Studies on the Role of Acid Sphingomyelinase and Ceramide in the Regulation of TACE Activity and TNFalpha Secretion in Macrophages

Abstract

Acid Sphingomyelinase (ASMase) activity mediates LPS signaling in various cell types. This study shows that in macrophages, ASMase is a negative regulator of LPS‐induced TNFα secretion. ASMase‐deficient (asm−/−) mice and peritoneal macrophages produce several fold more TNFα than their wild‐type (asm+/+) counterparts when stimulated with LPS, while the addition of exogenous ceramides or SMase 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 26kD precursor (proTNFα) to the active 17kD form (sTNFα). Asm−/− macrophages exhibited 2 to 3 fold higher TACE activity than asm+/+ macrophages and adding ceramide or SMase suppressed this difference. Our studies further showed that the activity of TACE was rate‐limiting factor for TNFα production and proTNFα that was not processed was internalized and degraded in the lysosomes. Indirect immunofluorescence experiments involving inhibitors of TACE, endocytosis, and lysosomal proteolysis showed that in asm−/− cells significant portion of proTNFα is sequestered within the early endosomes and instead of undergoing lysosomal proteolysis, it is recycled to the plasma membrane and processed to sTNFα. In conclusion, these experiments define a novel role of ASMase in regulation of TNFα secretion by macrophages.