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Apoptotic Cell-Derived Sphingosine-1-Phosphate Promotes HuR-Dependent Cyclooxygenase-2 mRNA Stabilization and Protein Expression¹

Axel M. Johann^*, Andreas Weigert^*, Wolfgang Eberhardt, Anne-Marie Kuhn^*, Vera Barra^*, Andreas von Knethen^*, Josef M. Pfeilschifter and Bernhard Brüne^2,*

^* Institute of Biochemistry I/Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit (ZAFES), Faculty of Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany; and Institute of Pharmacology/ZAFES, Faculty of Medicine, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from Deutsche Forschungsgemeinschaft (BR999, Excellence Cluster Cardiopulmonary System) and the European Community (PROLIGEN).

² Address correspondence and reprint requests to Dr. Bernhard Brüne, Institute of Biochemistry I/ZAFES, Faculty of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail address: bruene{at}zbc.kgu.de

³ Abbreviations used in this paper: AC, apoptotic Jurkat cell; S1P, sphingosine-1-phosphate; SphK2, sphingosine kinase 2; COX, cyclooxygenase; UTR, untranslated region; CM, conditioned medium; Cyt D, cytochalasin D; ARE, AU-rich element; wt, wild type; mt, mutant; DAPI, 4',6'-diamidino-2-phenylindole; DMS, dimethylsphingosine; VC, viable cell; NC, necrotic cell; siRNA, small interfering RNA.