INHIBITION OF I-KAPPA-B-ALPHA AND I-KAPPA-B-BETA PROTEOLYSIS BY CALPAIN INHIBITOR-I BLOCKS NITRIC-OXIDE SYNTHESIS

Lipopolysaccharide (LPS) stimulates the induction of the inducible iso form of nitric oxide synthase (iNOS) in part by inducing the nuclear t ranslocation of the transcription factor nuclear factor-kappa B (NF-ka ppa B). LPS induces ubiquination and phosphorylation of the I kappa B inhibitory subunit of NF-kappa B. Subsequently, the ubiquitin-proteaso me multicatalytic enzyme complex catalyzes the proteolytic degradation of I kappa B with resultant nuclear translocation of NF-kappa B. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW m acrophages, The inhibitor was found to block iNOS transcription and pr otein translation as noted by Northern analysis and Western blotting, respectively, LPS stimulated rapid proteolytic degradation of I kappa B-alpha which was inhibited by approximately 50% in the presence of ca lpain inhibitor I, In contrast, LPS induced the delayed proteolytic de gradation of I kappa B-beta which was almost totally inhibited by calp ain inhibitor I, Calpain inhibitor I also decreased the LPS-induced nu clear translocation of NF-kappa B. These results demonstrate that the ubiquitin-proteasome complex has an important role in induction of iNO S in response to stimuli which act via the NF-kappa B/I kappa B signal transduction pathway. Furthermore, the results suggest that the ubiqu itin-proteasome complex is important in the degradation of I kappa B-b eta as well as I kappa B-alpha. Finally, we have demonstrated that the re is a marked difference in the extent of proteolysis of I kappa B-al pha and I kappa B-beta when the ubiquitin-proteasome complex is inhibi ted with calpain inhibitor I. (C) 1996 Academic Press, Inc.