Peest Ulrike, Sensken Sven-Christian, Andréani Paul, Hänel Petra, Van Veldhoven Paul P, Gräler Markus H
Institute for Immunology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hanover, Germany.
J Cell Biochem. 2008 Jun 1;104(3):756-72. doi: 10.1002/jcb.21665.
Sphingosine 1-phosphate (S1P) is the natural ligand for a specific family of G protein-coupled receptors (-Rs). The type 1 S1P-R (S1P(1)) is important for lymphocyte egress, and blood-borne S1P as the natural ligand for S1P(1) is involved in the maintenance of lymphocyte circulation. This report reveals that extracellular S1P was cleared by all tested primary cells and cell lines with exponential progression. Clearance of S1P, but not sphingosine (Sph) was inhibited with the protein phosphatase inhibitor sodium orthovanadate. Fluorescence microscopy and flow cytometry using fluorescently labeled S1P and Sph showed a major cellular uptake of Sph, but not S1P. HPLC-analyses with C17-Sph demonstrated that cellular Sph accumulation was transient in tested cell lines, but enduring in mouse splenocytes. Sub cellular fractionation resulted in dephosphorylation of S1P to Sph by nuclear, membrane, and cytosolic fractions. Degradation of Sph however only occurred in combined membrane and cytosolic fractions. Inhibitors for Sph kinases 1/2, ceramide synthase, and S1P-lyase, as well as S1P-lyase deficiency did not block clearance of extracellular S1P. In vivo experiments revealed a transient increase in plasma S1P levels after single intravenous injection into C57BL/6 mice. This exogenously added S1P was cleared within 15-30 min in contrast to ex vivo incubation of whole blood which required more than 8 h for comparable clearance from plasma. Our data thus show that extracellular S1P is dephosphorylated and subsequently converted by cells, which appears to be important for clearance of the signaling molecule S1P in the local tissue environment after infections or injuries.
1-磷酸鞘氨醇（S1P）是一类特定的G蛋白偶联受体（GPCRs）的天然配体。1型S1P受体（S1P(1)）对淋巴细胞外流很重要，而作为S1P(1)天然配体的血源性S1P参与淋巴细胞循环的维持。本报告显示，所有测试的原代细胞和细胞系均以指数级进程清除细胞外S1P。蛋白磷酸酶抑制剂原钒酸钠可抑制S1P的清除，但不抑制鞘氨醇（Sph）的清除。使用荧光标记的S1P和Sph进行荧光显微镜检查和流式细胞术分析显示，细胞主要摄取Sph，而不是S1P。用C17-Sph进行的高效液相色谱分析表明，在测试的细胞系中细胞Sph积累是短暂的，但在小鼠脾细胞中是持久的。亚细胞分级分离导致核、膜和胞质部分将S1P去磷酸化为Sph。然而，Sph的降解仅发生在膜和胞质部分的组合中。鞘氨醇激酶1/2、神经酰胺合酶和S1P裂解酶的抑制剂，以及S1P裂解酶缺乏均未阻断细胞外S1P的清除。体内实验显示，向C57BL/6小鼠单次静脉注射后，血浆S1P水平短暂升高。与全血的体外孵育相比，这种外源性添加的S1P在15 - 30分钟内被清除，全血体外孵育从血浆中清除类似量的S1P需要超过8小时。因此，我们的数据表明，细胞外S1P被去磷酸化并随后被细胞转化，这对于感染或损伤后局部组织环境中信号分子S1P的清除似乎很重要。
