title: Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.
abstract: OBJECTIVE
  Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production.
  
  
  RESULTS
  We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown.
  
  
  METHODS
  These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production.
  
  
  CONCLUSIONS
  Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA ; Center for Muscle Biology, University of Kentucky, Lexington, KY, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Moylan
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA ; Center for Muscle Biology, University of Kentucky, Lexington, KY, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Smith
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA ; Center for Muscle Biology, University of Kentucky, Lexington, KY, USA ; Markey Cancer Center, University of Kentucky, Lexington, KY, USA.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Wolf Horrell
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA ; Center for Muscle Biology, University of Kentucky, Lexington, KY, USA.
authors.forename: Julie B
authors.initials: JB
authors.lastname: McLean
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA.
authors.forename: Gergana M
authors.initials: GM
authors.lastname: Deevska
authors.affiliation: Department of Surgery, University of Kentucky, Lexington, KY, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Bonnell
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA.
authors.forename: Mariana N
authors.initials: MN
authors.lastname: Nikolova-Karakashian
authors.affiliation: Department of Physiology, University of Kentucky, Lexington, KY, USA ; Center for Muscle Biology, University of Kentucky, Lexington, KY, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Reid
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism*; D010084:Oxidation-Reduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016877:Oxidants; D014409:Tumor Necrosis Factor-alpha; D013108:Sphingomyelin Phosphodiesterase; C547598:neutral sphingomyelinase 3, human
keywords: Free radicals; KDEL, ER retention amino acid sequence.; Neutral sphingomyelinase-3; Oxidants; PAGE, polyacrylamide gel electrophoresis; ROS; Reactive oxygen species; Skeletal muscle; Sphingolipid signaling; VDAC, voltage dependent anion channel; nSMase3