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Published ahead of print on January 27, 2005, doi:10.1165/rcmb.2004-0205OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 470-477, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0205OC

Keratinocyte Growth Factor Expression by Fibroblasts in Pulmonary Fibrosis

Poor Response to Interleukin-1ß

Sylvain Marchand-Adam, Laurent Plantier, Dominique Bernuau, Agnès Legrand, Murielle Cohen, Joëlle Marchal, Paul Soler, Guy Lesèche, Hervé Mal, Michel Aubier, Monique Dehoux and Bruno Crestani

INSERM unit 408 and INSERM unit 327, Faculté Xavier Bichat, Université Paris 7; Service de Pneumologie, and Laboratoire de Biochimie A, Hôpital Bichat; and Service de Chirurgie Thoracique et Vasculaire, and Service de Pneumologie, Hôpital Beaujon, Clichy, Assistance Publique-Hôpitaux de Paris, Paris, France

Correspondence and requests for reprints should be addressed to Dr. Bruno Crestani, Service de Pneumologie, Hôpital Bichat, 16 rue Henri Huchard, 75877, Paris cedex 18, France. E-mail: bruno.crestani{at}bch.ap-hop-paris.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1ß is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1ß stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1ß increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1ß. By contrast, in IPF fibroblasts, IL-1ß did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1ß similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1ß–stimulated IPF and control fibroblasts. IL-1ß increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1ß is associated with a defect of c-Jun expression and activation and a defect of JNK activation.

Key Words: human • lung • MAP kinase • transcription factors • usual interstitial pneumonia


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology associated with activation of fibroblasts and accumulation of extracellular matrix proteins, which result in irreversible distortion of the lung architecture. Alveolar epithelial injury and delayed repair are critical events in lung fibrogenesis (1). Epithelial–mesenchymal interactions are central to the lung repair process. The integrity of the alveolar epithelium is essential to limit activation and proliferation of fibroblasts (2). Pulmonary fibroblasts are important sources of cytokines, growth factors, and mediators that control type 2 pneumocyte proliferation and differentiation. The dysregulated secretion of epithelium-directed growth factors by fibroblasts could participate in the fibrotic process by delaying epithelium repair. Previous results of our laboratory support this hypothesis, as we showed that IPF fibroblasts secrete low levels of hepatocyte growth factor when compared with control fibroblasts (3).

Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7), has been identified as an important paracrine mediator of proliferation, migration, and differentiation of type II pneumocytes (4). KGF is produced by fibroblasts (5) and specifically binds to the KGF receptor, a splice variant of FGF receptor 2 (FGFR2-IIIb), which is essentially expressed by epithelial cells. Systemic administration of recombinant human KGF provides significant cytoprotection to epithelial tissues and improves survival in animal models of lung fibrosis induced by bleomycin instillation or exposure to radiation (6–9).

Despite the important function of KGF in lung homeostasis, the regulation of KGF production by lung fibroblasts is poorly known, and KGF secretion by IPF fibroblasts has not been previously studied. Inflammatory mediators are known stimulants of KGF in fibroblasts. Among these mediators, IL-1ß appears to be the most potent inducer of KGF expression in fibroblasts from multiple sources (10, 11). In the mesenchymal–epithelial interaction in the skin, IL-1ß secreted by epithelial cells is a key regulator of KGF secretion by dermal fibroblasts and has been demonstrated to activate the c-Jun pathway (12).

We hypothesized that a defect in KGF regulation in lung fibroblasts may contribute to the pathophysiology of IPF. Therefore, the aim of this study was to evaluate in vitro KGF expression by human lung fibroblasts from control subjects and from patients with IPF, at baseline and after IL-1ß stimulation, and to specifically analyze the role of the c-Jun pathway.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
This study was approved by the ethics committee of Paris-Bichat University Hospital.

Patients with IPF
Fibroblasts were derived from lung tissue samples from 10 patients with IPF. Lung samples were obtained by open lung biopsy (n = 5) or at the time of lung transplantation (n = 5). IPF was diagnosed according to the ATS-ERS consensus criteria (13), including the characteristic morphology of usual interstitial pneumonia (UIP). Patients (eight males; two females) had a median age of 50 yr (44–69). Seven were ex-smokers and three had never smoked. In all patients, lung function tests showed a restrictive functional pattern. At the time of lung sampling, three patients were treated with low dose oral corticosteroids, associated with azathioprin in one patient.

Control Patients
Fibroblasts were derived from lung samples from seven patients (four males, three females) undergoing lung surgery for removal of a primary lung tumor. Normal lung from a noninvolved segment, remote from the solidary lesion, was obtained. Median age was 56 yr (28–70). Three patients had never smoked, two were ex-smokers, and two were active smokers.

KGF Production by Fibroblasts
Human lung fibroblasts were cultured from lung explants as previously described (3) and used at passage 5. Fibroblasts were grown to confluence with Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum in 12-well tissue culture plates, then cultured in 0.5 ml of serum-free DMEM, either in basal conditions or with various concentrations (0.1–100 ng/ml) of IL-1ß (Sigma, Saint Quentin Fallavier, France) or with 10 ng/ml IL-1ß at various times (6–72 h). In some experiments, lung fibroblasts were incubated with 2.10–5 M SP600125 (a c-Jun N-terminal kinase [JNK] inhibitor) (Calbiochem-Novabiochem, Schwalbach, Germany) (14), 5.10–5 M PD 98059 (an inhibitor of ERK-1) (15), or 10–5 M SB 203580 (an inhibitor of P38 mitogen-activated protein [MAP] kinase) (16), with or without 10 ng/ml IL-1ß stimulation. IL-1ß was added 60 min after addition of the inhibitor. Inhibitors were dissolved in dimethylsulfoxide (DMSO). Cells cultured with DMSO (1{per thousand}) were used as controls. Because of the limited number of cells available, all the experiments could not be performed on all cell lines.

At the end of the incubation period, the supernatants were saved for KGF determination (KGF Quantikine ELISA kit; R&D Systems, Madison, WI). The cell monolayer was lysed in 500 µl of lysis buffer containing 50 mM Tris HCl, pH 7.4, 0.1 mM EGTA, 0.1 mM EDTA, 1 µM leupeptin, 1 µM aprotinin, and 1µM PMSF. The protein content of the cell monolayer was measured using the Bio-Rad protein assay (Marnes la Coquette, France). KGF concentrations were expressed as picograms of KGF per microgram of protein in the cell monolayer per milliliter of medium.

Quantitative Analysis of KGF, JunB, and c-Jun mRNA
Total RNA was extracted from fibroblasts cultured for 18 h. KGF mRNA was quantified with reverse transcriptase–real-time polymerase chain reaction (RT-PCR). The transcripts of Ubiquitin C served as endogenous RNA controls (17). Confluent fibroblasts were cultured in T-75 tissue culture flasks in 5 ml serum-free DMEM for 18 h. Total RNA was extracted and reverse transcribed as described previously (3). RT-PCR amplification mixtures (20 µl) contained 25 ng template cDNA, 2x SYBR Green Jumpstart TAQ readyMix (10 µl; Sigma) and 800 nM forward and reverse primer (Table 1). Reactions were run on an ABI PRISM 7700 (Applied Biosystems, Foster City, CA). Each assay included (in duplicate): a standard curve of four serial dilution points of cDNA (25 pg to 25 ng) from human fibroblasts (MRC5 cell line), a no-template control, and 25 ng of cDNA from IPF or control fibroblasts. All PCR efficiencies were above 96%. Sequence Detection Software (Applied Biosystems) results were exported as tab-delimited text files and imported into Microsoft Excel (Microsoft France, Courtaboeuf, France) for further analysis. The median coefficient of variation (based on calculated quantities) of duplicated samples was 12%. The results were expressed as KGF/Ubiquitin C mRNA ratio (10 IPF and 7 controls).


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  		TABLE 1. Primers and pcr cycling conditions

 
JunB and c-Jun mRNA were quantified by RNase protection assay, as previously described (18). The human S6 RNA was used as an internal control (18). Quantitative analysis was performed from gels directly counted with the Instant Imager (Packard, Groningen, Netherlands) and the ratio of the signals from protected fragments to S6 was calculated.

Western Blot Analysis
Nuclear extracts were prepared from fibroblasts cultured for 18 h. Cells were homogenized in a lysis buffer as previously described (18). Thirty micrograms of nuclear extracts were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Céra-Labo, Aubervilliers, France). Membranes were incubated overnight with a rabbit polyclonal antibody to c-Jun (SC-1694; Santa Cruz Biotechnology, Santa Cruz, CA), Jun B (SC-73; Santa Cruz Biotechnology), or human small nuclear ribonucleoprotein (Sn RNP, used as an internal control). All the antibodies were diluted to 1/500. Then the membranes were incubated with a peroxidase-labeled anti-rabbit IgG diluted to 1/5,000 for 1 h (Amersham Biosciences, Saclay, France), and visualized by chemiluminescence (Amersham). Immunoblots were scanned densitometrically to quantitate protein level (Candela software; Microvision Instruments, Evry, France).

Quantification of Phosphorylated c-Jun, and Total and Phosphorylated c-Jun N-terminal Kinase
Fibroblasts were grown to 80% confluence in 96-well tissue culture plates, then incubated for 15 min in basal conditions or with IL-1ß. After cell fixation by 4% formaldehyde in PBS, phosphorylated c-Jun was quantified using a commercially available assay (c-Jun [S73] ELISA kit; FACE, Active Motif Europe, Rixensart, Belgium) as were total JNK and phosphorylated JNK (JNK ELISA kit; FACE, Active Motif Europe), according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as the median (range). Differences between IPF and control fibroblasts were determined using the Mann-Whitney U test. To compare the effects of different agents on baseline conditions, we used the Wilcoxon's paired nonparametric test for group comparisons. The correlations were assessed with the Spearman's rank-order test. A P value < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Basal and IL-1ß–Stimulated KGF Secretion by Fibroblasts
KGF was detected in all fibroblast supernatants cultured for 48 h from control subjects and from patients with IPF. Basal KGF secretion by controls was 2.7 pg/µg protein/ml (1.5–3.1) and was strongly increased with IL-1ß stimulation (6.4 pg/µg protein/ml [3.4–13.2], P < 0.01) (Figure 1A).



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  		Figure 1. Effect of IL-1ß on KGF secretion and KGF mRNA content. (A) KGF concentration was similar in control and IPF fibroblasts at baseline. IL-1ß (10 ng/ml) increased KGF concentration in control fibroblasts. The effect of IL-1ß was weaker and heterogeneous in IPF fibroblasts. KGF concentrations were measured by ELISA in the cell culture supernatants obtained after 48 h of culture with or without IL-1ß stimulation, and expressed as pg/µg protein/ml. (B) KGF mRNA content was similar in control and IPF fibroblasts at baseline. IL-1ß increased KGF mRNA in control fibroblasts, but had no effect in IPF fibroblasts. KGF mRNA was measured by RT-PCR and expressed as the KGF/Ubiquitin-c mRNA ratio. The cells were cultured for 18 h with or without IL-1ß. Individual values and median (bar). *P < 0.05 and **P < 0.01 compared with basal conditions.

 
Basal KGF secretion by IPF fibroblasts was similar to control fibroblasts (1.7 pg/µg protein/ml [0.8–5.4], P = 0.10). However, IPF fibroblasts poorly responded to IL-1ß (2.1 pg/µg protein/ml [0.3–8.3], P = 0.04 versus basal). Three of the 10 IPF fibroblast lines did not show any increase of KGF secretion after IL-1ß stimulation. The median increase of KGF concentration with IL-1ß stimulation was higher in control fibroblasts (+156% [+55 to +342]) than in IPF fibroblasts (+40% [–50 to +76], P = 0.02 versus controls).

KGF concentrations did not correlate with any of the lung function test results in both control subjects and patients with IPF (data not shown). Among IPF fibroblasts, in vitro KGF secretion was not different in the three corticosteroid-treated patients (1.48 pg/µg protein/ml [0.82–5.37]) when compared with the seven untreated patients (1.98 pg/µg protein/ml [1.28–2.79], P = 0.91).

KGF mRNA was detected in all the lung fibroblast cultures. In nonstimulated fibroblasts, the KGF/Ubiquitin-C mRNA ratio was similar in control fibroblasts (0.17 [0.08 to 0.32]) and IPF fibroblasts (0.16 [0.05–0.29], P = 0.30) (Figure 1B). IL-1ß strongly increased KGF mRNA in control fibroblasts (+160%, P < 0.05 versus basal), whereas KGF mRNA level was not significantly modified in IPF fibroblasts.

Altogether these results point to a defect of transcriptional regulation of KGF expression by IL-1ß in IPF fibroblasts.

In view of the poor response to 10 ng/ml IL-1ß, we asked whether lower or higher doses of IL-1ß could stimulate KGF secretion by IPF fibroblasts. Dose–response experiments were performed in fibroblasts from three control subjects and four patients with IPF. In control fibroblasts, IL-1ß increased KGF secretion and KGF mRNA content at all the concentrations tested (0.1–100 ng/ml), the effect being maximal at 10 ng/ml (Figures 2A and 2B). In IPF fibroblasts, we observed no significant effect of IL-1ß at all the doses tested (Figures 2E and 2F).



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  		Figure 2. Time and dose responses of IL-1ß on KGF secretion and KGF mRNA content. KGF concentration (A) and KGF mRNA (B) increased with an increase of IL-1ß doses (0.1–100 ng/ml) to a top for 10 ng/ml dose in control fibroblasts. The response of IPF fibroblasts were heterogenous at various doses of IL-1ß (E and F). In control fibroblasts, the KGF secretion (C) and mRNA KGF content (D) were increased at all tested time of IL-1ß stimulation. In IPF fibroblasts, KGF increase were heterogenous and fickle (G and H).

 
Then, we asked whether the time-course of IL-1ß stimulation could be different in control and IPF fibroblasts and could explain the difference observed after a 48-h stimulation period. In control fibroblasts, IL-1ß increased KGF concentration and KGF mRNA content at all the times tested (Figures 2C and 2D), whereas we observed no significant effect in IPF fibroblasts, whatever the duration of IL-1ß stimulation (Figures 2G and 2H).

JunB and c-Jun Expression by Fibroblasts
IL-1ß has been shown to control KGF production through the activation of the c-Jun/AP-1 pathway (12). We hypothesized that the lack of KGF mRNA increase after IL-1ß stimulation was due to a defect in the c-Jun pathway. Therefore, we quantified c-Jun and JunB in control and IPF fibroblasts.

At baseline, c-Jun mRNA content was slightly higher in IPF than in control fibroblasts (P = 0.03, Figure 3), whereas JunB mRNA content was not different (P = 0.44, Figure 3). In control fibroblasts, IL-1ß increased both c-Jun and JunB mRNA (P < 0.05, Figure 3), whereas in IPF fibroblasts, IL-1ß increased JunB mRNA, but c-Jun mRNA remained unchanged (Figure 3). Western blot analysis of nuclear extracts from control and IPF fibroblasts showed that c-Jun and JunB contents were not different in unstimulated control and IPF fibroblasts (P = 0.77 and P = 0.32, respectively; Figure 4). IL-1ß increased both c-Jun and JunB in control fibroblasts (P = 0.02), whereas in IPF fibroblasts, Jun B increased but c-Jun was unchanged.



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  		Figure 3. Expression of c-Jun and JunB mRNA in IPF and control fibroblasts. C-Jun, JunB, and human S6 (hS6) mRNA content was quantified by RNase protection assay. (A) A representative RNase protection assay blot for two different control (C1 and C2) and two different IPF (IPF1 and IPF2) cultures. (B) c-Jun mRNA content, expressed as c-Jun/hS6 ratio, was higher in IPF fibroblasts than control fibroblasts at baseline. IL-1ß increased c-Jun content in control fibroblasts, but had no effect on IPF fibroblasts. (C) JunB mRNA content, expressed as JunB/hS6 ratio, was similar in control and IPF fibroblasts at baseline. IL-1ß increased JunB content in control fibroblasts and IPF fibroblasts. MW: molecular weight; tRNA (tRNA from brewer's yeast) and solvent (guanidine isothiocyanate) used as a negative control. Individual values and median (bar). *P < 0.05 compared with basal conditions; {dagger}P < 0.05 compared with control fibroblasts.

 


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  		Figure 4. Western blot analysis of c-Jun and JunB expression in IPF and control fibroblasts. (A) representative Western blot of c-Jun for two different control (C1 and C2) and two different IPF (IPF1 and IPF2) cultures. (B) c-Jun content, expressed as c-Jun/snRNP ratio, was similar in control and IPF fibroblasts at baseline. IL-1ß increased c-Jun content in control fibroblasts but had no effect in IPF fibroblasts. (C) Representative Western blot of JunB for two different control (C1 and C2) and two different IPF (IPF1 and IPF2) cultures. (D) JunB content, expressed as JunB/snRNP ratio, was similar in control and IPF fibroblasts at baseline. IL-1ß increased JunB content in control and IPF fibroblasts. Individual values and median (bar). *P < 0.05 compared with basal conditions.

 
Time– and dose–response experiments were performed to determine the effect of different doses of IL-1ß (0.1–100 ng/ml) or different duration of exposures (8–72 h) (Figure 5). Whatever the experimental conditions, IL-1ß failed to stimulate c-Jun mRNA expression in IPF fibroblasts (Figures 5E, 5F, 5G, and 5H).



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  		Figure 5. Time and dose responses of IL-1ß on mRNA c-Jun and JunB content. These was a dose and time response of IL-1ß on mRNA c-Jun in control fibroblasts, but not in IPF fibroblasts (A, B, E, and F). mRNA JunB content increase in presence of IL-1ß with a dose and time response in both IPF and control cultures (C, D, G, and H).

 
These results demonstrate a failure to increase c-Jun expression in IPF fibroblasts after IL-1ß stimulation.

Phosphorylated c-Jun in Fibroblasts
Phosphorylation of serine 63 and 73 in the amino-terminal region of c-Jun strongly enhances its transcriptional activity. To determine whether the failure to increase c-Jun mRNA with IL-1ß was associated with a defect of c-Jun phosphorylation, we quantified the phosphorylated form of c-Jun in lung fibroblasts cultures.

At baseline, phosphorylated c-Jun content was similar in IPF and control fibroblasts (Figure 6A). After IL-1ß stimulation, phosphorylated c-Jun content increased in all control fibroblast cultures (+60%, [+19 to +115] P = 0.02), whereas it was unchanged in IPF fibroblasts (–10% [–14 to +40], P = 0.31).



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  		Figure 6. Quantification of phosphorylated c-Jun in control and IPF fibroblasts. (A) IL-1ß (10 ng/ml) increased phosphorylated c-Jun (Ph-c-Jun) in control but not in IPF fibroblasts. c-Jun was measured in cell lysates by ELISA specific for S73 phosphorylated c-Jun, and expressed as c-Jun optical density (OD)/total protein OD ratio. Individual values and median (bar). *P < 0.05 compared with basal conditions. (B) Dose–response curves for phosphorylated c-Jun. IL-1ß induced a rapid increase of phosphorylated c-Jun in control fibroblasts with a top at 10 ng/ml dose, whereas phosphorylated c-Jun increase was weaker and inconstant in IPF fibroblasts.

 
The dose–response experiments demonstrate an increase of phosphorylated c-Jun content in control fibroblasts with 0.1 ng/ml IL-1ß and a maximum effect with 10 ng/ml. There was no significant effect in IPF fibroblasts (Figure 6B).

Quantification of JNK
JNK is the main regulator of c-Jun activation (19) through its capacity to phosphorylate c-Jun. We hypothesized that the failure to increase phosphorylated c-Jun was associated with a deficient activation of JNK. We quantified JNK and phosphorylated JNK, its active form, in IPF and control fibroblasts. Total JNK content was not different in IPF and control fibroblasts, either at baseline or after stimulation (data not shown).

At baseline, phosphorylated JNK content was similar in IPF and control fibroblasts (Figure 7A). IL-1ß (10 ng/ml) strongly increased phosphorylated JNK content in all control fibroblast cultures (+160% [+112 to +281], P = 0.03). IL-1ß increased phosphorylated JNK content only in four of the seven IPF fibroblasts studied. The median increase of JNK phosphorylation after IL-1ß was lower in IPF fibroblasts than in control fibroblasts (+60% [–80 to +120], P = 0.003 versus controls).



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  		Figure 7. Quantification of total and phosphorylated JNK in control and IPF fibroblasts. (A) IL-1ß increased phosphorylated JNK (Ph-JNK) in control fibroblasts. The effect of IL-1ß was heterogeneous in IPF fibroblasts. (B) There was a dose response with IL-1ß on JNK phosphorylation in control fibroblasts. After IL-1ß increase, the JNK phosphorylation was heterogenous in IPF fibroblasts. JNK was measured in cell lysates by ELISA specific for total and phosphorylated JNK, and expressed as JNK OD/ total protein OD ratio. Individual values and median (bar). *P < 0.05 compared with basal conditions.

 
Figure 7B shows the dose–response profiles for phosphorylated JNK content. IL-1ß increased phosphorylated JNK in control fibroblasts at all doses tested, whereas no significant effect could be observed in IPF fibroblasts, whatever the dose used. These results indicate that the deficient response to IL-1ß in IPF fibroblasts was associated with a defect of JNK activation.

Effect of MAP Kinase Inhibitors on KGF Secretion
To determine whether JNK inhibition could modulate KGF expression by control and IPF fibroblasts, we cultured fibroblasts with SP 600125, a pharmacologic inhibitor of JNK (Figure 8). In the same experiments, we tested the effect of an inhibition of P38 MAP kinase (SB 203580) (16) and an inhibitor of ERK-1 (PD 98059) (15), because IL-1ß as been shown to act through both MAP kinase pathways (20).



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  		Figure 8. Inhibition of JNK, ERK, and P38 MAP kinase modulates KGF secretion. PD 98059 (PD, an inhibitor of ERK-1) and SB 203580 (SB, an inhibitor of P38 MAPK) inhibited the KGF secretion at baseline in supernatants from control (A) and IPF (B) fibroblasts, whereas SP600125 (SP, an inhibitor of JNK) had no effect (A and B). Cells cultured with DMSO (1{per thousand}) served as controls because inhibitors were dissolved in DMSO (1{per thousand}, final concentration). SP600125 strongly inhibited the action of IL-1ß in control fibroblasts (A), whereas it had no effect on IPF fibroblasts (B). Individual values and median (bar). *P < 0.05 compared with basal DMSO condition; {dagger}P < 0.05 compared with cells stimulated with IL-1ß and DMSO.

 
In unstimulated fibroblast cultures, inhibition of ERK-1 reduced KGF secretion by control (–32% [–35 to +25], P = 0.04) and IPF fibroblasts (–26% [–59 to +78], P = 0.04) (Figure 8). The inhibition of P38 MAP Kinase also reduced KGF secretion by control (–49% [–72 to +16], P = 0.03) and IPF fibroblasts (–61% [–95 to +11], P = 0.01) (Figures 8A and 8B). By contrast, inhibition of JNK with SP 600125 did not modify basal KGF secretion by control or IPF fibroblasts. These results indicate that unstimulated KGF secretion by cultured lung fibroblasts is dependent upon ERK-1 and P38 MAP kinase pathways, but independent of JNK activation.

In IL-1ß–stimulated control fibroblasts, inhibition of ERK-1, P38 MAP kinase, or JNK similarly reduced KGF secretion (Figure 8A). This indicates that IL-1ß induces a JNK-dependent pathway of KGF secretion that was not active at baseline. By contrast, in IL-1ß–stimulated IPF fibroblasts, ERK-1 and P38 MAP kinase inhibition reduced KGF secretion, but JNK inhibition had no effect (Figure 8B).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The main findings of this study are (1) that human lung fibroblasts from patients with IPF have a decreased capacity to secrete KGF in vitro after IL-1ß stimulation, when compared with control fibroblasts; and (2) that the lack of IL-1ß response appears to be secondary to a defect of the c-Jun pathway, most likely due to a dysregulation of JNK activation.

Fibroblasts are involved in the complex repair process that follows lung injury and play a fundamental role in the pathophysiology of lung fibrosis (1). We studied KGF secretion by pulmonary fibroblasts isolated from normal and fibrotic lung by the explants technique. Fibroblasts were obtained from patients with different characteristics in terms of tobacco exposure or corticosteroid treatments. These factors could influence the biological cellular responses. This is a limitation of our study. Furthermore, the majority of our IPF fibroblast cell lines contained 5–10% myofibroblasts as assessed by the expression of the {alpha}–smooth –muscle actin. Myofibroblasts constitute one fibroblast subpopulation and are usually increased in human and experimental lung fibrosis (21). However, because myofibroblasts constitute a minority of the cells that we studied in vitro, it is unlikely that the reduced expression of KGF by pulmonary fibroblasts isolated from fibrotic lungs was completely linked to the myofibroblast phenotype.

To our knowledge, the current study is the first one to investigate the production of KGF by IPF fibroblasts. Although Bergeron and colleagues have shown that KGF mRNA was expressed in similar amounts in lungs from patients with IPF and control subjects (22), they did not specifically study the expression of KGF by lung fibroblasts. We observed that KGF concentration was not different in unstimulated IPF or control fibroblast supernatants. These findings contrast with decreased HGF production in IPF fibroblasts (3). This indicates that KGF and HGF are differentially regulated in lung fibroblasts.

We studied IL-1ß stimulated KGF secretion by fibroblasts because IL-1ß is known as the best inducer of KGF expression in fibroblasts (23). We observed that IPF fibroblasts responded poorly to exogenous IL-1ß when compared with control fibroblasts. This defect of IL-1ß response is not due to an abnormal IL-1ß receptor expression, because the type I IL-1 receptor has already been shown to be in similar number and affinity in IPF and control fibroblasts (24). Factor activator protein-1 (AP-1) sites have been found in the promoter region of the mouse and human KGF genes (12). We focused on the c-Jun pathway, a key AP-1 component, which has been shown to control KGF response to IL-1ß in mouse dermal fibroblasts. Specifically, Szabowski and colleagues demonstrated that the balance of c-Jun/JunB controlled IL-1ß–stimulated KGF secretion by dermal fibroblasts in mice, as KGF was hardly detectable in c-Jun–/– fibroblasts stimulated with IL-1ß, whereas KGF expression was enhanced in JunB–/– fibroblasts (12). These data might be interpreted as a demonstration that the KGF promoter is predominantly dependent on c-Jun regulation and not JunB regulation. Accordingly, in our study we show that the concomitant increase of c-Jun and JunB was associated with an increase of KGF expression and secretion in control fibroblasts, whereas the lack of increase of c-Jun was associated with a limited KGF response to IL-1ß in IPF fibroblasts despite an increase of JunB. Previous data suggested that JunB could substitute for c-Jun in some biological conditions (25). However, JunB is a weaker transcriptional activator compared with c-Jun (26–28) and has been shown to exert antagonistic activities against c-Jun (28, 29), particularly on KGF secretion (12). Altogether, these data suggest that the AP-1 site of the KGF promoter is predominantly regulated by c-Jun–containing dimers and that JunB cannot compensate for the lack of functional c-Jun.

We observed that c-Jun mRNA was slightly increased in IPF fibroblasts at baseline when compared with control fibroblasts. This could suggest that c-Jun was maximally stimulated in IPF fibroblasts. Clearly, this is not the case as we detected similar levels of c-Jun protein and phosphorylated c-Jun in IPF and control fibroblasts.

Previous studies have demonstrated that increased expression of c-Jun in the lung is concurrent to the onset of fibrosis in the amiodarone-treated rats or crocidolite asbestos–induced fibrosis (30, 31). Immunohistochemical observations have also indicated that fibroblasts contain c-Jun in areas of proliferation, whereas its expression is very weak in areas of complete fibrosis in rat lung after bleomycin-induced injury (32).

Our results indicate that IL-1ß–stimulated KGF secretion by control fibroblasts depends, at least in part, on the activation of the JNK pathway. Indeed, IL-1ß increased phospho-JNK content, and treatment with SP600125, a specific JNK inhibitor, inhibited the IL-1ß–stimulated KGF secretion. This pathway is not involved in the regulation of basal secretion of KGF by control and IPF fibroblasts because SP600125 had no effect on basal KGF secretion. This is the first demonstration of a role for JNK in the regulation of KGF expression in human lung fibroblasts. Our results also show that the inhibitor of JNK did not influence IL-1ß–stimulated secretion by IPF fibroblasts. After stimulation with IL-1ß, the variation in phosphorylated JNK was weaker and heterogenous in IPF fibroblasts, revealing a defect in JNK activation by IPF fibroblasts. The regulation of JNK pathway is extremely complex and depends on the phosphorylation and activation of different and specific kinases (33). Phospho-JNK has been shown to be significantly increased in lung homogenates from patients with IPF compared with control subjects (34). In addition, immunohistochemical analysis of the IPF lung has shown that phospho-JNK is increased in epithelial cells, endothelial cells, macrophages, smooth muscle cells, and lymphocytes, but not in fibroblasts (34). The absence of JNK activation may inhibit tissue repair, particularly in the lung, because mouse embryo JNK–/– fibroblasts exhibit significantly lower ability to close mechanically induced cell layer wounds (35). The absence of JNK activation may also promote fibrosis through increased alveolar epithelial cell apoptosis, as shown in JNK1–/– mice after exposure to hyperoxia (36).

We also demonstrate that basal secretion of KGF in control and IPF fibroblasts depends upon ERK-1 and P38 MAP kinase pathways, a previously unkown mechanism. It is possible that the culture of fibroblasts by itself stimulates ERK-1 and P38 MAP kinase pathways. The role of serum is unlikely because this was detected in cells cultured without serum for 48 h.

Much of the attention on the pathophysiology of pulmonary fibrosis has been placed on alterations in cellular phenotypes and mediator profiles that promote fibroblast activation and matrix protein deposition. Although generation of such profibrotic mediators is doubtless integral to the fibroproliferative responses, the importance of the failure to appropriately generate "good" mediators that promote repair and prevent fibrosis in the setting of injury should not be underestimated, as emphasized by our demonstration of a defect of KGF secretion by IPF fibroblasts.


   Acknowledgments
 
The expert technical assistance of Marie-Claire Castellotti and fruitful discussions with Dr. Christophe Quesnel and Dr. Aurélie Fabre are gratefully acknowledged. Some of the results of these studies have been previously reported in the form of an abstract (37).


   Footnotes
 
S.M.-A. is the recipient of a grant from the Fondation pour la Recherche Médicale (Prix Mariane Josso) and the Fondation Benaid. Part of this research program benefited from a grant of the Fondation de France.

Conflict of Interest Statement: S.M.-A. has no declared conflicts of interest; L.P. has no declared conflicts of interest; D.B. has no declared conflicts of interest; A.L. has no declared conflicts of interest; M.C. has no declared conflicts of interest; J.M. has no declared conflicts of interest; P.S. has no declared conflicts of interest; G.L. has no declared conflicts of interest; H.M. has no declared conflicts of interest; M.A. has no declared conflicts of interest; M.D. has no declared conflicts of interest; and B.C. has no declared conflicts of interest.

Received in original form June 24, 2004

Received in final form December 30, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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