Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43°C for 20 min) resulted in inhibition of H 2 O 2 -induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H 2 O 2 -induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homooligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H 2 O 2 -induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed serine-threonine kinase that was initially discovered as a mitogen-activated protein kinase kinase kinase (MAPKKK) in the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK signaling cascades (12, 13). ASK1 functions in apoptosis induced by diverse stimuli, including tumor necrosis factor alpha (TNF-␣), Fas, and many apoptotic stresses (12,13,30). Overexpression of a dominantnegative mutant of ASK1 thus prevents apoptosis induced by withdrawal of growth factors, DNA-damaging agents, TNF-␣, or agonistic antibodies to Fas (4,13,16,30). Activation of ASK1 is reported to induce apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 (11).Many cellular stresses that stimulate the stress-activated MAPK pathways can also induce expression of heat shock proteins. Heat shock protein 72 (Hsp72) is the major inducible heat shock protein (35). It plays a role in many cellular activities including protein synthesis, folding, and translocation into organelles as well as the assembly of multiprotein complexes (2,5,24,25,33). Hsp72 contains two conserved domains, an ATP-binding domain (ABD) and a peptide-binding domain (PBD), that are important for its chaperon function (7, 21). Hsp72 also prevents cell death initiated by various apoptotic stresses including heat shock, ceramide, ionizing irradiation, TNF-␣, and ischemia (8,14,20,23,32). Hsp72 suppresses several apoptotic signaling pathways, including caspase cascades and stress-activated MAPK pathways that include the JNK and p38 signaling cascades (3,8,9,15,17,19,22,23,29,31). Furthermore, Hsp72 has been shown to physically interact with and inhibit Apaf-1 and apoptosis-inducing factor, resulting in suppression of caspase-dependent and -independent apoptosis, respectively ...

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ, and induced T-cell-mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA(251-264)-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy.