title: Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression.
abstract: Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.
authors.affiliation: Institute of Biochemistry I/Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit (ZAFES), Faculty of Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany.
authors.forename: Axel M
authors.initials: AM
authors.lastname: Johann
authors.forename: Andreas
authors.initials: A
authors.lastname: Weigert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eberhardt
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Kuhn
authors.forename: Vera
authors.initials: V
authors.lastname: Barra
authors.forename: Andreas
authors.initials: A
authors.lastname: von Knethen
authors.forename: Josef M
authors.initials: JM
authors.lastname: Pfeilschifter
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D017209:Apoptosis; D002450:Cell Communication; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000235:genetics* / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D019169:Jurkat Cells; D008246:Lysophospholipids / Q000378:metabolism* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000099157:Sphingosine Kinase
chemical_list: D000954:Antigens, Surface; D017077:Culture Media, Conditioned; D051546:Cyclooxygenase 2; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D013110:Sphingosine; D000099157:Sphingosine Kinase; C098770:ELAVL1 protein, human; C060506:sphingosine 1-phosphate