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Volume 272, Number 26, Issue of June 27, 1996 pp. 16679-16687
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Suppression of Inducible Nitric-oxide Synthase (iNOS) Expression in Interferon (IFN)--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

(Received for publication, January 23, 1997, and in revised form, April 9, 1997)

Gaby Walker , Josef Pfeilschifter ^§ and Dieter Kunz

From the  Department of Pharmacology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the ^§ Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany

The murine macrophage cell line RAW 264.7 expresses inducible nitric-oxide synthase (iNOS) activity upon stimulation with interferon (IFN)- and/or bacterial lipopolysaccharide. We have studied the mechanisms by which the synthetic glucocorticoid dexamethasone suppresses IFN--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--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- 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.

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