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.     

Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element.

We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.     

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.