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Am J Physiol Lung Cell Mol Physiol 290: L1097-L1103, 2006; doi:10.1152/ajplung.00166.2005
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HGF synthesis in human lung fibroblasts is regulated by oncostatin M

Murielle Cohen,1,2 Sylvain Marchand-Adam,1,3 Véronique Lecon-Malas,2 Joëlle Marchal-Somme,1 Anne Boutten,1,2 Geneviève Durand,2 Bruno Crestani,1,3 and Monique Dehoux1,2

1Institut National de la Santé et de la Recherche Médicale Unité 700, Faculté de médecine Xavier Bichat, Université Paris 7, Paris; and 2Service de Biochimie and 3Service de Pneumologie, Hôpital Bichat-Claude Bernard, Assistance Publique-Hôpitaux de Paris, Paris, France

Submitted 13 April 2005 ; accepted in final form 28 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oncostatin M (OSM) is a IL-6 family cytokine locally produced in acute lung injury. Its profibrotic properties suggest a role in lung wound repair. Hepatocyte growth factor (HGF), produced by fibroblasts, is involved in pulmonary epithelial repair. We investigated the role of OSM in HGF synthesis by human lung fibroblasts. We showed that OSM upregulated HGF mRNA in MRC5 cells and in human lung fibroblasts, whereas IL-6 and leukemia inhibitory factor did not. OSM induced HGF secretion to a similar extent as IL-1beta in both a time- and dose-dependent manner. HGF was released in its cleaved mature form, and its secretion was completely inhibited in the presence of cycloheximide, indicating a de novo protein synthesis. OSM in combination with prostaglandin E2, a powerful HGF inductor, led to an additive effect. OSM and indomethacin in combination further increased HGF secretion. This could be explained, at least in part, by a moderate upregulation of specific OSM receptor beta mRNA expression through cyclooxygenase inhibition.These results demonstrate that OSM-induced HGF synthesis did not involve a PGE2 pathway. OSM-induced HGF secretion was inhibited by PD-98059 (a specific pharmacological inhibitor of ERK1/2), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor) by 70, 82, and 100%, respectively, whereas basal HGF secretion was only inhibited by SP-600125 by 30%. Our results demonstrate a specific upregulation of HGF synthesis by OSM, most likely through a MAPK pathway, and support the suggestion that OSM may participate in lung repair through HGF production.

oncostatin M receptor; prostaglandin E2; mitogen-activated protein kinase; lung repair; hepatocyte growth factor

ONCOSTATIN M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family (gp130 cytokines), which includes IL-6, leukemia inhibitory factor (LIF), IL-11, ciliary neurotrophic factor, and cardiotrophin 1. All these cytokines bind receptor complexes, which include the gp130 signaling subunit as well as receptor-specific chains (16). OSM is a 28-kDa glycoprotein originally identified for its inhibitory activity of tumor cell growth and is produced by activated monocytes, T lymphocytes, and polymorphonuclear neutrophils (14, 24, 51, 59). OSM has multiple biological activities such as modulation of proliferation, embryonic development, differentiation, inflammation, and regeneration of various tissues.

In human cells, OSM can interact with both type I OSM receptor (gp130/LIFR) and type II OSM receptor, composed of a gp130 and an OSM receptor beta-subunit (OSMRbeta) (31). The signal transduction pathway utilized by OSM has traditionally been thought to involve activation of the signal transducer and activator of transcription (STAT) family and/or tyrosine phosphorylation of a variety of components of the Ras-mitogen-activated protein kinase (MAPK) cascade (16, 18, 20, 21, 45, 60). MAPKs belong to a growing family of serine/threonine protein kinases that are important mediators of signal transduction from the cell surface to the nucleus. Three MAPK cascades have been identified: extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun NH2-terminal kinase/stress-activated protein kinases (JNK/SAPK) (4, 5, 8). All of them are believed to mediate activation of the MAPKs, most notably ERK1/2 from the activated gp130 receptor (52).

Epithelial-mesenchymal interaction represents a major component of the lung repair process. Pulmonary fibroblasts are an important source of cytokines, growth factors, and mediators that control alveolar epithelial cell proliferation and differentiation. Hepatocyte growth factor (HGF), otherwise known as scatter factor, is one of the key factors produced by fibroblasts and involved in lung repair (33, 53).

In humans, HGF was initially found in the plasma of patients with fulminant hepatic failure (10). Although HGF was originally thought to be a hepatocyte-specific mitogen, it is now known to be a multifunctional cytokine that acts as a mitogenic and morphogenic factor for a variety of cells, including pulmonary epithelial cells (11, 58). In patients with acute lung injury, elevated levels of HGF are found in both serum (23, 56) and bronchoalveolar lavage fluid, suggesting its involvement in human lung repair (46). The biological influence of HGF is mediated through its specific receptor, the c-Met tyrosine kinase (9).

HGF is secreted by stromal tissue as a 92-kDa single-chain proHGF. ProHGF is inactive and unable to elicit any biological activities. The mature bioactive HGF form consists of a disulfide-linked 69-kDa {alpha}-chain and a 34-kDa beta-chain (34). In pulmonary fibroblasts, IL-1beta, prostaglandin E2 (PGE2), and heparin have been shown to upregulate HGF synthesis, whereas transforming growth factor-beta1 and glucocorticoids downregulate HGF gene expression (28). Several studies have shown that OSM is able to enhance the expression of PGE2 and cyclooxygenase-2 (COX-2) and to induce growth factor such as basic fibroblast growth factor (bFGF) (2, 37, 54).

To date, little is known about HGF regulation by the IL-6 family cytokines. This led us to hypothesize that OSM may participate in alveolar repair through HGF synthesis. Thus the aim of this work was to study the regulation of HGF by the IL-6 family, focusing on the role of OSM in human pulmonary fibroblasts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cytokines, antibodies, and chemicals. Recombinant human OSM, LIF, IL-6, IL-1, and monoclonal anti-OSM receptor beta were purchased from R&D Systems (Abingdon, UK). PD-98059, SB-203580, and SP-600125 were purchased from Calbiochem (La Jolla, CA). Indomethacin, NS-398, PGE2, dimethyl sulfoxide, actinomycin D, and cycloheximide (CHX) were purchased from Sigma (Saint Quentin Fallavier, France). DMEM, L-glutamine, amphotericin B, penicillin G, streptomycin, TRIzol reagent, fetal calf serum (FCS), and phosphate-buffered saline (PBS) were purchased from Invitrogen (Cergy Pontoise, France). Aprotinin (Trazylol) was purchased from Bayer Pharma (Sens, France). Taq polymerase and molecular weight standards were purchased from Roche Diagnostics (Meylan, France). Vectastain ABC-alkaline phosphatase kit system was obtained from Vector (Burlingame, UK), and fast red substrate was obtained from Dako (Trappes, France). Nonspecific mouse IgG (32K9100) was purchased from Sigma.

Cell culture. Most experiments were performed with the human embryonic lung fibroblast cell line MRC5 (CCL MRCOO/OR; obtained from Eurobio, les Ulis, France). In some experiments, we used adult fibroblasts derived from lung samples from three patients undergoing lung surgery for the removal of a primary lung tumor, obtained by our laboratory as previously described (25) with approval by the ethics committee of Paris-Bichat University Hospital. Normal lung from a noninvolved segment, remote from the solitary lesion, was obtained. Human lung fibroblasts were isolated from lung explants as previously described (25). Human lung fibroblasts were used at passage 5. All fibroblasts were cultured with DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were kept in a humidified atmosphere of 5% CO2 in air at 37°C. Medium was routinely changed every 3–4 days.

HGF protein secretion. Cells were seeded on 12-well tissue culture plates at a density of 5 x 104 cells/well. Fibroblasts were grown to 80% confluence and then washed two times in PBS and cultured in 1 ml of serum-free DMEM, either in basal condition or stimulated by LIF (10 ng/ml), IL-6 (10 ng/ml), IL-1 (10 ng/ml; used as a positive control for HGF upregulation), or OSM. OSM was used at the concentration of 10 ng/ml, except in the OSM dose-response experiment. In selected experiments, cells were exposed to different pharmacological agents [pretreatment for 1 h with CHX (1 µg/ml) to inhibit protein synthesis; SB-203580 (20 µM), an inhibitor of p38 MAPK; SP-600125 (20 µM), an inhibitor of JNK/SAPK; or PD-98059 (30 µM), an inhibitor of ERK1/2 MAPK] before addition of OSM. After a 24-h incubation period, the supernatants were saved with aprotinin (50 µl/ml) and stored at –20°C before HGF protein determination. HGF was then measured using the HGF Quantikine ELISA kit (R&D Systems). The DNA content of the cell monolayer was measured as previously described (19). The results are expressed as picograms of HGF per microgram of DNA in the cell monolayer per milliliter of cell supernatants.

HGF Western blot analysis. Fibroblasts supernatants and recombinant human HGF (a mixture of proHGF and mature HGF, according to the manufacturer) were examined by Western blotting as previously described (12).

Analysis of HGF and OSM receptor beta mRNA with quantitative real-time PCR. MRC5 cells were cultured in DMEM-10% FCS at a density of 5 x 105 cells in a T-75 tissue culture flask. Confluent fibroblasts were stimulated in 5 ml of serum-free DMEM in the absence or presence of cytokines alone or in combination for 18 h. In some experiments, fibroblasts were cultured in the presence of the transcription inhibitor actinomycin D (5 µg/ml), which was added 30 min before the stimulation by OSM (10 ng/ml) over 3 h. Total RNA was extracted and reverse transcribed as described previously (12). A specific pair of primers designed for HGF, OSMRbeta, porphobilinogen deaminase (PBGD), and ubiquitin C was added to each complementary DNA reaction mix (Table 1). The transcripts of either PBGD or ubiquitin C served as endogenous RNA controls. RT-PCR amplification mixtures (25 µl) contained 25 ng of template cDNA, 2x SYBR Green I Master Mix buffer (12.5 µl) (Applera France, Applied Biosystems, Courtaboeuf, France), and 800 nM forward and reverse primers.


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  		Table 1. Primers

 
Reactions were run on an ABI PRISM 7000 (Applera). Each assay included (in duplicate) a standard curve of four serial dilution points of HGF cDNA (ranging from 25 ng to 25 pg), a no-template control, and 25 ng of each test cDNA. All PCR efficiencies were above 92%. Sequence Detection software (Applera) results were exported as tab-delimited text files and imported into Microsoft Excel for further analysis. The median coefficient of variation (based on calculated quantities) of duplicated samples was 18%. The results are expressed as HGF/PBGD mRNA or OSMRbeta/ubiquitin C mRNA ratios.

Statistical analysis. All data are expressed as means ± SE. To compare the effect of different agents, we used Wilcoxon’s paired nonparametric test for group comparison. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
OSM stimulates synthesis of HGF. We first evaluated the expression of HGF in MRC5 cells, stimulated or not with IL-6, LIF, and OSM. OSM induced a two- to threefold increase in HGF protein secretion, similar to that of IL-1beta (IL-1: 2,207 ± 996 pg HGF·µg DNA–1·ml–1 and OSM: 2,005 ± 1,211 pg HGF·µg DNA–1·ml–1 vs. 830 ± 504 pg HGF·µg DNA–1·ml–1 in unstimulated cells, P < 0.001) (Fig. 1A). No effect was observed with IL-6 and LIF. In a similar manner, OSM induced a twofold upregulation of HGF mRNA expression, whereas IL-6 and LIF did not (Fig. 1C).


Figure 1
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  		Fig. 1. Effects of leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6) on hepatocyte growth factor (HGF) expression by lung fibroblasts. MRC5 cells (A and C) or human lung fibroblasts (B and D) were cultured in the presence of cytokines at 10 ng/ml for 24 h (A and C) or 18 h (B and D). HGF protein was measured using ELISA (A and B), and HGF mRNA expression was measured using quantitative real-time RT-PCR (C and D). Results are expressed as means (SD). PBGD, porphobilinogen deaminase; N = no. of experiments. {dagger}P < 0.001 vs. basal control.

 
We confirmed these results in three different lines of control human adult lung fibroblasts. Indeed, OSM induced an upregulation of both HGF mRNA expression and protein secretion (253 ± 164 vs. 575 ± 187 pg HGF·µg DNA–1·ml–1), whereas other IL-6 family cytokines did not (Fig. 1, B and D).

Western blot analysis in human lung fibroblasts demonstrated that HGF detected in fibroblast supernatants was mainly in the cleaved mature form (presence of the 69-kDa {alpha}-chain) in either control or OSM-treated fibroblasts (Fig. 2).


Figure 2
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  		Fig. 2. Western blot analysis of HGF produced by OSM-stimulated fibroblasts. Human lung fibroblasts were cultured in the presence or absence of 10 ng/ml OSM for 24 h. Recombinant human HGF (rh-HGF, a mixture of proHGF and mature HGF) was loaded and blotted in parallel. The molecular masses of protein standards are indicated. HGF was in the cleaved mature form as evidenced by detection of the 69-kDa {alpha}-chain.

 
Because HGF is constitutively produced by fibroblasts, we examined whether OSM might induce HGF secretion by both a de novo protein synthesis and the release of an intracellular stock. In the presence of the transcription inhibitor actinomycin D (5 µg/ml), HGF mRNA expression either in basal conditions or after OSM stimulation (10 ng/ml) was significantly reduced compared with that in untreated fibroblasts (Fig. 3A). Cells were then pretreated for 1 h with CHX, an inhibitor of protein synthesis, and stimulated or not with OSM (10 ng/ml) for 24 h. In the presence of CHX, HGF protein concentration in the medium was reduced in unstimulated cells (71%), and the stimulated effect of OSM was completely inhibited (Fig. 3B). Together, our results show that the effect of OSM on HGF release may take place at the transcriptional level and that OSM-induced HGF secretion by fibroblasts is only due to a de novo protein synthesis.


Figure 3
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  		Fig. 3. OSM induces HGF synthesis. A: effect of actinomycin D (Act D) on induction of HGF. MRC5 cells were cultured in serum-free medium. Act D (5 µg/ml) was added 30 min before addition of OSM (10 ng/ml), and HGF mRNA expression was measured using real-time quantitative RT-PCR (n = 3) after 1 h of culture. B: effect of cycloheximide (CHX) on HGF secretion. MRC5 cells were cultured in serum-free medium. CHX (1 µg/ml) was added 1 h before addition of OSM (10 ng/ml), and HGF protein was measured using ELISA (n = 5) after 24 h of culture. *P < 0.001 vs. basal control. {dagger}P < 0.001 vs. OSM.

 
Stimulation of MRC5 cells with OSM at 10 ng/ml resulted in a time-dependent increase in HGF protein secretion. An increase in HGF release was observed as early as 12 h and was significant at 24 h posttreatment. The increase of HGF protein content lasted up to 72 h after stimulation (Fig. 4A).


Figure 4
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  		Fig. 4. Time- and dose-dependent effects of HGF induction. MRC5 cells were cultured in serum-free medium in the presence or absence of OSM (10 ng/ml) for the indicated times (A) or in the presence of increasing concentrations of OSM (1 to 50 ng/ml) for 24 h (B). HGF protein concentrations were measured using ELISA in the cell supernatants. {dagger}P < 0.05 vs. basal control.

 
OSM induced HGF secretion in a dose-dependent manner when MRC5 cells were cultured with increasing concentrations of OSM from 1 to 50 ng/ml. The cell response was observed until 50 ng/ml but was already detectable at a concentration of 5 ng/ml (Fig. 4B).

OSM does not act through a PGE2-dependent mechanism. PGE2, known as a powerful inductor of HGF, is produced by lung fibroblasts (7, 27). OSM enhances the expression of PGE2 and COX-2 in different cell types (2, 37). Therefore, we examined whether OSM might induce HGF through an endogenous PGE2 synthesis.

In our experiments, we confirmed that PGE2 (10–6 M) enhances HGF secretion. When PGE2 (10–6 M) and OSM (10 ng/ml) were simultaneously added, an additive effect on the HGF secretion was found (P < 0.05). When unstimulated MRC5 cells were cultured in the presence of indomethacin (2.5 µg/ml), a cyclooxygenase inhibitor, only a slight decrease of HGF release was observed. When OSM (10 ng/ml) and indomethacin (2.5 µg/ml) were simultaneously added, an increase in HGF secretion was found (P = 0.04) (Fig. 5).


Figure 5
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  		Fig. 5. Combined effect of OSM with IL-1beta (IL1), PGE2, or indomethacin (Indo) on HGF secretion. MRC5 cells were cultured in the presence of IL-1 (10 ng/ml), PGE2 (10–6 M), or Indo (2.5 µg/ml) either alone or in combination with OSM (10 ng/ml) for 24 h. HGF content was measured using ELISA in the cell supernatants and expressed as a percentage of basal secretion. *P < 0.05 vs. basal control. {dagger}P < 0.05 vs. OSM.

 
To explain this unexpected effect, we hypothesized that PGE2 or COX-2-derived prostaglandins may favor a downregulation of OSM receptor. We therefore analyzed the expression of OSMRbeta mRNA when fibroblasts were cultured in the presence of PGE2 (10–6 M), indomethacin (2.5 µg/ml), and NS-398 (25 µM), a specific COX-2 inhibitor, either alone or in combination with OSM. In four separate experiments, PGE2 alone had no significant effect on the basal expression of OSMRbeta mRNA after 18 h of stimulation, whereas indomethacin and NS-398 increased it (n = 4, P = 0.09 and P = 0.06, respectively). We found, as previously described (3), that OSMRbeta mRNA expression was increased in OSM-stimulated fibroblasts. OSM-stimulated cells in combination with PGE2 either slightly decreased or had no effect on OSMRbeta mRNA expression, whereas the combination of OSM with either indomethacin or NS-398 increased it (n = 4, P = 0.06) (Fig. 6).


Figure 6
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  		Fig. 6. Modulation of OSM receptor beta (OSMRbeta). MRC5 cells (n = 4) were cultured for 18 h in the presence of PGE2 (10–6 M), Indo (2.5 µg/ml), and NS-398 (25 µM), a specific cyclooxgenase-2 inhibitor, either alone or in combination with OSM (10 ng/ml). OSMRbeta mRNA expression was measured using quantitative real-time RT-PCR and expressed as the OSMRbeta /ubiquitin C mRNA ratio.

 
MAPK inhibitors block OSM-induced HGF secretion. Our later results excluded an effect of OSM on HGF secretion through a PGE2 pathway. Moreover, OSM (10 ng/ml) and IL-1beta (10 ng/ml) in combination did not lead to any additive effect in HGF secretion (P = 0.4) (Fig. 5), suggesting that OSM and IL-1 may act through the same signaling pathway. The MAPK pathway has been involved in several functions of both OSM and IL-1 in fibroblasts. Therefore, we tested specific inhibitors of the MAPK pathway for their ability to abrogate OSM-induced HGF secretion. PD-98059 (30 µM) an inhibitor of MEK1 and MEK2 activation, SB-203580 (10 µM), an inhibitor of p38 MAPK, and SP-600125 (20 µM), an inhibitor of the c-Jun pathway, were used at concentrations consistent with their IC50 values (1, 55). Cell viabilities were appreciated using lactate dehydrogenase activities and confirmed that the addition of these pharmacological inhibitors did not present any cytotoxic effect (data not shown). SP-600125 inhibited by 30% (P = 0.01) the HGF secretion in unstimulated fibroblast, whereas PD-98059 and SB-203580 had no effect (P > 0.5). In OSM-stimulated fibroblast, HGF secretion was completely inhibited by SP-600125 and reached the basal condition. The OSM-stimulating effect on HGF secretion was inhibited by 82 and 70% in the presence of SB-203580 and PD-98059, respectively (P = 0.001) (Fig. 7). Together, these results all suggest that HGF secretion induced by OSM involves the MAPK pathway.


Figure 7
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  		Fig. 7. Effect of kinase inhibitors on OSM-induced HGF release. MRC5 cells were pretreated for 1 h with SB-203580 (SB; 20 µM), SP-600125 (SP; 20 µM), or PD-98059 (PD; 30 µM) and then stimulated for 24 h with OSM (10 ng/ml). HGF content was measured using ELISA in the cell supernatants and expressed as a percentage of basal secretion. *P < 0.01 vs. basal control. {dagger}P < 0.001 vs. OSM.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies have raised a possible role of OSM in repair mechanisms. The present study demonstrated that OSM may participate in pulmonary repair through the induction of HGF synthesis in human lung fibroblasts. Indeed, we showed that OSM induced both the secretion of the active mature form of HGF and the transcriptionnal activation of HGF mRNA, whereas IL-6 and LIF did not. The de novo HGF protein synthesis induced by OSM did not involve the PGE2 pathway and required a MAPK signal transduction.

In this work, we showed that OSM induced HGF mRNA and protein upregulation in both human lung fibroblasts and MRC5 cells. This effect was not shared with LIF or IL-6. An IL-6 response element has been found near the transcription initiation site of human HGF gene (29). However, the effect of IL-6 on HGF synthesis remains controversial. Whereas it was previously shown that IL-6 was able to upregulate HGF synthesis notably in human liver myofibroblasts or in murine NIH/3T3 fibroblasts, our results, in agreement with those of Tamura et al. (48), showed a lack of IL-6 induction on HGF secretion in human lung fibroblasts (15, 22).

Tomida and Saito (49) recently demonstrated that HGF gene is transcriptionally activated by LIF in human melanoma and neuroepithelioma cells. In our study, using fibroblast cell types, we did not observe any LIF effect on HGF mRNA. OSM has been shown to induce cellular responses either by engaging and activating the LIF receptor (LIFR{alpha}) or via its specific type II receptor (OSMRbeta). Both LIFR{alpha} and OSMRbeta are expressed in lung fibroblasts (31). Scaffidi et al. (41) recently showed the presence of a great number of OSM type II receptors on the cell surface of human lung fibroblasts. Our results therefore suggest that OSM may act through its specific receptor to induce HGF.

In basal conditions, human lung fibroblasts and MRC5 cells are known to produce a significant amount of HGF. The possible release of an HGF intracellular pool induced by OSM has to be excluded. OSM induces HGF mRNA expression at the transcriptional level, a finding supported by the lack of HGF mRNA increase in the presence of actinomycin D. Moreover, OSM-induced HGF secretion is completely inhibited by CHX, suggesting that this secretion is only due to a de novo protein synthesis. The ability of OSM to induce the synthesis of growth factors has been poorly investigated. To our knowledge, only the OSM-induced synthesis of the bFGF in endothelial cells has been previously shown (54). It is thus the first study demonstrating the role of OSM in HGF synthesis. In agreement with previous studies (32), our Western blot experiments show that HGF is present in its mature form in cell culture supernatant of unstimulated fibroblasts. After OSM stimulation, HGF remains in this form. Parr and Jiang (36) demonstrated that HGFA, a membrane-bound serine protease that converts proHGF in mature HGF, is present in lung fibroblasts. Whether OSM modulates HGFA remains to be determined.

PGE2, which is produced by lung fibroblasts, is considered the most potent inducer of HGF mRNA activation and protein secretion by fibroblasts (7, 27). OSM is known to upregulate cyclooxygenase expression in different cell types and acts in synergy with PGE2 or IL-1 to enhance IL-6 production through endogenous PGE2 production (2, 37, 38, 40). This led us to consider the possibility that OSM induces HGF secretion through endogenous PGE2 synthesis. Our results excluded this hypothesis, because indomethacin, a cyclooxygenase inhibitor, not only did not decrease the OSM effect but led to an unexpected upregulation of HGF secretion. At least two hypotheses may explain this last result: either OSM may induce lipid mediators other than PGE2 that are able to inhibit HGF synthesis, or lipid mediators may downregulate OSM receptor. We confirmed that OSM was able to upregulate its own receptor (OSMRbeta) as previously described (3). Indomethacin or NS-398 alone and in combination with OSM increased OSMRbeta mRNA expression, whereas no effect or a slight decrease of OSMRbeta mRNA expression was observed when fibroblasts were treated with PGE2 alone or in combination with OSM (Fig. 6). Although the effect of cyclooxygenase inhibition was only moderate, these results might suggest that lipid mediators (likely other than PGE2) downregulate OSMRbeta. However, the modulation of OSMR could partly explain our results, and other mechanisms cannot be excluded.

In our experiments, when OSM and IL-1beta were added simultaneously, no additive effect on HGF secretion was found. This could suggest that OSM and IL-1beta may act through the same signal transduction. The three MAPKs (ERK1/2, p38, and JNK/SAPK) have been involved in IL-1beta signaling pathway (47). We showed that the OSM-induced HGF secretion involved a MAPK pathway by using selective pharmacological inhibitors. In agreement with our results, the MAPK pathway has been involved in many biological effects of OSM in lung fibroblast: OSM stimulates tissue inhibitor of metalloprotease-1 (TIMP-1) via the MEK-MAPK pathway in human myofibroblasts (44); OSM induced strong phosphorylation of ERK1/2 and p38 compared with LIF to stimulate TIMP-1 mRNA expression in primary mouse fibroblasts (50); and OSM stimulates the proliferation of human primary lung fibroblasts via the ERK1/2 MAPK pathway (41). Besides the ERK1/2 and p38 MAPK pathway, we found that JNK activation is likely to be of importance in HGF upregulation by OSM in lung fibroblast. The mechanisms involved in MAPK activation remain to be determined.

Biological effects of OSM include proinflammatory properties such as induction of adhesion molecules (P-selectin, E-selectin, VCAM-1, ICAM-1) and involvement in leukocyte migration (30). More recently, studies have implicated OSM as an anti-inflammatory cytokine given that OSM inhibits tumor necrosis factor-{alpha}, granulocyte-macrophage colony stimulating factor, and IL-8 production (39, 51). Among several biological effects, OSM is a potent mitogen for dermal fibroblasts and human lung fibroblast (18, 41). It has been shown to stimulate extracellular matrix production in dermal fibroblasts, hepatic stellate cells, and, more recently, in human lung fibroblasts (6, 17, 41). In lung fibroblasts, OSM also is a potent inducer of TIMP-1 and regulates matrix metalloprotease synthesis (40). Altogether, these properties of OSM, not shared by other cytokines from the IL-6 family, have raised a possible role of OSM in repair mechanisms, notably in the lung. OSM is produced by activated monocytes/macrophages, T lymphocytes, and polymorphonuclear neutrophils, cell types frequently found at sites of inflammatory wound repair. Furthermore, OSM can be detected in local tissues and body fluids affected by various inflammatory conditions including acute lung injury (13). Therefore, OSM may act locally to induce HGF synthesis by lung fibroblasts. HGF has a potent mitogenic effect, observed both in vitro (26, 42, 43) and in vivo in different animal models with lung injury (35). After lung injury, HGF promotes the proliferation of lung epithelial cells, thereby playing an important role in restoring the integrity of the alveolar and bronchial epithelium (57). In conclusion, our results indicate the potential role of OSM in the regulation of HGF expression in human lung fibroblast, therefore supporting alveolar epithelial repair after lung injury.


   ACKNOWLEDGMENTS
 
We thank Aurelie Fabre (INSERM U700) for valuable criticism of the manuscript and Francine Hochedez (Hopital Bichat, Paris) for helpful technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Dehoux, Service de Biochimie A, Hôpital Bichat-Claude Bernard, Assistance Publique-Hôpitaux de Paris, 46 rue Henri Huchard, 75877, Paris cedex 18, France (e-mail: monique.dehoux{at}bch.ap-hop-paris.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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