Division of Clinical Laboratory Medicine, Faculty of Medicine, Tottori University, 86 Nishi-Machi, Yonago 683-8503, Japan.
J Biol Chem. 2012 Nov 16;287(47):39898-910. doi: 10.1074/jbc.M112.416552. Epub 2012 Oct 3.
The role of "sphingolipid rheostat" by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(-)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(-) or C(2)-ceramide. AA(-) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(-). S1P exerts biological actions via cell surface receptors, and S1P(3) among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P(3) in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(-) or C(2)-ceramide. Whereas S1P treatment of S1P(3) overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(-) or C(2)-ceramide. These results indicate that S1P-S1P(3) plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(-)- or C(2)-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P(3) engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P(3) signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.
鞘脂“变阻器”在调节自噬中的作用尚不清楚。在人类白血病 HL-60 细胞中，氨基酸剥夺（AA(-)）引起自噬，酸性鞘氨醇酶（SMase）活性和神经酰胺增加，神经酰胺作为一种诱导自噬的脂质。酸性 SMase 的敲低显著抑制了自噬的诱导。S1P 处理可拮抗 AA(-)或 C2-神经酰胺诱导的自噬。AA(-)处理促进雷帕霉素靶蛋白（mTOR）去磷酸化/失活，诱导自噬。S1P 处理抑制 mTOR 失活和 AA(-)诱导的自噬。S1P 通过细胞表面受体发挥生物学作用，在五个 S1P 受体中，S1P(3)在 HL-60 细胞中表达最为丰富。我们评估了 S1P(3)在抑制自噬诱导中的作用。S1P 处理 CHO 细胞对 AA(-)或 C2-神经酰胺诱导的 mTOR 失活和自噬诱导没有影响。然而，S1P 处理 S1P(3)过表达的 CHO 细胞导致 mTOR 通路的激活，阻止细胞发生 AA(-)或 C2-神经酰胺诱导的自噬。这些结果表明，S1P-S1P(3)在拮抗介导 mTOR 控制的自噬的神经酰胺信号中起作用。此外，我们评估了神经酰胺激活的蛋白磷酸酶（CAPPs）在神经酰胺依赖性 mTOR 通路失活中的作用。AA(-)或 C2-神经酰胺处理细胞中 CAPP 的 okadaic 酸抑制 mTOR 的去磷酸化/失活、自噬诱导和自噬相关的细胞死亡，表明神经酰胺-CAPPs 在自噬诱导中的新作用。此外，S1P 通过 S1P(3)的结合拮抗细胞死亡。综上所述，这些结果表明，神经酰胺-CAPPs 和 S1P-S1P(3)信号中的鞘脂“变阻器”通过调节 mTOR 通路调节自噬及其相关的细胞死亡。
