Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site.

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.     

The oncoprotein Bcl-3 can facilitate NF-kappa B-mediated transactivation by removing inhibiting p50 homodimers from select kappa B sites.

Previously we have proposed a role for Bcl-3 in facilitating transactivation through kappa B sites by counteracting the inhibitory effects of bound, non-transactivating homodimers of the p50 subunit of NF-kappa B. Such homodimers are abundant for example in nuclei of unstimulated primary T cells. Here we extend the model and provide new evidence which fulfills a number of predictions. (i) Bcl-3 preferentially targets p50 homodimers over NF-kappa B heterodimers since the homodimers are completely dissociated from kappa B sites at concentrations of Bcl-3 which do not affect NF-kappa B. (ii) Select kappa B sites associate very strongly and stably with p50 homodimers, completely preventing binding by NF-kappa B. Such kappa B sites are likely candidates for regulation by p50 homodimers and Bcl-3. (iii) Bcl-3 and p50 can be co-localized in the nucleus, a requirement for active removal of homodimers from their binding sites in vivo. (iv) The ankyrin repeat domain of Bcl-3 is sufficient for the reversal of p50 homodimer-mediated inhibition, correlating with the ability of this domain alone to inhibit p50 binding to kappa B sites in vitro. Our data support the model that induction of nuclear Bcl-3 may be required during cellular stimulation to actively remove stably bound p50 homodimers from certain kappa B sites in order to allow transactivating NF-kappa B complexes to engage. This exact mechanism is demonstrated with in vitro experiments.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.     

Characterisation of NF-kappa B complexes in Theileria parva-transformedT cells

Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.