Involvement of Stat3 phosphorylation in mild heat shock-induced thermotolerance.

Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.

Nmi interacts with Hsp105ß and enhances the Hsp105ß-mediated Hsp70 expression.

The mammalian stress protein Hsp105α is expressed constitutively and is further induced under stress conditions, whereas the alternative spliced form, Hsp105ß is only expressed during mild heat shock. We previously reported that Hsp105α is localized mainly in the cytoplasm, whereas Hsp105ß is localized in the nucleus. Consistent with the different localization of these proteins, Hsp105ß but not Hsp105α induces the expression of the major stress protein Hsp70. We here identified N-myc and Stat interactor (Nmi), as an Hsp105ß-binding protein by yeast two-hybrid screening. Immunoprecipitation and pull-down assay showed that Nmi interacts with Hsp105ß in vivo and in vitro. Luciferase reporter gene assay and Western blotting showed that Nmi enhanced both the Hsp105ß-induced phosphorylation of Stat3 and the Hsp105ß-induced activation of the hsp70 promoter in a manner that is dependent on the Stat3-binding site, which results in an increase in Hsp70 protein levels. Most importantly, mild heat shock-induced Hsp70 expression, which is dependent on Hsp105ß, is suppressed by knockdown of endogenous Nmi. These results suggest that Nmi has a role as a positive regulator of Hsp105ß-mediated hsp70 gene expression along the Stat3 signaling pathway.