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Sphingomyelin Synthase Is Abnormal Expression Of The Discussion Of Its Relationship With Apoptosis

Posted on:2009-03-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:T B DingFull Text:PDF
GTID:1110360272958825Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Sphingomyelin synthase (SMS), the last key 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 constructed 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%), ceramide (44% or 42%), and DAG (35% or 31%), as well as the ceramide:SM ratio (18% or 18%), 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-α-mediated apoptosis. To confirm these results, 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%), with reducing LPS-mediated apoptosis compared with controls. These results indicate that SMS 1 and SMS2 are key factors in the control of SM and DAG levels within the cell and, thus, influence apoptosis.
Keywords/Search Tags:Sphingomyelin synthase, apoptosis, overexpresssion, knockdown
PDF Full Text Request
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