Taha Tarek A, Kitatani Kazuyuki, Bielawski Jacek, Cho Wonhwa, Hannun Yusuf A, Obeid Lina M
Division of General Internal Medicine, Ralph H. Johnson Veterans Administration Hospital, Charleston, South Carolina 29401, USA.
J Biol Chem. 2005 Apr 29;280(17):17196-202. doi: 10.1074/jbc.M413744200. Epub 2005 Feb 14.
Sphingosine kinase-1 (SK1) has emerged as a key component of cytokine responses, including roles in apoptosis, yet the specific mechanisms by which cytokines regulate SK1 in the apoptotic responses have not been studied. In this study, we show that prolonged treatment of MCF-7 cells with tumor necrosis factor (TNF) induces a dose- and time-dependent decrease in SK1 protein. Inhibition of the upstream caspase 8 by IETD significantly rescued TNF effects on SK1, yet the caspase 7 inhibitor DEVD failed to have any effect, suggesting that the decline in SK1 occurs downstream of the initiator caspase but upstream of the effector caspase. In addition to caspase activation, TNF caused disruption of lysosomes with relocation of the cysteine protease cathepsin B into the cytosol. Down-regulation of cathepsin B using small interfering RNA significantly restored SK1 levels following exposure to TNF, suggesting that SK1 loss was dependent on cathepsin B activity. The regulation of SK1 by the lysosomal protease was further supported by the colocalization of SK1 with the lysosome and cathepsin B in cells and the loss of the colocalization following exposure to TNF. The ability of cathepsin B to regulate SK1 was further corroborated by an in vitro approach where recombinant cathepsin B cleaved SK1 at multiple sites to produce several cleavage fragments. Therefore, these studies show that SK1 down-regulation by TNF is dependent on the "lysosomal pathway" of apoptosis and specifically on cathepsin B, which functions as an SK1 protease in cells.
鞘氨醇激酶-1（SK1）已成为细胞因子反应的关键组成部分，包括在细胞凋亡中发挥作用，然而细胞因子在凋亡反应中调节SK1的具体机制尚未得到研究。在本研究中，我们发现用肿瘤坏死因子（TNF）长时间处理MCF-7细胞会导致SK1蛋白呈剂量和时间依赖性下降。IETD对上游半胱天冬酶8的抑制显著挽救了TNF对SK1的影响，但半胱天冬酶7抑制剂DEVD却没有任何作用，这表明SK1的下降发生在起始半胱天冬酶的下游但效应半胱天冬酶的上游。除了半胱天冬酶激活外，TNF还导致溶酶体破坏，半胱氨酸蛋白酶组织蛋白酶B重新定位到细胞质中。使用小干扰RNA下调组织蛋白酶B可在暴露于TNF后显著恢复SK1水平，这表明SK1的缺失依赖于组织蛋白酶B的活性。细胞中SK1与溶酶体和组织蛋白酶B的共定位以及暴露于TNF后共定位的丧失进一步支持了溶酶体蛋白酶对SK1的调节。体外实验方法进一步证实了组织蛋白酶B调节SK1的能力，即重组组织蛋白酶B在多个位点切割SK1以产生几个切割片段。因此，这些研究表明TNF对SK1的下调依赖于细胞凋亡的“溶酶体途径”，特别是依赖于组织蛋白酶B，它在细胞中作为SK1蛋白酶发挥作用。
