Role of the Heat Shock Protein 90 in Immune Response Stimulation by Bacterial DNA and Synthetic Oligonucleotides

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) and Escherichia coli DNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN or E. coli DNA. These Hsp90 inhibitors, however, had no effect on Staphylococcus aureus Cowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN and E. coli DNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins