title: SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis.
abstract: 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-alpha-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.
authors.affiliation: Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA.
authors.forename: Tinbo
authors.initials: T
authors.lastname: Ding
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Li
authors.forename: Tiruneh
authors.initials: T
authors.lastname: Hailemariam
authors.forename: Sushmita
authors.initials: S
authors.lastname: Mukherjee
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Maxfield
authors.forename: Man-Ping
authors.initials: MP
authors.lastname: Wu
authors.forename: Xian-Cheng
authors.initials: XC
authors.lastname: Jiang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004075:Diglycerides / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D013109:Sphingomyelins / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology
chemical_list: D004075:Diglycerides; D007527:Isoenzymes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D013109:Sphingomyelins; C455440:SGMS1 protein, human; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase