The nuclear form of the NF‐kappa B transcription factor binds to DNA as a heterodimer of a 50 kDa (p50) and 65 kDa (p65) polypeptide. The two polypeptides are encoded by different genes but share a long region of homology, the NRD motif, encompassing domains required for DNA binding and dimerization. In this study we have analysed the contribution of the two subunits to the strong transactivating potential of NF‐kappa B. Transient expression of the p65 subunit alone resulted in a potent transactivation of a CAT reporter construct under the control of two NF‐kappa B binding sites in monkey COS and mouse L cells. The strongly DNA binding p50 subunit showed only very weak, if any, induction of gene expression. Co‐expression of p50 suppressed the transactivation by p65 presumably by competitive DNA binding of transcriptionally inactive p50 dimers (KBF1). Fusion of p65 sequences to DNA binding domain of the yeast GAL4 transcription factor allowed detection of the principal transactivation domain of p65 (TA1) in the C‐terminal 30 amino acid sequence. TA1 is likely to adopt an amphipathic alpha‐helical structure which clusters serine residues on the hydrophilic surface, a structural feature conserved between human, mouse and Xenopus p65. The unique C‐terminal third of p65 contained at least one more activation domain, TA2, within a 90 amino acid sequence directly adjacent to TA1. In two mammalian cell lines, TA1 and TA2 acted separately, while in an insect cell line, the two domains were inactive after their separation. Our study suggests that the p50 subunit in NF‐kappa B might only serve a helper function in DNA binding whereas the p65 subunit is responsible for initiating transcription. Homodimers of p50 seem to have the potential of down‐regulating kappa B‐specific gene expression.

Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-B plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-B-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple B-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a B-dependent reporter gene construct, without affecting the levels of NF-B binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 "one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 B subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-B, the p38 and extracellular signalregulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.

The transcription factor NF-B is one of the key regulators of genes involved in the immune and inflammatory response (for review, see Ref. 2). In mammalian cells, NF-B is composed of a homo-or heterodimer of various DNA-binding subunits. Five different DNA-binding subunits share a N-terminal homology domain, which confers DNA binding, dimerization, nuclear translocation, and interaction with the inhibitory IB proteins (for review, see Refs. 3 and 4). In most cell types these proteins sequester NF-B, which is frequently a heterodimer of the p50 and p65 (RelA) subunits, in the cytoplasm by masking their nuclear localization sequence. Constitutive NF-B activity in the cell nucleus can only be detected in certain neurons, some cells of the monocyte/macrophage lineage and B cells (for review, see Refs. 5 and 6

Full-length IL-33 is a member of the IL-1 family of cytokines, which can act in an autocrine or paracrine manner by binding to the IL-33R on several different target cell types. In addition, IL-33 can act in an intracrine fashion by translocating to the nucleus, where it binds to the chromatin and modulates gene expression. In this article, we report that full-length IL-33, but not mature IL-33, interacts with the transcription factor NF-κB. This interaction occurs between the N-terminal part of IL-33 from aa 66–109 and the N-terminal Rel homology domain of NF-κB p65. Coimmunoprecipitation experiments in cells overexpressing IL-33 or endogenously expressing IL-33 revealed rhIL-1β–stimulated association between IL-33 and p65, whereas binding to the p50 subunit was constitutive. The biological consequence of IL-33/NF-κB complex formation was reduction in NF-κB p65 binding to its cognate DNA and impairment of p65-triggered transactivation. Overexpression of IL-33 resulted in a reduction and delay in the rhIL-1β–stimulated expression of endogenous NF-κB target genes such as IκBα, TNF-α, and C-REL. We suggest that nuclear IL-33 sequesters nuclear NF-κB and reduces NF-κB–triggered gene expression to dampen proinflammatory signaling.

Glucocorticoids exert multiple anti-inf lammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-B. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IB molecule, which in turn would bind and remove activated, DNA-bound NF-B complexes in the cell nucleus.