title: Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.
abstract: OBJECTIVE
  Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway.
  
  
  METHODS
  Laboratory investigation.
  
  
  METHODS
  University medical center research laboratory.
  
  
  METHODS
  Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture.
  
  
  METHODS
  Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools.
  
  
  RESULTS
  Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism.
  
  
  CONCLUSIONS
  Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.
authors.affiliation: Department of Anesthesiology and Critical Care Medicine and the Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University School of Medicine, Jerusalem, Israel. weiss@hadassah.org.il
authors.forename: Yoram G
authors.initials: YG
authors.lastname: Weiss
authors.forename: Zohar
authors.initials: Z
authors.lastname: Bromberg
authors.forename: Nichelle
authors.initials: N
authors.lastname: Raj
authors.forename: Jacob
authors.initials: J
authors.lastname: Raphael
authors.forename: Pierre
authors.initials: P
authors.lastname: Goloubinoff
authors.forename: Yinon
authors.initials: Y
authors.lastname: Ben-Neriah
authors.forename: Clifford S
authors.initials: CS
authors.lastname: Deutschman
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000502:physiology*; D051550:I-kappa B Kinase / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012128:Respiratory Distress Syndrome / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D000072000:NF-KappaB Inhibitor alpha; D051550:I-kappa B Kinase; D046988:Proteasome Endopeptidase Complex