Activation of N-SMase (neutral sphingomyelinase) is an established part of the response of cytokines such as TNF (tumour necrosis factor)-α. However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, nSMase2 and nSMase3) are responsible for this activity. In MCF-7 cells, we found that TNF-α induces late, but not early, increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siRNA (small interfering RNA) knockdown of nSMase1 significantly decreased basal in vitro N-SMase activity of MCF-7 cells, whereas nSMase2 but not nSMase3 siRNA inhibited TNF-α-induced activity. Taken together, these results identify nSMase2 as the major TNF-α-responsive N-SMase in MCF-7 cells. Moreover, the results suggest that nSMase3 may not possess in vitro N-SMase activity and does not affect cellular sphingolipid levels in the cell lines evaluated. On the other hand, nSMase1 contributes to in vitro N-SMase activity, but does not affect cellular sphingolipids much.
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Research Article|
March 29 2011
Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-α in MCF-7 cells
Christopher J. Clarke;
Christopher J. Clarke
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, U.S.A.
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Emily A. Cloessner;
Emily A. Cloessner
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, U.S.A.
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Patrick L. Roddy;
Patrick L. Roddy
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, U.S.A.
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Yusuf A. Hannun
Yusuf A. Hannun
1
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, U.S.A.
1To whom correspondence should be addressed (email hannun@musc.edu).
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Publisher: Portland Press Ltd
Received:
October 25 2010
Revision Received:
February 03 2011
Accepted:
February 08 2011
Accepted Manuscript online:
February 08 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 435 (2): 381–390.
Article history
Received:
October 25 2010
Revision Received:
February 03 2011
Accepted:
February 08 2011
Accepted Manuscript online:
February 08 2011
Citation
Christopher J. Clarke, Emily A. Cloessner, Patrick L. Roddy, Yusuf A. Hannun; Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-α in MCF-7 cells. Biochem J 15 April 2011; 435 (2): 381–390. doi: https://doi.org/10.1042/BJ20101752
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