Heat shock inhibits phosphorylation of I-kappa B alpha

Previous studies demonstrated that induction of the heat shock response is associated with inhibition of the proinflammatory transcription factor NF-k appaB by a mechanism involving inhibition of I-kappaB alpha degradation. To provide further insight regarding the interactions of these fundamental ce llular responses, the present experiments were designed to elucidate the me chanism(s) by which heat shock inhibits degradation of I-kappaB alpha, In a n in vitro model of inflammatory cell signaling, treatment of RAW 264.7 mur ine macrophages with LPS (100 ng/mL) caused rapid degradation of I-kappaB a lpha, Heat shock, 1 h before treatment with LPS, completely inhibited LPS-m ediated degradation of I-kappaB alpha. Immunoprecipitation studies demonstr ated that heat shock inhibited LPS-mediated ubiquitination of I-kappaB alph a. Western-blot analyses using a phosphorylated I-kappaB alpha -specific an tibody 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 inhi bits LPS-mediated degradation of I-kappaB alpha involves specific inhibitio n of I-kappaB alpha phosphorylation and subsequent I-kappaB alpha ubiquitin ation. In addition, this mechanism does not involve activation of heat shoc k factor-1 or the heat shock proteins regulated by heat shock factor-1.