Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.
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-aSMase(WT) 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 C(16)-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-aSMase(WT) 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-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.
Jenkins
Russell W
RW
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Canals
Daniel
D
Idkowiak-Baldys
Jolanta
J
Simbari
Fabio
F
Roddy
Patrick
P
Perry
David M
DM
Kitatani
Kazuyuki
K
Luberto
Chiara
C
Hannun
Yusuf A
YA
D019943:Amino Acid Substitution; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology*; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D002518:Ceramides; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; C543066:SMPD1 protein, human; D013108:Sphingomyelin Phosphodiesterase
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