Interleukin 6 response factor binds co-operatively at two adjacent sites in the promoter upstream region of the rat alpha 2-macroglobulin gene.

Transcription of the alpha 2-macroglobulin gene (alpha 2M) in rat hepatocytes is strongly induced during acute inflammations by interleukin 6 (IL6). An IL6-response region has previously been mapped in the promoter upstream sequence of this gene. The region consists of two adjacent elements (IL6-REs), the IL6-RE core (CTGGGAA, -164 to -158 bp) and the core homology (CTGGAAA, -184 to -178 bp), elements, that are located 20 bp apart. Both elements bind nuclear factors with very similar protein-DNA contact patterns when they are contained in their original sequence context. A protein-DNA complex III was obtained in gel mobility shift experiments using a probe individually representing the core site. With probes containing both the core and core homology sites, a hormone inducible complex II of slower mobility was obtained. Complex II consisted of multiple copies of the same protein or proteins with very similar molecular masses bound at both sites. The core homology site was the weaker binding site. With a probe containing two tandem copies of the core site, binding at the second site occurred with 81 times greater affinity when the first site was occupied, than when it was free. Thus, the factor binding at the IL6-REs, the IL6-RE binding protein (IL6 RE-BP), was capable of co-operatively interacting with itself. Another factor, IL6-DBP/LAP, has recently been shown to be involved in the regulation of a major subgroup of acute phase genes by IL6. Using recombinant IL6-DBP/LAP and corresponding antisera, we demonstrated here that the IL6 RE-BP of the alpha 2M gene was distinct from IL6-DBP/LAP and from the related factor DBP. Thus, two major IL6-response elements can be distinguished: type 1 elements occurring in the human C-reactive protein, hemopexin and haptoglobin genes and utilizing IL6-DBP/LAP; and type 2 elements occurring in the rat alpha 2M, and alpha 1-acid glycoprotein genes, and utilizing a different IL6 RE-BP. The IL6 RE-BP of the alpha 2M gene was also shown to be distinct from the transcription factor NF kappa B. The IL6RE-BP had relative molecular mass of Mr = 46,000, distinct from IL6-DBP/LAP (Mr = 32,000) and NF kappa B (Mr = 50,000) and its overall DNA binding capacity was induced under acute phase conditions.