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Journal of Lipid Research, Vol. 49, 376-385, February 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis

Tinbo Ding^*, Zhiqiang Li, Tiruneh Hailemariam, Sushmita Mukherjee, Frederick R. Maxfield, Man-Ping Wu^1,* and Xian-Cheng Jiang^1,

Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203
^* School of Pharmacy, Fudan University, Shanghai, The People's Republic of China
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10065

Published, JLR Papers in Press, November 2, 2007.

¹ To whom correspondence should be addressed. e-mail: XJiang{at}downstate.edu (X-C.J.); mpwu{at}shmu.edu.cn (M.W.)

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 detergent-insoluble microdomains and significantly increased tumor necrosis factor--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.

Supplementary key words sphingomyelin synthase • siRNA • plasma membrane

Abbreviations: CHO, Chinese hamster ovary; DAG, diacylglycerol; FACS, fluorescence-activated cell sorting; KO, knockout; LPS, lipopolysaccharide; PC, phosphatidylcholine; PC-PLC, phosphatidylcholine-phospholipase C; PI-PLC, phosphatidylinositol-phospholipase C; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SMS, sphingomyelin synthase; SNK, Student-Newman-Keuls; SPT, serine palmitoyl-CoA transferase

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