Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Biochem J. 2011 Apr 15;435(2):381-90. doi: 10.1042/BJ20101752.
Activation of N-SMase (neutral sphingomyelinase) is an established part of the response of cytokines such as TNF (tumour necrosis factor)-α. However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, nSMase2 and nSMase3) are responsible for this activity. In MCF-7 cells, we found that TNF-α induces late, but not early, increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siRNA (small interfering RNA) knockdown of nSMase1 significantly decreased basal in vitro N-SMase activity of MCF-7 cells, whereas nSMase2 but not nSMase3 siRNA inhibited TNF-α-induced activity. Taken together, these results identify nSMase2 as the major TNF-α-responsive N-SMase in MCF-7 cells. Moreover, the results suggest that nSMase3 may not possess in vitro N-SMase activity and does not affect cellular sphingolipid levels in the cell lines evaluated. On the other hand, nSMase1 contributes to in vitro N-SMase activity, but does not affect cellular sphingolipids much.
中性鞘磷脂酶（N-SMase）的激活是细胞因子（如 TNF-α）反应的一个既定部分。然而，目前克隆的 N-SMase 同工酶（nSMase1、nSMase2 和 nSMase3）中，哪一种负责这种活性仍不清楚。在 MCF-7 细胞中，我们发现 TNF-α诱导晚期而非早期 N-SMase 活性增加，并且 nSMase2 是主要被激活的同工酶，最有可能通过转录后机制。令人惊讶的是，在多种细胞系中转染标记或未标记的 nSMase3 过表达对体外 N-SMase 活性没有显著影响。此外，只有 nSMase2 的过表达，而不是 nSMase1 或 nSMase3 的过表达，对细胞内鞘脂水平有显著影响，增加了神经酰胺并减少了鞘磷脂。此外，只有 nSMase1 的 siRNA（小干扰 RNA）敲低显著降低了 MCF-7 细胞体外基础 N-SMase 活性，而 nSMase2 而非 nSMase3 siRNA 抑制了 TNF-α诱导的活性。总之，这些结果表明 nSMase2 是 MCF-7 细胞中主要的 TNF-α 反应性 N-SMase。此外，结果表明 nSMase3 可能不具有体外 N-SMase 活性，并且在评估的细胞系中不影响细胞内鞘脂水平。另一方面，nSMase1 有助于体外 N-SMase 活性，但对细胞内鞘脂的影响不大。
