Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.