Mohamed Nesma Nabil Ibrahim, Okada Taro, Kajimoto Taketoshi, Nakamura Shun-Ichi
Division of Biochemistry, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Kobe J Med Sci. 2018 May 25;63(4):E123-E129.
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.
鞘氨醇-1-磷酸（S1P）是由鞘氨醇激酶（SphK）催化产生的一种具有生物活性的鞘氨醇磷酸化产物，参与包括囊泡运输在内的多种细胞功能。在本研究中，我们证明了SphK的一种亚型SphK2在调节人髓系白血病K562细胞释放的外泌体中的货物含量方面的重要性。首先，已证明SphK2在细胞中与N-Rh-PE阳性晚期内体共定位。其次，siRNA介导的Sphk2而非SphK1的敲低导致纯化外泌体中货物含量的降低。通过药理学方法进一步研究了SphK2在这一现象中的作用。当用N，N-二甲基鞘氨醇（DMS）处理细胞时，DMS是最常用的SphK抑制剂之一，纯化外泌体中的货物含量意外增加。最后，已证明DMS具有刺激SphK2活性的能力，这取决于底物鞘氨醇和抑制剂的剂量，这是通过使用纯化的SphK2的体外检测系统估计的。这些发现表明，SphK2/S1P信号通路在调节K562细胞外泌体中的货物含量方面发挥着重要作用。
