Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.
Essential Role of Sphingosine Kinase 2 in the Regulation of Cargo Contents in the Exosomes from K562 Cells.
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发表时间：
2018-05
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通讯作者：
N. Mohamed;T. Okada;T. Kajimoto;Shun-ichi Nakamura
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作者：
N. Mohamed;T. Okada;T. Kajimoto;Shun-ichi Nakamura
Sphingosine 1-phosphate （S1P）是由Sphingosine kinase （SphK）催化的具有生物活性的Sphingosine磷酸化产物，参与多种细胞功能，包括囊泡运输。在目前的研究中，我们已经证明了SphK亚型之一SphK2在调节人髓性白血病K562细胞释放的外泌体中的货物含量中的重要性。首先，SphK2已被证明定位于细胞中n - rh - pe阳性的晚期核内体。接下来，sirna介导的敲低Sphk2而非SphK1导致纯化外泌体中载货物含量的减少。SphK2在这一现象中的作用被药理学方法进一步研究。当细胞用最常用的SphK抑制剂之一N，N-二甲基鞘氨醇（DMS）处理时，纯化外泌体中的货物含量意外增加。最后，研究表明，DMS具有刺激SphK2活性的效力，这取决于底物鞘鞘醇和抑制剂的剂量，这是通过使用纯化的SphK2体外测定系统估计的。这些发现表明SphK2/S1P信号在K562细胞外泌体货物含量的调控中起重要作用。
Sphingosine 1-phosphate (S1P) is a bioactive phosphorylated product of sphingosine catalyzed by sphingosine kinase (SphK) and implicated in diverse cellular functions including vesicular trafficking. In the present study we have shown the importance of one of the subtypes of SphK, SphK2, in the regulation of cargo content in exosomes released from human myeloid leukemia K562 cells. First, SphK2 has been shown to localize with N-Rh-PE-positive late endosomes in the cells. Next, siRNA-mediated knockdown of Sphk2 but not SphK1 resulted in a reduction of cargo content in purified exosomes. The involvement of SphK2 in this phenomenon was further investigated by pharmacological approaches. When cells were treated with N,N-dimethylsphingosine (DMS), one of the most frequently used inhibitors for SphK, cargo contents in purified exosomes were enhanced unexpectedly. Finally, it has been shown that DMS has a potency to stimulate SphK2 activity depending on the substrate sphingosine- and the inhibitor-doses as estimated by in vitro assay systems using a purified SphK2. These findings suggest that SphK2/S1P signaling plays an important role in the regulation of cargo content in exosomes in K562 cells.