Redox-regulated NFkappaB activation following ischemia/reperfusion injury in the liver.

摘要

NFκB is a nuclear transcription factor involved in modulating cellular responses to stimuli such as ischemia/reperfusion injury. The most commonly studied pathway of NFκB activation involves the phosphorylation of IκBα on serine residues 32 and 36 by the IκB-kinase (IKK) complex. Alternatively, tyrosine 42 phosphorylation of IκBα without proteolytic degradation has also been shown to activate NFκB following hypoxia/reoxygenation (H/R), ischemia/reperfusion (I/R) or pervanadate (PV) treatment. In the present study, both in vitro and in vivo models were investigated to better understand the mechanisms involved in tyrosine kinase-mediated activation of NFκB and the downstream consequences of that activation. We have identified c-Src as the kinase leading to tyrosine phosphorylation IκBα and activation of NFκB through an IKK-independent mechanism. Furthermore, this pathway was regulated through a redox-dependent mechanism involving intracellular H₂O₂ production following H/R injury. The involvement of NFκB in apoptotic cellular responses to environmental injuries was also studied. To this end, we have constructed a recombinant adenoviral vector (Ad.IκBαAS) expressing antisense IκBα mRNA that is capable of augmenting NFκB activation prior to various cellular injuries such as TNF-α, UV, H/R, or pervanadate treatment. Using, an in vitro HeLa cell line model, we have also demonstrated that the constitutive activation of NFκB reduces apoptosis only following injuries associated with IκBα Y42, but not S32/36, phosphorylation. These findings demonstrate that the temporal regulation of NFκB and the apoptotic consequences of this activation are differentially influenced by whether the pathway mediating NFκB activation involves serine or tyrosine kinases. Studies evaluating IκB knock-in mice (AKBI), in which the IκBα gene is replaced by the IκBβ gene, were used to assess the role of tyrosine phosphorylation of IκBα in vivo. Our data indicated that following I/R injury in the liver, tyrosine 42 phosphorylation is required for NFκB activation, which mediated TNF-α secretion and subsequent pro-inflammatory responses. In contrast, no differences in any of these indicators were observed following a lethal injection of LPS. These results demonstrate that IκBα and IκBβ possess non-redundant biologic activities which adapt NF-κB-mediated proinflammatory responses to specific types of injury stimuli.