17-BETA-ESTRADIOL INHIBITS EXPRESSION OF HUMAN INTERLEUKIN-6 PROMOTER-REPORTER CONSTRUCTS BY A RECEPTOR-DEPENDENT MECHANISM

We previously reported that 17 beta-estradiol inhibits cytokine-stimul ated bioassayable IL-6 and the steady-state level of IL-6 mRNA. To det ermine the molecular basis of this effect, the transient expression of chloramphenicol acetyltransferase (CAT) reporter plasmid driven by th e human IL-6 promoter was studied here in HeLa or murine bone marrow s tromal cells (MBA 13.2). 17 beta-estradiol (10(-8) M) completely suppr essed stimulated CAT expression in HeLa cells cotransfected with IL-6/ CAT constructs and a human estrogen receptor (hER) expression plasmid; but had no effect on reporter expression in HeLa cells not transfecte d with hER. 17 beta-estradiol also inhibited stimulated expression in MBA 13.2 cells (which express the estrogen receptor constitutively) wi thout the requirement of cotransfection of the hER plasmid. The hormon al effects were indistinguishable between constructs containing a 1.2- kb fragment of the 5' flanking region of the IL-6 gene or only the pro ximal 225-bp fragment. However, yeast-derived recombinant hER did not bind to the 225-bp segment in DNA band shift assays, nor did the 225-b p fragment compete for binding of an estrogen response element oligonu cleotide to yeast-derived estrogen receptor. These data suggest that 1 7 beta-estradiol inhibits the stimulated expression of the human IL-6 gene through an estrogen receptor mediated indirect effect on the tran scriptional activity of the proximal 225-bp sequence of the prometer.