Heat shock inhibits phosphorylation of I-κBα

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-kappaB alpha 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-kappaB alpha, 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-kappaB alpha, Heat shock, 1 h before treatment with LPS, completely inhibited LPS-mediated degradation of I-kappaB alpha. Immunoprecipitation studies demonstrated that heat shock inhibited LPS-mediated ubiquitination of I-kappaB alpha. Western-blot analyses using a phosphorylated I-kappaB alpha -specific antibody demonstrated that heat shock inhibited LPS-mediated phosphorylation of I-kappaB alpha. 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-kappaB alpha. We conclude that the mechanism by which heat shock inhibits LPS-mediated degradation of I-kappaB alpha involves specific inhibition of I-kappaB alpha phosphorylation and subsequent I-kappaB alpha 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.