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) acti vates the NF-kappa B/Rel family of proteins and leads to the rapid ind uction of inflammatory gene products, including tissue factor (TP). TF is the primary cellular initiator of the coagulation protease cascade s. 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 w as activated by LPS with kinetics that preceded induction of the TF ge ne. In vitro binding studies demonstrated that the TF site bound trans lated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 hom odimers. Base-pair substitutions in the TF site indicated that the pre sence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migr ate with translated p50/p65 dimers but instead comigrated with c-Rel/p 65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV c ross-linking studies revealed the presence of c-Rel and p65 and the ab sence of p50 in the TF complex and further showed that c-Rel/p65 heter odimers selectively bound to the TF kappa B-like site. Functional stud ies indicated that the TF site conferred LPS inducibility on a heterol ogous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel KB like site mediated LPS induction of TF gene expression in mon ocytic cells.