Enhanced heat shock protein 70 expression alters proteasomal degradation of IκB kinase in experimental acute respiratory distress syndrome

OBJECTIVES: 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-κB (NF-κB) signal transduction pathway. NF-κB activation requires dissociation/degradation of a bound inhibitor, IκBα. IκBα degradation requires phosphorylation by IκB kinase, ubiquitination by the SCFβ (Skp1/Cullin1/Fbox β-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-κB activation at multiple points in the IκBα degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: 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-α and transfected. NF-κB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-κB and attenuated phosphorylation of IκBα. AdHSP treatment reduced, but did not eliminate, phosphorylation of the β-subunit of IκB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IκB kinase signalosome. This stabilized intermediary complexes containing IκB kinase components, IκBα, and NF-κB. Cellular studies indicate that although ubiquitination of IκBα was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-κB activation. This results from stabilization of intermediary NF-κB/IκBα/ IκB kinase complexes in a way that impairs proteasomal degradation of IκBα. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.