Regulated Secretion of Acid Sphingomyelinase: IMPLICATIONS FOR SELECTIVITY OF CERAMIDE FORMATION

• Published in: The Journal of biological chemistry 2010-11, Vol.285 (46), p.35706-35718
• Main Authors: Jenkins, Russell W, Canals, Daniel, Idkowiak-Baldys, Jolanta, Simbari, Fabio, Roddy, Patrick, Perry, David M, Kitatani, Kazuyuki, Luberto, Chiara, Hannun, Yusuf A
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Blotting, Western; Cell Line, Tumor; Ceramides - metabolism; Dose-Response Relationship, Drug; Extracellular Space - drug effects; Extracellular Space - enzymology; HEK293 Cells; Humans; Interleukin-1beta - pharmacology; Intracellular Space - drug effects; Intracellular Space - enzymology; Lipids; Lysosomes - enzymology; Mutation; Reverse Transcriptase Polymerase Chain Reaction; Serine - genetics; Serine - metabolism; Sphingomyelin Phosphodiesterase - genetics; Sphingomyelin Phosphodiesterase - metabolism; Time Factors; Transfection; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.125609

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMaseWT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C₁₆-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMaseWT exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMaseS⁵⁰⁸A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C₁₆-ceramide. Taken together, these data support a crucial role for Ser⁵⁰⁸ in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.