The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2 through the MAP kinases-NF-κB pathway

• UTP induced CCL2 production in cultured spinal microglia.
• CCL2 production induced by UTP is dependent on P2Y6 receptor stimulation.
• ERK and p38 activity are critical for CCL2 production.
• NF-κB is a crucial transcription factor in the production of CCL2.

Rat primary cultures of spinal microglia were stimulated by UTP, a known P2Y2/4 receptor agonist, which resulted in the production and release of the C–C chemokine CCL2 (monocyte chemoattractant protein-1; MCP-1) measured by real-time PCR and ELISA, respectively. In an in vitro preparation of rat spinal microglia, with regard to the P2Y subtypes, the expression of P2Y1, 2, 6, 12, 13 and P2Y14, but not P2Y4, were detected by RT-PCR. The subtype of microglial P2Y receptor which could be involved in the production of CCL2 was also determined. The UTP-induced production of CCL2 was significantly blocked by pretreatment with reactive blue 2 and suramin, nonselective P2Y receptor antagonists, and MRS2578, a selective P2Y6 receptor antagonist. By contrast, knockdown of the P2Y2 receptor by RNA interference had no effect. The stimulatory effect of UTP was inhibited by phospholipase C (PLC) inhibitor U73122 and Src tyrosine kinase inhibitor PP2. A potential role of mitogen activated protein kinases was suggested since UTP-induced CCL2 production was significantly blocked by both U0126 and SB 202190, which are potent inhibitors of extracellular signal-regulated kinase (ERK) and p38, respectively. Moreover, UTP-stimulated phosphorylation of these kinases involved the activation of the P2Y6 receptor. Lastly, activation of nuclear factor-κB (NF-κB) by UTP is likely to be essential in the expression of CCL2. Together, these findings suggest that stimulation of spinal microglia P2Y6 receptors induce the production of CCL2 through either PLC-mediated ERK or p38 phosphorylation and the subsequent activation of NF-κB.