The -300 region of the interleukin 10 (IL-10) promoter contains a functional NF-KB binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-KB site alone interacted with NF-KB proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50)

The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.

Multiple regulatory domains within the-100 region of the beta interferon (IFN-P) promoter control the inducible response of the IFN gene to virus infection. In this study, we demonstrate that the formation of NF-KB-specific complexes on the positive regulatory domain II (PRDII) precedes the onset of detectable IFN-I transcription in Sendai virus-infected cells. By using NF-KB subunit-specific antibodies, a temporal shift in the composition of NF-KB subunits in association with the PRDII domain is detected as a function of time after virus infection. Furthermore, a virus-induced degradation of IKBBa (MAD3) protein is observed between 2 and 8 h after infection; at later times, de novo synthesis of IKBa restores IKBC(to levels found in uninduced cells and correlates with the down regulation of IFN-0 transcription. In cotransfection experiments using various NF-KB subunit expression plasmids and two copies of PRDIIINF-KB linked to a chloramphenicol acetyltransferase reporter gene, we demonstrate that expression of p65, c-Rel, or p50 or combinations of p5O-p65 and p65-c-Rel differentially stimulated PRDII-dependent transcription. Coexpression of IK.Ba completely abrogated p65-, c-Rel-, or p65-p50-induced gene activity. When the entire IFN-4 promoter (-281 to +19) was used in coexpression studies, synergistic stimulation of IFN-f promoter activity was obtained when NF-KB subunits were coexpressed together with the IFN regulatory factor 1 (IRF-1) transcription factor. Overexpression of either IKB or the IRF-2 repressor was able to abrogate inducibility of the IFN-jI promoter. Thus, multiple regulatory events-including differential activation of DNA-binding NF-cB heterodimers, degradation of IKBa, synergistic interaction between IRF-1 and NF-KB, and decreased repression by IKB and IRF-2-are all required for the transcriptional activation of the IFN-, promoter.

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