Neutral Sphingomyelinase-2 Mediates Growth Arrest by Retinoic Acid through Modulation of Ribosomal S6 Kinase

Abstract

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.Sphingolipids such as ceramide (Cer) 2 are a class of bioactive lipids implicated in a variety of physiological processes. In particular, Cer is thought to be involved in apoptosis, inflammation, and the cellular response to cytokines and other stresses (1). Cer production can occur through multiple pathways and is under tight control within the cell. Sphingomyelinases (SMases) produce Cer through hydrolysis of sphingomyelin and are considered important mediators of stress-induced Cer production (2). Currently, three families of SMases have been identified and are classified by the pH optima of their activity. Of these, both acid and neutral SMases (N-SMases) have been reported to contribute to stress and cytokine-induced Cer production (2, 3).Currently, four mammalian N-SMases have been cloned and characterized: nSMase1, nSMase2, nSMase3, and the recently identified mitochondria-associated nSMase. Of these, studies have implicated nSMase2 in the acute responses to cytokines, chemotherapeutic drugs, and oxidative stress (2). Importantly, emerging evidence is beginning to suggest a more protracted mode of nSMase2 regulation involving changes in nSMase2 gene expression. For example, increased nSMase2 expression has been demonstrated in confluence-induced growth arrest of MCF-7 cells (4) and in rat 3Y1 cells where the CCA1 gene (for cell confluence arr...

Cardiac dysfunction in β-carotene-15,15′-dioxygenase-deficient mice is associated with altered retinoid and lipid metabolism

“…We observed elevated levels of both FFAs and ceramides in the hearts of Bco1 Ϫ/Ϫ mice. Interestingly, ATRA treatment of cultured human SK-N-SK and SK-N-AS neuroblastoma cells or MCF-7 mammary epithelial cells results in increased cellular ceramide levels (8,22). For these cell model systems, this caused an inhibition of cell growth.…”

ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation

“…Notably, as both CBP and p300 seem to be upstream of nSMase2, this would imply that nSMase2 might have roles within all of these outcomes. Thus, we speculate that nSMase2 may be a "core" gene of ATRA responses, which would be consistent with its early induction time in MCF7 cells (5). According to this hypothesis, additional targets of CBP and p300 would then determine specific cellular responses.…”

“…Previously, we characterized nSMase2 as an early ATRA response gene (<6 h of ATRA treatment) that was important for ATRA-induced growth arrest of breast epithelial cells (5). Here, we have extended this work to define the mechanism by which ATRA regulates nSMase2 expression.…”

“…Indeed, emerging evidence has begun to point to a more protracted regulation of at the transcriptional level. Increases in nSMase2 expression have been reported in response to bone morphogenetic protein-2 (BMP2) (17,18), daunorubicin (19), cigarette smoke (20), confluence (6), the hedgehog signaling mediator cyclopamine (21), and all-trans retinoic acid (ATRA) (5,22,23). Notably, in the latter two cases, increased expression of nSMase2 was required for ATRA-induced growth arrest (23) and cyclopamine-induced apoptosis, respectively (21).…”

“…However, recent emerging evidence has pointed to a more protracted regulation of nSMase2 occurring at the level of expression. Indeed, increased expression of nSMase2 has been reported in response to chemotherapies (19,21), BMP2 (17,18), confluence (6), and ATRA (5,22,23).…”