AN ACUTE-PHASE RESPONSE FACTOR NF-KAPPA-B SITE DOWNSTREAM OF THE JUNBGENE THAT MEDIATES RESPONSIVENESS TO INTERLEUKIN-6 IN A MURINE PLASMACYTOMA

The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect , we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and eval uated the IL-6 inducibility of these se quences by transient expressio n in an IL-6 dependent plasmacytoma cell line, Although a 6.5 kilobase fragment of upstream flanking sequence did not increase the IL-6 indu cibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down stream flanking sequence that both up regula tes the promoter and confers inducibility by IL-6. Point mutation of a n acute phase response factor (APRF) site within this region significa ntly reduced up-regulation of the promoter in cells grown continuously in IL-6, as well as inducibility upon restimulation of cells with IL- 6 after withdrawal from the growth factor. Point mutation of an NF-kap pa B site sharing five nucleotides with the APRF site reduced up-regul ation of the promoter but not inducibility by IL-6, whereas mutation o f two other NF-kappa B sites in the 222-base pair fragment had no effe ct on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and const itutive binding of NF-kappa B p65 to the APRF/NF-kappa B site.