Department of Cardiology, the First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun road, Hangzhou 310003, China.
Department of Cardiology, the First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun road, Hangzhou 310003, China.
Exp Cell Res. 2018 May 15;366(2):114-120. doi: 10.1016/j.yexcr.2018.03.003. Epub 2018 Mar 6.
The DEAD box protein DDX5, an ATP-dependent RNA helicase, plays an important role in transcriptional regulation and is associated with solid tumors and leukemia. However, its role in oxLDL-induced lipid uptake in macrophages remains unclear. In this study, we detected the expression of DDX5 mRNA and protein in oxidized low-density lipoprotein (oxLDL)-treated human primary macrophages that were induced from monocytes isolated from human peripheral blood with or without several chemical inhibitors using quantitative real-time PCR (qRT-PCR) or Western blotting. We found that oxLDL induced DDX5 expression to be independent of both the MAPK and NF-κB pathways. We also found that DDX5 promoted macrophage lipid uptake by evaluating the fluorescence intensity of engulfed dil-oxLDL. Various scavenger receptors that participate in lipid uptake were detected in siR-DDX5 transfected macrophages using qRT-PCR and Western blotting. Macrophage scavenger receptor A (MSR1) was found to be involved the upregulation of DDX5-mediated lipid uptake. Through the use of a dual luciferase reporter assay system and incubation with cycloheximide (CHX) MG132 and actidione (ActD), we found that DDX5 promoted MSR1 protein expression by stabilizing MSR1 mRNA. Moreover, the mechanism involved in DDX5 regulation of MSR1 mRNA was also explored using mass spectrum analysis; Immunoprecipitations (IPs) and RNA- Immunoprecipitations (R-IPs) revealed that mettl3 was involved in DDX5-mediated MSR1 mRNA stabilization. In addition, we also demonstrated that DDX5 inhibited mettl3 to catalyze ma methylation in MSR1 mRNA, which contributed to the maintenance of MSR1 mRNA stability. In conclusion, ox-LDL promotes DDX5 expression in macrophages, which interacts with mettl3 to stabilize MSR1 mRNA by decreasing the ma modification of MSR1 mRNA, ultimately promoting lipid uptake in macrophages.
DEAD 框蛋白 DDX5 是一种 ATP 依赖性 RNA 解旋酶，在转录调控中发挥重要作用，与实体瘤和白血病有关。然而，其在氧化低密度脂蛋白（oxLDL）诱导的巨噬细胞脂质摄取中的作用尚不清楚。在这项研究中，我们使用定量实时 PCR（qRT-PCR）或 Western blot 检测了用 oxLDL 处理的人原代巨噬细胞中 DDX5 mRNA 和蛋白的表达，这些巨噬细胞是从人外周血中的单核细胞诱导而来的，单核细胞分别用或不用几种化学抑制剂处理。我们发现 oxLDL 诱导的 DDX5 表达独立于 MAPK 和 NF-κB 途径。我们还发现，通过评估吞噬 dil-oxLDL 的荧光强度，DDX5 促进了巨噬细胞的脂质摄取。使用 qRT-PCR 和 Western blot 在 siR-DDX5 转染的巨噬细胞中检测到参与脂质摄取的各种清道夫受体。发现巨噬细胞清道夫受体 A（MSR1）参与了 DDX5 介导的脂质摄取的上调。通过使用双荧光素酶报告基因检测系统和用环己酰亚胺（CHX）、MG132 和放线菌酮（ActD）孵育，我们发现 DDX5 通过稳定 MSR1 mRNA 促进 MSR1 蛋白表达。此外，还通过质谱分析探讨了 DDX5 调节 MSR1 mRNA 的机制；免疫沉淀（IP）和 RNA-免疫沉淀（R-IP）表明，mettl3 参与了 DDX5 介导的 MSR1 mRNA 稳定。此外，我们还证明 DDX5 抑制了 mettl3 对 MSR1 mRNA 中 ma 甲基化的催化作用，有助于维持 MSR1 mRNA 的稳定性。总之，ox-LDL 促进巨噬细胞中 DDX5 的表达，DDX5 与 mettl3 相互作用，通过减少 MSR1 mRNA 中的 ma 修饰来稳定 MSR1 mRNA，最终促进巨噬细胞中的脂质摄取。
