Inhibition of IkappaB-alpha and IkappaB-beta proteolysis by calpain inhibitor I blocks nitric oxide synthesis.
Lipopolysaccharide (LPS) stimulates the induction of the inducible isoform of nitric oxide synthase (iNOS) in part by inducing the nuclear translocation of the transcription factor nuclear factor-kappa B (NF-kappaB). LPS induces ubiquination and phosphorylation of the IkappaB inhibitory subunit of NF-kappaB. Subsequently, the ubiquitin-proteasome multicatalytic enzyme complex catalyzes the proteolytic degradation of IkappaB with resultant nuclear translocation of NF-kappaB. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW macrophages. The inhibitor was found to block iNOS transcription and protein translation as noted by Northern analysis and Western blotting, respectively. LPS stimulated rapid proteolytic degradation of IkappaB-alpha which was inhibited by approximately 50% in the presence of calpain inhibitor I. In contrast, LPS induced the delayed proteolytic degradation of IkappaB-beta which was almost totally inhibited by calpain inhibitor I. Calpain inhibitor I also decreased the LPS-induced nuclear translocation of NF-kappaB. These results demonstrate that the ubiquitin-proteasome complex has an important role in induction of iNOS in response to stimuli which act via the NF-kappaB/IkappaB signal transduction pathway. Furthermore, the results suggest that the ubiquitin-proteasome complex is important in the degradation of IkappaB-beta as well as IkappaB-alpha. Finally, we have demonstrated that there is a marked difference in the extent of proteolysis of IkappaB-alpha and IkappaB-beta when the ubiquitin-proteasome complex is inhibited with calpain inhibitor I.
Milligan
S A
SA
Department of Medicine, VA Medical Center and Louisiana State University Medical Center, Shreveport 71101, USA.
Owens
M W
MW
Grisham
M B
MB
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006023:Glycoproteins / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; C497594:Relb protein, mouse; D014157:Transcription Factors; C039304:calpain inhibitors; D051841:Transcription Factor RelB; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex