By differential screening of tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS)- activated endothelial cells (ECs), we have identified a cDNA clone that turned out to be a member of the inhibitor of apoptosis (iap) gene family. iap genes function to protect cells from undergoing apoptotic death in response to a variety of stimuli. These iap genes, hiap1, hiap2, and xiap were found to be strongly upregulated upon treatment of ECs with the inflammatory cytokines TNF-α, interleukin 1β, and LPS, reagents that lead to activation of the nuclear transcription factor κB (NF-κB). Indeed, overexpression of IκBα, an inhibitor of NF-κB, suppresses the induced expression of iap genes and sensitizes ECs to TNF-α–induced apoptosis. Ectopic expression of one member of the human iap genes, human X-chromosome–linked iap (xiap), using recombinant adenovirus overrules the IκBα effect and protects ECs from TNF-α– induced apoptosis. We conclude that xiap represents one of the NF-κB–regulated genes that counteracts the apoptotic signals caused by TNF-α and thereby prevents ECs from undergoing apoptosis during inflammation.

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Tumor necrosis factor (TNF) ␣ has been shown to be a major therapeutic target in rheumatoid arthritis with the success of anti-TNF␣ antibody clinical trials. Although signaling pathways leading to TNF␣ expression have been studied in some detail, there is evidence for considerable differences between individual cell types. This prompted us to investigate the intracellular signaling pathways that result in increased TNF␣ synthesis from macrophages in the diseased synovial joint tissue. Using an adenoviral system in vitro we report the successful delivery of genes to more than 95% of normal human macrophages. This permitted us to show, by using adenoviral transfer of IB␣, the natural inhibitor of NF-B, that induction of TNF␣ in normal human macrophages by lipopolysaccharide, but not by some other stimuli, was inhibited by 80%. Furthermore the spontaneous production of TNF␣ from human rheumatoid joint cell cultures was inhibited by 75%, indicating that the NF-B pathway is an essential step for TNF␣ synthesis in synovial macrophages and demonstrating that NF-B should be an effective therapeutic target in this disease.

We aimed to investigate the dynamics of the NF-B signaling pathway in living cells using GFP variants of p65-NF-B, IB␣, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-B inducing kinase (NIK) and IB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IB␣ enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-B and IB␣ revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795-805, which counteracts the inherent NLS at amino acids 143-149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-B⅐IB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-B.