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A more recent version of this article appeared on February 1, 2008

Papers In Press, published online ahead of print November 2, 2007
J. Lipid Res., doi:10.1194/jlr.M700401-JLR200
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Submitted on September 5, 2007
Revised on October 29, 2007
Accepted on November 1, 2007

Sphingomyelin synthase (SMS) overexperssion and knockdown: Impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apopotosis

Tinbo Ding, Zhiqiang Li, Tiruneh Hailemariam, Sushmita Mukherjee, Frederick R. Maxfield, Manping Wu, and Xian-Cheng Jiang

Anatomy and Cell Biology, SUNY, Brooklyn, NY 11203

Corresponding Author: xjiang{at}downstate.edu

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine (PC) as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary (CHO) 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 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 detergent-insoluble microdomains, and significantly increased tumor necrosis factor(TNF)-a–mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 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 (LPS)-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.


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