Expression of neutral sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes modulates IL-beta-induced JNK activation

FASEB J. 2004 Jun;18(9):968-70. doi: 10.1096/fj.03-0875fje. Epub 2004 Apr 1.


Neutral sphingomyelinase (NSMase) has been proposed to mediate interleukin (IL)-1beta signaling in liver. In this paper, we used adenovirus-mediated gene transfer to inducibly express FLAG-tagged mouse NSMase-2 in primary rat hepatocytes in order to further elucidate the molecular nature of the NSMase involved. Initial studies confirmed that the EST clone used in these experiments encoded a Mg2+-dependent NSMase. The in vitro activity of the heterologously expressed enzyme was inhibited in the presence of 0.5% Triton or 50 mM EDTA. In addition, the expression of this NSMase-2 clone in primary hepatocytes led to increased cellular levels of ceramide, indicating that the enzyme is active in situ. Immunofluorescence studies in Hep G2 cells infected with NSMase-2 expressing adenoviruses showed that the FLAG-tagged NSMase-2 was localized to the plasma membrane. Cell viability remained unchanged 72 h following infection and induction. The effect of NSMase-2 expression on IL-1beta-induced activation of c-Jun N-terminal kinase (JNK) was tested. Expression of NSMase-2 increased JNK phosphorylation between 1.5- and 2-fold over the basal level. Furthermore, NSMase-2 was found to strongly increase the ability of IL-1beta to phosphorylate JNK. This potentiation was mediated by a phosphatase from the PP2A family, possibly by modulating the phosphorylation pattern of IL-1beta receptor-associated kinase (IRAK). In conclusion, the data presented suggest that NSMase-2 could be involved in IL-1beta-induced JNK activation in hepatocytes.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Membrane / enzymology
  • Cells, Cultured
  • Cloning, Molecular
  • Enzyme Activation / drug effects
  • Expressed Sequence Tags
  • Hepatocytes / drug effects
  • Hepatocytes / enzymology
  • Hepatocytes / metabolism
  • Humans
  • Interleukin-1 / pharmacology*
  • Interleukin-1 Receptor-Associated Kinases
  • JNK Mitogen-Activated Protein Kinases
  • Male
  • Mice
  • Mitogen-Activated Protein Kinases / metabolism*
  • Models, Biological
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation / drug effects
  • Protein Kinases / metabolism
  • Protein Transport
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Rats
  • Rats, Inbred F344
  • Sphingomyelin Phosphodiesterase / chemistry
  • Sphingomyelin Phosphodiesterase / genetics*
  • Sphingomyelin Phosphodiesterase / metabolism*
  • Tyrphostins / pharmacology


  • Interleukin-1
  • Tyrphostins
  • AG 127
  • Protein Kinases
  • Protein-Tyrosine Kinases
  • Interleukin-1 Receptor-Associated Kinases
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases
  • Phosphoprotein Phosphatases
  • SMPD3 protein, human
  • Smpd3 protein, mouse
  • Smpd3 protein, rat
  • Sphingomyelin Phosphodiesterase