Inhibition of sphingomyelin synthase (SMS) affects intracellular sphingomyelin accumulation and plasma membrane lipid organization

Abstract

Sphingomyelin plays a very important role both in cell membrane formation that may well have an impact on the development of diseases like atherosclerosis and diabetes. However, the molecular mechanism that governs intracellular and plasma membrane SM levels is largely unknown. Recently, two isoforms of sphingomyelin synthase (SMS1 and SMS2), the last enzyme for SM de novo synthesis, have been cloned. We have hypothesized that SMS1 and SMS2 are the two most likely candidates responsible for the SM levels in the cells and on the plasma membrane. To test this hypothesis, cultured cells were treated with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of SMS, or with SMS1 and SMS2 siRNAs. Cells were then pulsed with [ 14 C]-L-serine (a precursor of all sphingolipids). SMS activity and [ 14 C]-SM in the cells were monitored. We found that SMS activity was significantly decreased in cells after D609 or SMS siRNA treatment, compared with controls. SMS inhibition by D609 or SMS siRNAs significantly decreased intracellular [ 14 C]-SM levels. We measured cellular lipid levels, including SM, ceramide, phosphatidylcholine, and diacylglycerol and found that SMS1 and SMS2 siRNA treatment caused a significant decrease of SM levels (20% and 11%, respectively), compared to control siRNA treatment; SMS1 but not SMS2 siRNA treatment caused a significant increase of ceramide levels (10%). There was a decreasing tendency for diacylglycerol levels after both SMS1 and SMS2 siRNA treatment, however, it was not statistical significant. As shown by lipid rafts isolation and lipid determination, SMS1 and SMS2 siRNA treatment led to a decrease of SM content in detergent-resistant lipid rafts on the cell membrane. Furthermore, SMS1 and SMS2 siRNA-treated cells had a stronger resistance than did control siRNA-treated cells to lysenin (a protein that causes cell lysis due to its affinity for plasma membrane SM). These results indicate that both SMS1 and SMS2 contribute to SM de novo synthesis and control SM levels in the cells and on the cell membrane including plasma membrane, implying an important relationship between SMS activity and cell functions.

Tales and Mysteries of the Enigmatic Sphingomyelin Synthase Family

“…74 Since in HeLa cells, SMS1 localizes exclusively at the Golgi and SMS2 localizes at the Golgi and plasma membrane, these observations indicate that the unique plasma membrane localization of SMS2 did not specifically facilitate SM resynthesis from plasma membrane derived-ceramide, thus suggesting that the pool of SMS2 in the Golgi may be of significant activity. In support of this conclusion, over-expression of either SMS1 or SMS2 in HeLa cells comparably increased de novo SM biosynthesis, known to occur in the Golgi, 74 whereas down-regulation of either one significantly inhibited it in HeLa, 74,80 Huh, 72 HEK 293 cells and macrophages isolated from KO mice. 76 In HeLa cells down-regulation of either SMS1 or SMS2 inhibited TNF-mediated NF-gB activation by altering plasma membrane receptor activation, similarly to HEK 293 cells (Luberto and Marimuthu, unpublished observations).…”

“…77 An increase of ceramide levels was also observed upon down-regulation of either SMS1 or SMS2 in HeLa and Huh cells, 66,72,74 suggesting that changes in ceramide levels due to modulation of SMSs might be differently regulated depending on the specific cellular context.…”

“…Whereas the levels of PC do not seem to change following modulation of SMSs, 66,72,74,77 altering the expression of SMS1 or SMS2 exerts diverse responses on DAG levels. In CHO cells over-expression of SMS1 or SMS2 induced a significant accumulation of total DAG.…”

“…72 In these cells, it was shown that siRNA-mediated down-regulation of either SMS1 or SMS2 induced a significant reduction of SM content in detergent-resistant fractions. Confirmation of the involvement of SMS2 in the physiological maintenance of plasma membrane SM has come from studies using macrophages from SMS2 knock out (KO) mice.…”

“…Interestingly, expression of both SMS1 and SMS2 has been observed in all adherent cell lines analyzed so far (breast cancer MCF-7 cells, cervical cancer HeLa cells, hepatocellular carcinoma HepG2 cells, colon cancer CaCo 2 cells, human lung fibroblasts, human epithelial HEK-293 cells, mouse melanoma MEB4, mouse fibroblasts) whereas expression of SMS2 seems to be very low or absent in the majority of suspension cell lines (S49, WR19L/Fas, Ramos and U937 lymphoma cells, HL-60, Molt-4 and K562 leukemia cells and primary human B, T and monocytic cells) 58,62,[72][73][74] and Luberto, unpublished observations). Since SMS2 is in part localized at the plasma membrane, with its catalytic site oriented toward the outside of the cell and it is expressed mainly in adherent cells, it may serve a specialized function in cell-cell or cell to matrix interactions.…”