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 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 . In general, UTP preferentially binds to and activates P2Y2, P2Y4, and P2Y6 receptors , and FLS1 cells strongly express P2Y2, P2Y4, P2Y12, P2Y13, and P2Y14 receptors , thereby indicating that UTP enhances the expression of α-SMA mRNA via interaction with either P2Y2 or P2Y4 receptors in these cells. Altogether, these findings indicate that UTP-induced specific intracellular signaling mediated by P2Y2 and P2Y4 receptors does not enhance the expression of MCP-1/CCL2 in FLS1 cells.
Given that ADP typically binds to P2Y1, P2Y12, and P2Y13, we sought to determine which subtypes of P2Y receptor mediate the promotive function of ADP in MCP-1/CCL2 expression. In this regard, we found that the P2Y1 antagonist MRS2179 (10–100 µM) had no effect on the ADP-promoted expression of MCP-1/CCL2 in FLSs (Fig. 2A). Previously, Bynagari et al. demonstrated that MRS2179 (100 µM) significantly downregulates the 2MeSADP-induced phosphorylation of nPKCeta in human platelets , and Atterbury-Thomas et al. reported that MRS2179 (10 µM) suppresses the ADP-induced calcium increase in mouse glial cells . These findings together indicate that MRS2179 concentrations in the range 10–100 µM would be optimal for antagonizing ADP-activated P2Y1 signaling. In addition, we also showed that the P2Y12 antagonist ARC-66096 (10–100 µM) had no effects on the ADP-promoted expression of MCP-1/CCL2 in FLSs (Fig. 2B). Bélanger et al. reported that ARC-66096 (10 µM) significantly inhibits platelet aggregation , and Quintas et al. demonstrated that ARC-66096 (10 µM) can significantly abrogate adenosine 5ʹ-O-(2-thio)-diphosphate-induced astroglial proliferation , thus indicating 10 µM to be the optimal concentration of ARC-66096 for antagonizing ADP-activated P2Y12 signaling. Notably, we established that the P2Y13 antagonist MRS2211 (50–100 µM) significantly abrogated the ADP-promoted expression of MCP-1/CCL2 in a concentration-dependent manner (Fig. 2C). In contrast, Kim et al. demonstrated that when applied at a concentration of 30 µM, MRS2211 inhibited ADP-induced inositol triphosphate production in human astrocytoma cells . In addition, Zeng et al. demonstrated that MRS2211 (100 µM) abrogated ADP-induced Ca2+ mobilization in primary cultured microglia . Therefore, the application MRS2211 in the concentration range 30–100 µM is optimal for antagonizing ADP-activated P2Y13 signaling. Collectively, these findings provide convincing evidence to indicate that extracellularly applied ADP promotes the expression of MCP-1/CCL2 mRNA via the activation of P2Y13 receptors in FLS1 cells.
Earlier, Inose et al. demonstrated that levels of MCP-1/CCL2 mRNA expression and those of its receptor CCR2 were significantly increased in response to lysophosphatidylcholine (LPC) stimulation, and that the LPC-mediated increase in MCP-1/CCL2 transcripts was reduced by blocking the P2X receptor P2X7 in a microglial-derived cell line . Moreover, Satonaka et al. reported that ADP upregulated the expression of MCP-1/CCL2 mRNA in cultured rat vascular smooth muscle cells (VSMCs), which was significantly inhibited by a P2Y12 inhibitor, thereby indicating that ADP promotes MCP-1/CCL2 mRNA expression via P2Y12 receptors in VSMCs . Taken together, these observations imply that intracellular mechanisms underlying the extracellular nucleotide-mediated regulation of MCP-1/CCL2 expression may differ according to cell type.
In the present study, we established that ADP also promotes ERK1/2 signaling in FLS1 cells (Fig. 3A) and confirmed that U0126 abrogates this ADP-mediated upregulation of ERK1/2 phosphorylation (Fig. 3B). Moreover, U0126 partially (nevertheless significantly) abrogated the ADP-mediated upregulation of MCP-1/CCL2 mRNA expression (Fig. 4A), thereby providing convincing evidence that ADP promotes MCP-1/CCL2 mRNA expression in FLSs in an MEK/ERK-dependent manner, which positively regulates the infiltration of monocytes/macrophages into the mouse TMJ.
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 . Moreover, Satonaka et al. reported that JNK/SAPK inhibition attenuates the ADP-induced upregulation of MCP-1/CCL2 mRNA and protein in VSMCs , whereas Ip et al. demonstrated that IL-1 and IL-13 induce MCP-1/CCL2 expression in ERK- and p38 MAPK-dependent manners in human bronchial epithelial cells . 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 . 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, and we failed to detect JNK/SAPK, even in response ADP stimulation (data not shown). 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 inflammation in TMJ-OA, which in turn enabled us to establish the potential therapeutic significance of TMJ-OA-related 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.