Studies on the Role of Acid Sphingomyelinase and Ceramide in the Regulation of Tumor Necrosis Factor α (TNFα)-converting Enzyme Activity and TNFα Secretion in Macrophages

• Published in: The Journal of biological chemistry Vol. 285; no. 27; pp. 21103 - 21113
• Main Authors: Rozenova, Krasimira A., Deevska, Gergana M., Karakashian, Alexander A., Nikolova-Karakashian, Mariana N.
• Format: Journal Article
• Language: English
• Published: 9650 Rockville Pike, Bethesda, MD 20814, U.S.A Elsevier Inc 02-07-2010; American Society for Biochemistry and Molecular Biology
• Subjects: Ceramide; Cytokines/Tumor Necrosis Factor; Endosomes; Inflammation; Lipids; Lipopolysaccharide (LPS); Lysosomes; Secretion; Sphingomyelinase; TACE; Toll-like Receptors (TLR); Toll-like Receptors (TLR); Secretion; Cytokines/Tumor Necrosis Factor; Lysosomes; TACE; Ceramide; Inflammation; Sphingomyelinase; Lipopolysaccharide (LPS); Endosomes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.080671

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 α (TNFα) secretion in macrophages. ASMase-deficient (asm−/−) mice and isolated peritoneal macrophages produce severalfold more TNFα 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 TNFα-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFα) to an active 17-kDa form (soluble (s)TNFα). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFα production. A substantial portion of the translated pro-TNFα was not processed to sTNFα; 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 TNFα-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-TNFα was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFα.