Chang Na, Ge Jingjing, Xiu Lei, Zhao Zhongxin, Duan Xianghui, Tian Lei, Xie Jieshi, Yang Lin, Li Liying
Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, No.10 Xitoutiao, You An Men, Beijing, 100069, China.
J Mol Med (Berl). 2017 Jan;95(1):69-82. doi: 10.1007/s00109-016-1460-x. Epub 2016 Aug 20.
UNLABELLED: Sphingosine 1-phosphate (S1P) participates in migration of bone marrow (BM)-derived mesenchymal stem cells (BMSCs) toward damaged liver via upregulation of S1P receptor 3 (S1PR3) during mouse liver fibrogenesis. But, the molecular mechanism is still unclear. HuR, as an RNA-binding protein, regulates tumor cell motility. Here, we examined the role of HuR in migration of human BMSCs (hBMSCs) in liver fibrosis. Results showed that HuR messenger RNA (mRNA) level was increased in human or mouse fibrotic livers, and correlated with S1PR3 mRNA expression. Using immunofluorescence, we found that HuR mainly localized in the nuclei of hepatocytes and non-parenchymal cells in normal livers. However, in fibrotic livers, we detected an increased HuR cytoplasmic localization in non-parenchymal cells. In chimeric mice of BM cell-labeled by EGFP, significant numbers of EGFP-positive cells (BM origin) were positive for HuR in fibrotic areas. Meanwhile, HuR-positive cells were also positive for α-SMA (myofibroblasts). In vitro, S1P induced hBMSCs migration via S1PR3 upregulation. HuR involved in S1P-induced hBMSCs migration and increased stabilization of S1PR3 mRNA via competing with miR-30e. RNA immunoprecipitation showed that HuR interacted with S1PR3 mRNA 3'UTR. Moreover, S1P resulted in phosphorylation and cytoplasmic translocation of HuR via S1PR3 and p38MAPK. Furthermore, we transplanted EGFP BMSCs with or without HuR small interfering RNA (siRNA) into carbon tetrachloride-treated mice and found that knockdown of HuR inhibited the migration of BMSCs toward injured livers by flow cytometric analysis in vivo. We identified a positive feedback regulation mechanism between HuR and S1PR3 in S1P-induced BMSCs migration. HuR participates in upregulation of S1PR3 induced by S1P. S1P results in phosphorylation and translocation of HuR via S1PR3. Our results provide a new regulatory manner to the mechanism of liver fibrogenesis. KEY MESSAGE: HuR expression and cytoplasmic localization were increased in fibrotic livers. S1P induced migration of human bone marrow Mesenchymal Stem Cells via S1PR3 and HuR. HuR regulated S1PR3 mRNA expression by binding with S1PR3 mRNA 3'UTR. S1P induced HuR phosphorylation and cytoplasmic translocation via S1PR3. HuR regulated S1PR3 expression by competing with miR-30e.
未标记：在小鼠肝纤维化过程中，1-磷酸鞘氨醇（S1P）通过上调S1P受体3（S1PR3）参与骨髓来源的间充质干细胞（BMSC）向受损肝脏的迁移。但是，其分子机制仍不清楚。HuR作为一种RNA结合蛋白，可调节肿瘤细胞的运动。在此，我们研究了HuR在肝纤维化过程中对人BMSC迁移的作用。结果显示，人或小鼠纤维化肝脏中HuR信使核糖核酸（mRNA）水平升高，且与S1PR3 mRNA表达相关。通过免疫荧光，我们发现正常肝脏中HuR主要定位于肝细胞和非实质细胞的细胞核中。然而，在纤维化肝脏中，我们检测到非实质细胞中HuR的细胞质定位增加。在经EGFP标记骨髓细胞的嵌合小鼠中，纤维化区域有大量EGFP阳性细胞（源自骨髓）呈HuR阳性。同时HuR阳性细胞也呈α -平滑肌肌动蛋白（α - SMA，肌成纤维细胞）阳性。在体外，S1P通过上调S1PR3诱导hBMSC迁移。HuR参与S1P诱导的hBMSC迁移，并通过与miR - 30e竞争增加S1PR3 mRNA的稳定性。RNA免疫沉淀显示HuR与S1PR3 mRNA的3'非翻译区（3'UTR）相互作用。此外，S1P通过S1PR3和p38丝裂原活化蛋白激酶（p38MAPK）导致HuR磷酸化和细胞质易位。此外，我们将携带或不携带HuR小干扰RNA（siRNA）的EGFP BMSC移植到四氯化碳处理的小鼠中，通过体内流式细胞术分析发现敲低HuR可抑制BMSC向受损肝脏迁移。我们确定了HuR和S1PR3在S1P诱导的BMSC迁移中存在正反馈调节机制。HuR参与S1P诱导的S1PR3上调。S1P通过S1PR3导致HuR磷酸化和易位。我们的结果为肝纤维化机制提供了一种新的调节方式。 关键信息：纤维化肝脏中HuR表达及细胞质定位增加。S1P通过S1PR3和HuR诱导人骨髓间充质干细胞迁移。HuR通过与S1PR3 mRNA的3'UTR结合调节S1PR3 mRNA表达。S1P通过S1PR3诱导HuR磷酸化和细胞质易位。HuR通过与miR - 30e竞争调节S1PR3表达。
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