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.
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.
Rozenova
Krasimira A
KA
Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Deevska
Gergana M
GM
Karakashian
Alexander A
AA
Nikolova-Karakashian
Mariana N
MN
D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D000818:Animals; D002518:Ceramides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005838:Genotype; D006579:Heterozygote; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013108:Sphingomyelin Phosphodiesterase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D002518:Ceramides; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D012313:RNA; D013108:Sphingomyelin Phosphodiesterase; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse