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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To study the mechanisms by which hepatocyte growth factor (HGF) down-regulates collagen and connective tissue growth factor (CTGF) in scleroderma (systemic sclerosis [SSc]) lung fibroblasts.

Methods

CTGF, type I collagen, and IκBα expression, together with MAPK phosphorylation, were studied by immunoblotting of lung fibroblasts derived from white SSc patients. Matrix metalloproteinase 1 (MMP-1) expression in cell culture medium samples was measured by enzyme-linked immunosorbent assay, MMP-1 activity was studied using an MMP-1 assay, and NF-κB DNA binding activity was determined using a transcription factor assay.

Results

In lung fibroblasts from white SSc patients, HGF activated MAPK (ERK-1/2) signaling pathways and MMP-1, while it inhibited NF-κB and significantly down-regulated CTGF and collagen in a time- and dose-dependent manner. Small interfering RNA (siRNA)–mediated depletion of Grb2 expression disrupted c-Met receptor downstream signaling, which resulted in diminished HGF-induced ERK-1/2 phosphorylation and the recovery of HGF-inhibited expression of MMP-1, NF-κB, collagen, and CTGF. The MAPK inhibitor, U0126, blocked MMP-1 activity and restored HGF-inhibited collagen and CTGF accumulation. Inhibition of MMP activity by MMP inhibitor GM1489 and inhibition of MMP-1 expression by siRNA did not prevent HGF-induced ERK-1/2 phosphorylation and NF-κB activity, but significantly restored HGF-inhibited collagen and CTGF accumulation. NF-κB inhibitor BAY 11-7082 did not interfere with MAPK phosphorylation or MMP-1 expression and activation, but significantly inhibited NF-κB DNA binding activity and acted synergistically with HGF to completely diminish the expression of CTGF.

Conclusion

In lung fibroblasts from white SSc patients, HGF down-regulates the accumulation of CTGF via MAPK/MMP-1 and NF-κB signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1–dependent pathway.

Pulmonary fibrosis is a frequent complication and a major cause of death in systemic sclerosis (SSc; scleroderma); however, the pathogenesis is unclear and no safe and effective therapy exists (1–3). The pathology of pulmonary fibrosis in SSc includes features of dysregulated and abnormal repair, fibroproliferation, deposition of collagen, connective tissue growth factor (CTGF), and other extracellular matrix (ECM) proteins (1–4). Extensive deposition of ECM impairs the ability of lung epithelial cells to regenerate, which results in accelerated lung tissue damage and respiratory failure (4).

Hepatocyte growth factor (HGF) was initially identified and cloned as a mitogen for mature hepatocytes (5). Later studies revealed that HGF is widely expressed in many different organs, including the lungs, and has multiple biologic activities (6, 7). In the lungs, HGF acts as a potent regenerative and cytoprotective factor during organogenesis or following acute injury (8–11). Numerous studies have implicated HGF as an endogenous antifibrotic factor, ameliorating fibrotic lesions and preserving organ function in a wide variety of experimental animal models (12–15). However, the mechanisms by which HGF exerts its antifibrotic effects are not yet fully understood.

In vitro studies have shown that HGF specifically counteracts many profibrotic actions of transforming growth factor β (TGFβ), suggesting that a balance between HGF and TGFβ may play a decisive role in the pathogenesis of fibrosis (16, 17). Using electroporation-mediated gene transfer in the bleomycin-injured lung, Gazdhar et al showed that HGF decreased TGFβ1 levels and increased alveolar epithelial cell proliferation and survival (18). Mizuno and colleagues identified HGF as a key ligand to elicit myofibroblast apoptosis and ECM degradation in bleomycin-treated murine lungs (13). Wu et al reported that HGF prevented and ameliorated the symptoms of dermal sclerosis in a mouse model of SSc (19). Several recent studies have characterized antifibrotic effects of HGF on collagen, matrix metalloproteinase 1 (MMP-1), and CTGF in SSc skin fibroblasts (20, 21), but the mechanisms underlying its antifibrotic effects in human lung fibroblasts remain to be clarified.

Increased levels of serum HGF have been observed in scleroderma patients (22). Recently, we showed that HGF protein expression was significantly up-regulated in bronchoalveolar lavage (BAL) fluid and plasma from white but not from African American SSc patients as compared with healthy controls (23). We also showed that the antifibrotic activity of HGF was diminished in lung fibroblasts isolated from African Americans due to limitations in c-Met receptor phosphorylation, whereas in lung fibroblasts from white SSc patients HGF readily down-regulated type I collagen and CTGF accumulation (23). The present study was undertaken to investigate the signaling pathways underlying HGF's effects on type I collagen and CTGF in lung fibroblasts isolated from white SSc patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Materials.

Recombinant human HGF and the Quantikine human proMMP-1 immunoassay kit were purchased from R&D Systems (Minneapolis, MN); an EnzoLytePlus 520 MMP-1 assay kit was obtained from AnaSpec (San Jose, CA); a NoShift transcription factor assay kit, NoShift NF-κB (p65) reagents, and a NucBuster protein extraction kit were purchased from Novagen (Madison, WI); MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and NF-κB inhibitor BAY 11-7082 were purchased from EMD Biosciences (San Diego, CA); Grb2 siRNA, MMP-1 siRNA, control siRNA-A, anti-CTGF, and fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti–type I collagen was obtained from Southern Biotechnology (Birmingham, AL); and anti–p44/p42 MAPK (anti–ERK-1/2), anti–phospho–p44/p42 MAPK, and anti-IκBα were purchased from Cell Signaling Technology (Danvers, MA).

Cell culture.

Lung fibroblasts were derived from lung tissue obtained at autopsy from 5 white patients with end-stage SSc lung disease. Lung tissue was diced (0.5 × 0.5–mm pieces) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, 2 mML-glutamine, gentamicin sulfate (50 μg/ml), and amphotericin B (5 μg/ml) at 37°C in 10% CO2. The medium was changed every 3 days to remove dead and nonattached cells until fibroblasts reached confluence. Monolayer cultures were maintained in the same medium. Lung fibroblasts were used between the second and fourth passages in all experiments. The purity of isolated lung fibroblasts was determined by crystal violet staining and by immunofluorescence staining using monoclonal antibody 3C4 against human fibroblasts (obtained from Dr. J. H. Korn, University of Connecticut School of Medicine, Farmington, CT), followed by FITC-conjugated goat anti-mouse IgG staining, as previously described (24).

Depletion of Grb2 and MMP-1 expression mediated by siRNA.

Lung fibroblasts were grown overnight to 60–70% confluence on 6-well culture plates and transfected with Grb2 or MMP-1 siRNA (40 pmoles per well) using siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions. On the day following transfection, medium was changed to a serum-free medium, and cells were incubated with or without HGF for up to 72 hours. Control siRNA with a scrambled sequence that did not lead to the specific degradation of any known cellular messenger RNA were used as a control. Transfection efficiency was routinely tested by immunoblotting for expression of Grb2 and by enzyme-linked immunosorbent assay (ELISA) for expression of MMP-1.

ELISA.

Levels of proMMP-1 in samples consisting of 50 μl of cell culture medium were measured using the Quantikine human proMMP-1 immunoassay according to the manufacturer's instructions. Briefly, samples and standards were incubated for 2 hours in 96-well plates precoated with proMMP-1 monoclonal antibody. After washing away any unbound materials, an enzyme-linked polyclonal antibody was added to the wells for another 2 hours. The unbound antibody–enzyme reagent was then removed, and substrate solution was added to the wells for 30 minutes to develop the color intensity in proportion to the amount of proMMP-1 bound in the initial step. The optical density of each well was determined within 30 minutes, using a microplate reader set to 450 nm. Concentrations were calculated using a standard curve generated with specific standards provided by the manufacturer.

MMP-1 assay.

MMP-1 activity was measured in samples consisting of 100 μl of cell culture medium using the EnzoLytePlus 520 assay kit according to the manufacturer's instructions. An anti–human MMP-1 monoclonal antibody was used to pull down both proMMP-1 and active MMP-1 from the cell culture supernatant, and the proteolytic activity of MMP-1 was measured using fluorophore 5-FAM quenched by QXL 520 (AnaSpec) in the intact fluorescence resonance energy transfer (FRET) peptide. The fluorescence intensity of 5-FAM was monitored, with excitation at 490 nm and emission at 520 nm. The result in blank controls containing FRET substrate only was subtracted from the total from all wells.

NF-κB DNA binding activity assay.

Nuclear protein extracts were prepared using the NucBuster protein extraction kit in accordance with the manufacturer's instructions; protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL). Nuclear extracts (25 μg each) from lung fibroblasts or from HeLa epithelial cells provided by the manufacturer (Novagen) as a control were incubated with various combinations of biotinylated NF-κB wild-type double-stranded DNA (dsDNA), specific NF-κB competitor dsDNA without biotin end labels, and nonspecific, nonbiotinylated dsDNA with a mutant NF-κB consensus-binding motif. Negative controls consisted of reactions performed in the absence of a binding sequence. Anti–NF-κB (p65) mouse monoclonal antibody was used to pull down DNA-bound NF-κB within 1 hour at 37°C. After washing away any unbound materials, a horseradish peroxidase–conjugated secondary antibody was added to the wells for another 30 minutes. The unbound antibody–enzyme reagent was removed, and tetramethylbenzidine substrate solution was added to the wells to develop the color intensity in proportion to the amount of dsDNA bound in the initial step. The reaction was quenched with 1N HCl, and the optical density of each well was determined using a microplate reader set to 450 nm.

Immunoblotting.

Confluent cultures of lung fibroblasts were serum-starved for 24 hours, then incubated with HGF (50 ng/ml or indicated concentrations) for 72 hours or for the indicated lengths of time, with or without the various inhibitors listed above. For CTGF assays, cells were harvested with phosphate buffered saline (PBS) and solubilized in heparin–Sepharose binding buffer (100 mM Tris [pH 7.4], 10 mM trisodium citrate, 100 mM NaCl, and 1% Triton X-100 [Amersham Biosciences, Piscataway, NJ]), and protease inhibitor cocktail (Sigma, St. Louis, MO). Cell lysates were cleared by centrifugation, and the protein concentration was determined using the BCA protein assay kit. CTGF was selected from 500 μg of cell lysate per sample using heparin–Sepharose and subjected to 4–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using anti-CTGF in accordance with the manufacturer's instructions. To measure type I collagen and IκBα expression, cells were collected with lysis buffer containing 50 mM Tris (pH 7.4), 10 mM EDTA, 150 mM NaCl, 1% Nonidet P40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitor cocktail. Forty micrograms of protein per sample was resolved by 4–20% gradient SDS-PAGE and analyzed by Western blotting using anti–type I collagen or anti-IκBα antibodies. The immunoblots were then stripped and reblotted with anti–β-actin (Sigma) as a loading control.

The phosphorylation of p44/p42 MAPK isoforms was measured by Western blot analysis using anti–phospho-p44/42 in accordance with the manufacturer's instructions. Briefly, SSc lung fibroblasts were cultured on 6-well plates (2 × 106 cells/well) to 90% confluence, synchronized with serum-free DMEM for 24 hours, and then pretreated for 40 minutes with or without U0126 (1 μM), GM1489 (10 nM), or BAY 11-7082 (10 μM). For one series of experiments, cells were transfected with Grb2 siRNA and MMP-1 siRNA, as described above. Next, cells were incubated with or without HGF (5–500 ng/ml) for various periods of time, rapidly washed with ice-cold PBS, and collected in 1× SDS sample buffer (100 μl/well). Twenty microliters of sample was separated on 4–20% SDS–polyacrylamide gel and immunoblotted with anti–phospho-p44/42. The total amount of ERK-1/2 was evaluated by reblotting with anti–p44/42 MAPK polyclonal antibody.

Statistical analysis.

Results are expressed as the mean ± SD. Statistical analyses were performed with KaleidaGraph 4.0 (Synergy Software, Reading, PA) using analysis of variance with post hoc testing. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

HGF-induced reduction of CTGF and collagen accumulation in SSc lung fibroblasts.

Conditioned media from SSc lung fibroblasts that had been cultured for 72 hours contained significant amounts of collagen and CTGF. Treatment of cultured SSc lung fibroblasts with HGF resulted in down-regulation of collagen and CTGF expression. To study the mechanisms by which HGF reduced both extracellular matrix proteins, we used siRNA-based technology and knocked down Grb2 and MMP-1 expression. The MMP-1 protein level was reduced by 60–80% in cells transfected with MMP-1 siRNA (data not shown). Additionally, we used several commercially available inhibitors, such as MAPK inhibitor U0126, MMP inhibitor GM1489, and NF-κB inhibitor BAY 11-7082. Using these various reagents, we observed that depletion of Grb2 expression by Grb2 siRNA prevented HGF-induced down-regulation of CTGF and collagen. U0126 and GM1489, as well as siRNA-mediated depletion of MMP-1, significantly reduced HGF's inhibitory effect on collagen and CTGF, whereas BAY 11-7082 had no effect on collagen expression. The basal level of collagen was not affected by any of the above-listed inhibitors, whereas CTGF was significantly down-regulated by BAY 11-7082, similar to the inhibition of CTGF by HGF. Treating lung fibroblasts with HGF and BAY 11-7082 demonstrated a synergistic effect and completely diminished expression of CTGF (Figures 1A and B).

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Figure 1. Effect of hepatocyte growth factor (HGF) and selected inhibitors on expression of connective tissue growth factor (CTGF) and type I collagen in lung fibroblasts from systemic sclerosis patients. A, Confluent cultures of lung fibroblasts were serum-starved for 24 hours, then incubated for 72 hours with or without HGF (50 ng/ml), MEK-1/2 inhibitor U0126 (1 μM), matrix metalloproteinase (MMP) inhibitor GM1489 (GM; 10 nM), or NF-κB inhibitor BAY 11-7082 (BAY; 10 μM). In some experiments, cells were transfected with Grb2 small interfering RNA (siRNA), MMP-1 siRNA, or control siRNA, as described in Materials and Methods. The cells were collected and then analyzed by Western blotting using anti-CTGF and anti–type I collagen antibodies. Anti–β-actin was used as a sample loading control; anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Immunoblots are representative of 3 independent experiments. B, The results of immunoblot analysis were quantified. This analysis represents bands corresponding to the α1 and α2 chains of type I collagen; numbers below the columns correspond to the lanes in A. Values are the mean and SD from 3 independent experiments. ∗ = P < 0.05 versus unstimulated cells.

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HGF-induced ERK-1/2 phosphorylation.

Previously we showed that the basal level of phospho–ERK-1/2 is elevated in the lung fibroblasts of SSc patients (25). HGF further induced ERK-1/2 phosphorylation, in a time- and dose-dependent manner (Figures 2A and B). We observed that within 2 minutes of HGF treatment (50 ng/ml), ERK-1/2 phosphorylation was significantly increased, with maximum phosphorylation reached within 10 minutes of HGF stimulation, and the phosphorylation persisted for 72 hours (Figure 2A). Treating cells with varying concentrations of HGF (5–500 ng/ml) for 10 minutes resulted in dose-dependent ERK-1/2 phosphorylation (Figure 2B). The basal level of nonphosphorylated ERK-1/2 in SSc lung fibroblasts was not affected by HGF stimulation.

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Figure 2. Effect of HGF on p44/p42 MAPK (ERK-1/2) phosphorylation in lung fibroblasts from systemic sclerosis (SSc) patients. A, HGF-induced ERK-1/2 phosphorylation was determined in cells treated for various time periods with HGF (50 ng/ml). Cell extracts were immunoblotted with anti–phospho–ERK-1/2 (ph-ERK-1/2) or anti–ERK-1/2 (as a control) (see Materials and Methods). B, Dose-dependence of ERK-1/2 phosphorylation was determined by adding HGF at varying doses (5–500 ng/ml) to SSc lung fibroblasts for 10 minutes. C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA or pretreated for 40 minutes with or without MEK-1/2 inhibitor U0126 (1 μM), MMP inhibitor GM1489 (10 nM), or NF-κB inhibitor BAY 11-7082 (10 μM), then incubated with HGF (50 ng/ml) for 10 minutes. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. ∗ = P < 0.05 versus unstimulated cells. See Figure 1 for other definitions.

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To examine the pathways involved in HGF-induced ERK-1/2 phosphorylation, we treated SSc lung fibroblasts with various inhibitors. Depletion of Grb2 with siRNA did not affect the basal level of total or phospho–ERK-1/2, while it prevented HGF-induced ERK-1/2 phosphorylation. GM1489, MMP-1 siRNA, and BAY 11-7082 did not affect either total or phosphorylated basal or HGF-induced ERK-1/2. U0126, at a concentration of 1 μM, decreased basal and HGF-induced levels of phospho–ERK-1/2 but did not affect the total level of ERK-1/2 (Figure 2C). Higher doses of U0126 (5 and 10 μM) reduced not only the phosphorylated but the basal level of ERK-1/2 in SSc lung fibroblasts; therefore, we subsequently used only a 1 μM concentration of U0126 in this study.

HGF-induced MMP-1 expression and activity in SSc lung fibroblasts.

Basal expression and activity of MMP-1 in SSc lung fibroblasts is reduced compared with that of normal fibroblasts (26). It has been reported that HGF induces expression and activity of MMP-1 in SSc skin fibroblasts (20, 21). To examine whether HGF affects MMP-1 expression in SSc lung fibroblasts, we incubated cells with various concentrations of HGF (5–500 ng/ml) for 72 hours. We observed that HGF, at doses of 50 and 500 ng/ml, was a potent inducer of proMMP-1 in SSc lung fibroblasts (Figure 3A). At a dose of 50 ng/ml, HGF significantly induced proMMP-1 expression in SSc lung fibroblasts over a period of 24–72 hours (Figure 3B). To examine the pathway responsible for HGF-induced expression of MMP-1 in SSc lung fibroblasts, we used several inhibitors described above. Both Grb2 siRNA and U0126 prevented HGF-induced expression of proMMP-1, while GM1489 and BAY 11-7082 had no effect on proMMP-1 expression (Figure 3C). Transfection of SSc lung fibroblasts with MMP-1 siRNA resulted in a reduction in proMMP-1 levels of 60–80% in these cells (data not shown).

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Figure 3. Effect of HGF on proMMP-1 expression in lung fibroblasts from systemic sclerosis patients. A, HGF increased proMMP-1 levels in lung fibroblast culture medium, in a dose-dependent manner, within 72 hours. B, HGF, administered for 72 hours at a dose of 50 ng/ml, induced significant increases in proMMP-1 expression after 24–72 hours of treatment. C, Grb2 siRNA and MEK-1/2 inhibitor U0126 prevented HGF-induced expression of proMMP-1, while MMP inhibitor GM1489 and NF-κB inhibitor BAY 11-7082 had no effect on HGF-induced proMMP-1 expression. MMP-1 siRNA reduced both basal and HGF-induced proMMP-1 protein expression. Findings with control siRNA treatment were identical to findings with serum-free medium (SFM). Each bar represents the mean and SD of duplicate determinations in 4 independent experiments. Note that the proteolytic activity of GM1489 did not affect MMP-1 expression. ∗ = P < 0.05 versus unstimulated cells. See Figure 1 for other definitions.

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To measure MMP-1 activity, we performed a fluorescence analysis of conditioned media from SSc lung fibroblasts, incubated with or without HGF and various inhibitors. We found that HGF significantly increased MMP-1 activity in a time- and dose-dependent manner (Figures 4A and B). Grb2 siRNA and U0126 prevented HGF-induced activity of MMP-1, while BAY 11-7082 had no effect on MMP-1 activity. GM1489 and MMP-1 siRNA significantly reduced both basal and HGF-induced MMP-1 activity in SSc lung fibroblasts (Figure 4C).

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Figure 4. Effect of HGF on proteolytic activity of MMP-1 in lung fibroblasts from systemic sclerosis (SSc) patients. Proteolytic activity of MMP-1 was studied by fluorescence analysis, as described in Materials and Methods. Values are presented in relative fluorescence units (RFUs), recorded with a fluorescence microplate reader with excitation set at 485 nm and emission set at 520 nm. A, HGF increased the proteolytic activity of MMP-1 in lung fibroblast culture medium in a dose-dependent manner within 72 hours. B, HGF, at a dose of 50 ng/ml, induced proteolytic activity of MMP-1 in SSc lung fibroblasts in a time-dependent manner. C, Grb2 siRNA and MEK-1/2 inhibitor U0126 prevented HGF-induced proteolytic activity of MMP-1, and MMP inhibitor GM1489 and MMP-1 siRNA significantly reduced the proteolytic activity of MMP-1. NF-κB inhibitor BAY 11-7082 had no effect on the proteolytic activity of MMP-1. Each bar represents the mean and SD of duplicate determinations in 4 independent experiments. ∗ = P < 0.05 versus serum-free medium (SFM) from unstimulated cells. See Figure 1 for other definitions.

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HGF-inhibited NF-κB DNA binding activity in SSc lung fibroblasts.

Since HGF, together with BAY 11-7082, synergistically inhibited CTGF expression in SSc lung fibroblasts, we examined whether HGF affects NF-κB activation. NF-κB DNA binding activity was measured by binding NF-κB to oligonucleotides containing the consensus-binding site. Nuclear extract from HeLa cells stimulated with tumor necrosis factor α (TNFα) was used as a positive control. Nuclear extracts from SSc lung fibroblasts, incubated with or without HGF and various inhibitors, were prepared as described in Materials and Methods. To assess sequence-specific binding activity, nuclear extracts were incubated with NF-κB wild-type DNA, with or without either a specific NF-κB competitor DNA or a nonspecific mutant NF-κB consensus-binding motif. Nuclear extracts incubated with NF-κB wild-type DNA expressed actual NF-κB binding activity. The specific NF-κB competitor DNA reduced the binding activity in all cell lines, which confirmed the sequence-specificity of the assay for NF-κB binding, but the binding activity of a nonspecific mutant did not significantly differ among the various conditions used in this experiment (Figure 5).

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Figure 5. Effect of HGF on DNA binding activity of NF-κB in lung fibroblasts from systemic sclerosis (SSc) patients. A competitive analysis of NF-κB DNA binding activity in SSc lung fibroblasts and HeLa cell nuclear extracts was performed, as described in Materials and Methods. A, HGF added to the cells for 72 hours inhibited NF-κB DNA binding activity in a dose-dependent manner. B, Nuclear extract from SSc lung fibroblasts treated with HGF significantly reduced NF-κB DNA binding activity in a time-dependent manner. C, Grb2 siRNA prevented HGF-induced inhibition of NF-κB, whereas MEK-1/2 inhibitor U0126, MMP inhibitor GM1489, and MMP-1 siRNA had no effect on NF-κB DNA binding activity. Lightly shaded bars = binding activity assessed with biotinylated NF-κB wild-type (WT) double-stranded DNA (dsDNA); darkly shaded bars = binding activity assessed with WT dsDNA plus nonspecific, nonbiotinylated dsDNA with a mutant NF-κB consensus-binding motif; solid bars = binding activity assessed with WT dsDNA plus specific NF-κB competitor dsDNA without biotin end labels. Each bar represents the mean and SD of duplicate determinations in 3 independent experiments. ∗ = P < 0.05 versus nuclear extracts from unstimulated cells. Neg = negative control; SFM = serum-free medium (see Figure 1 for other definitions).

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Interestingly, we observed that the basal level of active NF-κB in SSc lung fibroblasts was higher than in TNFα-induced HeLa cells used as a positive control (Figure 5). Additionally, the basal level of active NF-κB was significantly increased when compared with normal lung fibroblasts (data not shown). Nuclear extract prepared from SSc lung fibroblasts and treated with HGF demonstrated a significant reduction of NF-κB DNA binding activity, in a time- and dose-dependent manner (Figures 5A and B). Grb2 siRNA prevented the inhibition of NF-κB binding activity by HGF. U0126, GM1489, and MMP-1 siRNA did not affect NF-κB binding activity, while BAY 11-7082 significantly reduced both basal and HGF-induced NF-κB binding activity in SSc lung fibroblasts (Figure 5C).

HGF-induced accumulation of IκBα in SSc lung fibroblasts.

The IκBα protein represents the major proximal regulator of NF-κB. In an activated state, cellular IκBα undergoes degradation in the proteasome, releasing NF-κB for nuclear translocation. We observed that IκBα was barely detectable in untreated SSc lung fibroblasts (Figure 6). HGF at a dose as low as 5 ng/ml and applied to the cells for 72 hours significantly induced accumulation of IκBα (Figure 6A). A statistically significant increase in expression of IκBα was noted within 40 minutes of HGF treatment (50 ng/ml), followed by a >10-fold increase within 72 hours (Figure 6B). Grb2 siRNA prevented HGF-induced accumulation of IκBα, while U0126, GM1489, MMP-1 siRNA, and BAY 11-7082 had no effect on HGF-induced accumulation of IκBα in SSc lung fibroblasts (Figure 6C).

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Figure 6. HGF-induced accumulation of IκBα in lung fibroblasts from systemic sclerosis (SSc) patients. A, SSc lung fibroblasts were incubated for 72 hours with various doses of HGF (5–500 ng/ml) to determine the dose-dependence of IκBα expression. B, HGF-induced IκBα accumulation was determined in cells treated for various time periods with HGF (50 ng/ml). C, Lung fibroblasts were transfected with or without Grb2 siRNA, MMP-1 siRNA, and control siRNA, or with or without 40-minute pretreatment with MEK-1/2 inhibitor U0126 (1 μM), MMP inhibitor GM1489 (10 nM), or NF-κB inhibitor BAY 11-7082 (10 μM), then incubated with HGF (50 ng/ml) for 72 hours. Anti-Grb2 was used to validate the knockdown effect of Grb2 siRNA. Cell culture supernatant was collected and subjected to enzyme-linked immunosorbent assay to measure the depletion of MMP-1 siRNA. Findings with control siRNA treatment were identical to findings with serum-free medium (lane 1). Anti–β-actin was used as a loading control. Immunoblots are representative of 3 independent experiments. Of note, BAY 11-7082 did not affect HGF-induced IκBα accumulation. Quantitative results of densitometric analysis of immunoblots are also shown; values are the mean and SD from 3 independent experiments. ∗ = P < 0.05 versus unstimulated cells. See Figure 1 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

We recently showed that the antifibrotic activity of HGF is significantly reduced in lung fibroblasts isolated from African Americans (23), a population known to have significantly higher mortality rates and a higher prevalence of the more severe diffuse cutaneous SSc compared with white patients (27, 28). The results were consistent for different cell lines in SSc lung fibroblasts or in normal lung fibroblasts stimulated with TGFβ (23). HGF considerably inhibited type I collagen and CTGF accumulation in lung fibroblasts from white patients. However, HGF had no effect on either collagen or CTGF expression in lung fibroblasts isolated from African Americans; we showed that this was a result of a deficiency of HGF-receptor phosphorylation (23). The present study was undertaken, therefore, to investigate signaling pathways underlying HGF's antifibrotic effects in lung fibroblasts isolated from white SSc patients.

In a previous study (23), we showed that HGF protein expression was significantly up-regulated in BAL fluid and plasma from white but not from African American SSc patients as compared with healthy controls. HGF levels in serum from scleroderma patients ranged from 2.5 to 6.0 ng/ml. In the study of HGF effects in human lung fibroblasts, HGF was administered in a range of doses from 5 to 500 ng/ml. HGF at a dose of 5 ng/ml had clear antifibrotic effects; however, not all of the findings were statistically significant. Therefore, we used 50 ng/ml of HGF to demonstrate the profound antifibrotic effects of HGF in human lung fibroblasts.

Although earlier studies implicated HGF in embryo development and in promoting tissue regeneration after acute injury, evidence is now emerging that HGF is also an intrinsic antifibrotic factor that plays a critical role in preventing tissue fibrosis in various animal models (8–15). Over the past several years, progress has been made in identifying the cellular targets of HGF and in unraveling the molecular mechanisms that underlie its action in tissue fibrosis (13, 14, 16, 17, 21). The biologic effects of HGF are mediated by a membrane-spanning c-Met tyrosine kinase receptor (29). After binding of HGF, the c-Met receptor undergoes autophosphorylation at tyrosine residues in its cytoplasmic domain, recruits a group of downstream molecules or adapter proteins such as Grb2, GAB-1, STAT-3, Shc, SH2-containing inositol phosphatase, and Src tyrosine kinase to its multidocking sites, and initiates a cascade of signal transduction events that eventually leads to specific cellular responses (7). The binding of Grb2 to the guanine nucleotide–releasing factor human son of seven less homolog 1 establishes a connection between receptor tyrosine kinase and Ras signaling (30). The depletion of Grb2 by siRNA disrupts the downstream signaling of c-Met and completely prevents HGF-induced inhibition of CTGF and collagen, while MAPK and MMP inhibitors significantly reduce the antifibrotic effect of HGF.

Previously we showed that the basal levels of phospho–ERK-1/2 are elevated in SSc lung fibroblasts (25). Here we show that HGF further induces ERK-1/2 phosphorylation in a time- and dose-dependent manner and that this persists up to 72 hours. Basal levels of nonphosphorylated ERK-1/2 in SSc lung fibroblasts were not affected by HGF stimulation. Grb2 siRNA prevented HGF-induced ERK-1/2 phosphorylation, indicating that HGF phosphorylates ERK-1/2 via the c-Met/Grb2/Ras pathway. A similar pathway of HGF-induced ERK-1/2 phosphorylation has been reported for several other cell lines (for review, see ref. 31).

It is known that basal expression and activity of MMP-1 are reduced in SSc fibroblasts compared with normal fibroblasts (26, 32). It has been reported that HGF induces expression and activity of MMP-1 in bleomycin-induced pulmonary fibrosis (13) and kidney fibrosis (33). Recently it has been shown that HGF enhanced MMP-1 production in SSc skin fibroblasts (20, 21). We now report that HGF increases MMP-1 expression and activity in SSc lung fibroblasts in a time- and dose-dependent manner. Up-regulation of MMP-1 expression and activity by HGF in SSc lung fibroblasts appears to be regulated by MAPK-dependent and NF-κB–independent pathways.

NF-κB is known to play an essential role in the regulation of a variety of genes involved in immune and inflammatory reactions leading to fibrogenic responses (34, 35). HGF was reported to prevent vascular endothelial growth factor–induced and TNFα-induced NF-κB activation in endothelial cells (36, 37). In an animal model of renal tubular interstitial fibrosis, HGF reduced NF-κB activation by lowering its expression and translocation to the nuclei of tubular epithelial cells (38). It has been postulated that the antiinflammatory action of HGF can be mediated by its inhibition of NF-κB signaling (36–38). We observed that the basal level of active NF-κB in SSc lung fibroblasts was even higher than in TNFα-stimulated HeLa cells. Additionally, the basal level of active NF-κB in SSc lung fibroblasts was significantly increased when compared with that in normal lung fibroblasts (Bogatkevich GS: unpublished observations). HGF considerably reduced NF-κB DNA binding activity, in a time- and dose-dependent manner. Although the depletion of Grb2 by siRNA prevented the inhibitory effect of HGF on NF-κB activity, the MAPK pathway did not appear to be involved in HGF-induced NF-κB activity in SSc lung fibroblasts.

The activation of NF-κB is tightly regulated in living cells (35). In resting cells, NF-κB is retained in the cytoplasm by a family of inhibitory proteins (IκB). The IκBα isoform represents the major proximal regulator of NF-κB. In an activated state, IκBα undergoes degradation in the proteasome, releasing NF-κB for nuclear translocation (35). Here we show that HGF markedly enhances IκBα protein expression in SSc lung fibroblasts in a time- and dose-dependent manner. Similar to NF-κB activity, HGF-induced IκBα accumulation was regulated by a Grb2-dependent pathway and was independent of MAPK signaling.

In summary, we conclude that in lung fibroblasts from white SSc patients, HGF down-regulates the accumulation of CTGF via MAPK/MMP-1 and NF-κB signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1–dependent pathway. More studies clarifying the mechanisms of HGF signaling pathways in lung fibroblasts are required. Also, any systemic effect of HGF must be taken into consideration because of the oncogenic potential of overexpressed or activated HGF receptor (39, 40). Nevertheless, the antifibrotic, antiinflammatory, and proregenerative properties of HGF suggest that it could be a promising therapeutic agent for some patients with fibrosing disorders. Our recent observation of differences in the responsiveness of SSc lung fibroblasts to HGF according to race (23) indicates the importance of careful characterization of future study patients and their response to this antifibrotic agent.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Dr. Bogatkevich had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Bogatkevich, Ludwicka-Bradley, Highland, Nietert, Silver.

Acquisition of data. Bogatkevich, Hant, Singleton.

Analysis and interpretation of data. Bogatkevich, Ludwicka-Bradley, Nietert, Silver.

Manuscript preparation. Bogatkevich, Ludwicka-Bradley, Highland, Hant, Nietert, Silver.

Statistical analysis. Bogatkevich, Nietert.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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