Neutral sphingomyelinase-2 mediates growth arrest by retinoic acid through modulation of ribosomal S6 kinase

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 G₀/G₁ 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 G₀/G₁ 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.