Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation

J Biol Chem. 2010 Nov 12;285(46):35706-18. doi: 10.1074/jbc.M110.125609. Epub 2010 Aug 31.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • 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*

Substances

  • Ceramides
  • Interleukin-1beta
  • Tumor Necrosis Factor-alpha
  • Serine
  • SMPD1 protein, human
  • Sphingomyelin Phosphodiesterase