Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures.
The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.
Franzen
Rochus
R
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany.
Pfeilschifter
Josef
J
Huwiler
Andrea
A
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005920:Glomerular Mesangium / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D050356:Lipid Metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D055576:Neutral Ceramidase; D009569:Nitric Oxide / Q000378:metabolism*; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism*
D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D025801:Ubiquitin; C067713:lactacystin; D009569:Nitric Oxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000581:Amidohydrolases; C526748:Asah2 protein, rat; D055572:Ceramidases; D055576:Neutral Ceramidase; D000111:Acetylcysteine