title: Biological activity of truncated C-terminus human heat shock protein 72.
abstract: Heat shock protein 72 (Hsp72), a canonical intracellular molecular chaperone, may also function as an extracellular danger signal for the innate immune system. To further delineate the biological role of Hsp72 in the innate immune system, we generated two truncated versions of the full length human Hsp72 (N-terminus Hsp72, amino acids 1-430; and C-terminus Hsp72 amino acids 420-641) and directly compared their ability to activate cells from the macrophage/monocyte lineage. In RAW 264.7 macrophages transfected with a NF-κB-dependent luciferase reporter plasmid, C-terminus Hsp72 was a more potent inducer of NF-κB activity than N-terminus Hsp72, and this effect did not seem to be secondary to endotoxin contamination. C-terminus Hsp72-mediated activation of the NF-κB pathway was corroborated by increased activation of IκB kinase, degradation of IκBα, and increased NF-κB-DNA binding. C-terminus Hsp72 was a more potent inducer of tumor necrosis factor-α (TNFα) expression in RAW 264.7 macrophages and in primary murine peritoneal macrophages from wild-type mice. C-terminus Hsp72 did not induce TNFα expression in primary murine peritoneal macrophages from Toll-like receptor (TLR4) mutant mice, indicating a role for TLR4. In human THP-1 mononuclear cells, C-terminus Hsp72 induced tolerance to subsequent LPS stimulation, whereas N-terminus Hsp72 did not induce tolerance. Finally, control experiments using equimolar amounts of N-terminus or C-terminus Hsp72 demonstrated a higher biological potency for C-terminus Hsp72. These data demonstrate that the ability of human Hsp72 to serve as an activator for cells of the macrophage/monocyte lineage primarily lies in the C-terminus region spanning amino acids 420-641.
authors.affiliation: Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center and Cincinnati Children's Research Foundation, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States.
authors.forename: Derek S
authors.initials: DS
authors.lastname: Wheeler
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Dunsmore
authors.forename: Alvin G
authors.initials: AG
authors.lastname: Denenberg
authors.forename: Larissa
authors.initials: L
authors.lastname: Muething
authors.forename: Sue E
authors.initials: SE
authors.lastname: Poynter
authors.forename: Hector R
authors.initials: HR
authors.lastname: Wong
mesh_terms: D000818:Animals; D002460:Cell Line; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects / Q000276:immunology*; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D009000:Monocytes / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D050884:HSP72 Heat-Shock Proteins; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4