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.     

Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.