title: The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2 through the MAP kinases-NF-κB pathway.
abstract: 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.
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address: mnori@hiroshima-u.ac.jp.
authors.forename: Norimitsu
authors.initials: N
authors.lastname: Morioka
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Tokuhara
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Sakura
authors.initials: S
authors.lastname: Harano
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Yoki
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Kazue
authors.initials: K
authors.lastname: Hisaoka-Nakashima
authors.affiliation: Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakata
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017879:Isothiocyanates / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013116:Spinal Cord / Q000166:cytology*; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D017879:Isothiocyanates; C541384:N,N''-1,4-butanediylbis(N'-(3-isothiocyanatophenyl))thiourea; D016328:NF-kappa B; D058921:Purinergic P2Y Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; C099845:purinoceptor P2Y6; D020929:Mitogen-Activated Protein Kinase Kinases; D013890:Thiourea; D014544:Uridine Triphosphate
keywords: CCL2; MAP kinases; NF-κB; P2Y(6); Spinal microglia; UTP