Adenosine inhibits TNFα-induced MMP-3 Production in MH7A RA Synoviocytes via A2A Receptor Signaling

Adenosine is the effector molecule mediating the antirheumatic effects of methotrexate, but the contributions of synoviocyte adenosine receptors (AdoRs) are unknown. Matrix metalloproteinase 3 (MMP-3) is released by broblast-like synoviocytes in response to inammatory signaling, and serum MMP-3 is elevated during active rheumatoid arthritis. To elucidate the therapeutic mechanisms of methotrexate, we investigated the effects of A 2A AdoR activation and inhibition on tumor necrosis factor-alpha (TNFa)-induced MMP-3 release by MH7A human rheumatoid synovial cells.


Background
Rheumatoid arthritis (RA) is a chronic in ammatory disorder characterized by joint pain, stiffness, and immobility, typically starting in smaller peripheral joints and eventually a icting the larger joints [1]. While the underlying etiology is still unclear, these symptoms are associated with in ammatory proliferation of synovial cells induced by cytokines released from in ltrating lymphocytes and macrophages, eventually resulting in pannus formation and joint destruction [2,3].
Matrix metalloproteinases (MMPs) are a family of enzymes that catalyze the degradation of extracellular matrix. Most MMPs are secreted as inactive preproteins that are activated when cleaved by extracellular proteinase [4]. The human enzyme MMP-3, also known as stromelysin-1, is expressed and secreted by broblast-like synoviocytes (FLSs) and chondrocytes within joints upon stimulation by in ammatory cytokines such as TNFa [5]. Once released and activated, MMP-3 degrades a wide variety of extracellular matrix proteins, including collagen types II, III, IV, IX, and X, proteoglycan, bronectin, laminin, and elastin [6]. Moreover, MMP-3 can activate several other MMPs, including MMP-1, MMP-7, and MMP-9, thereby amplifying matrix proteolysis [7]. It is thought that these proteases contribute to joint destruction in RA by degrading cartilage extracellular matrix [8,9]. Indeed, joint MMP-3 concentration is markedly elevated in RA compared to other MMPs [8,10]. Further, increased production by FLS cells is frequently associated with a parallel increase in serum MMP-3 concentration. Elevated serum MMP-3 is observed in 80%-90% of RA patients, and re ects the degree of synoviocyte proliferation. Thus, serum MMP-3 may be a prognostic indicator of disease progression, especially in the early phase of RA [11]. When serum MMP-3 concentrations are high, joint destruction is expected to progress rapidly. Conversely, MMP-3 levels decrease when the condition stabilizes in response to antirheumatic drugs, including biologics [12].
Methotrexate (MTX) is recommended as a rst-line drug for the initial treatment of RA [13,14]. The antirheumatic effects of MTX are thought to be mediated by both inhibition of dihydrofolate reductase [15,16] and by increasing the release of adenosine [17][18][19][20]. Dihydrofolate reductase is an enzyme that produces tetrahydrofolate required for nucleic acid synthesis, resulting in the suppression of immunocyte proliferation and enhanced apoptosis [17,18]. Adenosine is a purine metabolite produced by most cells and required for adenosine triphosphate (ATP) synthesis. Further, adenosine acts as an intercellular signaling factor by stimulating four G protein-coupled adenosine receptors (A 1 , A 2A , A 2B , and A 3 ), all of which are expressed on synoviocytes [21,22]. Among these, A 2A AdoR signaling via the G protein Gs has been shown to stimulate cAMP formation via adenylyl cyclase activation, which antagonizes T cell receptor signaling [23,24]. In addition, intracellular cAMP may modulate MMP-3 expression [25]. RA patients treated with MTX exhibited reduced blood MMP-3 levels [26], suggesting that adenosine negatively regulates MMP-3 production by FLSs. However, the speci c functions of AdoRs expressed on synovial cells have not been clari ed. We therefore investigated how adenosine A 2A AdoR signaling

Statistical analysis
All experiments were repeated at least three times, and representative results are shown. Data are presented as mean ± standard deviation (SD). Treatment group means were compared using two-tailed unpaired t-tests. A P < 0.05 (two-tailed) was considered signi cant for all tests. In accord with previous studies [27,28], MH7A cells constitutively expressed MMP-3 mRNA, and expression was reduced dose-dependently by HENECA (Figure 3). In addition, MMP-3 protein release into the extracellular medium was accelerated by TNFa stimulation (Figure 4, white bars). This response was partially inhibited in a concentration-dependent manner by co-treatment with HENECA (p < 0.05 at 10 or suppression of MMP-3 mRNA expression (Figure 3). Pretreatment of MH7A cells with the A 2A AdoR antagonist ZM241385 blocked the inhibitory effects of HENECA on TNFa-induced MMP-3 release ( Figure   4, black bars). Consistent with a contribution of A 2A AdoR/Gs/AC signaling to these responses, the cAMP analog dbcAMP also blocked TNFa-induced augmentation of MMP-3 production (p < 0.01 at 50 or 100 mM dbcAMP vs. TNFa alone) ( Figure 5). Collectively, these results indicate that adenosine signaling via the A 2A AdoR/Gs/AC/cAMP pathway can block TNFa-mediated MMP-3 production in MH7A synoviocytes.

Stimulation of A 2A AdoRs reverses TNFa-mediated activation of p38 MAPK signaling in MH7A cells
Stimulation of MH7A cells with TNFa for 10 min and 1 h signi cantly enhanced phosphorylation of p38 MAPK and ATF-2 compared to untreated negative control cells ( Figure 6A&B). Enhanced phosphorylation of both proteins by TNFa was inhibited by co-application of HENECA (although p-p38 remained above the basal level). These ndings suggest that activation of A 2A AdoR/Gs/AC/cAMP signaling by HENECA may reduce TNFa-mediated MMP-3 release by partially suppressing p38 MAPK and ATF-2 activation.

Discussion
In clinical practice, serum MMP-3 levels often remain positive despite successful MTX treatment of RA, whereas serum C-reactive protein (CRP) levels fall within the reference range when MTX treatment is successful. In the present study, we provide evidence that MMP-3 remains positive following successful MTX treatment due to incomplete (although substantial) suppression of TNFa-induced production by synovial cells via A 2A AdoR signaling.
We rst con rmed that MH7A rheumatoid synoviocytes expressed all four AdoR mRNAs expressed by primary RA FLSs [11] and that A 2A AdoR mRNA and cell-surface protein expression were markedly enhanced by TNFa, also in accordance with a previous report on primary RA FLSs [29]. Primary RA FLSs, however, may act differently in vitro due to differences in patient treatment history [30], so the RA FLS line MH7A was chosen for this study.
This study focused on A 2A AdoR for several reasons. First, expression of A 2A AdoR mRNA was dramatically enhanced by 1000 pg/ml TNFa, while expression levels of other AdoR mRNAs were not altered ( Figure 1A). Furthermore, A 2A AdoR is coupled to AC via Gs and thus activation results in intracellular cAMP accumulation and ensuing PKA signaling (Figure 3), which is reported to suppress MMP-3 production in human chondrocytes [25]. Alternatively, A 1 and A 3 AdoRs are coupled to AC via Gi, leading to reduced cAMP. The selective A 2A AdoR agonist HENECA partially suppressed TNFa-stimulated MMP-3 production (Figure 4), a response mimicked by the cell-permeable cAMP analog dbcAMP ( Figure  5). This apparent inhibitory effect was not due to HENECA, DMSO, or dbcAMP cytotoxicity, as none of these agents reduced viable cell number (data not shown). Moreover, the inhibitory effect of HENECA was signi cantly blocked by the selective A 2A AdoR antagonist ZM241385 (Figure 5), con rming that A 2A AdoR is the primary mediator of reduced TNFa-stimulated MMP-3 production.
We then provide evidence that this suppressive effect of A 2A AdoR activation stems from partial inhibition of p38 MAPK/ATF-1 signaling. In ammatory cytokines including TNFa are known to induce rapid activation of the MAPK signaling pathway, and several studies have reported that inhibition of MAPK phosphorylation suppresses MMP-3 production [31,32]. Further, p38 MAPK signaling is reportedly also involved in MMP-3 production by MH7A cells [33]. Expression levels of the AP-1 family transcription factor ATF-2 were higher in FLSs from RA patients than non-patients [34] and phosphorylation of p38 and ATF-2 drives the production of MMP-3 [35,36]. HENECA partially inhibited TNFa-induced p38 and ATF-2 phosphorylation (Figure 6), in accordance with HENECA-mediated partial inhibition of MMP-3 production by TNFa.
In RA, blood cell components such as macrophages and lymphocytes in ltrate joints and induce persistent synovitis through secretion of in ammatory cytokines, which leads to the production of MMP-3 [23,37]. In contrast, Gs-coupled A 2A AdoRs increase intracellular cAMP and suppress in ammation [38,39]. However, activation of A 2A AdoR signaling alone using HENECA did not reduce TNFa-induced MMP-3 production to basal levels, which may explain why MTX usually decreases but does not eliminate serum MMP-3 [40]. Shiozawa et al. reported that 87.0% of RA patients treated with MTX for 3 years still exhibited blood MMP-3 levels above 103.7 mg/ml as well as ongoing joint destruction (de ned as a change in van der Heijdi modi ed total Sharp score > 3.0) [26]. They also revealed that 94.3% of patients treated with MTX and still exhibiting rapid radiographic progression (de ned as a change in van der Heijdi modi ed total Sharp score > 5.0) had blood MMP-3 concentrations above 103.7 mg/ml [26]. Similarly, Ma et al. reported that patients with continuously elevated serum MMP-3 levels for 3-6 months had showed radiographic progression even when the therapeutic target, including CRP levels, was achieved [41]. Their prospective cohort study also showed that the serum MMP-3 level was signi cantly higher in progressive patients than in nonprogressive patients for an entire year and that elevated serum MMP-3 levels at baseline and the rst, third, and sixth months were signi cant predictors of 1-year radiographic progression with cutoff points of 159 ng/ml, 264 ng/ml, 178 ng/ml, and 161 ng/ml, respectively [41]. Furthermore, matrix metalloproteinase inhibitors prevented the progression of joint destruction in rats with collagen-induced arthritis [42,43]. These ndings suggest that residual MMP-3 production is a crucial contributor to further joint destruction in MTX-treated RA patients.
The results presented here show for the rst time the important contribution of FSL A 2A AdoRs signaling to the therapeutic mechanisms of MTX. These ndings may also explain, at least in part, why MTX treatment alone does not normalize blood MMP-3 in most RA patients, and suggest that additional anti-TNFa treatments may be necessary to achieve long-term remission. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All the authors have no con icts of interest to declare.

Funding
The study was funded by the Hyogo College of Medicine.
Authors' contributions HK and MK contributed to study design, acquisition and analysis of the data, and statistical calculations as well as manuscript preparation. SK, RY, MI, MS, NY, and MN contributed to study design and analysis of data. HK conducted laboratory experiments. All authors revised and approved the manuscript for submission.

43.
Ishikawa T, Nishigaki F, Miyata S, Hirayama Y, Minoura K, Imanishi J, et al. Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase Western blotting (B), respectively. Both A2A AdoR mRNA and membrane protein expression were enhanced by TNFα compared to untreated controls. ß-actin was used as an internal control. Experiments were repeated three times and representative data are shown.

Figure 2
Adenosine A2A receptors in MH7A cells are functionally coupled to adenylyl cyclase and intracellular cAMP production. Cells were stimulated for 30 min with the indicated concentration of A2A AdoR agonist HENECA. Experiments were repeated three times, and representative data are shown. Intracellular cAMP levels were measured using an enzyme immunoassay.

Figure 6
Activation of A2A receptor signaling suppressed TNFα-induced activation of p38 MAPK and ATF-2. Cells were incubated for 10 min (A) or 1 h (B) in TNFα (1 ng/ml) with or without HENECA (1 μM). Expression levels of p-p38 and p-ATF-2 were estimated by western blotting and densitometry. HENECA suppressed TNFα-induced phosphorylation (activation) of p38 MAPK (A) and ATF-2 (B). ß-actin served as the gel loading control. Experiments were repeated three times, and representative data are shown. The results are expressed as the mean ± SD. p < 0.01 vs. TNFα alone.