Mechanisms of suppression of inducible nitric-oxide synthase (iNOS) expression in interferon (IFN)-gamma-stimulated RAW 264.7 cells by dexamethasone. Evidence for glucocorticoid-induced degradation of iNOS protein by calpain as a key step in post-transcriptional regulation.
The murine macrophage cell line RAW 264.7 expresses inducible nitric-oxide synthase (iNOS) activity upon stimulation with interferon (IFN)-gamma and/or bacterial lipopolysaccharide. We have studied the mechanisms by which the synthetic glucocorticoid dexamethasone suppresses IFN-gamma-stimulated iNOS expression in RAW 264.7 cells. Treatment of cells with dexamethasone reduces the formation of nitrite, one of the stable end products of NO production measured in culture supernatants with an IC50 of 9 nM. The reduction of iNOS activity is caused by decreased iNOS protein levels as assessed by immunoblotting using a specific anti-iNOS antibody. Dexamethasone treatment also reduces the formation of iNOS mRNA steady state levels to about 50% in IFN-gamma-stimulated cells. This is due to decreased iNOS gene transcription and iNOS mRNA stability. More importantly, dexamethasone reduces the amount of iNOS protein by two additional mechanisms: reduction of the translation of iNOS mRNA and increased degradation of the iNOS protein. Using a specific protease inhibitor for the cysteine protease calpain I, N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I), the enhanced proteolysis of the iNOS protein can efficiently be blocked, whereas other protease inhibitors such as tosyl-L-lysine chloromethyl ketone have no effect. Dexamethasone does not significantly alter calpain gene expression. Northern blot analyses reveal that calpain mRNA steady state levels are virtually not affected upon incubation of the cells with IFN-gamma and dexamethasone. Immunoprecipitation using a polyclonal anti-calpain antibody reveals that calpain protein levels are also not affected by the glucocorticoid. This is the first evidence that the iNOS protein is a molecular target for the cysteine protease calpain.
Walker
G
G
Department of Pharmacology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Pfeilschifter
J
J
Kunz
D
D
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D012333:RNA, Messenger; D014107:Tosyllysine Chloromethyl Ketone; D003907:Dexamethasone; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D002154:Calpain