title: LPS potentiates nucleotide-induced inflammatory gene expression in macrophages via the upregulation of P2Y2 receptor.
abstract: Sepsis is a severe systemic inflammatory response that is associated with high morbidity and mortality. A previous study using an animal model of sepsis showed that survival was significantly lower in WT mice than in P2Y(2) receptor (P2Y(2)R)-deficient mice, suggesting that P2Y(2)R plays a role in septic death. We therefore investigated the role of P2Y(2)R in the inflammatory responses of RAW264.7 murine macrophages to LPS. LPS time-dependently upregulated P2Y(2)R mRNA levels, with a prominent increase observed at 4 h. In addition, LPS increased ATP release in a time dependent manner (5-120 min post LPS treatment). Accordingly, we pretreated cells with LPS for 4 h to induce P2Y(2)R expression and then stimulated the cells with UTP or ATP for 16 h. Interestingly, ATP- or UTP-dependent P2Y(2)R activation in LPS-pretreated cells resulted in dramatically enhanced HMGB1 secretion, COX-2 and iNOS expression, and furthermore PGE2 and NO production compared to LPS treatment alone (4 h) or ATP or UTP treatment alone (16 h), an effect that was inhibited by P2Y(2)R silencing. In addition, these increases in HMGB1 secretion, COX-2 and iNOS expression and PGE(2) and NO production commonly involved the JNK, PKC and PDK pathways. Taken together, these data demonstrate that LPS-dependent upregulation of P2Y(2)R plays a critical role in facilitating the inflammatory responses induced by LPS.
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
authors: So Young
authors: SY
authors: Eun
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
authors: Jihye
authors: J
authors: Seo
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
authors: Sang Won
authors: SW
authors: Park
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
authors: Jae Heun
authors: JH
authors: Lee
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
authors: Ki Churl
authors: KC
authors: Chang
authors: Department of Pharmacology, School Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea. Electronic address: hyejungkim@gnu.ac.kr.
authors: Hye Jung
authors: HJ
authors: Kim
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D015870:Gene Expression; D024243:HMGB1 Protein / Q000235:genetics; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics*; D015854:Up-Regulation; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D058473:Receptors, Purinergic P2Y2; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D064413:3-Phosphoinositide-Dependent Protein Kinases; D011493:Protein Kinase C; D048670:MAP Kinase Kinase 4; D015232:Dinoprostone; D014544:Uridine Triphosphate
keywords: ANOVA; COX-1/PGE(2); COX-2; HEPES; HMGB1; LPS; N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid; NO; Nucleotides; P2Y(2) receptor; P2Y(2)R; PDK; PGE2; PI3K; PKC; RT-PCR; SDS-PAGE; TBS-T; Tris-buffered saline-Tween-20; analysis of variance; cyclooxygenase-2; iNOS; iNOS/NO; inducible nitric oxide synthase; lipopolysaccharide; nitric oxide; phosphatidylinositol 3-kinase; phosphoinositide-dependent kinase; prostaglandin E2; protein kinase C; reverse transcription-polymerase chain reaction; sodium dodecyl sulfate-polyacrylamide gel electrophoresis