Rt. Brown et al., AN ACUTE-PHASE RESPONSE FACTOR NF-KAPPA-B SITE DOWNSTREAM OF THE JUNBGENE THAT MEDIATES RESPONSIVENESS TO INTERLEUKIN-6 IN A MURINE PLASMACYTOMA, The Journal of biological chemistry, 270(52), 1995, pp. 31129-31135
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.