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 NF-kappa B precursor p105 and the proto-oncogene product Bcl-3 are I kappa B molecules and control nuclear translocation of NF-kappa B.

We have examined the interaction of the NF-kappa B precursor p105 with NF-kappa B subunits. Similar to an I kappa B molecule, p105 associates in the cytoplasm with p50 or p65. Through this assembly, p105 efficiently blocks nuclear transfer of either subunit. Moreover, the p105 protein inhibits DNA binding of dimeric NF-kappa B subunits in a similar, but not identical, manner to its isolated C-terminal domain, which contains an ankyrin-like repeat domain (ARD). The proto-oncogene product Bcl-3 also controls nuclear translocation of p50, but not of p65. Hence, p50 can be retained in the cytoplasm via at least three distinct interactions: through direct interactions either with its own precursor, with Bcl-3 or indirectly through I kappa B alpha or -beta when attached to p65. We discuss a function of p105 as a cytoplasmic assembly unit for homo- and heteromeric NF-kappa B complexes and of Bcl-3 as an I kappa B with novel subunit specificity.     

DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65.     

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.     

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.     

Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.