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Am J Physiol Lung Cell Mol Physiol. Oct 2008; 295(4): L603–L611.
Published online Aug 1, 2008. doi:  10.1152/ajplung.00530.2007
PMCID: PMC2575950

Proteomic analysis of CTGF-activated lung fibroblasts: identification of IQGAP1 as a key player in lung fibroblast migration

Abstract

Connective tissue growth factor (CTGF, CCN2) is overexpressed in lung fibroblasts isolated from patients with interstitial lung disease (ILD) and systemic sclerosis (SSc, scleroderma) and is considered to be a molecular marker of fibrosis. To understand the significance of elevated CTGF, we investigated the changes in lung fibroblast proteome in response to CTGF overexpression. Using 2-dimensional gel electrophoresis followed by in-gel proteolytic digestion and mass spectrometric analysis, we identified 13 proteins affected by CTGF. Several of the CTGF-induced proteins, such as pro-α (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, are known to be elevated in pulmonary fibrosis, whereas 9 of 13 proteins have not been studied in pulmonary fibrosis and are, therefore, novel CTGF-responsive molecules that may have important roles in ILD. Our study demonstrates that 1 of the novel CTGF-induced proteins, IQ motif containing GTPase activating protein (IQGAP) 1, is elevated in lung fibroblasts isolated from scleroderma patients with ILD. IQGAP1 is a scaffold protein that plays a pivotal role in regulating migration of endothelial and epithelial cells. Scleroderma lung fibroblasts and normal lung fibroblasts treated with CTGF demonstrated increased rate of migration in a wound healing assay. Depletion of IQGAP1 expression by small interfering RNA inhibited CTGF-induced migration and MAPK ERK1/2 phosphorylation in lung fibroblasts. MAPK inhibitor U0126 decreased CTGF-induced cell migration and did not interfere with CTGF-induced IQGAP1 expression, suggesting that MAPK pathway is downstream of IQGAP1. These findings further implicate the importance of CTGF in lung tissue repair and fibrosis and propose that CTGF-induced migration of lung fibroblasts to the damaged tissue is mediated via IQGAP1 and MAPK signaling pathways.

Keywords: scleroderma, lung fibrosis, connective tissue growth factor, cell migration

interstitial lung disease (ILD) is the most serious complication and a leading cause of mortality in systemic sclerosis (SSc, scleroderma) (20). Fibroblasts isolated from SSc patients, also called activated fibroblasts or myofibroblasts, continue in vitro to generate the excess of ECM proteins and to produce various proinflammatory and profibrotic mediators (7, 24). One of the major cytokines constitutively elevated in scleroderma fibroblasts in vivo and in vitro is connective tissue growth factor (CTGF; Refs. 2, 23, 33). Fonseca et al. (12) recently demonstrated a direct genetic association between CTGF and SSc, identifying a CTGF polymorphism within a novel Sp3-dependent transcriptional repressor site that is significantly associated with the susceptibility to SSc. Increased amounts of CTGF are found in scleroderma patients with more extensive skin involvement and severe pulmonary fibrosis (31). In addition to increased tissue expression, CTGF levels are increased in the sera of patients with SSc compared with healthy controls and patients with other connective tissue diseases; also, patients with diffuse scleroderma exhibit higher levels of CTGF than those with the limited form of the disease (31). Furthermore, bronchoalveolar lavage fluid from SSc patients with active lung fibrosis was found to contain much higher levels of CTGF compared with patients without lung involvement (33).

Profibrotic properties of CTGF have been lately well established. It was shown that CTGF promotes deposition of several ECM proteins such as collagen, fibronectin, and tenascin C (4, 9, 16, 19, 23, 38). Using CTGF-specific small interfering RNA (siRNA), Xiao et al. (36) found that silencing of CTGF significantly decreases expression of collagen in SSc skin fibroblasts. In addition, CTGF was demonstrated to promote fibroblast proliferation, collagen gel contraction, cell adhesion, and migration (2, 10, 13, 17, 23). The molecular mechanisms by which CTGF exerts its profibrotic effects have been intensively investigated; however, an analysis of the overall effect of CTGF on cell proteome has not been reported.

Proteome analysis has generated much interest in recent years and is presently used as a modern tool for determination of biochemical processes involved in diseases and for characterization of protein expression levels (8, 11, 25, 34). In the present work, a proteomic approach has been used to analyze CTGF-induced protein expression profile in human lung fibroblasts. We observed that lung fibroblasts isolated from healthy individuals when transfected with CTGF produce more ECM and cytoskeletal proteins compared with control cells. We also identified a novel CTGF-induced protein, IQGAP1, which is inherently elevated in lung fibroblasts isolated from SSc patients. We show that expression of IQGAP1 in SSc lung fibroblasts correlates with the migration rate of the cells in a wound healing assay and that IQGAP1 via ERK1/2 mediates cell migration induced by CTGF in normal lung fibroblasts.

MATERIAL AND METHODS

Cell culture and transfection.

Lung fibroblasts were derived from lung tissues obtained at autopsy from SSc patients and from age-, race-, and sex-matched normal subjects who died from nonpulmonary causes. Lung tissue was diced (0.5- × 0.5-mm pieces) and cultured in DMEM (GIBCO, Grand Island, NY) supplemented with 10% FCS, 2 mM l-glutamine, gentamicin sulfate (50 μg/ml), and amphotericin B (5 μg/ml) at 37°C in 10% CO2. 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 2nd and 4th passages in all experiments. Normal lung fibroblasts were transfected with CTGF in pcDNA3 (a kind gift of Dr. D. R. Brigstock, Ohio State University) or pcDNA3 (as a control) by Effectene reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Briefly, cells were grown to 70% confluence on 100-mm dishes, washed with PBS, and incubated overnight with 10 ml of growth medium containing 5 μg of DNA and 60 μl of Effectene reagent. The fibroblasts were grown for another 48 h, harvested in PBS, and subjected to 2-dimensional gel electrophoresis (2-DE). Transfection efficiency is routinely checked by immunoblotting using anti-CTGF antibody from Santa Cruz Biotechnology (Santa Cruz, CA).

Sample preparation and 2-DE.

Lung fibroblasts were solubilized in sample rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 0.5% ZOOM Carrier Ampholytes) containing protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentration was determined by bicinchoninic acid protein estimation method (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. 2-DE was carried out in ZOOM IPGRunner System (Invitrogen, Carlsbad, CA). Immobilized pH gradient (IPG) strips with pH ranges of 4–7 were rehydrated overnight in 155 μl of the sample rehydration buffer containing traces of bromophenol blue, 20 mM DTT, and 100-μg protein samples. The first dimension was performed using a multistep protocol (25 min at 200 V, 20 min at 450 V, 20 min at 750 V, and 50 min at 2,000 V). By the end of isoelectric focusing, each strip was equilibrated in two steps (15 min each) in 5 ml of NuPAGE LDS Sample Buffer supplemented with 10 mg/ml DTT and 40 mg/ml iodoacetamide, respectively. The second dimension was carried out at 200 V using NuPAGE Novex 7% Tris-Acetate gels. Gels were stained with SimplyBlue SafeStain or SilverQuest Silver Staining Kit (both from Invitrogen) in accordance with the manufacturer's instructions. The stained gels were scanned by the Molecular Imager FX System and analyzed with PDQuest Software (Bio-Rad, Hercules, CA) running under Windows XP on a PC workstation. Background was subtracted, and peaks for the protein spots were located and counted.

In-gel digestion and mass spectrometric analysis.

Gel plugs were excised and placed in an Eppendorf tube. Each plug was washed with 50 mM ammonium bicarbonate for 10 min. Next, the plugs were destained using 25 mM ammonium bicarbonate in 50% acetonitrile for 15 min, repeated twice. The plugs were dehydrated with 100% acetonitrile for 15 min and then dried in a SpeedVac. Each gel plug was covered with Proteomics Grade Trypsin (Sigma) and incubated at 37°C overnight. The supernatant was collected in a clean, dry Eppendorf tube. Peptides were further extracted with one wash of 25 mM ammonium bicarbonate for 20 min and three washes of 5% formic acid and 50% acetonitrile for 20 min each. The supernatant was collected and pooled after each wash and then dried down in a SpeedVac to ~1 μl for Matrix Assisted Laser Desorption Ionization Time-of-Flight/Time-of-Flight (MALDI TOF/TOF) analysis and to ~2 μl for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Before MALDI TOF/TOF analysis, the samples were reconstituted with 10 μl of 0.1% trifluoracetic acid. After the spots were dried completely, the insert was placed in a holding sled and subsequently loaded into the Applied Biosystems 4800 Proteomics Analyzer. An external calibration was preformed before analyzing samples using the manufacturer's standards and protocol. Samples were analyzed in batch mode using 3,000–5,000 laser shots per spectrum. First, peptide mass maps were acquired over the mass-to-charge ratio (m/z) range of 800–3,000 in reflectron mode with a delayed extraction time optimized for m/z 1,500 by averaging 3,000–5,000 scans to locate peaks of peptide origin. The next batch run performed MS-MS analyses to obtain sequence data on the 15 most abundant peaks from the MS analysis. On completion of the batch processing, the data was exported into the GPS Explorer data processing system for interpretation and identification. The MASCOT database-searching algorithm analyzed the data and summarized the results in report format. Database searches were preformed using 2 missed cleavages and 1 differential modifications of methionine oxidation. The top 20 matches were reviewed before assigning confident protein identifications. Tandem MS spectra were reviewed manually to confirm assignments. Assigned protein identifications had at minimum 2 sequenced peptides with each peptide containing at least 4 consecutive b- or y-ions.

Trypsin-digested samples were also analyzed via LC-ESI-MS/MS on a linear ion trap mass spectrometer (LTQ, Thermo Finnigan) coupled to an LC Packings Nano LC system. A 75-μm C18 reverse-phase LC column (Microtech Scientific) was used with a 30-min gradient from 2% acetonitrile and 0.2% formic acid to 70% acetonitrile and 0.2% formic acid. Data-dependent analysis was used on the LTQ to perform MS/MS on all ions above an ion count of 1,000. Dynamic exclusion was set to exclude ions from MS/MS selection for 3 min after being selected two times in a 30-s window.

The MS/MS data was searched against the National Center for Biotechnology Information (NCBI) human database using Thermo Finnigan Bioworks 3.3 software. Variable modifications of methionine oxidation were considered. Protein identifications met the minimum criteria of a Protein Probability of 1.0 E-3 or better and had an Xcorr vs. charge state >1.5, 2.0, 2.5 for +1, +2, and +3 ions, with at least 2 unique peptides matching the protein, and a good match for at least 4 consecutive y- or b-ion series from the MS/MS spectra.

Cell migration wound healing “scratch” assay.

Lung fibroblasts were cultured to form a monolayer on fibronectin-coated six-well plates. Since high fibronectin concentration promotes cell spreading and reduces cell motility (30), the concentration of fibronectin was always low (10 μg/ml). Cells were serum-deprived overnight and then mechanically “wounded” by scraping with a Fisherbrand ready-tip (1–200 μl). Cell monolayers were washed twice with PBS and treated with CTGF (BioVendor, Candler, NC). For one series of experiments, cells were incubated with or without MEK-1/2 inhibitor U0126 (EMD Biosciences, San Diego, CA) or cell proliferation inhibitor 5-fluorouracil (5-FU; Sigma).

After 6 h of incubation, fibroblasts were washed with PBS and stained by Diff-Quick solution. Pictures were taken at ×2.5 magnification. Migration rate was calculated by counting the cells that cross into the “scratch” (wounded) area and presented as a percentage of total cells on identical “nonscratch” area (100%). In some experiments, IQGAP1 siRNA from Santa Cruz Biotechnology was transfected into cells according to the manufacturer's instructions 48 h before scratch assay. Briefly, lung fibroblasts were seeded on fibronectin-coated six-well plates (2 × 105 cells per well) in antibiotic-free growth medium supplemented with FBS. On the next day, cells were washed once with siRNA Transfection Medium and transfected with IQGAP1 siRNA (40 pmol per well) using siRNA Transfection Reagent. siRNAs containing a scrambled sequence that does not lead to the specific degradation of any known cellular mRNA were used as a control. Transfection efficiency was routinely tested by immunoblotting with anti-IQGAP1 antibody (Santa Cruz Biotechnology).

MAPK assay.

The phosphorylation of p42/p44 MAPK isoforms was analyzed by Western blot using anti-phospho-p42/44 antibody (Cell Signaling Technology, Danvers, MA) as previously described (3). Briefly, lung fibroblasts were cultured on six-well plates (2 × 106 cells per well) to 90% confluence, synchronized with serum-free DMEM for 24 h, and then treated with CTGF. For part of the experiments, cells were transfected with IQGAP1 siRNA or control siRNA as described above. After incubation with or without CTGF (10–100 ng/ml) for various times, cells were rapidly washed with ice-cold PBS and collected in 1× SDS sample buffer (100 μl per well). Twenty microliters of sample was separated on 4–20% SDS-polyacrylamide gels and immunoblotted with anti-phospho-p42/44 antibody. Total amount of ERK1/2 was evaluated by reblotting with anti-p42/44 polyclonal antibody.

Statistical analysis.

For 2-DE analysis, statistical approach was applied when determining differentially expressed proteins using the PDQuest software. Student's t-test was performed with 95% significance level to determine which proteins were differently expressed between vector- and CTGF-transfected lung fibroblasts. A minimum of 1.8-fold change was considered for the upregulated proteins, and 0.7-fold for downregulated proteins.

For all other experiments, statistical analyses were performed with KaleidaGraph 4.0 (Synergy Software, Reading, PA). All data were analyzed using ANOVA with post hoc testing. The results were considered significant if P < 0.05.

RESULTS

Proteome analysis.

Six different lung fibroblast cell lines [3 scleroderma-associated ILD (SSc-ILD) and 3 normal lung fibroblasts isolated from matched controls] were used in this study. CTGF protein level in scleroderma lung fibroblasts was extremely high, whereas in normal lung fibroblasts it was hardly detectable (Fig. 1). To investigate CTGF-induced proteins in normal lung fibroblasts, we transfected the cells with CTGF DNA (5 μg per 100-mm plate). The level of CTGF in transfected cells increased ~10 times compared with nontransfected cells and was comparable with the level of CTGF in SSc lung fibroblasts. Protein extracts from normal lung fibroblasts transfected with vector or CTGF and from SSc lung fibroblasts were analyzed by 2-DE using NuPAGE 7% Tris-Acetate gel designed to study high molecular weight proteins. We reproducibly detected ~50 protein spots after SimplyBlue SafeStain (data not shown) and >800 protein spots after silver staining (Fig. 2). The molecular weight distribution of proteins was between 50 and 250 kDa. Five normalized protein spots corresponding to vimentin, which did not change on CTGF transfection, were selected as housekeeping proteins. Quantification of the individual protein spots from the gels revealed that a majority of total protein spots on each gel were quantitatively similar regardless of whether they represented CTGF- or vector-transfected normal lung fibroblasts or scleroderma lung fibroblasts. The protein spots of interest were cut out from the gel and identified either by MALDI TOF/TOF or by LC-ESI-MS/MS analysis. We analyzed at least 3 gels for each experimental condition and identified 13 proteins that were affected by CTGF overexpression in normal lung fibroblasts (Table 1; also see Supplemental Table 1 available in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site with peptides for all identified proteins). Several of the CTGF-induced proteins, such as pro-α (I) collagen and cytoskeletal proteins vinculin, moesin, and ezrin, were previously known to be elevated in pulmonary fibrosis (15, 24, 32), whereas 9 of 13 proteins have not been reported in lung fibrosis and are, therefore, novel CTGF-responsive molecules. We identified 8 CTGF-induced proteins; 6 of them are elevated in scleroderma lung fibroblasts, whereas expression of cytoskeleton-associated protein-4 and vinculin in SSc lung fibroblasts is comparable to that of normal lung fibroblasts. We also detected 5 proteins that were downregulated by CTGF in normal lung fibroblasts (Table 1, D9-D13). All of them were expressed at a low level in scleroderma lung fibroblasts. These data suggest that normal lung fibroblasts transfected with CTGF resemble SSc lung fibroblasts.

Fig. 1.
Connective tissue growth factor (CTGF) expression in normal and systemic sclerosis (SSc) lung fibroblasts. Confluent cultures of lung fibroblasts were collected with lysis buffer and analyzed by Western blotting using anti-CTGF antibody. Anti-β-actin ...
Fig. 2.
A: 2-dimensional gel electrophoresis (2-DE) of proteins from lung fibroblasts. Lung fibroblasts transfected with pcDNA3 (left) or CTGF (middle) and nontransfected SSc lung fibroblasts (right) were collected with PBS and solubilized in sample rehydration ...
Table 1.
Identified proteins that are differently expressed in vector and CTGF-transfected human lung fibroblasts

Analysis of CTGF-induced IQGAP1 in human lung fibroblasts.

We selected IQGAP1 from the CTGF-induced proteins for further study because of its ability to bind and modulate multiple signaling and structural molecules. Figure 3 demonstrates full MS scan of IQGAP1. We detected a total of 47 unique peptides of this protein (for complete list, see online data supplement).

Fig. 3.
The identification of IQ motif containing GTPase activating protein (IQGAP1) using Matrix Assisted Laser Desorption Ionization Time-of-Flight/Time-of-Flight (MALDI TOF/TOF) analysis. Presented is a full-scan MALDI MS of the tryptic digest of the spot ...

To confirm the effect of CTGF on IQGAP1 expression, we incubated normal lung fibroblasts with various doses of CTGF (5–100 ng/ml) for 24 h and then detected IQGAP1 by immunoblotting using anti-IQGAP1 polyclonal antibody. CTGF notably induced IQGAP1 in normal lung fibroblast in a dose-dependent manner (Fig. 4A). The most profound effect of CTGF on IQGAP1 expression was observed using 50 ng/ml CTGF. Therefore, this dose was chosen for all other experiments. CTGF also induced IQGAP1 in a time-dependent manner (Fig. 4B). IQGAP1 protein expression was induced after 2 h of incubation with 50 ng/ml CTGF with a maximum increase noted after 24 h. The noticeable increase of IQGAP1 expression by CTGF was observed in three different cell lines of normal lung fibroblasts. In contrast, IQGAP1 was inherently elevated in all three SSc lung fibroblast cell lines, and exogenous CTGF only slightly increased the level of IQGAP1 in these SSc cells (Fig. 4C).

Fig. 4.
Analysis of CTGF-induced IQGAP1 in human lung fibroblasts. A: normal lung fibroblasts were incubated with various doses of CTGF for 24 h and analyzed by Western blot using anti-IQGAP1 antibody. B: CTGF (50 ng/ml) induced IQGAP1 in normal lung fibroblasts ...

IQGAP1 mediates CTGF-induced migration in normal and SSc lung fibroblasts.

IQGAP1 is known as a regulator of endothelial and epithelial cell migration, but little is known about its effect of fibroblast migration. To study the role of IQGAP1 in lung fibroblast migration, we used a wound healing scratch assay that measures cell migration toward the injured sites. Confluent monolayers of normal and SSc lung fibroblasts were scratched, and migrating cells were counted after 6 h. We observed that SSc lung fibroblasts migrate more intensely than normal lung fibroblasts. The migration rate of SSc lung fibroblasts reached 42.9% in 6 h, whereas it was equal to only 16.8% in normal lung fibroblasts (Fig. 5). CTGF significantly increased the migration rate of normal lung fibroblasts from 16.8% to 50.7% (Fig. 6, A and B). To determine whether IQGAP1 is involved in CTGF-induced migration of normal lung fibroblasts, we employed siRNA-based technology and knocked down IQGAP1 expression. Transfection of lung fibroblasts with IQGAP1 siRNA resulted in significant reduction of IQGAP1 protein level as demonstrated by immunoblotting with anti-IQGAP1 antibody (Fig. 6C). The depletion of IQGAP1 decreased the CTGF-induced migration rate of normal lung fibroblasts from 50.7% to 24.8% while having no effect on control cells. The migration rate in scleroderma lung fibroblasts after knockout of IQGAP1 was decreased to a level similar to that of control normal lung fibroblasts (Fig. 6, A and B). These data suggest that migration of SSc lung fibroblasts and CTGF-induced migration in normal lung fibroblasts have a similar mechanism that is regulated by IQGAP1.

Fig. 5.
Cell migration wound healing “scratch” assay in normal and SSc lung fibroblasts. A: lung fibroblasts were cultured to form a monolayer on fibronectin-coated 6-well plates. Cells were serum-deprived overnight and then mechanically “wounded” ...
Fig. 6.
IQGAP1 mediates CTGF-induced migration in normal and SSc lung fibroblasts. A: lung fibroblasts were seeded on fibronectin-coated 6-well plates (2 × 105 cells per well) in antibiotic-free growth medium supplemented with FBS and transfected with ...

MAPK mediates CTGF-induced migration in lung fibroblasts downstream of IQGAP1.

To determine other signaling molecules involved in IQGAP1-mediated CTGF-induced migration of lung fibroblasts, we studied effects of MAPK inhibitor U0126 and general inhibitor of cell proliferation 5-FU on CTGF-stimulated migration. We found that U0126 inhibits CTGF-induced migration (Fig. 7, A and B), whereas 5-FU does not change CTGF-stimulated migration. Inhibitors U0126 and 5-FU do not affect basal cell migration when applied alone without CTGF (data not shown). Next, we investigated whether MAPK inhibitor U0126 will interfere with CTGF-induced IQGAP1 expression. We incubated lung fibroblasts with or without CTGF and with or without U0126 for 24 h and then determined IQGAP1 level in cells by immunoblotting. We observed that U0126 does not affect either CTGF-induced or basal expression of IQGAP1 (Fig. 7C). We also observed that CTGF stimulates MAPK p44/42 phosphorylation in a time- and dose-dependent manner (Fig. 7D). The depletion of IQGAP1 by IQGAP1 siRNA notably reduced CTGF-stimulated MAPK p44/42 phosphorylation suggesting that IQGAP1 is required for MAPK p44/p42 activation by CTGF (Fig. 7E).

Fig. 7.
MAPK mediates CTGF-induced migration in human lung fibroblasts downstream of IQGAP1. A: MAPK inhibitor U0126 reduces CTGF-stimulated migration of lung fibroblasts. Wounded lung fibroblasts were treated with proliferation inhibitor 5-fluorouracil (5-FU; ...

DISCUSSION

The overexpression of CTGF in fibrotic lesions is well documented (2, 23, 31, 33). However, the biological significance of excess CTGF in fibrosis remains unclear. To better understand the consequences of elevated CTGF in pulmonary fibrosis, particularly SSc-ILD, we investigated the changes in the proteome of lung fibroblasts in response to overexpression of CTGF. Proteomic analysis of SSc lung fibroblasts and CTGF-stimulated normal lung fibroblasts demonstrates increased amounts of ECM and cytoskeletal proteins compared with control cells. The overexpression of collagen and other ECM proteins in fibrotic lesions is considered to be a molecular marker of fibrosis (1, 7, 22, 24). Accumulation of collagen type I, in particular, is a hallmark of SSc (1, 16, 24, 36). Therefore, we consider detection of collagen I by proteomics as a proof of accuracy of this method. Another CTGF-induced protein detected in this study is the prolyl 4-hydroxylase β-subunit, which is the major subunit of the collagen prolyl 4-hydroxylases (P4Hs), key enzymes of collagen biosynthesis. P4Hs are located within the endoplasmic reticulum and catalyze the formation of 4-hydroxyproline residues in X-Pro-Gly sequences of collagens and >15 other proteins that have collagen-like domains (29). Several investigators have demonstrated that CTGF induces collagen I in various cell lines by stimulating transcription and promoter activity of collagen I (9, 16). Our data suggest that CTGF may also facilitate the hydroxylation of collagens and thereby increase their accumulation. Additionally, we observed elevated levels of five cytoskeletal proteins after CTGF overexpression. Three of them, ezrin, moesin, and vinculin, have been shown previously to be involved in fibrosis (15, 32). However, they have not previously been reported to be upregulated by CTGF. We observed that ezrin and moesin were elevated in SSc lung fibroblasts, whereas the expression of vinculin was similar to that in normal lung fibroblasts. This is in agreement with a previous report that vinculin is elevated only in areas of early intra-alveolar fibrosis (15), since in this study we used lung fibroblasts from scleroderma patients with late stage of lung fibrosis. Another CTGF-induced cytoskeleton protein, caldesmon, is a multifunctional ubiquitous regulator of the actin cytoskeleton that may affect actin polymerization (18). CTGF-induced cytoskeleton-associated protein-4 is an integral membrane protein that links the endoplasmic reticulum to microtubules (35). This protein was not increased in SSc lung fibroblasts, and its role in pulmonary fibrosis is not known.

Five proteins downregulated by CTGF in normal lung fibroblasts included molecular chaperones (BiP glucose-regulated protein and stress-induced phosphoprotein-1), ER-60 protease, and proteins participating in nucleic acid synthesis (heterogeneous ribonucleoprotein U) and posttranslational modification (valosin-containing protein). All of them were expressed at low levels in scleroderma lung fibroblasts. These data suggest that overexpression of CTGF in normal lung fibroblasts results in a phenotype resembling that of SSc lung fibroblasts.

Among all CTGF-affected proteins, IQGAP1 is perhaps the most interesting molecule because of its ability to bind and modulate multiple signaling and structural molecules. Our proteomic studies demonstrate that in normal lung fibroblasts, CTGF significantly increases the expression of IQGAP1, which is inherently elevated in SSc lung fibroblasts. The effect of CTGF on IQGAP1 expression was verified by immunoblotting analysis. Using anti-IQGAP1 antibody, we found that CTGF increases IQGAP1 in normal lung fibroblasts in both a dose- and time-dependent manner. The increase of IQGAP1 expression by CTGF was observed in three different cell lines of normal lung fibroblasts, whereas it was inherently elevated in all three tested SSc lung fibroblast cell lines.

IQGAP1 is a ubiquitous 189-kDa protein that contains several protein-interacting domains (Fig. 8). Calponin homology domain binds to F-actin and mediates IQGAP1-induced actin polymerization in vitro (28). The WW domain is an interaction module for proline-rich ligands characterized by 2 conserved tryptophan residues (26). The calmodulin-binding IQ domain is a tandem repeat of 4 IQ motifs, each containing ~25 amino acids with conserved isoleucine and glutamine residues (21). The GAP-related domain mediates the binding of the Cdc42 and Rac1 GTPases (21). The RasGAP COOH terminus domain interacts with microtubule-binding protein CLIP-170 necessary for binding β-catenin and E-cadherin (14). Through interaction with its target proteins, IQGAP1 participates in multiple cellular functions including Ca2+-calmodulin signaling, definition of cytoskeletal architecture, regulation of Cdc42- and Rac1-dependent cytoskeletal changes, and control of E-cadherin-mediated intercellular adhesion (reviewed in Ref. 6). Lung fibroblasts contain 2 IQGAP proteins, IQGAP1 and IQGAP2. Interestingly, that CTGF induces IQGAP1 only and does not interfere with IQGAP2 expression (data not shown).

Fig. 8.
Structure of IQGAP1. The numbers indicate the position of amino acid residues. CHD, calponin homology domain; WW, domain with 2 conserved Trp residues; IQ, domain containing tandem repeats of 4 IQ motifs with conserved Ile and Gln residues; GRD, GAP-related ...

IQGAP1 plays a pivotal role in the control of cell migration essential for tissue homeostasis, morphogenesis, and wound repair (5, 6). Studies of Mataraza et al. (27) demonstrated that overexpression of IQGAP1 in epithelial cells increases cell motility, whereas knockdown of the protein reduces cell migration. Additionally, IQGAP1 was shown to directly interact with VEGF receptor and mediate reactive oxygen species-dependent endothelial cell migration and proliferation, which may contribute to the regeneration of endothelial cells after vascular injury (37). However, the role of IQGAP1 in fibroblast migration has not been reported. Using a wound healing scratch assay, we observed that SSc lung fibroblasts migrate more intensely than normal lung fibroblasts. The migration rate of SSc lung fibroblasts reached 42.9% in 6 h, whereas it was equal to only 16.8% in normal lung fibroblasts. CTGF significantly increased the migration rate of normal lung fibroblasts from 16.8% to 50.7%. The depletion of IQGAP1 by siRNA significantly decreased migration of scleroderma lung fibroblasts and CTGF-induced migration of normal lung fibroblasts. MAPK inhibitor U0126 significantly reduced CTGF-stimulated lung fibroblast migration but had no effect on CTGF-induced IQGAP1 expression. CTGF induced ERK1/2 phosphorylation in an IQGAP1-dependent manner. These data suggest that CTGF-induced migration of normal lung fibroblasts is regulated by IQGAP1 and MAPK signaling. IQGAP1 can possibly mediate other effects of CTGF in lung fibroblasts, such as cell spreading, which need to be addressed in future studies.

We conclude that lung fibroblasts isolated from healthy individuals when transfected with CTGF produce more ECM and cytoskeletal proteins compared with control cells. The majority of CTGF-induced proteins, including IQGAP1, are inherently elevated in lung fibroblasts isolated from SSc patients. The expression of IQGAP1 in lung fibroblasts correlates with the migration rate of the cells in a wound healing assay, suggesting that IQGAP1 may mediate the migration of lung fibroblasts, particularly myofibroblasts, to damaged tissue.

Cell migration is a fundamental cellular process for normal development and homeostasis of tissue and organ. Fibroblasts of normal stroma are relatively stationary despite the absence of architectural boundaries such as basement membranes. Explant culture is selective for migratory fibroblasts and can be used as a model in vitro to study the migration of myofibroblasts in vivo to the foci of lung fibrosis. Although the functional relationship of CTGF-induced proteomics in vivo remains unknown, our in vitro data imply that it may contribute to the tissue repair in SSc-ILD and, in particular, may provide insight into an important role of IQGAP1 in pulmonary fibrosis.

GRANTS

This work was supported by National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants P60 AR049459 (Multidisciplinary Clinical Research Center, to R. M. Silver) and K01AR051052 (to G. S. Bogatkevich) and a New Investigator Grant from the Scleroderma Foundation (to G. S. Bogatkevich).

Supplementary Material

[Supplemental Figure]

Notes

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.

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