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Clin Exp Immunol. May 2006; 144(2): 335–341.
PMCID: PMC1809657

Imatinib mesylate inhibits proliferation of rheumatoid synovial fibroblast-like cells and phosphorylation of Gab adapter proteins activated by platelet-derived growth factor

Abstract

Receptors for platelet-derived growth factor (PDGF) are abundantly expressed on synovial fibroblast-like (SFL) cells from patients with rheumatoid arthritis (RA), and stimulation with PDGF enhances both the anchorage-dependent and -independent growth of RA–SFL cells. To elucidate the molecular mechanisms responsible for the excessive growth of RA–SFL cells and to seek a novel molecular-targeting therapy for RA, we examined the expression of adapter proteins and the effect of the specific inhibition of PDGF receptor activation by imatinib mesylate. Cultured SFL cells were used in the present study after 2–5 passages. The anchorage-dependent and -independent growth patterns of the SFL cells were evaluated using a tetrazolium-based assay and colony formation in 0·3% agar, respectively. Adapter proteins Gab1 and Gab2 were expressed in RA–SFL cells, and both proteins were rapidly (< 1 min) tyrosine-phosphorylated after the stimulation of RA–SFL cells with 10 ng/ml of PDGF and, to a lesser extent, after stimulation with 100 ng/ml of epidermal growth factor (EGF). The inhibition of PDGF receptor tyrosine kinase activation by 1 µM or less of imatinib mesylate specifically suppressed the PDGF-dependent, but not EGF-dependent, tyrosine phosphorylation of various proteins. Moreover, imatinib mesylate abolished both the anchorage-dependent and -independent proliferation of RA–SFL cells induced by PDGF stimulation. These results suggest that Gab adapter proteins are expressed and likely to be involved in the growth signalling of rheumatoid synovial cells and that imatinib mesylate, a key drug in the treatment of chronic myeloid leukaemia, may also be effective for the treatment of RA.

Keywords: adapter proteins, arthritis, growth factors, signal transduction

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive arthropathy associated with synovitis that eventually leads to disability and reduced life expectancy in the majority of affected patients [1]. The synovial membrane in patients with RA is characterized by the excessive proliferation of synoviocytes, increased vascularity and the infiltration of inflammatory cells including, CD4+ T lymphocytes [1].

However, accumulating evidence suggests that T cell-independent pathways play an indispensable role in the pathophysiology of RA [2]. Indeed, morphological and functional examinations show that synovial fibroblasts are activated and transformed into aggressive, matrix-degrading cells in patients with RA [3,4]. Consequently, cultured synovial fibroblast-like (SFL) cells from RA patients exhibit anchorage-independent growth and a loss of contact inhibition, especially in the presence of platelet-derived growth factor (PDGF) [5], as well as oncogene activation [6], monoclonal or oligoclonal cell expansion [7] and, most importantly, cartilage invasion in severe combined immunodeficient mice [8]. The above characteristics are not observed typically in SFL cells from patients with osteoarthritis (OA) [38].

Research on PDGF produced some of the first evidence that signals from polypeptide growth factors act through the cell surface tyrosine kinase receptor to stimulate various cellular functions, including proliferation and differentiation [9,10]. PDGF consists of four polypeptide chains that form five disulphide-bonded dimeric PDGF isoforms: AA, AB, BB, CC and DD. PDGF receptor (PDGF-R), in turn, may occur as α or β homodimers or as α/β heterodimers [11]. The binding of the dimeric ligand to the extracellular portions of the two PDGF-R chains elicits the dimerization of PDGF-Rs and the autophosphorylation of their cytoplasmic tyrosine residues. Consequently, multiple src-homology 2 (SH2) domain-containing signal transduction molecules are recruited to the cell surfaces, including adapter proteins Grb2, Nck and Shc [9,10].

Recently, the overactivity of the PDGF/PDGF-R axis has been implicated in various human tumours, including glioblastomas, prostate cancer and chronic myeloproliferative diseases [12,13]. Imatinib mesylate (STI571) is an inhibitor of Abl, Bcr-Abl tyrosine kinase, PDGF-R, and the tyrosine kinase receptor for stem cell factor (c-Kit) at IC50 values of 0·1–0·3 µM, 0·25 µM, 0·12–0·15 µM and < 1 µM, respectively [14,15]. Based on its inhibition of Bcr-Abl, imatinib mesylate has been used successfully in the treatment of chronic myeloid leukaemia (CML) [16,17]. Subsequently, imatinib mesylate has been used in many clinical trials against other human tumours, such as prostate cancer, in the hope that it might suppress PDGF-R signalling [11,12].

PDGF-Rs are expressed abundantly on the surface of RA–SFL cells, and stimulation with PDGF enhances both the anchorage-dependent and -independent growth of RA–SFL cells [5,1825], implicating PDGF in the activation and transformation of RA–SFL cells. Indeed, PDGF immunostaining of RA synovia is more extensive and intense than that of osteoarthritis (OA) or normal synovia, and PDGF-R expression is also elevated in RA synovia compared with OA and normal synovia [18,22,23]. Moreover, PDGF, together with tumour necrosis factor (TNF)-α, was identified as the major growth factors of RA–SFL cells [26]. Furthermore, thrombin activity in synovial fluid is significantly higher in the patients with RA than in patients with OA, and the mitogenic activity of thrombin toward RA–SFL cells is associated with an increase in the expression of mRNA of PDGF-Rs [27].

Recently, accumulating evidence demonstrates the necessity of adapter proteins, including the Gab family, in the PDGF signalling pathway [9,10,2830]. Therefore, in the present study, we examined the expression of adapter proteins in RA–SFL cells to elucidate the molecular mechanisms responsible for the excessive growth of RA–SFL cells upon PDGF stimulation. Moreover, we demonstrated that imatinib mesylate may be a candidate drug for novel molecular-targeting therapies for the treatment of RA through its inhibition of PDGF-R activation and the subsequent tyrosine phosphorylation of adapter proteins, including Gab1 and Gab2.

Materials and methods

Materials

RPMI-1640, 0·05% trypsin-ethylenediamine tetraacetic acid (EDTA) and gentamicin were purchased from (GIBCO, Grand Island, NY, USA). Collagenase and albumin from bovine serum (Cohn fraction V, pH 7·0) was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Non-fat dry milk, acrylamide and bisacrylamide were purchased from Bio-Rad (Hercules, CA, USA). BCA protein assay reagent was purchased from Pierce (Rockford, IL, USA). The immunoglobulin conjugates, Hyperfilm and enhanced chemiluminescence (ECL) reagents were purchased from Amersham (Piscataway, NJ, USA). Anti-Gab1 and anti-Gab2 were obtained from Upstate Biotechnology (Lake Placid, NY, USA), while anti-phosphotyrosine (anti-p-Tyr) antibody (PY99) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Imatinib mesylate (STI571) was kindly provided by Novartis Pharma AG (Basel, Switzerland). Fetal bovine serum (FBS), PDGF-BB, epidermal growth factor (EGF) anti-PDGF-BB, tyrphostin AG1478 and all other reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Preparation of SFL cells

Synovial membranes were obtained from RA and OA patients undergoing total knee replacement surgery. Written informed consent was obtained from all patients before their inclusion in the study, which was approved by the Ethics Committee of our institute. The tissues were minced and digested sequentially with 10 ml of 0·05% trypsin/EDTA added to an isovolume of RPMI-1640 and then with 10 mg/ml of collagenase in RPMI-1640. The dissociated cells were filtrated through a sterile gauze and cultured in RPMI-1640 containing 10% FBS and gentamicin (10 µg/ml). Cultured SFL cells with typical morphological and immunocytochemical features, as described previously [25], from passages 2–5 were used in our experiments.

Immunoprecipitation and Western blot analysis

After reaching 60–70% confluence on 150-mm dishes, the cells were serum-deprived for 20 h and then challenged with 10 ng/ml of PDGF or 100 ng/ml of EGF at 37°C for the indicated times. The detailed protocols for the immunoprecipitation and Western blot analysis were described previously [31,32].

Cell proliferation assay

RA–SFL cells (1 × 103 cells/well) were seeded onto 96-well plates and cultured. The proliferation rate of the cells was determined using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]-based kit (Roche, Indianapolis, IN, USA), according to the manufacturer's instructions, in which tetrazolium salts are cleaved to form a formazan dye by metabolic active cells. The results from the MTT assay showed a good correlation to those from a [3H]-thymidine incorporation assay using fibroblastic cell lines, as described previously [31]. All incubation protocols were performed eight times.

Cell growth in soft agar

A soft agar assay was performed as described previously [33]. Briefly, 2 × 103 cells were suspended in 0·3% bacto-agar (Gibco) containing 0·1% bactopeptone and 10% FBS on a 0·6% bactoagar layer containing 0·1% bactopeptone and 10% FBS in RPMI-1640 medium using six-well plates. The plates were incubated at 37°C for 21 days and scored for colonies that were greater than 60 µm in diameter (greater than 30 cells). For some assays, 10 ng/ml of PDGF or 100 ng/ml of EGF were added to the top layer of the agar.

Results

Functional adapter proteins downstream of PDGF-R are expressed in RA–SFL cells

First, we examined whether synovial cells from patients with RA were positive for the expression of functional adapter proteins Gab1 and Gab2, which have been reported to be involved in PDGF-R signalling in fibroblasts and other cell types. Both Gab1 (110–120 kDa) and Gab2 (97–100 kDa) exhibited rapid tyrosine phosphorylation that peaked at 1 min after PDGF stimulation and decayed gradually thereafter (Fig. 1a,b). Both Gab1 and Gab2 rapidly showed mobility shifts after PDGF stimulation, due probably to phosphorylation, and their expression level did not decay (Fig. 1a,b). Therefore, the adapter proteins Gab1 and Gab2 were thought to be involved in the PDGF-R signalling pathway in RA–SFL cells.

Fig. 1
Gab1 and Gab2 were rapidly tyrosine-phosphorylated upon the stimulation of synovial fibroblast-like cells with platelet-derived growth factor (PDGF). Rheumatoid arthritis–synovial fibroblast-like (RA–SFL) cells were challenged with 10 ...

Imatinib mesylate (STI571) specifically inhibits the PDGF-induced tyrosine phosphorylation of Gab1 and Gab2

We next compared the relative importance of PDGF to EGF in the activation and proliferation of RA–SFL cells, as these two growth factors shares many signalling molecules, including Gab1 adapter protein, and because EGF has been shown to be a very potent stimulator of the proliferation of some fibroblastic cell lines, including Syrian hamster embryo fibroblasts and NIH3T3 [28,3032,34]. PDGF stimulation enhanced the proliferation of RA–SFL cells under basal conditions (cultured in 5% FBS-containing medium), while EGF showed minimal up-regulation (data not shown). Based on the above results indicating a pivotal role of PDGF in RA–SFL cells, we investigated whether STI571, a potent inhibitor of PDGF-R activation, could inhibit the signalling activation of RA–SFL cells induced by PDGF stimulation. Preincubation with 1 µM of STI571 almost abolished the tyrosine phosphorylation of many proteins including ~185 kDa of PDGF-R (Fig. 2a). In contrast, STI571 did not show any inhibition of the EGF-induced modest tyrosine phosphorylation of signalling molecules. Furthermore, 1 µM of STI 571 abolished or significantly reduced the tyrosine phosphorylation of Gab1 and Gab2 upon PDGF stimulation (Fig. 2b). Thus, a specific inhibition of PDGF-R activation and subsequent tyrosine phosphorylation of Gab adapter proteins by STI571 raised the possibility that the drug might be a potent inhibitor of RA–SFL cell proliferation.

Fig. 2
STI571 inhibits tyrosine phosphorylation of signalling proteins including Gab1 and Gab2 induced by platelet-derived growth factor (PDGF) stimulation. (a) Rheumatoid arthritis–synovial fibroblast-like (RA–SFL) cells were challenged with ...

STI571 effectively inhibits both anchorage-dependent and -independent growth patterns of PDGF-stimulated RA–SFL cells

Finally, we examined the up-regulating effect of PDGF on the anchorage-dependent (Fig. 3a) and -independent (Fig. 3b) proliferation of RA–SFL cells, as well as the inhibitory effect of STI571. Both growth conditions showed similar results. Namely, PDGF enhanced significantly the growth of RA–SFL cells grown on both plates and in 0·3% soft agar. Furthermore, STI571 completely suppressed the anchorage-dependent and -independent proliferation of RA–SFL cells stimulated by PDGF.

Fig. 3
STI571 inhibited completely both the anchorage-dependent and -independent growth of rheumatoid arthritis–synovial fibroblast-like (RA–SFL) cells stimulated by platelet-derived growth factor (PDGF). (a) RA–SFL cells were cultured ...

Discussion

This is the first report on the expression of adapter proteins in RA synovial cells. More importantly, the present study is the first description of the inhibitory effect of STI571 on the abnormal growth of RA synovial cells.

Several lines of evidence, including the overexpression of PDGF/PDGF-R in RA synovial cells and the PDGF-responsive, anchorage-independent growth of RA–SFL cells [5,1825], indicate that adapter proteins are involved in the aggressive growth of RA–SFL cells in response to PDGF and, possibly, the acquisition of this feature. Among the various adapter proteins implicated in PDGF-R signalling, we focused on Gab adapter proteins because accumulating reports suggest strongly that Gab1 and Gab2 have crucial functions in cellular transformation. Two Gab1-overexpressing NIH3T3 cell clones were shown to exhibit significant colony formation in soft agar in the presence of additional growth factors, with EGF producing a more potent response than insulin [34]. Gab1 was also required for cellular transformation induced by Met [35] and polyoma middle T antigen [36]. Moreover, the altered expression of Gab1 lacking a PH domain drove neoplastic progression in an in vitro transformation system of carcinogen-treated Syrian hamster embryo cell cultures [32]. As for Gab2, the activated form of ErbB2-mediated transformation was suppressed strongly when Gab2-deficient cells were transfected with active ErbB2 [37], and Gab2-deficient cells expressing Bcr-Abl exhibited defective PI3K-Akt and extracellular signal-regulated kinase (ERK) activation as well as resistance to transformation by Bcr-Abl [38].

In the present study, we confirmed the expression of Gab1 and Gab2 in RA–SFL cells. The expression level of Gab1 and Gab2 is not high enough to be evaluated by Western analysis without the condensation by immunoprecipitation of target proteins. Thus, in a preliminary study, we compared the mRNA expression level of Gab1 and Gab2 between synoviocytes from five patients each with RA and OA by reverse transcription–polymerase chain reaction (RT–PCR) using commercially available primer settings (SC-35431PR for Gab1 and SC-40606PR for Gab2, respectively; Santa Cruz Biotechnology, Inc.), which did not reveal significant differences (data not shown). Furthermore, protein expression levels of other adapter proteins, Nck and Shc, did not differ substantially between RA and OA synoviocytes (data not shown). Therefore, alterations in the expression of these adapter proteins were considered to be unlikely causes of the transformed features of RA–SFL cells. Instead, an alteration in other signalling elements of cellular growth, such as PDGF-R overexpression [18,23,25], for example, may be primarily responsible for transformation, and adapter proteins may play a pivotal role in this mechanism.

Further investigation is required to clarify the molecular mechanisms responsible for PDGF-R signalling in RA–SFL cells and the possible implication of this process in cell transformation. Very recently, PDGF was shown to phosphorylate both Akt and extracellular signal-regulated kinase (ERK) in synovial cells, and PDGF-pretreatment markedly suppressed tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in synovial cells [39]. Therefore, the inhibition of PDGF-R activation and the subsequent suppression of Gab adapter proteins and ERK by STI571 should result in the down-regulation of synovial hyperplasia in vivo. Indeed, Eklund et al. [40] recently described three patients with severe RA who were treated for 12 weeks with escalating daily doses (from 100 to 400 mg) of STI571. All three patients had failed to respond to prior anti-rheumatic medications, including methotrexate and infliximab. All the patients reported less pain and disease activity, and their health assessment questionnaire scores improved after the STI571 treatment. In addition, Miyachi et al. [41] reported a case with RA and chronic myeloid leukaemia, both of which were treated successfully using STI571. These in vivo findings are consistent with the fact that STI571 inhibits Bcr-Abl and PDGF-R at comparable IC50 values, as described previously [14,15].

During the last decade, efforts to develop more effective treatments for RA based on an improved understanding of the role of inflammatory mediators have led to successful therapies involving monoclonal antibodies against TNF-α. However, such immunomodulatory treatments are accompanied inevitably by an elevated risk of contracting opportunistic infections. Thus, the present study is likely to provide a novel and powerful molecular-targeting therapy that would complement current immunosuppressive treatments for RA. To elucidate the importance of Gab adapter proteins in the proliferative signalling via PDGF-R, further investigations determining whether the specific inhibition of Gab adapter proteins mimics the effect of imatinib mesylate appear warranted.

Acknowledgments

We thank Yumiko Setoyama for excellent laboratory assistance. This work was supported by research grants from the Japanese Ministry of Health, Labour and Welfare (H13-Immunology-003) and the Japanese Ministry of Education, Culture, Sports, Science and Technology (13670471).

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