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J Biol Chem. 2010 Jul 2;285(27):21103-13. doi: 10.1074/jbc.M109.080671. Epub 2010 Mar 17.

Studies on the role of acid sphingomyelinase and ceramide in the regulation of tumor necrosis factor alpha (TNFalpha)-converting enzyme activity and TNFalpha secretion in macrophages.

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Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, USA.


Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor alpha (TNFalpha) secretion in macrophages. ASMase-deficient (asm(-/-)) mice and isolated peritoneal macrophages produce severalfold more TNFalpha than their wild-type (asm(+/+)) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNFalpha-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFalpha) to an active 17-kDa form (soluble (s)TNFalpha). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFalpha production. A substantial portion of the translated pro-TNFalpha was not processed to sTNFalpha; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2-3-fold higher in asm(-/-) macrophages as compared with asm(+/+) macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNFalpha-positive structures in the close vicinity of the plasma membrane in asm(-/-) but not in asm(+/+) macrophages. asm(-/-) cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm(-/-) cells a significant portion of pro-TNFalpha was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFalpha.

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