Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2011 Jun 17;286(24):21565-76. doi: 10.1074/jbc.M110.193375. Epub 2011 May 2.
All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.
全反式维甲酸（ATRA）可诱导许多细胞类型的生长停滞。先前的研究报告称，ATRA 可以调节细胞鞘脂，但鞘脂在 ATRA 反应中的作用尚不清楚。本研究以 MCF-7 细胞作为模型系统，结果表明 ATRA 可刺激神经酰胺水平升高，随后引发 G0/G1 期生长停滞。值得注意的是，nSMase2 的诱导是 ATRA 对鞘脂网络的主要作用，并且与时间和剂量呈依赖性。重要的是，nSMase2 siRNA 的预处理可显著抑制 ATRA 对神经酰胺水平和生长停滞的影响。相反，nSMase2 的过表达足以增加神经酰胺水平并诱导异步 MCF-7 细胞的 G0/G1 期生长停滞。令人惊讶的是，ATRA 刺激或 nSMase2 过表达均未对经典细胞周期调节剂（如 p21/WAF1 或视网膜母细胞瘤）产生显著影响。相反，ATRA 抑制核糖体 S6 激酶（S6K）及其下游靶标 S6 和 eIF4B 的磷酸化。重要的是，这些作用可被 nSMase2 siRNA 显著抑制。相反，nSMase2 的过表达足以抑制 S6K 的磷酸化和信号转导。值得注意的是，ATRA 对 S6K 磷酸化的作用以及 nSMase2 的作用均不需要先前鉴定为 S6K 调节所必需的神经酰胺激活的蛋白磷酸酶 PP2A。最后，nSMase2 的过表达足以降低翻译，如通过甲硫氨酸掺入和多核糖体谱分析来衡量。总之，这些结果表明 nSMase2 是 ATRA 诱导 MCF-7 细胞生长停滞的主要组成部分，并确定 S6K 是 nSMase2 的一个新的下游靶标。
