title: Expression of neutral sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes modulates IL-beta-induced JNK activation.
abstract: 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.
authors.affiliation: Department of Physiology, University of Kentucky, A. B. Chandler Medical Center, Lexington, Kentucky 40536, USA.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Karakashian
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Giltiay
authors.forename: George M
authors.initials: GM
authors.lastname: Smith
authors.forename: Mariana N
authors.initials: MN
authors.lastname: Nikolova-Karakashian
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D020224:Expressed Sequence Tags; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D053592:Interleukin-1 Receptor-Associated Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D008297:Male; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D011916:Rats, Inbred F344; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D007375:Interleukin-1; D020032:Tyrphostins; C069028:AG 127; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; C491484:SMPD3 protein, human; C491483:Smpd3 protein, mouse; C107180:Smpd3 protein, rat; D013108:Sphingomyelin Phosphodiesterase