Extracellular adenosine 5ʹ-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes

Background Temporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear. Methods and results Reverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5ʹ-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C–C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression. Conclusion ADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.


Introduction
The temporomandibular joint (TMJ) comprises the mandibular condyle and fossa [1], the osteoarthritis (OA) of which is characterized by bone cavitation, cartilage deterioration, and fibrosis, ultimately resulting in TMJ pain and stiffness [2]. Previous histological studies revealed the pathogenesis of fibrosis in TMJ-OA [3,4], indicating that the formation of fibrotic tissue can cause restricted joint movements [5]. Moreover, Zang et al. demonstrated that chronic aseptic inflammation is associated with macrophage recruitment into inflammatory lesion, which contributes to onset of fibrosis [6].
Necroptosis cell death leads to pronounced detrimental effects on tissue homeostasis by promoting the release of damage-associated molecular patterns (DAMPs), including nucleotides, high mobility group box 1, and uric acid [7]. However, endogenous ligands of purinergic receptors, such as extracellular adenine nucleotide (ATP) and adenosine 5′-diphosphate (ADP), which function as paracrine signaling molecules, have considerable potential in both pathological and physiological control of such processes [8]. Zhou et al. reported that platelet-derived ADP functions as an important mediator that promotes chondrocyte-based cartilage repair and proliferation in OA [9]. In addition, ATP and uracil nucleotide (UTP) have been shown to stimulate calcium mobilization from intercellular stores in human rheumatoid synovial cells [10].
We previously established that the fibroblast-like synoviocyte synovial cell line FLS1 (obtained from the TMJ of mouse) exhibits myofibroblastic characteristics [11]. We also demonstrated that FLS1 cells vigorously expressed the purinergic receptors P2X 3 , P2X 7 P2Y 2 , P2Y 4 , P2Y 12 , P2Y 13 , and P2Y 14 [12]. Typically, P2X receptors comprise ion channels that mediate the passage of cations, such as sodium, potassium, or calcium; P2Y receptors couple with the G proteins involved in the modulation of cytoplasmic Ca 2+ concentrations and regulation of intracellular adenylyl cyclase [13]. Moreover, Jacobson et al. reported that ATP activates P2X receptors and that five or more nucleotides (including ATP, ADP, and UTP) activate P2Y receptors [14], whereas in macrophage lineage cells, extracellular ADP has also been found to promote the expression of monocyte chemotactic protein 1 (MCP-1)/C-C motif chemokine ligand 2 (CCL2) via extracellular signal-regulated kinase (ERK) signaling [15].
Chemokines play major roles in the selective recruitment of neutrophils, lymphocytes, and monocytes as well as in the activation of G protein-coupled receptors, among which MCP-1/CCL2 regulates the infiltration and migration of monocytes/macrophages [16]. However, it is yet to be established whether extracellular nucleotides affect the expression levels of MCP-1/CCL2 in FLSs.
In this study, we examined the mechanism by which DAMPs, such as ATP, ADP, and UTP, influence the expression of MCP-1/CCL2 in FLS1 cells. Specifically, we assessed whether ATP, ADP, and UTP affect the activity of mitogen-activated protein kinases (MAPKs), such as ERK, p38 MAPK, and c-Jun N-terminal kinase (JNK), in FLS1 cells. Moreover, we investigated whether ATP-, ADP-, or UTP-activated MAPKs influence the status of MCP-1/CCL2 expression in FLS1 cells. Our findings contribute to a better understanding of the signaling pathway that underlies the development of inflammation in TMJ-OA, and provide important insights regarding the potential therapeutic significance of TMJ-OA-related inflammatory activity.

Cell culture
FLS1 cells were cultured in Ham's F-12 medium containing 10% FBS (Invitrogen, Carlsbad, CA, USA), 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. To evaluate MCP-1/CCL2 mRNA expression, these cells were treated with different concentrations of ATP, ADP, and UTP for the indicated time periods. Prior to ADP administration, the cells were pre-treated for 30 min with P2Y purinergic receptor antagonists MRS 2179, AR-C 66096, and MRS 2211 to evaluate the effects of these antagonists on the ADP-induced promotion of MCP-1/CCL2 expression. In addition, prior to ADP stimulation, some cells were pretreated for 30 min with U0126 or SB203580.

Statistical analysis
All experiments were performed in triplicate. The data are presented as the means ± standard deviation (n = 3). Statistical significance was evaluated using Tukey's multiple comparison test using SPSS software (version 24.0). P-values were considered significant if *P < 0.01 and **P < 0.05.

ADP is more effective than ATP in promoting the expression of MCP-1/CCL2 mRNA in FLSs
As shown in Fig. 1, compared to the control cells, cells treated with ATP (100 µM) and ADP (100 µM) showed 1.57fold and 2.31-fold upregulation of MCP-1/CCL-2 mRNA expression in FLS1 cells, respectively, thereby indicating greater efficacy of ADP over ATP. In contrast, treatment with UTP (100 µM) was found to have no discernable effect on the expression of MCP-1/CCL2 mRNA in FLS1 cells.

ADP upregulates ERK1/ERK2 phosphorylation through P2Y 13 receptor in FLSs
Using western blotting, we evaluated the phosphorylation status of ERK1/2 following stimulation of FLS1 cells with ADP. As shown in Fig. 3 A, ADP (100 µM) treatment led to an upregulation of ERK1/2 phosphorylation, with peak expression levels being detected 10 min after the treatment, following which, expression levels declined between 30 and 120 min post-treatment. Moreover, we established that such ADP-promoted upregulation of ERK1/2 phosphorylation could be abrogated by the administration of U0126 (10 µM) (Fig. 3B). We also confirmed that ADP (100 µM) had no significant effects on p38 MAPK phosphorylation levels (Fig. 3 C) and that SAPK/JNK remained undetectable in FLS1 cells (Fig. 3D), even after ADP stimulation. We further found that MRS 2211 (100 µM) significantly reduced the ADP-promoted upregulation of ERK1/2 phosphorylation (Fig. 3E).

Discussion
In this study, we demonstrated that extracellularly applied ADP is more effective than ATP in promoting the expression of MCP-1/CCL2 in FLSs derived from the mouse Fig. 1 ADP promotes the expression of MCP-1/CCL2 in fibroblastlike synoviocytes. Cells were cultured with or without ATP (100 µM), ADP (100 µM), or UTP (100 µM) for 24 h. The relative expression of MCP-1/CCL2 was determined using RT-qPCR. Data are presented as the means ± standard deviation (SD) (n = 3). *P < 0.01 TMJ (Fig. 1), thereby providing evidence to indicate that P2X receptor-mediated signaling might also positively regulate MCP-1/CCL2 expression in FLSs. Given that the intercellular mechanisms underlying the ATP-mediated induction of MCP-1/CCL2 expression have yet to be identified, elucidating the mechanisms associated with ATP stimulation in FLS1 cells will be the focus of our future studies. However, we established herein that UTP has no appreciable effects on MCP-1/CCL2 expression in FLS1 cells (Fig. 1). In our previous studies, we demonstrated that UTP significantly reduces mRNA expression of the fibrogenic marker α-SMA in FLS1 cells [12]. In general, UTP preferentially binds to and activates P2Y 2 , P2Y 4 , and P2Y 6 receptors [13], and FLS1 cells strongly express P2Y 2 , P2Y 4 , P2Y 12 , P2Y 13 , and P2Y 14 receptors [12], thereby indicating that UTP enhances the expression of α-SMA mRNA via interaction with either P2Y 2 or P2Y 4 receptors in these cells. Altogether, these findings indicate that UTP-induced specific intracellular signaling mediated by P2Y 2 and P2Y 4 receptors does not enhance the expression of MCP-1/CCL2 in FLS1 cells.
In the present study, we established that ADP also promotes ERK1/2 signaling in FLS1 cells (Fig. 3 A) and confirmed that U0126, and MRS 2211 inhibits this ADP-mediated upregulation of ERK1/2 phosphorylation (Fig. 3B, and E, respectively). Moreover, U0126 partially (nevertheless significantly) abrogated the ADP-mediated upregulation of MCP-1/CCL2 mRNA expression (Fig. 4 A), thereby providing convincing evidence that ADP promotes MCP-1/CCL2 mRNA expression through P2Y 12 receptor in FLSs in a MEK/ERK-dependent manner, which positively regulates the infiltration of monocytes/macrophages into the mouse TMJ. Zang et al. demonstrated that chronic aseptic inflammation is associated with macrophage recruitment into inflammatory lesion, which contributes to onset of fibrosis [6]. Extracellular nucleotide such as ADP derived from necrotic cells in inflammatory lesion in TMJ-OA possibly induces expression of MCP-1/CCL2 in FLSs around TMJ, which leaded to the recruitment of macrophages into the inflammatory TMJ lesion, further resulting in progression of fibrosis in TMJ-OA.
Previously, Liao et al. demonstrated that the expression of interleukin-17 (IL-17), which plays an essential role in the immune system and in the development of infectious and inflammatory diseases, upregulates MCP-1/CCL2 in RAW264.7 cells via p38 MAPK [25]. Moreover, Satonaka et al. reported that JNK/SAPK inhibition attenuates the ADP-induced upregulation of MCP-1/CCL2 mRNA and protein in VSMCs [24], whereas Ip et al. demonstrated that IL-1 and IL-13 induce MCP-1/CCL2 expression in ERKand p38 MAPK-dependent manners in human bronchial epithelial cells [26]. Moreover, Wuyts et al. reported that IL-1β promotes the expression of MCP-1/CCL2 protein in ERK-, p38 MAPK-, and JNK/SAPK-dependent manners in human airway smooth muscle cells [27]. Collectively, these findings suggest that MAPK (ERK, p38 MAPK, and JNK/SAPK)mediated intracellular signaling plays important roles in the expression of MCP-1/CCL2. However, in the present study, western blot analysis enabled us to confirm that ADP (100 µM) does not significantly affect the phosphorylation of p38 MAPK in FLS1 cells (Fig. 3 C), and we failed to detect JNK/ SAPK, even in response ADP stimulation (Fig. 3D). These findings, therefore, provide a strong indication that at least in case of FLSs, ADP does not promote the expression of MCP-1/CCL2 via p38 MAPK or JNK/SAPK signaling pathways. We also established that the p38 inhibitor SB 203580 abrogates the ADP-promoted mRNA expression of MCP-1/ CCL2 (Fig. 4B), thereby indicating that the basal activity of p38 MAPK plays an important role in the ADP-induced promotion of MCP-1/CCL2 expression in FLS1 cells.
Based on the findings of this study, we identified inflammatory molecules underlying the development of stimulation. The relative expression of MCP-1/CCL2 were determined using RT-qPCR. Data are presented as the means ± SD (n = 3). *P < 0.01. **P < 0.05 inflammation in TMJ-OA, which in turn enabled us to establish the potential therapeutic significance of TMJ-OArelated inflammatory activity. Herein, we provide convincing evidence to indicate that ADP might serve as an effective molecular target for preventing OA-related inflammation around the TMJ.