 3 min), with a final elongation cycle of 72°C for 10 min. The PCR product was cloned into the TA cloning vector (Invitrogen, Carlsbad, CA) and confirmed to be mPGES by sequence analysis.

Statistical Analysis
Data were analyzed using two-way or three-way ANOVA or two-sample t test depending on the size of the experiment. Analyses were performed using PROC GLM in SAS. Comparisons were considered significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Arachidonic Acid Release from Lung Fibroblasts
Mammalian cells contain several types of PLA2 that can potentially mediate arachidonic acid release for eicosanoid production. Fibroblasts were isolated from lungs of wild-type or cPLA2{alpha}-deficient mice to investigate the role of this PLA2 in mediating arachidonic acid release and PGE2 production. MLF+/+ and MLF-/- exhibited morphology and growth characteristics consistent with fibroblasts, and showed characteristic staining with antibodies to vimentin (not shown). To obtain a continuous source of cells, MLF+/+ and MLF-/- were immortalized with SV40.

A variety of agents were tested for their ability to induce arachidonic acid release from primary and immortalized fibroblasts. cPLA2 is regulated by an increase in intracellular calcium and by phosphorylation on Ser505 by mitogen-activated protein kinases (MAPK) (14, 21). The fibroblasts were treated with the calcium ionophore A23187 together with the protein kinase C (PKC) activator, PMA, to maximally activate cPLA2 by inducing a sustained increase in intracellular calcium, and activation of MAPK, which are downstream of PKC (22). The effect of A23187 and PMA was compared with arachidonic acid release induced by serum, a complex mixture of mediators which lung cells are exposed to during acute lung injury as a result of increased vascular permeability. The responses of primary and immortalized fibroblasts were very similar (Figures 1A and 1B). Serum and the combination of A23187 and PMA strongly stimulated arachidonic acid release, which was increased over 10-fold in MLF+/+ and IMLF+/+. The effect of two mediators found in serum, LPA and PDGF, which are known to induce biological responses through specific receptors on fibroblasts, was also tested (23). When LPA and PDGF were tested individually on MLF+/+, PDGF induced a small increase (2-fold) in arachidonic acid release. This response was enhanced by the addition of LPA resulting in a 3-fold increase in release of arachidonic acid in MLF+/+. In IMLF+/+, the combination of LPA and PDGF stimulated arachidonic acid release by 6-fold. Arachidonic acid release from cPLA2{alpha}-deficient cells (MLF-/- or IMLF-/-) was not above basal levels in cells treated with LPA/PDGF. The amount of arachidonic acid released from MLF-/- and IMLF-/- treated with A23187/PMA and serum was reduced by 80–90% compared with wild-type cells. The results demonstrate a primary role for cPLA2{alpha} in mediating agonist-induced arachidonic acid release from mouse lung fibroblasts. Because arachidonic acid release was similar in primary and immortalized fibroblasts, the immortalized cells were used to compare the time course of arachidonic acid release in response to mouse serum and A23187/PMA. As shown in Figure 2, the greatest rate of arachidonic acid release from IMLF+/+ occurred in the first 10 min after adding agonists. Although the rate of arachidonic acid release was similar to IMLF+/+, the amount of arachidonic acid release from IMLF-/- in response to mouse serum or A23187/PMA was 5- to 10-fold lower than from IMLF+/+.



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  		Figure 1. Agonist-induced arachidonic acid release from (A) primary mouse lung fibroblasts from wild-type (MLF+/+) and cPLA2-deficient (MLF-/-) mice, and from (B) immortalized mouse lung fibroblasts from wild-type (IMLF+/+) and cPLA2-deficient (IMLF-/-) mice. Fibroblasts were labeled with [3H]arachidonic acid in serum-free medium overnight and then stimulated for 30 min with 0.5 µg/ml A23187 together with 20 ng/ml PMA, 10% mouse serum (MS), 10% FBS, 10 µM LPA, and 30 ng/ml PDGF. [3H]arachidonic acid released into the medium from stimulated and unstimulated (US) cells is expressed as a percentage of the total incorporated radioactivity. Gray bars correspond to MLF+/+ and IMLF+/+, whereas MLF-/- and IMLF-/- are indicated by black bars. Data shown are the average ± SE of three independent experiments.

 


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  		Figure 2. Time course of arachidonic acid release from IMLF+/+ and IMLF-/-. Fibroblasts were labeled with [3H]arachidonic acid in serum free medium overnight and then stimulated with 0.5 µg/ml A23187 together with 20 ng/ml PMA, or 10% mouse serum (MS). The amount of [3H]arachidonic acid released into the medium from stimulated and unstimulated (US) cells was measured at the times indicated and expressed as a percentage of the total incorporated radioactivity. Data shown are average ± SE of three independent experiments.

 
The results demonstrate an important role for cPLA2{alpha}; however, there was a small amount of arachidonic acid release from IMLF-/- and MLF-/- particularly in response to A23187/PMA and mouse serum. This suggests that another PLA2 also plays a role in agonist-induced arachidonic release from lung fibroblasts. To investigate this in more detail, the effect of A23187 (without PMA) on arachidonic acid release from IMLF-/- and IMLF+/+ was compared. Arachidonic acid release was stimulated by similar concentrations of A23187 in both IMLF+/+ and IMLF-/- (Figure 3A). However, the response in the IMLF-/- was 40% of the IMLF+/+ response. As shown in Figure 3, the relative amount of arachidonic acid release by IMLF-/- compared with IMLF+/+ was greater when the cells were treated with A23187 without PMA than when the two agonists were combined (compare with Figure 1). This suggests that the PLA2 involved on arachidonic acid release in IMLF+/+ and IMLF-/- are both activated by an increase in intracellular calcium but differentially regulated by PMA. To further investigate this, the effect of PMA alone and together with A23187 on arachidonic acid release from IMLF+/+ and IMLF-/- was evaluated (Figure 3B). PMA alone stimulated arachidonic acid release from IMLF+/+, although the response was less than with A23187 alone. A synergistic response was observed in IMLF+/+ when treated with PMA together with low concentrations of A23187 (0.5 µg/ml) consistent with the known properties of cPLA2{alpha}. In contrast, arachidonic acid release from IMLF-/- was not stimulated by PMA, nor was the response to A23187 further enhanced by PMA.



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  		Figure 3. Effect of A23187 and PMA on arachidonic acid release from IMLF+/+ and IMLF-/-. Fibroblasts were labeled with [3H]arachidonic acid in serum free medium overnight and then stimulated for 45 min with (A) the indicated concentrations of A23187 or with (B) 0.5 µg/ml A23187, 20 ng/ml PMA, or both combined. The amount of [3H]arachidonic acid released into the medium from stimulated and unstimulated (US) cells was measured and expressed as a percentage of the total incorporated radioactivity. Data shown are average ± SE of three independent experiments. Gray bars, IMLF+/+; black bars, IMLF-/-.

 
cPLA2 Expression in Primary and Immortalized Mouse Lung Fibroblasts
The relative amounts of [3H]arachidonic acid released from IMLF+/+ and MLF+/+ were similar, suggesting that they contain comparable levels of cPLA2. This was confirmed by Western blot analysis as shown in Figure 4. It has previously been reported that levels of cPLA2 are increased in embryonic fibroblasts from human lung (WI38 cells) and human rheumatoid synovial fibroblasts treated with interleukin (IL)-1 (24, 25). The effect of a variety of cytokines on the level of cPLA2 expression was investigated in IMLF+/+ and MLF+/+ (Figure 4). IL-1 did not affect levels of cPLA2 expression and only a very slight increase was observed in IMLF+/+ treated with PDGF and in MLF+/+ treated with FBS.



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  		Figure 4. cPLA2 expression in IMLF+/+ and MLF+/+. Fibroblasts were treated with 2 ng/ml IL-1ß, 2 ng/ml TGF-ß, 2 ng/ml TNF-{alpha}, 10% FBS, or 30 ng/ml PDGF as indicated for 20 h in serum-free medium. Cell lysates (30 µg protein) from stimulated or unstimulated (US) cells were separated by SDS-PAGE and analyzed for cPLA2 by Western blotting.

 
COX2 Upregulation and PGE2 Production
Upregulation of COX2 in lung fibroblasts has been shown to result in enhanced PGE2 production. In addition, it has previously been reported that cPLA2{alpha} can play a role in augmenting expression of COX2 (26). To investigate this in MLF, the effect of agonists that stimulate arachidonic acid release on COX2 expression was compared in IMLF+/+ and IMLF-/-. When analyzed by Western blotting, the amount of COX2 was found to increase in a time-dependent manner in both IMLF+/+ and IMLF-/- in response to mouse serum, FBS, and LPA/PDGF (Figure 5A). An increase in COX2 protein occurred between 1 and 8 h after adding the agonists. IMLF-/- expressed higher amounts of COX2 at early times (1–3 h) than IMLF+/+. The effect of various cytokines on levels of COX2 was also compared (Figure 5B). COX2 increased to the greatest extent in IMLF+/+ and IMLF-/- treated overnight with TGF-ß and only slightly by IL-1ß and TNF-{alpha}.



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  		Figure 5. COX2 expression in IMLF+/+ and IMLF-/-. (A) Fibroblasts were incubated in serum-free medium overnight and then treated with 10% mouse serum (MS), 10% FBS, or 10 µM LPA together with 30 ng/ml PDGF for the times indicated. (B) Fibroblasts were treated with 2 ng/ml IL-1ß, 2 ng/ml TGF-ß, 2 ng/ml TNF{alpha}, or 30 ng/ml PDGF for 20 h in serum-free medium. Cell lysates (30 µg protein) from stimulated or unstimulated (US) cells were separated by SDS-PAGE and analyzed for COX2 by Western blotting.

 
Experiments were performed comparing the ability of lung fibroblasts from wild-type and cPLA2{alpha} null mice to produce PGE2 (Table 1). The cells were first incubated in serum-free medium overnight without or with TGF-ß to upregulate COX2 and then treated for 1 h with agonists (LPA/PDGF and A23187/PMA) that induce arachidonic acid release. MLF+/+ produced PGE2 only when the cells were stimulated with PLA2 agonists, A23187/PMA or LPA/PDGF. The levels of PGE2 produced were significantly higher (6-fold) in stimulated cells preincubated with TGF-ß. In contrast, MLF-/- not preincubated with TGF-ß did not produce detectable amounts of PGE2. MLF-/- produced detectable levels of PGE2 only when they were pretreated with TGFß and then stimulated with A23187/PMA. However, the level of PGE2 produced was significantly lower (20-fold) than the amount of PGE2 produced by MLF+/+ similarly treated. Unlike MLF+/+, IMLF+/+ did not produce detectable amounts of PGE2 in the absence of TGF-ß. When IMLF+/+ were pretreated with TGF-ß a low level of PGE2 was produced in response to A23187/PMA or LPA/PDGF, but this was significantly lower (30-fold) than the amount of PGE2 produced by MLF+/+. IMLF-/- did not produce detectable amounts of PGE2 under any conditions. In additional experiments, preincubation of primary and immortalized fibroblasts with the specific COX2 inhibitor NS398 (10 µM) completely inhibited the production of PGE2 under all the conditions shown in Table 1 (data not shown). The results demonstrate that cPLA2 plays an important role in providing arachidonic acid to COX2 for the production of PGE2 in MLF.


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  		TABLE 1 PGE2 production by primary and immortalized mouse lung fibroblasts from wild-type and cPLA2{alpha} null mice

 
The low level of PGE2 produced by IMLF+/+ was surprising, considering that they exhibited similar abilities to release arachidonic acid as MLF+/+ and expressed similar amounts of cPLA2{alpha}. The relative level of COX2 expression in primary and immortalized lung fibroblasts was also compared under conditions used for measuring PGE2. The cells were incubated overnight with or without TGF-ß, and then the TGF-ß–treated cells were incubated for an additional 1 h with mouse serum, A23187/PMA, or LPA/PDGF. As shown in Figure 6A, both MLF+/+ and IMLF+/+ expressed a low level of COX2 that increased after TGF-ß treatment. The level of COX2 was further increased in MLF+/+ by treatment with LPA/PDGF (2.4 ± 0.1-fold, n = 3). The cPLA2{alpha}-deficient fibroblasts (MLF-/- and IMLF-/-) also expressed a low basal level of COX2 that increased after TGF-ß treatment. COX1 was also expressed in the primary and immortalized lung fibroblasts, although lower levels were consistently observed in IMLF+/+ (Figure 6A). Unlike COX2, COX1 expression was not increased by TGF-ß.



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  		Figure 6. Expression of COX2, COX1, and PGES in primary and immortalized mouse lung fibroblasts. (A) MLF+/+ and IMLF+/+ were incubated overnight in serum-free DMEM without (unstimulated, US) or with 2 ng/ml TGF-ß. Some of the cells treated with TGF-ß were incubated an additional 1 h with 10% mouse serum (MS), 0.5 µg/ml A23187 together with 20 ng/ml PMA, or 10 µM LPA together with 30 ng/ml PDGF. Cell lysates (30 µg protein) were separated by SDS-PAGE and analyzed for COX2 and COX1 by Western blotting. (B) Primary and immortalized mouse lung fibroblasts were incubated overnight in serum-free DMEM. Total RNA was isolated and cDNA generated for PCR analysis of mPGES and GAPDH as described in MATERIALS AND METHODS. (C) Primary and immortalized mouse lung fibroblasts were treated as described above for (A) and cell lysates analyzed for mPGES and cPGES by Western blotting.

 
PGES Expression Levels in MLF and IMLF
There are two forms of PGES in cells that can convert prostaglandin D2 to PGE2. It has been reported that COX2 functionally couples to mPGES for production of PGE2 (27). mPGES was originally identified as a protein induced by the tumor suppressor gene, p53 (28). A hallmark of SV40 transformation is the inactivation of p53 by T antigen (29). Experiments were performed to determine if differences in the levels of PGE2 produced by immortalized and primary mouse lung fibroblasts correlated with differences in expression of mPGES. As shown in Figures 6B and 6C, mPGES message, and protein were present in MLF+/+ and MLF-/- but not detectable in either IMLF+/+ or IMLF-/-. Western blot analysis showed that mPGES was constitutively expressed in the primary fibroblasts and was not increased in cells treated with TGFß (Figure 6C). In contrast, IMLF+/+ expressed slightly higher levels of cPGES than MLF+/+ (Figure 6C).

Phenotypic Properties of MLF+/+ and MLF-/-
Experiments were performed to determine if MLF+/+ and MLF-/- exhibited phenotypic differences that correlated with levels of PGE2 production. The fibroblasts were plated in medium containing 10% FBS and growth monitored over 4 d. The cells were plated at a density to achieve ~ 50% confluence at the onset of the experiment (6 h) because low-density cultures exhibited poor survival. The plating efficiency of both MLF+/+ and MLF-/- was 80%, and they both exhibited a 2.4-fold increase in cell number at 24 h, at which time the cultures were ~ 90% confluent (Figure 7A). Thereafter, the number of adherent MLF+/+ and MLF-/- decreased, which was due to loss of adherent cells during the washing before trypsinization. The viability of the cells that remained adherent was > 90% at all time points. The amount of PGE2 produced by the cells was compared at 24 and 48 h after plating in serum-containing media (Figure 7B). The amount of PGE2 in the culture medium increased from 24 h to 48 h, and significantly more was produced by MLF+/+ than MLF-/- at both times. The amount of collagen in deposited ECM was found to be inversely proportional to the level of PGE2 (Figure 7C). The level of collagen produced by MLF-/- was significantly higher (2.4-fold) than the amount produced by MLF+/+.



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  		Figure 7. MLF+/+ and MLF-/- growth properties and collagen production. (A) MLF+/+ and MLF-/- were plated in 12-well plates (1.25 x 104 cells/well) in DMEM containing 10% FBS. The cells were washed twice to remove the serum-containing medium, trypsinized and cell numbers determined 6 h after plating and every 24 h for 4 d. The data are the average of three separate experiments ± SE. The plating efficiency (cell count at 6 h) and cell numbers for MLF+/+ and MLF-/- were not significantly different. (B) The culture media from MLF-/- and MLF+/+ cultured for 24 and 48 h in DMEM containing 10% FBS were analyzed by mass-spectrometry for PGE2 content. The data represent the average of two experiments ± SD. PGE2 production by MLF+/+ and MLF-/- is significantly different at 24 h (P = 0.02) and 48 h (P = 0.01). (C) Incorporation of [3H]proline into collagen was quantified as described in MATERIALS AND METHODS and expressed as a percentage of total ECM proteins. Collagen production by MLF+/+ and MLF-/- is significantly different (P = 0.0002).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PGE2 has been implicated as a negative regulator of fibroblast function. Although the role of PGE2 in promoting vascular changes (vasodilation, increased vascular permeability) is documented, there is evidence that it can also play a role in attenuating acute inflammation (30). Therefore, PGE2 may play a protective role in the normal lung, and participate in the resolution of inflammation and control of fibrotic changes. Numerous studies have investigated the role of cyclooxygenases in PGE2 production in lung fibroblasts; however, the involvement of specific PLA2s has not been well studied (8–10). The cPLA2{alpha}-/- mouse model has established the importance of this enzyme in various diseases and in providing arachidonic acid for eicosanoid production in cells involved in inflammation (15, 18). cPLA2{alpha} has been shown to be responsible for releasing arachidonic acid for prostanoid and leukotriene production in mouse peritoneal macrophages, and for immediate and delayed eicosanoid production in bone marrow–derived mast cells (15, 26). It has recently been shown that cPLA2{alpha} is required for most of the thromboxane A2 produced by collagen-treated platelets (31). By using cells from wild-type and cPLA2{alpha}-deficient mice, we provide evidence in this study that cPLA2{alpha} plays a predominant role in providing arachidonic acid for prostanoid production in primary lung fibroblasts. In addition, the data demonstrate that lung fibroblasts lacking cPLA2{alpha} produce more collagen than wild-type fibroblasts and suggests a role for PGE2 in regulating collagen production.

cPLA2{alpha} is constitutively expressed in most cells, and post-translational processes are important for activation of cPLA2{alpha}; however, transcript levels can be increased in some cells by cytokines (32, 33). In human fibroblasts derived from fetal and neonatal lung, cPLA2 mRNA is constitutively present but can be increased by IL-1ß, TNF-{alpha}, and interferon-{gamma}, but not by TGF-ß (32). It also has been reported that the amounts of cPLA2{alpha} protein are increased by IL-1{alpha} in human fibroblasts from embryonic lung (WI-38), and this correlated with increased production of PGE2 (24). However, IL-1{alpha} had no effect on COX levels in WI-38 cells. In contrast, we found that levels of cPLA2{alpha} protein were not significantly affected by IL-1ß (or TGF-ß), and that increased PGE2 production correlated with increased COX2 expression.

cPLA2 is strongly activated by serum in the lung fibroblasts indicating the potential for large amounts of arachidonic acid release and PGE2 production during acute lung injury. Multiple signaling events act together for full activation of cPLA2{alpha}. An increase in intracellular calcium induces translocation of cPLA2 from cytosol to Golgi, endoplasmic reticulum, and nuclear envelope for access to membrane phospholipid substrate (34). cPLA2{alpha} is also regulated by phosphorylation, and several functionally important phosphorylation sites have been identified (35, 36). Serum contains many potential agonists that act together to provide signals for cPLA2 activation. Although two components in serum, LPA and PDGF, together stimulated cPLA2-mediated arachidonic acid release, the response was considerably less than with serum, implicating the presence of additional agonists.

Although cPLA2{alpha} plays an important role in mediating arachidonic acid release, the involvement of an additional PLA2 is also indicated by data showing that cPLA2{alpha}-deficient fibroblasts release arachidonic acid in response to A23187 and to a lesser extent with serum, although at much lower levels than MLF+/+. It has been shown in platelets from cPLA2{alpha}-/- mice that another unidentified PLA2 mediates thromboxane A2 production in response to ADP, but thromboxane A2 production induced by collagen is primarily due to cPLA2{alpha} (31). Mammalian cells contain multiple PLA2s that are differentially regulated providing alternative pathways for arachidonic acid release and eicosanoid production. The unidentified PLA2 in cPLA2{alpha}-deficient lung fibroblasts is regulated by calcium but, unlike cPLA2{alpha}, is not activated by PMA. The mouse strains used for generating the cPLA2{alpha} knockout have a naturally occurring disruption in the gene for Group IIA secretory PLA2, eliminating this enzyme as a possible candidate (26). However, there are at least ten other calcium-regulated PLA2s in mammalian cells that could be involved. The arachidonic acid released by this enzyme can couple to COX2 for PGE2 production as observed in MLF-/- pretreated with TGF-ß and then stimulated with A23187. The potential role of this PLA2 in lung fibroblasts will await its identification and understanding its mode of regulation.

Primary fibroblasts from cPLA2{alpha}-deficient mice expressed similar levels of COX2 and mPGES as primary lung fibroblasts from wild-type mice, indicating that their inability to produce PGE2 is through a lack of cPLA2{alpha}-mediated arachidonic acid release. The importance of COX2 in PGE2 production by mouse lung fibroblasts was demonstrated by its sensitivity to inhibition by NS398 (data not shown). Recent characterization of the terminal glutathione-dependent synthases involved in the conversion of prostaglandin H2 (PGH2) to PGE2 has provided additional insight into the regulation of PGE2 production. The ubiquitous cPGES is constitutively expressed and found to be identical to the hsp90-associated p23 protein (37). cPGES preferentially converts PGH2 derived from COX1 to PGE2. In contrast, the membrane bound mPGES is an inducible enzyme that is involved in delayed PGE2 production and primarily uses PGH2 derived from COX2 (27). We found that MLF+/+ and MLF-/- express similar levels of mPGES, but unlike COX2 the expression of the synthase was not affected by TGF-ß. However, there is evidence that mPGES can be coordinately upregulated with COX2 by lipopolysaccharide or inflammatory cytokines (27, 38). The importance of mPGES in coupling with COX2 for PGE2 production has also recently been demonstrated in KAT-50 thyrocytes. These cells constitutively express COX2 and cPGES but produce comparatively low levels of PGE2 because of a lack of mPGES (39).

A role for mPGES in PGE2 production in mouse lung fibroblasts is suggested from comparisons of primary and SV40 immortalized cells. IMLF+/+ produced several-fold lower levels of PGE2 than MLF+/+, and this correlated with the lack of mPGES but not with cPLA2 function (arachidonic acid release) or levels of COX2 expression. Although the immortalized cells expressed slightly higher levels of cPGES than primary fibroblasts, arachidonic acid metabolized by COX2 did not efficiently couple to cPGES for PGE2 production. Another possibility that may contribute to the differences in PGE2 production is that the immortalized cells contain less esterified arachidonic acid than the primary fibroblasts and therefore have less substrate for making PGE2. Although the relative amount of labeled arachidonic acid released by MLF+/+ and IMLF+/+ is similar, it is possible that there are differences in the specific activity of the label released if they contain different amount of arachidonic acid. In addition, IMLF+/+ expressed lower levels of COX1. The basis for this is not known, because it was only observed in IMLF+/+ and not in IMLF-/-. However, PGE2 production by IMLF+/+ and MLF+/+ was inhibited by NS398, suggesting a primary role for COX2. Therefore it seems unlikely that the lower PGE2 production by IMLF+/+ is due to the lower expression of COX1. Although these are alternative possibilities for the low PGE2 production by IMLF+/+, the data are consistent with a role for mPGES in the primary fibroblasts since arachidonic acid metabolized through COX2 has been shown to preferentially couple to mPGES rather than cPGES (27).

The functional consequences of cPLA2{alpha} activation are complex because it can provide arachidonic acid for both proinflammatory/profibrotic and anti-inflammatory/antifibrotic eicosanoids. During lung injury that is accompanied by an influx of inflammatory cells, the production of proinflammatory eicosanoids such as leukotriene B4, leukotriene C4, and thromboxane A2 would predominate. Leukotrienes are increased in patients with idiopathic pulmonary fibrosis, and leukotrienes and thromboxane are increased in bronchoalveolar lavage fluid in mice during bleomycin-induced pulmonary fibrosis (11, 12, 17). The protection from bleomycin-induced fibrosis in the 5-LO and cPLA2{alpha}-deficient mouse models supports an important role for products from the 5-LO pathway in promoting fibrosis. However, increased levels of PGE2 are evident in bronchoalveolar lavage fluid of 5-LO–deficient mice treated with bleomycin, suggesting that the increased production of antifibrotic mediators together with a decrease of profibrotic leukotrienes may both contribute to protection from fibrosis in the 5-LO–deficient mice (12). Increased production of PGE2 would not be expected in the bleomycin-induced fibrosis model in cPLA2{alpha}-deficient mice, suggesting that the decrease in profibrotic eicosanoids plays a dominant role in protection from fibrosis. In addition to activation of cPLA2 in inflammatory cells during fibrosis, cPLA2{alpha} would also be activated in fibroblasts, and this would promote production of PGE2. The increased susceptibility of COX2-deficient mice to pulmonary fibrosis points to a protective role for prostanoids (8, 10). However, there is evidence suggesting that PGE2 production is suppressed in fibroblasts from fibrotic lung, which may be due to a defect in upregulation of COX2 (8, 9).


   Acknowledgments
 
This work was supported by National Institutes of Health Grants HL34303 (C.L. and R.M.), HL61378 (C.L.), a Michael and Eleanore Stobin Pediatric Fellowship (M.G.), DK38452, NS10828, and DK39773 (J.B.). The authors acknowledge Chris A. Johnson for mass spectrometry analysis and Dr. Mathew Strand for statistical analysis.

Received in original form January 7, 2003

Received in final form June 24, 2003


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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