Heat shock inhibits phosphorylation of I-kappaBalpha.
Previous studies demonstrated that induction of the heat shock response is associated with inhibition of the proinflammatory transcription factor NF-kappaB by a mechanism involving inhibition of I-kappaBalpha degradation. To provide further insight regarding the interactions of these fundamental cellular responses, the present experiments were designed to elucidate the mechanism(s) by which heat shock inhibits degradation of I-kappaBalpha. In an in vitro model of inflammatory cell signaling, treatment of RAW 264.7 murine macrophages with LPS (100 ng/mL) caused rapid degradation of I-kappaBalpha. Heat shock, 1 h before treatment with LPS, completely inhibited LPS-mediated degradation of I-kappaBalpha. Immunoprecipitation studies demonstrated that heat shock inhibited LPS-mediated ubiquitination of I-kappaBalpha. Western-blot analyses using a phosphorylated I-kappaBalpha-specific antibody demonstrated that heat shock inhibited LPS-mediated phosphorylation of I-kappaBalpha. In contrast, heat shock induced phosphorylation of c-jun. In murine fibroblasts having genetic ablation of the heat shock factor-1 gene, heat shock inhibited tumor necrosis factor-alpha mediated degradation of I-kappaBalpha. We conclude that the mechanism by which heat shock inhibits LPS-mediated degradation of I-kappaBalpha involves specific inhibition of I-kappaBalpha phosphorylation and subsequent I-kappaBalpha ubiquitination. In addition, this mechanism does not involve activation of heat shock factor-1 or the heat shock proteins regulated by heat shock factor-1.
Shanley
T P
TP
Division of Critical Care Medicine, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.
Ryan
M A
MA
Eaves-Pyles
T
T
Wong
H R
HR
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response / Q000502:physiology*; D020672:I-kappa B Proteins; D007249:Inflammation / Q000209:etiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D014157:Transcription Factors; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha