Background-NFB has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFB activation. Methods and Results-To investigate the role of SMS2 in NFB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA-treated HEK 293 cells after tumor necrosis factor (TNF)-␣ simulation. In line with attenuated NFB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF-␣-stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFB activation. Conclusions-SMS2 is a modulator of NFB activation, and thus it could play an important role in NFB-mediated proatherogenic process. Key Words: sphingomyelin synthase 2 Ⅲ sphingomyelin Ⅲ lipid rafts Ⅲ NFB Ⅲ atherosclerosis A therosclerosis is an inflammatory disease. The accumulation of macrophage-derived foam cells in the vessel wall is always accompanied by the production of a wide range of chemokines, cytokines, and growth factors. 1 These factors regulate the turnover and differentiation of immigrating and resident cells, eventually influencing plaque development. One of the key regulators of inflammation is NFB, 2 which has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. 3,4 Sphingomyelin (SM) is one of the major lipids on the plasma membrane and is enriched in lipid rafts, which are considered microdomains of plasma membrane critical for signal transduction. 5,6 Depletion of cholesterol from rafts causes a redistribution of TNF-␣ receptor 1 to nonraft plasma membrane, preventing NFB activation 7 or ligand-induced RhoA activation, 8 and such treatment also inhibits proinflammatory signals mediated by TLRs. 9 Studies also suggest that NFB activation is triggered by SM-derived ceramide. 10,11 On the contrary, it has been shown that ceramide is not necessary or even inhibits NFB activation. 12 SM biosynthesis might also affect NFB activation. SM is synthesized by sphingomyelin synthase (SMS), which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide, producing SM and diacylglycerol (DAG). 13 Luberto et al 14 found that D609, a nonspecific SMS inhibitor, blocks TNF-␣-and phorbol ester-mediated NFB activation that was c...

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.

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergentinsoluble microdomains and significantly increased tumor necrosis factor-a-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.-Ding, T., Z. Li, T. Hailemariam, S. Mukherjee, F. R. Maxfield, M-P. Wu, and X-C. Jiang. SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis. J. Lipid Res. 2008. 49: 376-385.