Atypical mechanism of NF-kappaB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases.
The transcription factor nuclear factor kappaB (NF-kappaB) regulates genes that contribute to acute inflammatory reactions in cytokine-activated endothelium. Tumor necrosis factor activates NF-kappaB through serine phosphorylation, induced by inhibitor kappaB kinases (IKK), and subsequent degradation of inhibitor kappaB (IkappaB). 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-kappaB activation during H/R stress without IkappaBalpha degradation. This mechanism involves activation of protein tyrosine kinases (PTK) that phosphorylate IkappaBalpha 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-kappaB activation. Tyrosine phosphorylation alters the association between IkappaBalpha and NF-kappaB with sufficient intensity to allow transient NF-kappaB translocation to the cell nuclei within 45 min of onset of reoxygenation stress. Immunofluorescence imaging of NF-kappaB protein reveals it to be shuttled between the nucleus and cytoplasm within 90 min of reoxygenation. Furthermore, IkappaBalpha appears to be associated with NF-kappaB during the nucleo-cytoplasmic shuttling and is thus protected from degradation. Overall, these studies suggest that tyrosine phosphorylation of IkappaBalpha represents a proteolysis-independent mechanism of NF-kappaB activation that can be targeted for preventing H/R-mediated injury without affecting normal inflammatory responses.
D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase; D008833:Microcirculation / Q000502:physiology*; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D010101:Oxygen Consumption / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014020:Tissue Extracts / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D014020:Tissue Extracts; D014409:Tumor Necrosis Factor-alpha; D030421:Peroxynitrous Acid; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human