Positive feedback regulation of heat shock protein 70 (Hsp70) is mediated through Toll-like receptor 4-PI3K/Akt-glycogen synthase kinase-3β pathway.

Alarmins, the endogenous molecules that recruit and activate innate immune cells, are considered as subgroups of damage-associated molecular patterns. Heat shock protein 70 (Hsp70) is one of putative alarmins together with high mobility group box 1, S100s, interleukin-1α, and annexins. It has cytokine-like functions as well as molecular chaperone functions. However, the cytokine function of Hsp70 has not been clear. Here, we demonstrated that there exists the positive feedback regulation of Hsp70 induction in innate immune cells. Heat stress (HS) increased intracellular Hsp70 (iHsp70) and it was actively released into extracellular space through the Golgi complex. Human recombinant Hsp70 (rhHsp70) up-regulated iHsp70 expression and induced pro-inflammatory cytokine secretion via Toll-like receptor 4 (TLR4). rhHsp70 rapidly activated Akt, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Moreover, glycogen synthase kinase-3β (GSK-3β) was inactivated by rhHsp70-induced Akt activation. Knockdown of TLR4 and overexpression of dominant negative TLR4 (DN-TLR4) suppressed the above effects of rhHsp70. The effects of rhHsp70 were not due to endotoxin contamination. Akt-dependent GSK-3β inactivation was responsible for iHsp70 induction by rhHsp70. Overexpression of DN-Akt or constitutively active GSK-3β or pretreatment of LY294002 inhibited rhHsp70-induced iHsp70 up-regulation, which was similar to the mechanism of HS-mediated induction of Hsp70. Thus, these data suggest the positive feedback regulatory mechanism of iHsp70 induction.