Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.

中文翻译：

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鞘氨醇激酶对人胚胎肾细胞中p53，p38 MAPK，JNK和XBP-1s的调节。

由于鞘氨醇激酶的抑制剂（SK1，SK2）已显示出诱导p53介导的细胞死亡的作用，因此我们进一步研究了它们在调节HEK293T细胞中p53，应激激活的蛋白激酶和XBP-1s中的作用。用鞘氨醇激酶抑制剂SKi处理这些细胞不能诱导凋亡，它促进了p53转化为两种分子量分别为63和90 kDa的蛋白质，并且由于泛素的过度表达而得以增强。SK1的siRNA敲低也增强了SKi诱导的p53向p63 / p90的转化，但SK2或二氢神经酰胺去饱和酶（Degs1）却没有，这表明SK1是该过程的负调控因子。相反，另一种鞘氨醇激酶抑制剂ABC294640仅非常弱地刺激p63 / p90的形成并诱导HEK293T细胞凋亡。我们以前已经表明，SKi促进Degs1的多聚泛素化，并且这些形式正调控p38 MAPK / JNK通路，从而促进HEK293T细胞的存活/生长。敲低SK1的siRNA可以增强p38 MAPK / JNK通路对SKi的响应，这表明SK1的功能与HEK293T细胞中这些促存活通路相反。SKi还增强了蛋白酶体抑制剂MG132对促存活蛋白XBP-1s表达的刺激作用，这被siRNA敲除SK2所降低，而被p53敲除所增加。这些发现表明SK1和SK2在调节HEK293T细胞中的p53依赖性功能中具有相反的作用。敲低SK1的siRNA可以增强p38 MAPK / JNK通路对SKi的响应，这表明SK1的功能与HEK293T细胞中这些促存活通路相反。SKi还增强了蛋白酶体抑制剂MG132对促存活蛋白XBP-1s表达的刺激作用，这被siRNA敲除SK2所降低，而被p53敲除所增加。这些发现表明SK1和SK2在调节HEK293T细胞中的p53依赖性功能中具有相反的作用。敲低SK1的siRNA可以增强p38 MAPK / JNK通路对SKi的响应，这表明SK1的功能与HEK293T细胞中这些促存活通路相反。SKi还增强了蛋白酶体抑制剂MG132对促存活蛋白XBP-1s表达的刺激作用，这被siRNA敲除SK2所降低，而被p53敲除所增加。这些发现表明SK1和SK2在调节HEK293T细胞中的p53依赖性功能中具有相反的作用。