Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression
Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression
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DOI：
10.4049/jimmunol.180.2.1239
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发表时间：
2008-01-15
影响因子：
4.4
通讯作者：
Bruene, Bernhard
中科院分区：
文献类型：
--
作者：
Johann, Axel M.;Weigert, Andreas;Bruene, Bernhard
吞噬细胞清除凋亡细胞被认为是关键的免疫调节过程。尽管人们对吞噬后巨噬细胞表型已经有了相当多的了解，但有关引起巨噬细胞极化的分子机制的信息却很少。在这项研究中，我们发现人凋亡 Jurkat 细胞 (AC) 或 AC 条件培养基 (CM) 通过 1-磷酸鞘氨醇 (S1P) 快速诱导小鼠 RAW264.7 巨噬细胞中环氧合酶-2 (COX-2) 的表达。 AC 释放 S1P 的药理学抑制或使用人 MCF-7 细胞中鞘氨醇激酶 2 敲低的细胞的 CM 可以消除这种效应。 COX-2 的表达是由于 3'-非翻译区 (UTR) 的 mRNA 稳定性增加所致，COX-2-3'-UTR 和富含 AU 元件驱动的报告基因检测显示。 Western 分析证实了 CM 处理后 RNA 结合蛋白 HuR 的核质穿梭增加。 RNA EMSA 分析揭示了 S1P 和 CM 介导的 HuR-RNA 与 COX-2 特异性 UTR 结合的增加，而 HuR 敲低表明其对 S1P 在 CM 诱导的 COX-2 表达中的重要性。磷脂酶 A(2) (PLA(2)) 的免疫荧光显微镜检查和 PGE(2) 的 ELISA 分析揭示了 PLA(2) 的激活和 PGE(2) 的产生响应 CM 而不是 S1P。 S1P 由 AC 释放，利用 HuR 稳定 COX-2 mRNA，从而增加 COX-2 蛋白表达。然而，只有 CM 也能激活 PLA(2) 为 COX-2 提供底物。我们的数据强调了 S1P 通过稳定巨噬细胞中的 COX-2 mRNA（PGE(2) 形成的先决条件）在 AC 介导的免疫调节中的重要性。
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 A(2) (PLA(2)) and ELISA analysis of PGE(2) revealed activation of PLA(2) and production of PGE(2) 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 PLA(2) 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 PGE(2) formation.