Inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway in rheumatoid synovial fibroblasts using small molecule compounds

Authors


Summary

Janus kinase (JAK) inhibitors have been developed as anti-inflammatory agents and have demonstrated clinical efficacy in rheumatoid arthritis (RA). We investigated if JAK-3-selective inhibition alone could disrupt cytokine signalling in rheumatoid synovial fibroblasts. In-vitro studies were performed using synovial fibroblasts isolated from patients with RA. Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target-gene expression levels were measured by reverse transcription–polymerase chain reaction (RT–PCR) or real-time PCR. The JAK inhibitors CP-690,550 and INCB028050 both suppressed activation of JAK-1/-2/-3 and downstream STAT-1/-3/-5, as well as the expression levels of target proinflammatory genes (MCP-I, SAA1/2) in oncostatin-M (OSM)-stimulated rheumatoid synovial fibroblasts. In contrast, the JAK-3-selective inhibitor, PF-956980, suppressed STAT-1/-5 activation but did not affect STAT-3 activation in OSM-stimulated rheumatoid synovial fibroblasts. In addition, PF-956980 significantly suppressed MCP-1 gene expression, but did not block SAA1/2 gene expression in OSM-stimulated rheumatoid synovial fibroblasts. These data suggest that JAK-3-selective inhibition alone is insufficient to control STAT-3-dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK-1/-2, is needed to control the proinflammatory cascade in RA.

Introduction

The Janus kinase (JAK) family of cytoplasmic tyrosine kinases mediates signalling by association with type 1 and type II cytokine receptors [1]. JAK activation leads to activation of their downstream substrates, the signal transducer and activator of transcription (STAT) proteins, followed by their nuclear translocation and subsequent activation of target genes [2]. Dysfunctional JAK/STAT signalling has been implicated in various haematological and immunological disorders [3] and other pathological inflammatory conditions, such as rheumatoid arthritis (RA) [4]. Because JAKs play an essential role in cellular signalling pathways involved in regulating the immune and inflammatory process [5, 6], targeting of the JAK family members may cause immunosuppression or anti-inflammatory effects [7]. Clarification of the modification of downstream signalling cascades induced by JAK inhibition is thus important for elucidating the molecular mechanisms whereby JAK inhibitors might exert their beneficial effects against RA. JAK-3 is important in proinflammatory cytokine-mediated signalling [8, 9], which is involved in the pathogenesis of RA. The use of kinase inhibitors with wide-ranging effects on immune/inflammatory mediators may have a more beneficial response than biological agents that target a single cytokine [10, 11].

Small-molecule inhibitors of JAKs are emerging as promising therapies for RA [12]. However, the inhibitory activities responsible for the beneficial effects of these inhibitors against RA are unknown. The JAK-3 inhibitor CP-690,550 has demonstrated efficacy in clinical trials of RA [13-15]. Although CP-690,550 inhibits JAK-3, it also exerts overlapping activities against JAK-1 and JAK-2 [16]. Moreover, the potent selective JAK-1/-2 inhibitor INCB028050 was shown to be efficacious in animal models relevant to RA and in clinical trials in patients with RA [17]. These findings raise the question of whether inhibition of JAK-3 alone is sufficient to disrupt cytokine signalling and ameliorate the rheumatoid inflammatory processes. Although the importance of JAK-3 in the development and activation of the lymphoid lineage has been well characterized [5, 6], its role in non-lymphoid-cell activation has not been explored fully. We therefore analysed the role of JAK-3 in rheumatoid synovitis using synovial fibroblasts isolated from patients with RA.

Material and methods

Reagents

JAK inhibitors, CP-690,550 and INCB028050 were obtained from Sellck (Houston, TX, USA). PF-956980 was obtained from Sigma-Aldrich Japan (Tokyo, Japan). Human oncostain-M (OSM) was purchased from Peprotech (Rocky Hills, NJ, USA). Phosphospecific antibodies against JAK-1 (Tyr1022/1023), JAK-2 (Tyr1007/1008), STAT-1 (Tyr701), STAT-3 (Tyr705) and STAT-5 (Tyr694) were purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphospecific antibody against JAK-3 (Tyr980) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunohistochemistry

For immunohistochemical analysis, formalin-fixed and paraffin-embedded tissue blocks were cut into 4-μm-thick sections. The sections were deparaffinized in xylene and subsequently rehydrated in sequential ethanol (100–70%). After washing three times with 10 mM phosphate-buffered saline (PBS, pH 7·4), antigen retrieval was carried out in a microwave at 95°C for 20 min in 10 mM citrate buffer (pH 6·0), then by washing three times in PBS for 10 min. The sections were treated with peroxidase-blocking solution (Dako Japan, Kyoto, Japan) for 5 min, and incubated with 1:1000 dilution of anti-phospho-JAK-1,-2,-3, anti-CD3, CD68 and anti-vimentin (Dako Japan) antibodies. A standardized two-step method with Envision plus (Dako) was used for detection. The reaction products were visualized using diaminobenzidine as a chromogen (Dako) and counterstained with Mayer's haematoxylin (Dako).

Preparation of rheumatoid synovial fibroblasts

Synovial tissue was obtained from patients with RA or osteoarthritis (OA) at the time of total joint replacement or synovectomy. Synovium was minced and incubated with 1 mg/ml collagenase type VIII (Sigma-Aldrich, St Louis, MO, USA) in serum-free RPMI-1640 medium (Life Technologies, Grand Island, NY, USA) for 1 h at 37°C, filtered, washed extensively and cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO2 atmosphere. Fibroblast-like synoviocytes (synovial fibroblasts) were used from passages 4 to 7, during which time they are a homogeneous population of cells (<1% CD45-positive). The whole study was approved by the Ethics Committees Nagasaki Medical Center and informed consent was obtained from each of the individuals.

Cell lysis and Western blotting

Serum-starved rheumatoid synovial fibroblasts were stimulated with OSM for the times indicated in the figure legends and the cells were washed with ice-cold PBS and lysed with a lysis buffer [1% Nonidet P 40, 50 mM Tris, pH 7·5, 100 mM NaCl, 50 mM NaF, 5 mM ethylenediamine tetraacetic acid (EDTA), 20 mM β-glycerophosphate, 1·0 mM sodium orthovanadate, 10 μg/ml aprotinin and 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, Hercules, CA, USA). An identical amount of protein (50 μg) for each lysate was subjected to 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. Western blot analysis using phosphospecific anti-JAKs and STATs antibodies was performed with an ECL Western blotting kit (Amersham, Little Chalfont, UK).

Reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was extracted from fibroblast-like synoviocytes (FLS) using the RNeasy total RNA isolation protocol (Qiagen, Crawley, UK). Total cellular RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. First-strand cDNA was synthesized from 1 μg of total cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. Thereafter, cDNA was amplified using specific primers for acute phase-SAA (SAA1 + SAA2), respectively. The specific primers used were as follows: A-SAA: forward primer 5′-CGAAGCTTCTTTTCGTTCCTT-3′, reverse primer 5′-CAGGCCAGCAGGTCGGAAGTG-3′; β-actin; and forward primer 5′-GTGGGGCGCCCCAGGCACCA-3′, reverse primer 5′-CTCCTTAATGTCACGCACGATTTC-3′.

The product sizes were 300 base pairs (bp) for A-SAA and 234 bp for β-actin. The thermocycling conditions (35 cycles) for the targets were as 94°C for 60 s and 53°C for 60 s, and 72°C for 60 s. The PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.

The amplification of the MCP-1 transcripts was performed on a Light Cycler (Roche Diagnostics, Mannheim, Germany) using specific primers. The housekeeping gene fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for verification of equal loading.

Results

JAK-3 phosphorylation status in synovial infiltrating cells

To study the role of the JAK-3 pathway in rheumatoid synovitis, we examined JAK-3 phosphorylation levels using immunohistochemical staining of synovial tissues isolated from RA and OA patients. Fig. 1a shows a representative section of synovial tissues from seven independent patients with RA and two with OA. Brown phospho-JAK-3 staining was observed in the rheumatoid synovium, indicating that infiltrating mononuclear cells in the synovial sublining area expressed high levels of phospho-JAK-3. In contrast, few infiltrating cells in the OA synovium expressed phospho-JAK-3. In immunohistochemical analysis using the serial sections, the immunophenotype of the infiltrates expressing phospho-JAK-3 was found to be predominantly CD3+ T cells, however, some of which expressed vimentin partiality in sublining infiltrating cells (Fig. 1b). These findings indicate that in addition to lymphoid cells, infiltrating non-lymphoid cells, such as fibroblasts, expressed phospho-JAK-3.

Figure 1.

Phospho-Janus kinase (JAK)-3 expressions in synovial tissues. (a) Synovial tissue sections obtained from independent rheumatoid arthritis (RA) patients (n = 7) and osteoarthritis (OA) patients (n = 2) were stained using antibodies that were specific for phospho-JAK-3 protein. (original magnification ×200). (b) Staining synovial tissue sections with anti-CD3, anti-CD68 and anti-vimentin antibodies showed that phospho-JAK-3-expressing cells were CD3+ T cells and vimentin+ synovial fibroblasts. (original magnification ×200). A representative result of three independent experiments.

JAK inhibitors CP-690,550 and INCB028050 inhibit OSM-induced JAK-3 activation

To elucidate the role of JAK-3 phosphorylation, we examined the effects of JAK-3 inhibition in cytokine-stimulated rheumatoid synovial fibroblasts in vitro. OSM has been shown to activate synoviocytes to produce proinflammatory mediators, and JAK-3 phosphorylation has been demonstrated in OSM-stimulated synovial fibroblasts [18].

CP-690,550 is a potent inhibitor of JAK-3, while ICNB028050 is a selective JAK1/2 inhibitor. We therefore examined the effects of JAK-3 inhibition on OSM-induced synovial fibroblasts by comparing their differential effects on JAK/STAT signalling. We stimulated synovial fibroblasts with OSM to activate JAK1/2/3. OSM stimulation also induced STAT-1/-3/-5 phosphorylation in synovial fibroblasts. CP-690,550 blocked OSM-induced JAK-1/-2/-3 and STAT-1/-3/-5 phosphorylation. Unexpectedly, INCB028050 also inhibited JAK-3 in addition to JAK-1/-2 (Fig. 2). These findings demonstrate that CP-690,550 and INCB028050 had similar potencies in terms of suppressing JAK-3, as well as JAK-1/-2 and downstream STAT-1/-3/-5.

Figure 2.

Janus kinase (JAK) inhibitors suppressed oncostatin-M (OSM)-induced JAK-1/-2/-3 activations in rheumatoid synovial fibroblasts. Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550 or INCB028050 or 2 h, then stimulated with OSM (20 ng/ml) for 20 min. Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Biological effects of JAK-3 inhibition in rheumatoid synovial fibroblasts

We assessed the role of JAK-3 in the biological functions of rheumatoid synovial fibroblasts using the JAK-3-specific inhibitor PF-956980 [19]. To evaluate the selectivity of JAK family inhibition, we compared the effects of PF-956980 with CP-690,550 and INCB028050 in OSM-stimulated synovial fibroblasts. As shown in Fig. 3, PF-956980 efficiently blocked OSM-induced JAK-3 phosphorylation, but had no effect on OSM-induced JAK-1 or JAK-2 phosphorylation in synovial fibroblasts. In contrast, CP-690,550 and INCB028050 blocked JAK-1/-2/-3 phosphorylation in OSM-stimulated synovial fibroblasts. We further examined the effect of JAK-3 inhibition on downstream STATs activation. Selective JAK-3 inhibition caused by PF-956980 prevented OSM-induced STAT-1/-5 activation, but had no effect on OSM-induced STAT-3 activation. In contrast, CP-690,550 and INCB028050 pretreatments blocked activation of all STAT members (STAT-1, -3 and -5) in synovial fibroblasts (Fig. 3). These results suggest that pharmacological JAK-1/-2 inhibition specifically blocks downstream STAT-3 activation, which is involved in the proinflammatory pathway.

Figure 3.

PF956980 suppressed oncostatin-M (OSM)-induced Janus kinase (JAK)-3 activation in rheumatoid synovial fibroblasts. Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, 028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 20 min. Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Biological effects of JAK inhibition on OSM-induced gene expressions

OSM induces gene expression of cytokines/chemokines or acute phase serum amyloid A (SAA) [20, 21]. To gain insights into the mechanism of OSM signalling leading to induction of MCP-I and acute-phase SAA1/2 gene expression, we extracted total RNA from synovial fibroblasts after treatment with OSM or OSM plus JAK inhibitors for 6 h and subjected it to PCR analysis. As shown in Fig. 4a, PF-956980, CP-690,550 and INCB028050 suppressed OSM-induced MCP-I gene expression. However, although CP-690,550 and INCB028050 also completely blocked OSM-induced SAA1/2 mRNA induction, PF-982560 failed to suppress this induction (Fig. 4b). These results are in agreement with previous reports that demonstrated a pivotal role for STAT-3 in SAA1 induction [22], and suggest that the STAT-3 pathway is a key signalling mediator for acute-phase SAA induction by interleukin (IL)-6-type cytokines.

Figure 4.

PF956980 did not inhibit oncostatin-M (OSM)-induced A-SAA mRNA expression in synovial fibroblasts. (a) Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, INCB028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 6 h, after which, MCP-1 and GAPDH mRNA expression was determined by the real-time polymerase chain reaction (PCR) method. *P < 0·01 compared to OSM-stimulated synovial fibroblasts; **P < 0·0001 compared to OSM-stimulated synovial fibroblasts. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown. (b) Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, INCB028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 6 h. A-SAA mRNA expression was analysed by PCR following reverse transcription. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Biological effects of JAK inhibition in osteoarthritis synovial fibroblasts

Finally, we examined the biological effects of JAK inhibition using OA synovial fibroblasts. As shown in Fig. 5, phospho-JAK-2 staining was observed in monocyte-like cells and phospho-JAK-3 was observed in infiltrating mononuclear cells into rheumatoid synovial tissues. Whereas phospho-JAK-2 staining was barely detected in synovial tissues isolated from OA patients. When synovial fibroblasts isolated from OA synovial tissues were stimulated with OSM, phosphorylation of JAK-1/-2/-, as well as STAT-1/-3/-5, was observed. CP-690,550 or INCB028050 pretreatment efficiently blocked OSM-induced JAK-1/-2/-3 and downstream STAT-1/-3/-5 phosphorylation (Fig. 6).

Figure 5.

Phospho-Janus kinase (JAK)-1/-2/-3 expressions in synovial tissues. Synovial tissue sections obtained from rheumatoid arthritis (RA) (n = 1) or osteoarthritis (OA) patients (n = 1) were stained using antibodies that were specific for phospho-JAK-1, JAK-2 and JAK-3 protein (original magnification ×200).

Figure 6.

Janus kinase (JAK) inhibitors suppressed oncostatin-M (OSM)-induced JAK-1/-2/-3 activations in osteoarthritis (OA) synovial fibroblasts. Quiescent synovial fibroblasts isolated from OA patients were pretreated with various concentrations of CP-690,550 or INCB028050 for 2 h, then stimulated with OSM (20 ng/ml). Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Two experiments were performed using different OA synovial fibroblasts and a representative result is shown.

Discussion

Several JAK inhibitors are currently in development for therapy of RA [23]. JAK-3 expression is restricted to immune cells, and selective JAK-3 inhibition thus represents a potential new strategy for immunosuppression [10]. The clinical efficacy of CP-690,550 for treating RA suggests that targeting JAK-3 is useful for suppressing autoimmune, as well as inflammatory diseases [7]. The inhibition of JAK-3 signalling in lymphocytes has been the main focus of research [24], and little is known about the effects of JAK inhibitors on the innate immune system. In addition to myeloid cells, such as lymphocytes and monocytes, rheumatoid synovial fibroblasts have also been shown to express phospho-JAK-3 in vivo. OSM, an IL-6-type proinflammatory cytokine, is a multi-functional cytokine affecting the growth and differentiation of numerous cell types [25]. It is produced by activated T lymphocytes and monocytes, and can induce the expression of various proinflammatory molecules [26]. It is present in the synovial fluid of RA patients and has been implicated in rheumatoid synovitis [27]. OSM had been shown to activate JAK and STAT pathways in primary human rheumatoid synoviocyte systems [18]. However, the mechanisms resulting in JAK activation and the downstream signalling events whereby active STATs may lead to rheumatoid inflammatory processes are still unclear. Because OSM is likely to play a role in rheumatoid inflammation, we used this cytokine to analyse the mechanisms by which cytokine signalling contributes to inflammatory cascades, and to establish the feasibility of using JAK inhibitors to control inflammation. Previous reports suggested a role for CP-690,550-mediated T cell signalling blockade [28]. It is also possible that inhibition of non-lymphoid cells, such as synovial cells, may contribute to the efficacy of JAK inhibitors. Using a primary rheumatoid synovial fibroblast culture system, we investigated the effects of specific JAK inhibition on proinflammatory signalling. The results of the current study showed that the JAK inhibitors, CP-690,550 and INCB028050, effectively inhibited OSM-induced JAK-1/-2/-3 activation and subsequent STAT-1/-3/-5 activation in rheumatoid synovial fibroblasts, suggesting that CP-690,550 and INCB028050 are pan-JAK inhibitors that inhibit the JAK/STAT signalling in synovium, thus contributing to the efficacy of RA treatment. However, identification of the JAK responsible for the therapeutic effectiveness of JAK inhibitors against rheumatoid synovitis remains a key question. CP-690,550 and INCB028050 both blocked OSM-induced JAK-1/-2/-3 phosphorylation, as well as STAT-3 activation and subsequent acute-phase SAA mRNA expression. In contrast, the JAK-3-selective inhibitor, PF-956980, failed to inhibit OSM-induced STAT-3 activation and acute-phase SAA mRNA expression. In addition to STAT-3, STAT-1 and STAT-5 have also been shown to exert potent immune-activation actions and to contribute to rheumatoid synovitis [29]. In agreement with previous reports, this study showed that JAK-3 plays an important role in downstream STAT-1/-5 activation and subsequent MCP-I mRNA expression [20]. However, JAK-3 inhibition alone was insufficient to control STAT-3-mediated proinflammatory cascades.

JAKs are fundamental components of diverse signalling pathways, including immune cells [30]. It appears likely that this new class of immunomodulatory drug will have an impact on the treatment of immune-mediated diseases. In relation to JAK-specific inhibition, CP-690,550 was reported recently to have modest selectivity against JAK-1/-2 in addition to JAK-3 [16], while the JAK-1- and JAK-2-selective inhibitor INCB028050 has also demonstrated efficacy in an RA mouse model mice, as well as in the treatment of RA [17]. These findings suggest that JAK-1/-2 signalling may also contribute to the rheumatoid proinflammatory process, and that pan-JAK inhibitors also effectively suppress STAT-3-mediated rheumatoid inflammation. Our results revealed that selective inhibition of JAK-3 alone resulted in abortive STAT-1/-5 activation in rheumatoid synoviocytes, but did not affect OSM-induced STAT-3 activation. Additionally, JAK-3-selective inhibition did not down-regulate OSM-induced acute-phase SAA mRNA expression, in which STAT-3 activation plays a critical role [22]. Research into JAK inhibitors is at an interesting phase, with several selective and non-selective inhibitors in various stages of clinical trials [31]. It seems logical to target a single JAK, if possible, in order to minimize the adverse effects [32]. However, non-selective JAK inhibitors may have advantages against multi-factorial disorders with proinflammatory characteristics.

In conclusion, the results of this study indicate that JAK inhibition can affect multiple steps of cytokine-induced proinflammatory pathways by targeting downstream STATs in rheumatoid synovial fibroblasts. However, suppression of JAK-3 alone did not affect STAT-3 activation or STAT-3-dependent proinflammatory gene expression. These results suggest that the proinflammatory responses induced by IL-6-type cytokines may be blocked by non-selective JAK inhibitors such as CP-690,550 and INCB028050.

Acknowledgements

This work was supported by a grant from the Japan Society for the Promotion of Science (Grant-in-Aid for Scientist Research C23591452).

Disclosures

The authors declare that they have no competing interests.

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