KAPPA-B BINDING-ACTIVITY IN A MURINE MACROPHAGE-LIKE CELL-LINE - SEQUENCE-SPECIFIC DIFFERENCES IN KAPPA-B BINDING AND TRANSCRIPTIONAL ACTIVATION FUNCTIONS

The role of two distinct kappa B sequence motifs found in the promoter of the murine IP-10 gene was studied in the transcriptional response of macrophages to lipopolysaccharides (LPS). When the murine macrophag e cell line RAW 264.7 was stimulated with LPS, at least three differen t kappa B sequence specific complex-forming activities were observed i n nuclear extracts as assayed by electrophoretic mobility shift assay (EMSA). These three complexes were distinguished from one another in t erms of time of appearance following stimulation and selectivity for o ne of the two different kappa B sequence motifs. The participation of individual members of the Rel homology family of kappa B sequence bind ing factors was assessed by use of specific antibodies in combination with either EMSA or UV-cross-linking to radiolabeled, BrdUrd-substitut ed oligonucleotide probes. The C1 complex contained predominantly NF k appa B1 (p50). The C2 complex contained NF kappa B1, RelA (p65), and p erhaps other factors. The C3 complex contained predominantly c-Rel. Bo th kappa B sequences were able to mediate reporter gene transcription in LPS-stimulated macrophages, but the sites behaved differentially in cells co-transfected with expression vectors encoding different membe rs of the Rel homology family. The results indicate that LPS activates several different forms of kappa B binding activity in murine macroph ages which are composed of at least three different members of the Rel homology family. These binding activities exhibit differential recogn ition of and functional activation through the two distinct kappa B se quence motifs.