title: Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70.
abstract: OBJECTIVE
  Alveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress protein response (SPR) in alveolar macrophages on iNOS-dependent NO production in response to interferon gamma (IFNgamma), a major cytokine present in the airspace of patients with ALI, was investigated.
  
  
  METHODS
  The SPR was activated in murine and primary human alveolar macrophages prior to analysis of signal transducer and activator of transcription factor 1 (STAT1) activation, iNOS mRNA and protein synthesis, and NO production.
  
  
  RESULTS
  SPR activation resulted in inhibition of IFNgamma-mediated NO production (p=0.001) with >95% detergent insolubilisation of the STAT1 protein. Its subsequent proteasomal degradation was partially reversed with pretreatment of cells with the chemical chaperone glycerol. This early effect of the SPR was caused by the complete disruption of heat shock protein 90 (Hsp90)-STAT1 binding, as shown by immunoprecipitation. Recovery of STAT1 activation and recovery of iNOS synthesis occurred within 12 h after SPR activation (p=0.02). NO production (as compared with non-SPR controls) did not occur until 48 h later (p=0.02). SPR-induced Hsp70 (Hsp70i) expression caused a late inhibition of NO production (p=0.02). Inhibiting >50% Hsp70i expression recovered NO production to control levels whereas overexpressing Hsp70i in the absence of the SPR inhibited NO production (p=0.02).
  
  
  CONCLUSIONS
  Early inhibition of STAT1 following its dissociation from Hsp90, and later inhibition of iNOS activity by Hsp70i, represent novel mechanisms by which SPR activation modulates the IFNgamma signalling in alveolar macrophages. These results highlight a potential clinical application for Hsp90 inhibitors in modulating NO signalling during the early phase of acute lung injury.
authors.affiliation: Laboratory of Surgical Research, Department of Anesthesia, University of California, San Francisco, CA 94110, USA. howardm@anesthesia.ucsf.edu
authors.forename: Marybeth
authors.initials: M
authors.lastname: Howard
authors.forename: Jérémie
authors.initials: J
authors.lastname: Roux
authors.forename: Hyon
authors.initials: H
authors.lastname: Lee
authors.forename: Byron
authors.initials: B
authors.lastname: Miyazawa
authors.forename: Jae-Woo
authors.initials: JW
authors.lastname: Lee
authors.forename: Brandi
authors.initials: B
authors.lastname: Gartland
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Howard
authors.forename: Michael A
authors.initials: MA
authors.lastname: Matthay
authors.forename: Michel
authors.initials: M
authors.lastname: Carles
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Pittet
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D006358:Hot Temperature; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013312:Stress, Physiological / Q000187:drug effects / Q000502:physiology
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II
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