Regulation of rheumatoid synoviocyte proliferation by endogenous p53 induction

Abstract

The p53 tumour suppressor protein protects cells from tumorigenic alterations by inducing either cell growth arrest or apoptosis. In the present study, we investigated the role of endogenous p53 expressed in rheumatoid arthritis synovial fibroblasts which show transformed-appearing phenotypes. Type B synovial cells (fibroblast-like synovial cells) were exposed to a proteasome inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132). During this process, the expressions of p53 and p21 were examined by Western blot. Cell cycle analysis of the synovial cells was determined by DNA staining using propidium iodide (PI). Inhibition of proteasome resulted in the accumulation of p53 which was followed by an increase in the amount of a cyclin-dependent kinase (CDK)-inhibitor, p21. As a consequence, the retinoblastoma gene product, Rb, remained in the hypophosphorylated state, thus preventing PDGF-stimulated synovial cells from progressing into S-phase. This study shows that endogenous p53, which is inducible in rheumatoid synovial cells, is functionally active based on the findings that its expression blocks the G1/S transition by inhibiting the CDK-mediated phosphorylation of Rb via p21 induction. Thus the induction of p53 using proteasome inhibitor may provide a new approach in the treatment of RA.

Introduction

Rheumatoid arthritis (RA) is characterized by pronounced synovial hyperplasia and transformed-appearing synovial fibroblasts [1,2]. Tissue homeostasis is maintained by means of a balance between cell proliferation and apoptotic cell death [3]. Apoptotsis of synovial cells has been identified in rheumatoid synovium [4,5], suggesting that synovial tissue hyperplasia may be a result of cell proliferation rather than that of apoptotic cell death. Therefore, the control of cell cycle progression of synovial fibroblasts is one of the therapeutic strategies for the treatment of RA.

p53 is the tumour suppressor gene product that serves as a critical regulator of cell survival and proliferation [6]. It functions as a transcriptional activator of genes that block the progression from G1 to S phase in cell cycle [7]. p53-dependent G1 arrest occurs largely through the induction of p21^WALF/CIP1, which prevents entry into S phase by inhibiting the G1 cyclin-dependent kinase (CDK) complex [8]. These complexes phosphorylate proteins of the retinoblastoma (Rb) family and activate transcriptional factors, such as E2F, that are essential for DNA synthesis during S phase [9]. Loss of p53 function, either through mutation, deletion or other mechanisms, is often associated with neoplasma, whereas reinduction of the gene suppresses tumour growth [10,11].

Recent publications report that the p53 gene, which shows a specific mutation pattern, may play an important role in rheumatoid synovial hyperplasia [12–15]. In contrast, a detailed investigation of p53 gene mutations has failed to detect any mutations in synovial fibroblasts from RA patients [16]. At present, whether p53 mutations occur in the rheumatoid synovium remains to be determined. Furthermore, the physiological function of endogenous p53 expressed in rheumatoid synovium has not been clarified. In the present report we show that p53 expression is inducible in rheumatoid synovial fibroblasts by inhibiting proteasome. Furthermore, this endogenous p53 protein accumulation interfered with the growth factor-mediated cell cycle progression of rheumatoid synovial fibroblasts.

Materials and method

Antibodies and reagents

Mouse anti‐human p53 (DO-7), anti‐human p21 (F-5) and anti‐human Rb (G3–245) monoclonal antibodies were purchased from PharMingen Japan (Osaka, Japan). Rabbit anti‐phospho-MEK1 and anti‐phospho-ERK1/2 were purchased from New England Biolabs (Boston, MA, USA). MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) and PSI (carbobenzoxy-l-Isoleucyl-l-t-Butyl-l-Glutamyl–l-Alanyl-l-Leucinal) were obtained from the Peptide Institute (Osaka, Japan).

Synovial cell culture

Synovial tissue samples from eight patients with RA and one patient with OA were obtained at the time of surgery for total knee replacement. All patients fulfilled the American College of Rheumatology criteria for RA. The experimental protocol was approved by the local ethics committee and a signed consent form was obtained from each patient. Synovial tissue samples were obtained from patients with RA during synovectomy. The synovial membranes were minced aseptically, then dissociated enzymatically using collagenase (4·0 mg/ml, Sigma) in RPMI 1640 for 4 h at 37°C. The obtained cells were plated on culture dishes and allowed to adhere. To eliminate non-adherent cells from synovial cell preparations, the plated cells were cultured for 18 h with RPMI 1640 supplemented with 10% FCS at 37°C in humidified 5% CO₂ in air. Cells were then washed thoroughly with phosphate-buffered saline (PBS) solution. Adherent synovial cells were removed by adding trypsin-EDTA followed by washing with PBS containing 2% FCS. Collected synovial cells were used at the third or fourth passages for subsequent experiments. Synovial cell preparations were less than 1% reactive with monoclonal antibodies CD3, CD20, CD68 (Coulter Immunology, FL, USA) and anti‐human von Willebrand factor (Immunotech, Marseille, France), indicating that these preparations were almost free of mature T lymphocytes, B lymphocytes, monocytes/macrophages and vascular endothelial cells.

Analysis of cell cycle

Synovial cells (5 × 10⁵) were plated in culture dishes (Falcon 3003, Becton Dickinson) in RPMI 1640 supplemented with 10% FCS. For serum starvation, cells were washed with PBS and maintained in RPMI 1640 with 0·3% BSA for 24 h. After starvation and washing the medium, synovial cells were stimulated with PDGF (50 ng/ml) in the presence or absence of MG132 for 24 h. Synovial cells were fixed at − 20°C with 70% ethanol, then washed and incubated at 37°C for 30 min with ribonuclease (100 µg/ml, Sigma). After centrifugation, cells were resuspended in 2·0 ml of propidium iodide (100 µg/ml, Sigma) in PBS for at least 1 h then analysed by flow cytometry. An argon-ion laser flow cytometer (Profile model, Coulter) was used, with an excitation of 488 nm. Red fluorescence was collected using a photomultiplier masked with a 610-nm long-band pass filter. Cells (2 × 10⁴) were collected at a sample flow rate of 10 µl/min.

Western blot analysis

Synovial cells were cultured in the presence or absence of MG132 for 24 h, In some experiments, synovial cells were starved by serum-free medium in the presence or absence of MG132 for 24 h. After starvation, synovial cells were stimulated with PDGF (50 ng/ml). Cells were washed with cold PBS and lysed by the addition of a lysis buffer (1% Nonidet-P 40, 0·1% SDS, 50 mm Tris, pH 7·5, 150 mm NaCl, 50 mm NaF, 5 mm EDTA, 20 mm β-glycerophosphate, 1·0 µm sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin) for 20 min at 4°C. Insoluble material was removed by centrifugation at 15 000 g for 15 min at 4°C. The supernatant was saved and the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). An identical amount of protein (50 µg) for each lysate was subjected to 10% SDS-PAGE. Western blotting was performed with the following primary antibodies at indicated dilutions: anti‐p53 (1 : 1000), anti‐p21 (1 : 2000), anti-Rb (1 : 500), anti‐phospho-MEK1 (1 : 2000) and anti‐phospho-ERK1/2 (1 : 4000), followed by horseradish peroxidase-conjugated anti‐mouse or anti‐rabbit IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and were developed with ECL Western blotting kit (Amersham, Arlington Heights, IL, USA).

Results

Recent studies have indicated that the proteasome-dependent proteolysis takes place in the turnover of wild-type p53 [17]. To evaluate the involvement of proteasome in p53 degradation, endogenous p53 levels were examined in the rheumatoid synovial cells exposed to specific proteasome inhibitor MG132. p53 protein expression was analysed by Western blot analysis. As shown in Fig. 1a, non-treated synovial cells express small amounts of p53. In contrast, p53 protein expression was markedly increased in MG132-treated synovial cells. This MG132-mediated p53 induction was observed in all synovial cells isolated from three different RA patients. As well, the accumulation of p53 was dependent upon the concentrations of MG132 and reached a maximum above 5 µm of MG132 (Fig. 1b). We performed the same experiments using the synovial fibroblasts isolated from OA patients. Similarly, p53 was up-regulated by proteasome inhibitors in OA synovial fibroblasts (Fig. 1c). This p53 induction was also observed when synovial fibroblasts were treated with an another proteasome inhibitor, PSI (Fig. 1d).

Fig. 1.

p53 protein expression of rheumatoid synovial cells. (a) Rheumatoid synovial fibroblasts isolated from three different RA patients (RA1, 2, 3) were cultured with a proteasome inhibitor, MG132 (5 µm), for 24 h. Cells were lysed and cellular lysates were analysed by anti‐p53 Western blot. (b) Rheumatoid synovial fibroblasts were cultured with various concentrations of MG132 for 24 h. Cells were lysed and cellular lysates were analysed by anti‐p53 Western blot. (c) Synovial fibroblasts isolated rheumatoid arthritis (RA) or osteoarthritis (OA) were cultured with various concentrations of MG132 for 24 h. Cells were lysed and cellular lysates were analysed by antip53 Western blot. (d) Rheumatoid synovial fibroblasts were cultured with PSI or MG132 for 24 h. Cells were lysed and cellular lysates were analysed by anti‐p53 Western blot.

p21 is transcriptionally up-regulated by p53 and its induction is suggested to be involved in the p53-mediated growth arrest. To test whether MG132 induces p21 through the induction of p53, we examined the effects of MG132 on the levels of p21 in rheumatoid synovial cells. Although p21 expression was not detected in non-treated synovial cells, its expression was induced in MG132-treated synovial cells in a dose-dependent manner (Fig. 2). p21 has been shown to block cell cycle progression by inhibiting cyclin-dependent kinase (CDK) complex. Therefore, we investigated the effects of MG132 on the cell cycle progression of PDGF-stimulated synovial cells. Synovial cells were synchronized in the early G1 phase by starvation for 24 h. Quiescent cells were stimulated with 50 ng/ml of PDGF for another 24 h. Although PDGF stimulation induced cell cycle progression, MG132 produced a virtual inhibition of S-phase entry and arrested the cell cycle progression of synovial cells in G1 phase (Fig. 3).

Fig. 2.

Accumulation of p21 in rheumatoid synovial cells treated with proteasome inhibitor. Rheumatoid synovial fibroblasts were cultured with various concentrations of MG132 for 24 h. Cells were lysed and cellular lysates were analysed by anti‐p21 Western blot. Results shown are representative examples from four independent experiments.

Fig. 3.

MG132 arrests platelet-derived growth factor (PDGF)-induced cell cycle progression of rheumatoid synovial cells. Rheumatoid synovial fibroblasts were pretreated with or without MG132 (5 µm) in serum free condition for 24 h. The cells were stimulated with PDGF (50 ng/ml) for 24 h. Cell cycle analysis was performed as described in Material and methods. Results shown are representative examples from three independent experiments.

It is generally accepted that p21 is a potent inhibitor of CDK and can inhibit the phosphorylation of the retinoblastoma gene product, pRb. We next examined the phosphorylation state of pRb of synovial cells in response to PDGF. PDGF treatment led to the phosphorylation of pRb, resulting in a shift to lower electrophoretic mobility. However, in the presence of MG132, we observed the hypophosphorylated pRb and PDGF-induced pRb phosphorylation was abolished in synovial cells (Fig. 4).

Fig. 4.

Effects of MG132 on pRb phosphorylation of PDGF-stimulated rheumatoid synovial cells. Rheumatoid synovial fibroblasts were pretreated with or without MG132 (5 µm) in serum-free conditions for 24 h. The cells were stimulated with PDGF (50 ng/ml) for 12 h. Cells were lysed and cellular lysates were analysed by anti‐pRb Western blot. The arrows indicate hyperphosphorylated (114 KD) forms of pRb. Results shown are representative examples from four independent experiments.

Finally, we determined whether MG132 blocks PDGF-induced cell cycle progression by affecting other growth-promoting signal transduction pathways. PDGF stimulation induced the phosphorylation of MEK1 and ERK1/2 (Fig. 5), which are involved in cell growth or survival [18]. MG132 treatment did not influence this PDGF-induced MEK or ERK1/2 activation. This result suggests that MG132 may arrest cell cycle progression by inducing cyclin-dependent kinase (CDK) inhibitor, p21, but not by affecting other growth promoting signals.

Fig. 5.

MG132 did not affect PDGF-induced ERK1/2 and MEK1 kinase activation of PDGF-stimulated rheumatoid synovial cells. Rheumatoid synovial fibroblasts were pretreated with or without MG132 (5 µm) in serum free condition for 24 h. The cells were stimulated with PDGF (50 ng/ml) for 5 min. Cells were lysed and cellular lysates were analysed by anti‐phospho-ERK1/2 (a) or anti‐phospho-MEK1 (b) Western blot. Results shown are representative examples from three independent experiments.

Discussion

Rheumatoid arthritis (RA) is a disease of unknown aetiology which involves excessive growth of the synovial membrane, leading to destruction of cartilage and bone [1]. The molecular and cellular basis of this synovial hyperplasia is characterized by abnormal expression of oncogenes and tumour suppressor genes modulating cellular proliferations [19]. p53 is a tightly regulated transcription factor that induces cell cycle arrest or apoptosis in response to cellular stresses such as DNA damage [6]. Inactivation of p53 function may lead to dysregulated cell proliferation and resistance to cell death [11]. High levels of p53 expression have been reported in the synovial membranes of patients with long-standing RA [13]. p53 gene mutation in these tissues were also demonstrated [12,13]. Moreover, p53 mutation was shown to be dominant negative and to suppress wild-type p53, suggesting an explanation for the tumour-like growth of synoviocytes [20]. However, an another study did not detect high p53 expression [21]. More recently, Kullmann et al. reported that no p53 mutation was detected in synovial fibroblasts from European RA patients [16]. At present it is unclear whether p53 is mutated or overexpressed in the RA synovium. Moreover, the regulation of endogenous p53 expression in rheumatoid synovium is poorly understood, and there is no information on physiological function of endogenous p53 expressed in rheumatoid synovial cells.

In the present study, we investigated the roles of endogenous p53 in rheumatoid synovial fibroblasts. p53 is normally degraded by the ubiquitin–proteasome system [22]. Therefore, proteasome inhibitors are conventional tools for the induction of endogenous p53, thereby permitting studies to assess its function in the rheumatoid synovium. We demonstrated that rheumatoid synovial fibroblasts are capable of expressing p53 by inhibiting proteasome. Moreover, this induction of endogenous p53 resulted in up-regulated p21 expression. It was demonstrated that the expression of p21 is inducible by wild-type but not by mutated p53 [8]. These results indicate that endogenous p53, which is inducible in rheumatoid synovial cells, has a normal function and could be involved in the regulation of synovial cell growth.

p53 is normally degraded by proteasome through the interaction with MDM2 and p53 phosphorylation is implicated in its stability, which is associated with the inhibition of MDN2-mediated p53 degradation [23]. p53 is phosphorylated by stress-activated protein kinases, such as JNK [24], which have been shown to be activated in rheumatoid synovium [25]. Therefore, it is possible that activated stress-activated protein kinases may regulate synovial overgrowth by stabilizing antiproliferative protein, p53.

p21^WALF/CIP1, a transcriptional target of P53, blocks cell cycle progression by inhibiting CDK that phosphorylates and activates the retinoblastoma gene product, Rb [8]. Phosphorylated Rb activates E2F transcriptional factor complexes that are required for cell cycle progression [9]. To determine whether accumulated p21 induced by proteasome inhibitor are biologically active, we analysed the cell cycle progression of PDGF-stimulated rheumatoid synovial fibroblasts. Consistent with the function of p21, we found that the induction of p21 resulted in cell cycle arrest at G1 phase. As a consequence, this p21 induction was associated with the blockage of Rb phosphorylation of PDGF-stimulated rheumatoid synovial fibroblasts. Present studies indicate clearly that the regulated degradation of p53, which is the substrate for ubiqutin–proteasome, is linked to the control of synovial cell proliferation. In contrast, it appears possible that proteasome inhibition may act on growth-promoting signal pathways such as MAP kinases via other molecules. However, the inhibition of proteasome did not affect PDGF-induced MEK or ERK1/2 kinase activation.

The therapeutic strategy of RA is to suppress synovial overgrowth, since it forms the pannus that invades the cartilage of affected joints. Therefore, the blockage of cell cycle progression should effectively prevent the articular destruction observed in RA. In the present study, we found that treatment with proteasome inhibitor resulted in p53 accumulation in rheumatoid synovial cells. This endogenous p53 was biologically active based on the finding of p21 induction and cell cycle arrest.