Atypical mechanism of NF-κB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases

The transcription factor nuclear factor κB (NF-κB) regulates genes that contribute to acute inflammatory reactions in cytokine-activated endothelium. Tumor necrosis factor activates NF-κB through serine phosphorylation, induced by inhibitor κB kinases (IKK), and subsequent degradation of inhibitor κB (IκB). In contrast to cytokine stress, our studies show that oxidative stress, generated by exposure to hypoxia followed by reoxygenation (H/R), failed to activate IKK in human microvascular endothelial cells (HMEC-1). We report an alternative mechanism for NF-κB activation during H/R stress without IκBα degradation. This mechanism involves activation of protein tyrosine kinases (PTK) that phosphorylate IκBα with peak phosphorylation occurring after 30 min of reoxygenation. Involvement of PTK was reinforced by the demonstration that the PTK inhibitor, herbimycin A, prevented H/R-mediated NF-κB activation. Tyrosine phosphorylation alters the association between IκBα and NF-κB with sufficient intensity to allow transient NF-κB translocation to the cell nuclei within 45 min of onset of reoxygenation stress. Immunofluorescence imaging of NF-κB protein reveals it to be shuttled between the nucleus and cytoplasm within 90 min of reoxygenation. Furthermore, IκBα appears to be associated with NF-κB during the nucleo-cytoplasmic shuttling and is thus protected from degradation. Overall, these studies suggest that tyrosine phosphorylation of IκBα represents a proteolysis-independent mechanism of NF-κB activation that can be targeted for preventing H/R-mediated injury without affecting normal inflammatory responses.