An acute phase response factor NF-kappa B site downstream of the junB gene 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 evaluated the IL-6 inducibility of these se quences by transient expression in an IL-6 dependent plasmacytoma cell line, Although a 6.5 kilobase fragment of upstream flanking sequence did not increase the IL-6 inducibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down stream flanking sequence that both up regulates the promoter and confers inducibility by IL-6. Point mutation of an acute phase response factor (APRF) site within this region significantly 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-kappa B site sharing five nucleotides with the APRF site reduced up-regulation of the promoter but not inducibility by IL-6, whereas mutation of two other NF-kappa B sites in the 222-base pair fragment had no effect on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and constitutive binding of NF-kappa B p65 to the APRF/NF-kappa B site.