Published ahead of print on May 8, 2003, doi:10.1165/rcmb.2002-0243OC
American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 537-544, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0243OC
Prostaglandin E2 Inhibits Fibroblast to Myofibroblast Transition via E. Prostanoid Receptor 2 Signaling and Cyclic Adenosine Monophosphate Elevation
Jill E. Kolodsick,
Marc Peters-Golden,
José Larios,
Galen B. Toews,
Victor J. Thannickal and
Bethany B. Moore
Department of Internal Medicine, Pulmonary and Critical Care Medicine Division, University of Michigan School of Medicine, Ann Arbor, Michigan
Address correspondence to: Bethany B. Moore, Ph.D., University of Michigan School of Medicine, Internal Medicine/Pulmonary and Critical Care Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0642. E-mail: Bmoore{at}umich.edu
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Abstract
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Myofibroblasts, the hallmark of fibrotic disease, contribute to the pathology of fibrosis by secreting large amounts of extracellular matrix and contributing to alveolar contraction. Myofibroblasts are characterized by the expression of  -smooth muscle actin (-SMA), a contractile protein normally associated with smooth muscle cells. Transforming growth factor-ß1 (TGF-ß1) is a well characterized profibrotic cytokine that induces myofibroblast transformation both in vitro and in vivo. We report here that the lipid mediator prostaglandin E2 (PGE2) inhibits TGF-ß1–induced expression of -SMA in primary fetal and adult lung fibroblasts. This inhibition of -SMA expression is associated with a reduction in the expression of collagen I. Inhibitory actions of PGE2 are mediated via E prostanoid receptor 2 (EP2) signaling, but not by EP3 signaling, and increases in cyclic adenosine monophosphate production. The inhibitory effects of PGE2 on TGF-ß1–induced -SMA expression are mimicked by an EP2 selective agonist, butaprost, and by forskolin-induced direct activation of adenyl cyclase. An EP2 antagonist blocks the inhibitory effects of PGE2, and an EP3 agonist does not inhibit TGF-ß1–mediated increases in -SMA expression. Our results demonstrate that PGE2 inhibits transition of fibroblasts to myofibroblasts by an EP2 receptor-activated pathway. Augmenting this pathway may serve as a potent antifibrotic therapeutic strategy.
Abbreviations: -smooth muscle actin, -SMA • cyclic adenosine monophosphate, cAMP • cyclooxygenase, COX • E prostanoid receptor, EP • monocyte chemoattractant protein-1, MCP-1 • prostaglandin E2, PGE2 • transforming growth factor-ß1, TGF-ß1 • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR • sodium dodecylsulfate, SDS • tumor necrosis factor, TNF
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Introduction
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Idiopathic pulmonary fibrosis is a fibrotic disease of the lung involving dysregulated repair and remodeling in response to an injurious agent. The dysregulated tissue repair is characterized by increased production of fibrogenic cytokines, fibroblast proliferation, and an excessive growth of fibrous connective tissue, resulting in diminished lung volumes and compromised gas exchange (1, 2). Pulmonary fibrosis is often irreversible and fatal, with a 5-yr survival rate of only 30% (1), and current treatments are generally not effective.
A critical feature of fibrotic diseases involving the lung as well as other organs is the appearance of fibroblast-like cells expressing -smooth muscle actin (-SMA) within areas of active fibrosis (3, 4). The expression of -SMA suggests the fibroblasts have acquired morphological and biochemical features of contractile cells, and such cells are termed myofibroblasts (5–7). Myofibroblasts have been identified as the predominant source of increased collagen gene expression (8), and their emergence directly correlates with the development of decreased lung compliance (4, 6). In addition to altering tissue contractility and increasing extracellular matrix synthesis, the myofibroblast secretes substances with autocrine and paracrine effects in fibrogenesis. Specifically, myofibroblasts secrete two key mediators with inflammatory and fibrogenic actions in fibrosis; namely, monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-ß1 (TGF-ß1) (9, 10). These properties endow the myofibroblast with a pluripotent capacity to promote fibrosis (11).
Fibroblasts transition into myofibroblasts through a process regulated by cytokines and extracellular matrix components (7). Among the -SMA inducing factors, TGF-ß1 is thought to be the most efficient (12). TGF-ß1 stimulates the expression of -SMA mRNA and protein in myofibroblasts (4, 6, 13). In contrast to TGF-ß1, interleukin-1ß and interferon- downregulate the expression of -SMA mRNA and protein as well as decrease fibroblast proliferation and collagen deposition (5, 14, 15). The mechanism underlying the interleukin-1ß effect involves induction of apoptosis of myofibroblasts via nitric oxide production (5), but the mechanism by which interferon- exerts its effects on the fibroblast remains unclear. Thus, the mechanisms by which myofibroblast transition is regulated are incompletely understood.
Because such information might provide important insights into the mechanisms of fibrosis and suggest potential therapeutic targets, we sought to identify additional factors that might regulate myofibroblast transition. An antifibrotic mediator of growing interest is prostaglandin E2 (PGE2). PGE2 is a lipid mediator that can be derived from cell membrane phospholipids via the cyclooxygenase (COX) pathway of arachidonic acid metabolism. Addition of PGE2 to lung fibroblast cultures suppresses fibroblast proliferation (16, 17) and decreases transcription of collagen mRNA (18, 19). Importantly, levels of PGE2 in lung lavage fluid from patients with idiopathic pulmonary fibrosis are 50% lower than in normal subjects (20) and fibroblasts isolated from the lungs of these patients were found to have a defect in their capacity to synthesize PGE2 (21, 22).
The purpose of this study was to determine if PGE2 could modulate the transition of lung fibroblasts to myofibroblasts as assessed by -SMA protein expression. In addition, we examined the receptor and signal transduction mechanism by which PGE2 modulates -SMA protein production. Our results provide novel insights into the regulation of myofibroblast transition and suggest new therapeutic avenues for fibrotic lung disease.
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Materials and Methods
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Cell Culture
Most experiments were performed on early passage normal human fetal lung fibroblasts (IMR-90; Institute for Medical Research, Camden, NJ). The cells were grown in Dulbecco's Modified Eagle's Medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), 100 U/ml penicillin/streptomycin, and fungizone (GIBCO, Grand Island, NY). Medium was changed every three days and cells were expanded and plated into 35-mm and 100-mm dishes. Once the cells reached 85% confluency, they were serum starved for 48 h before the addition of medium alone or medium containing 2 ng/ml TGF-ß1 (R&D Systems, Minneapolis, MN) and PGE2, AH6809, butaprost, or sulprostone (Cayman Chemicals, Ann Arbor, MI). Forskolin used in some experiments was purchased from Sigma (St. Louis, MO). The solvent for PGE2 was ethanol. The solvent for all other drugs was dimethyl sulfoxide (DMSO).
In some experiments, normal human adult lung fibroblasts (Clonetics normal human lung fibroblasts [NHLF]) obtained from Cambrex (Walkersville, MD) were cultured and treated the same way as the fetal lung fibroblasts. Murine lung fibroblasts from C57Bl/6 mice purchased from Jackson Laboratories (Bar Harbor, ME) were also used. Fibroblasts were grown as previously described (23) from lung minces for 14–21 d (3–5 passages) before being cultured and treated as described above.
Cyclic Adenosine Monophosphate Assays
Serum-starved IMR-90 cells grown in 100-mm dishes were treated for 5 min with PGE2, or 15 min with butaprost or forskolin. Within any given experiment, cells were studied at 85% confluence ( 4.5 x 106 cells). After treatment, medium was removed and the cells were washed with phosphate-buffered saline (PBS). Immediately after washing, 2 ml of ice-cold ethanol was added to every dish. After 5 min, cells were scraped into a centrifuge tube and spun for 10 min at 3,000 x g to pellet the cell debris. The ethanol supernatant was then evaporated under a stream of dry nitrogen and the sample was reconstituted in 0.5 ml phosphate buffer and acetylated according to manufacturer's directions. The cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay (1:100 dilution) was performed according to manufacturer's instructions (Cayman Chemicals). Samples treated with the solvent controls were identical to media alone and are not shown.
Western Blotting
Cells were grown and serum-starved in 35-mm dishes. Cultures were exposed to treatments for 24 h and then were washed with ice-cold PBS and 200 µl of cold lysis buffer (1% Igepal-CA-630 [Sigma Chemicals], 1% sodium deoxycholate, 0.1% sodium dodecylsulfate [SDS], 0.15 M sodium chloride, 0.01 M sodium phosphate NaH2PO4, 0.02 M ethylenediaminetetraacetate, 0.05 M sodium fluoride [NaF], 0.002 M sodium orthovanadate [NA3VO4], 1:100 dilution of Calbiochem Protease Cocktail Set III [Calbiochem-Novabiochem Corporation, San Diego, CA]) was added to each sample. Lysates were assayed for total protein concentration using the DC Protein Assay (Bio-Rad, Hercules, CA). Equal amounts of protein from each experimental condition were mixed with a 1:5 vol/vol ratio of 6x electrophoresis sample buffer (0.2 M ethylenediaminetetraacetate, 40 mM dithiothreitol, 6% SDS, 0.6 mg/ml pyronin, pH 6.8) and boiled for 7 min. SDS–polyacrylamide gel electrophoresis was performed using a 4–20% polyacrylamide gradient gel. Protein samples were electrophoretically transferred to polyvinylidene fluoride membrane (Immobilon-P; Millipore Inc., Bedford, MA) and incubated in blocking buffer containing 75 mM sodium phosphate, 70 mM sodium chloride, and 0.1% Tween-20, pH 7.4, with 5% BSA for at least 1 h at room temperature. -SMA protein was detected by incubating for 1 h with a monoclonal antibody (Clone 1A4; Dako, Carpinteria, CA) diluted 1:1,000 in blocking buffer, followed by three washes (75 mM sodium phosphate, 70 mM sodium chloride, and 0.1% Tween-20, pH 7.4) and a horseradish peroxidase–coupled secondary antibody (ImmunoPure goat anti-mouse immunoglobulin G, [H+L] Peroxidase Conjugated, Pierce). The membrane was then washed five times before incubation with SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL) for 5 min and exposed to chemiluminescent-sensitive Kodak X-OMAT AR film (Kodak, Rochester, NY). EP2 protein was detected using a rabbit polyclonal primary antibody (Cayman Chemical, Ann Arbor, MI) and a goat anti-rabbit horseradish peroxidase–coupled secondary antibody (Pierce). Western blots for the expression of EP2 and EP4 were performed as described above using primary antibodies available from Cayman Chemical. Western blotting for total intracellular type I collagen was performed as described for -SMA with the following exceptions. A 4–12% gradient gel was used for SDS–polyacrylamide gel electrophoresis, 5% nonfat milk was used as the blocking buffer, the primary antibody was a 1:500 dilution of purified rabbit anti-mouse collagen type I (Cedarlane Laboratories Limited, Ontario, Canada) in 5% nonfat milk, and the secondary was a horseradish peroxidase–coupled goat anti-rabbit (ImmunoPure Goat Anti-Rabbit immunoglobulin G, [H+L] peroxidase conjugated; Pierce). Samples treated with the solvent controls were identical to media alone and are not shown.
Immunofluorescence Microscopy for -SMA
Cells grown on cover slips were initially rinsed with Dulbecco's modified Eagle's medium (BioWhittaker) for 30 s at ambient temperature and then fixed in 4% formaldehyde for 5 min. Cells were then washed three times in PBS before permeabilization and following each subsequent step. Permeabilization was performed in buffer consisting of 0.1% Triton in 50 mM piperazine-N, N'-bis-2-ethanesulfonic acid (pH 7.0), 90 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.0), 0.5 mM magnesium chloride, 0.5 mM ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 75 mM potassium chloride for 30 s at room temperature. Cover slips were sequentially incubated with mouse monoclonal anti–-SMA (clone 1A4, Dako) and fluorescein isothiocyanate–labeled goat anti-mouse antibody (Jackson Immuno Research, West Grove, PA) each for 60 min at room temperature. Cells were then visualized and photographed using a Zeiss fluorescence microscope at 40x magnification.
RNA Analysis
To prepare RNA, IMR-90 cells were resuspended in 2 ml Trizol reagent (Life Technologies, Gaithersburg, MD) and total RNA was extracted according to manufacturer's instructions. Complementary DNA was reverse-transcribed, followed by polymerase chain reaction (PCR) amplification using 1 µg of total RNA and the Promega Access reverse transcriptase (RT)-PCR kit (Madison, WI) according to manufacturer's instructions. The annealing temperature was 55°C. ß-Actin samples were subjected to 25 cycles, while EP 1–3 receptors were amplified for 35 cycles. Samples testing EP4 mRNA were amplified for 50 cycles. Sequences of primers used are given in Table 1. PCR amplification of whole-lung RNA using these primers generated products of the expected sizes: ß-actin 510 bp, EP1 336 bp, EP2 401 bp, EP3 437 bp, and EP4 423 bp. The specificity of the amplified product was confirmed by Southern blotting using 32P-labeled internal oligos (Table 1) that were hybridized at 63°C and washed at 65°C.
PCR Southern Blotting
Amplified DNA was subjected to electrophoresis in a 1.5% agarose gel for Southern filter hybridization analysis. After electrophoresis, DNA was transferred in 0.4 N NaOH onto Zetaprobe membrane (Bio-Rad Inc, Hercules, CA). Southern filter hybridization was performed in 6x saline sodium citrate buffer (SSC), 0.5% SDS, and 5x Denhardt's. 32P end-labeled oligonucleotides (sequences detailed in Table 1) were utilized as probes. Autoradiography was performed and digital images of the autoradiographs were captured using the Kodak Digital Science (Eastman Kodak Co., Rochester, NY) 1 hardware and software package.
Densitometry Analysis
A digital picture of each autoradiograph was taken and band intensities were analyzed using NIH Image public domain software (developed at the Research Services Branch of the National Institute of Mental Health, Bethesda, Maryland; available for download at http://rsb.info.nih.gov/nih-image).
Statistical Analysis
Statistical significance was analyzed using the InStat 2.01 program (Graphpad Software, San Diego, CA) on a Power Macintosh G3. Analysis of variance analysis with a post-hoc Bonferroini test was used to determine which groups showed significant differences. P < 0.05 was considered significant.
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Results
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PGE2 Inhibits TGF-ß1–Induced -SMA Expression
We first examined if PGE2 could inhibit the transition of fibroblasts to myofibroblasts, as denoted by the expression of increased levels of -SMA protein. A well described nontransformed fetal lung cell line, IMR-90, was used as the source of lung fibroblasts (24). When serum-starved, these cells express little -SMA at baseline but exhibit marked induction following incubation with TGF-ß1 (12). In these experiments, IMR-90 fibroblasts were serum-starved for 48 h before being incubated with TGF-ß1 ± PGE2 for 24 h, and their ability to transition to myofibroblasts was assessed. As illustrated in Figure 1A, a concentration-dependent decrease in TGF-ß1–stimulated -SMA protein expression was observed in cells treated with PGE2, as compared with cells that were treated with TGF-ß1 alone. Inhibition of TGF-ß1–induced -SMA expression was 32% by 10 nM PGE2, and ranged from 60–79% inhibition at higher PGE2 concentrations. We confirmed that this reduction in -SMA protein expression seen by Western blot correlated with a reduction of -SMA protein organized into cellular stress fibers (Figure 1B). These results demonstrate that PGE2 inhibits the transition of fibroblasts to myofibroblasts. To confirm that TGF-ß1–stimulated IMR-90 cells exhibited the high level of collagen synthesis characteristic of myofibroblasts, we analyzed the expression of collagen protein by immunoblot analysis in untreated cell lysates as well as lysates from cells treated with TGF-ß1 ± PGE2 (Figure 1C). As expected, TGF-ß1 increased the synthesis of collagen I in IMR-90 cells. At the lowest dose of PGE2 used in this experiment (10 nM), we observed an 55% decrease in collagen protein production relative to the TGF-ß1 control cell lysate. Therefore, analysis of both -SMA and collagen I protein levels indicates that PGE2 inhibits the transition of IMR-90 fibroblasts to myofibroblasts.
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Figure 1. PGE2 inhibits TGF-ß1–induced -SMA Expression. (A) IMR-90 cells were grown to 85% confluence before being serum-starved for 48 h. After serum starvation, cells were treated with serum-free media alone, 2 ng/ml TGF-ß1, or TGF-ß1 with increasing concentrations of PGE2 (10, 100, 500, or 1,000 nM) for 24 h. Cell lysates were assayed for -SMA protein expression by Western blot. (B) IMR-90 cells were grown to confluence on glass microscope slides before being serum-starved for 48 h. Following serum starvation, cells were treated with serum-free media, TGF-ß1 (2 ng/ml) or TGF-ß1 + 10 nM PGE2, for 24 h. Cells were stained and examined by immunofluorescence for expression of -SMA organized into cellular stress fibers (fluorescein isothiocyanate stain-green immunofluorescence). Cells were also stained with DAPI to distinguish nuclei (blue staining). (C) IMR-90 cells were grown to confluence before being serum-starved for 48 h. After serum starvation, cells were incubated with serum-free media alone, TGF-ß1 or TGF-ß1 + 10 nM PGE2 for 24 h before cellular lysates were prepared and analyzed for collagen I expression by Western blot. Data are representative of at least three separate experiments that showed similar results.
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PGE2 and Forskolin Increase cAMP
PGE2 has been shown to have suppressive effects on proliferation and collagen production in fibroblasts and it has been suggested that these effects are mediated by increases in cAMP (16, 17, 25, 26). Furthermore, increased cAMP has been shown to inhibit hepatic stellate cell transdifferentiation, which is characterized by expression of -SMA (27). We hypothesized, therefore, that the ability of PGE2 to decrease -SMA expression would correlate with the ability of PGE2 to stimulate cAMP generation in IMR-90 cells. Therefore, we determined cAMP levels in IMR-90 cells treated with PGE2 (Figure 2A). PGE2 increased cAMP at the same concentrations that were effective in limiting -SMA and collagen 1 protein expression (see Figure 1). Forskolin, an agent that increases cAMP in a receptor-independent manner by directly activating adenylate cyclase, also increased cAMP (Figure 2B) and decreased levels of -SMA protein by 40% at a concentration of 25 µM (Figure 2C).
PGE2 Receptor Expression on IMR-90 Cells
The biological effects of PGE2 are exerted via four E prostanoid receptor types, EP1, EP2, EP3, and EP4. Two of these receptors, EP2 and EP4, are known to be coupled to the activation of adenyl cyclase. Therefore, we hypothesized that EP2 and/or EP4 were the likely receptors on IMR-90 cells mediating the inhibitory effects. We assessed the PGE2 receptors expressed by the IMR-90 cells used in this study. RT-PCR and Southern blot analysis of IMR-90 cells revealed the presence of EP2 and EP3 mRNA at 35 cycles of amplification, but no expression of EP1 or EP4. When amplified for 50 cycles, there was a faint EP4 band detectable upon Southern blot analysis; however, EP1 was still not identified (Figure 3A). The presence of the EP2 receptor protein at the predicted molecular mass was confirmed by Western blotting (Figure 3B). EP4 receptor expression was not detectable at the protein level (not shown). Effective antibodies for the analysis of EP3 protein by Western blot are not available. Therefore, given the levels of mRNA and protein expression, it is likely that EP2 is responsible for the inhibition of TGF-ß1–induced -SMA expression.
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Figure 3. IMR-90 cells express EP2, EP3 and possibly low-level EP4 receptors. (A) Total RNA was prepared from IMR-90 cells and analyzed by RT-PCR and Southern blotting for expression of EP1–EP4 and ß-actin. EP2 and EP3 mRNA were detected at 35 cycles of amplification. EP4 was faintly visible by Southern blotting after 50 cycles of amplification. (B) EP2 protein expression was verified in IMR-90 cells by Western blot analysis. EP4 protein was not detectable. IMR-90 receptor expression did not change when cells were grown in complete media versus serum-free media.
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Increases in cAMP are Mediated through the EP2 Receptor
We wanted to confirm that EP2 receptor stimulation was responsible for the activation of adenyl cyclase. Our experimental results presented in Figure 2A demonstrate that PGE2 (which can bind all EP receptors) increased cAMP in these cells. To prove that this was due to EP2 receptor signaling, we tested the ability of the selective EP2 agonist, butaprost, to increase cAMP levels in IMR-90 cells. Butaprost (median effective concentration = 5,000 nM) elicited a dose-dependent increase in cAMP levels (Figure 4A). Sulprostone, an EP3 agonist, did not stimulate cAMP levels in IMR-90 cells (data not shown). These results implicate EP2, and not EP3, as the receptor expressed on IMR-90 cells that mediates adenyl cyclase activation in response to PGE2.
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Figure 4. An EP2 agonist mimics the effects of PGE2 on cAMP, -SMA, and collagen I. (A) Butaprost increases cAMP levels in IMR-90 cells. IMR-90 cells were grown to confluence and serum-starved for 48 h before being incubated with serum-free media ± increasing concentrations of butaprost for 15 min. Cellular lysates were harvested as described and analyzed for cAMP levels. Values represent mean ± SEM, n = 3. Butaprost at concentrations of 500 and 5,000 nM significantly increased cAMP levels compared with baseline (P < 0.05). (B) Butaprost decreases -SMA expression. IMR-90 cells were grown to confluence, serum-starved, and incubated with serum-free media, TGF-ß1 (2 ng/ml) or TGF-ß1 + 5,000 nM butaprost, for 24 h; 5,000 nM butaprost is the median effective concentration. Cellular lysates were harvested and analyzed for -SMA expression by Western blot. (C) Butaprost inhibits collagen expression. IMR-90 cells were grown and treated as in (B). Lysates were analyzed for collagen I protein expression by Western blot. Data are representative of at least three separate experiments that showed similar results.
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Inhibition of TGF-ß1–Induced -SMA and Collagen I Is Mediated through the EP2 Receptor
Once we had determined that the increase in cAMP was attributed to activation of the EP2 receptor, we evaluated whether the inhibition of -SMA by PGE2 seen in Figure 1A was due to signaling through the EP2 receptor. To address this question, we examined the ability of the selective EP2 agonist, butaprost, to limit TGF-ß1–induced expression of -SMA. Coincubation of IMR-90 cells with TGF-ß1 and butaprost resulted in a 33% decrease in -SMA (Figure 4B) and a 34% decrease in collagen protein (Figure 4C) expression as compared with IMR-90 cells treated with TGF-ß1 alone. These results were analogous to those seen in Figures 1A and 1C, where PGE2 decreased the levels of these myofibroblastic proteins. These data indicate that the inhibition by PGE2 of TGF-ß1–induced myofibroblast transition is associated with EP2 receptor activation.
An EP2 Receptor Antagonist Blocked the Inhibitory Effects of PGE2
To confirm that EP2 receptor signaling was necessary for the inhibitory effects of PGE2, we assessed the ability of PGE2 to inhibit TGF-ß1–stimulated -SMA expression in the presence of AH6809, a well established selective EP2 receptor antagonist. Figure 5 demonstrates that 10 nM PGE2 inhibited TGF-ß1–induced -SMA expression by 50% in the absence of AH6809 (compare lanes 2 and 3) as seen previously in Figure 1. The presence of AH6809 alone in serum-free media (lane 4) had no effect on basal -SMA expression. However, the presence of AH6809 prevented PGE2 from inhibiting the TGF-ß1–stimulated increase in -SMA expression. AH6809 was effective at blocking the inhibitory effect of even 100 nM PGE2. Because AH6809 could fully block the inhibitory effects of PGE2, these results suggested that in IMR-90 cells, EP2 and not EP4 was the predominant receptor mediating the inhibitory effect. As noted in Figure 3, this is likely due to the paucity of EP4 receptors present in IMR-90 cells.
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Figure 5. An EP2 receptor antagonist blocks the inhibitory effects of PGE2. IMR-90 cells were grown to confluence, serum-starved, and incubated with serum-free media, TGF-ß1 (2 ng/ml), or TGF-ß1 + PGE2. Samples containing the EP2 receptor antagonist, AH6809 (10 µM) were pretreated with AH6809 for 30 min before the addition of TGF-ß1 + PGE2.
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An EP3 Agonist Does Not Inhibit TGF-ß1–Induced -SMA or Collagen 1 Expression
As mentioned previously, sulprostone, an EP3 agonist, did not increase cAMP levels in IMR-90 cells (data not shown). We next assessed the effect of sulprostone treatment on the TGF-ß1–mediated stimulation of -SMA and collagen I. Figure 6 demonstrates that sulprostone at the median effective concentration of 10 nM did not inhibit TGF-ß1–induced expression of -SMA or collagen 1. In fact, sulprostone had a slight stimulatory effect. Therefore, EP3 signaling was not associated with the inhibitory effects of PGE2 on myofibroblast transition.
PGE2 Can Inhibit TGF-ß1–Mediated Increases in -SMA Expression in Adult Lung Fibroblasts
The data presented thus far involved the use of a human fetal lung fibroblast cell line, IMR-90. Because most fibrotic disorders occur in adults, we wanted to determine whether the inhibitory effects of PGE2 on TGF-ß1–mediated transition of fibroblasts to myofibroblasts were active in adult fibroblasts as well. Figure 7A demonstrates that normal adult human lung fibroblasts could be induced to express higher levels of -SMA when incubated with TGF-ß1. PGE2 was able to inhibit this induction by 60% at a concentration of 100 nM. Similarly, fibroblasts isolated from adult murine lungs (Figure 6B) were also stimulated to produce -SMA in the presence of TGF-ß1. PGE2 could inhibit this induction by 50% at a concentration of 100 nM. Thus, PGE2 was effective in limiting TGF-ß1–induced fibroblast to myofibroblast transition in both fetal and adult primary lung fibroblasts.
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Discussion
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We provide evidence for the first time that PGE2 can inhibit the transition of both fetal and adult fibroblasts to myofibroblasts by TGF-ß1 as assessed by -SMA protein levels. The inhibition of -SMA expression by PGE2 correlates with reduced collagen synthesis in PGE2-treated cells. In addition, this study identifies the EP2 receptor and increased cAMP as critical components of this inhibitory process. Previous studies have documented the important role of -SMA-expressing myofibroblasts in the pathogenesis of fibrotic disease (3, 6, 8, 10, 28). Numerous factors have been identified as promoting the transition of fibroblasts to myofibroblasts, specifically TGF-ß1, granulocyte macrophage-colony stimulating factor, heparin, and TNF- (12, 29, 30). Few factors have been identified that inhibit the transformation of myofibroblasts (5, 14, 15, 31). Thus, these results provide evidence that strategies to activate EP2 receptors on fibroblasts may have therapeutic benefit in limiting pulmonary fibrotic disease.
Our studies document the importance of PGE2 as an inhibitor of myofibroblast transition and help to explain the manifestations that occur clinically as a result of reduced PGE2 synthesis in fibrotic lungs. Normally, lung injury leads to upregulation of profibrotic mediators. Many of these profibrotic mediators, including TGF-ß1, TNF-, and platelet-derived growth factor, induce synthesis of PGE2, the major eicosanoid produced by fibroblasts, via induction of COX-2 (32, 33). PGE2 plays an important counter-regulatory role by suppressing fibroblast proliferation and collagen production (18, 34). In this manuscript, we show that the inhibitory actions of PGE2 on these functional outcomes correlate with the ability of PGE2 to inhibit myofibroblast transition. In fibrotic disease, however, there is an inadequate increase in PGE2 synthesis in response to profibrotic mediators. This is reflected in lung lavage PGE2 levels being lower than in normal individuals (20). Moreover, fibroblasts cultured from idiopathic pulmonary fibrosis patients exhibit an impaired ability to synthesize PGE2 (21, 22), which has been attributed to an inability to appropriately upregulate COX-2 mRNA and protein levels (22, 35). Therefore, these biochemical alterations seen in pulmonary fibrosis result in the cellular consequence of increased myofibroblast transformation after fibrotic lung injury. These findings support the prevailing hypothesis that fibrosis is the consequence of dysregulated repair after some form of lung injury (2).
Our data demonstrate that PGE2 inhibits myofibroblast differentiation via activation of EP2 receptors and elevations in cAMP. Given this observation, it is perhaps not surprising that previous studies have linked the inhibitory actions of PGE2 on fibroblast production of collagen mRNA to activation of EP2 and cAMP (26). Similarly, previous work has suggested that elevating cAMP levels directly in IMR-90 cells suppresses collagen production (18, 34). Our work demonstrates that PGE2 generates increased cAMP via stimulation of the EP2 receptor and that this results in inhibition of TGF-ß1–mediated expression of -SMA. Our data demonstrating that forskolin-mediated activation of adenyl cyclase also inhibits -SMA expression indicates that cAMP elevation is the proximal second messenger generated by EP2 signaling, which is responsible for the inhibition of myofibroblast differentiation. The fact that EP2 receptor antagonists can block the inhibitory effects of relatively high doses (100 nM) of PGE2 suggests that in IMR-90 cells at least, EP2 and not EP4 is the major inhibitory receptor. This is likely due to the paucity of EP4 receptors found in IMR-90 cells. Furthermore, our data demonstrate that EP3 receptor stimulation does not increase cAMP, nor does it inhibit TGF-ß1–mediated increases in -SMA or collagen I. Collectively, this body of work demonstrates that PGE2 can potently downregulate fibrogenesis by inhibiting myofibroblast transformation and that the pathway involved in this regulatory process is PGE2 binding the EP2 receptor and activating adenyl cyclase. Although increasing cAMP is an important mechanism for the inhibition of fibroblast to myofibroblast transition, the ability of various agonists to inhibit -SMA is not precisely correlated with their ability to increase cAMP. It is possible that other as yet unidentified PGE2-induced signaling pathways distinct from or distal to cAMP may contribute to this inhibitory process.
PGE2 is one of only a few factors that have been identified that can inhibit myofibroblast transition (5, 14, 15, 31). Furthermore, we have characterized the specific receptor (EP2) and signaling molecule (cAMP) that regulate the inhibitory actions of PGE2 with regards to TGF-ß1 activation of myofibroblasts. Under normal circumstances, injury involves an elegant feedback loop in which TGF-ß1 induces the synthesis of matrix to close the wound and, ultimately, PGE2 is produced as a counter-regulatory mechanism to inhibit further matrix deposition. Fibrosis represents a situation where sufficient induction of counter-regulatory PGE2 synthesis does not occur. Animal models have proven that pharmacologic or genetic blockade of prostaglandin production during the evolution of bleomycin-induced pulmonary fibrosis results in worse fibrotic outcomes (35, 36). Because strategies aimed at blocking TGF-ß1 expression are likely to have multiple deleterious effects, selective therapy to inhibit myofibroblast transformation offers an exciting alternative approach. In this regard, it is particularly important to note that our results demonstrate the ability of PGE2 to inhibit TGF-ß1–induced myofibroblast transition in adult lung cells as well as in fetal lung fibroblasts. Our results do suggest, however, that slightly higher concentrations of PGE2 may be needed in adult cells (100 nM) compared with fetal cells (10 nM) to achieve the inhibition. Our results demonstrate that exogenous addition of PGE2 or of EP2 selective agonists may represent a viable therapeutic means to achieve the dual goals of antifibrotic therapy: (i) to limit the synthesis of matrix by myofibroblasts already present in situ and (ii) to prevent the transdifferentiation of newly recruited fibroblasts into effector myofibroblasts.
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Acknowledgments
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The authors thank Raquel Angobaldo and Deirdra Williams for technical assistance and advice. This work was supported by National Institutes of Health grants HL071586 (B.B.M.), CA79046 (B.B.M.), P50HL56402 (B.B.M., G.B.T., and M.P.G.) and P50HL60289 (B.B.M. and G.B.T.). J.K. is supported by the National Institutes of Health Multidisciplinary Training Program in Lung Disease, 2T32HL07749.
Received in original form November 8, 2002
Received in final form April 16, 2003
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Abstract
Introduction
