A Multiple Cytokine- and Second Messenger-Responsive Element in the Enhancer of the Human Interleukin-6 Gene: Similarities with c-fosGene Regulation

Interleukin-6 (IL-6) is a major systemic alarm signal that indicates the occurrence of tissue damage. The IL-6 gene is induced in various cell types by serum, inflammation-associated cytokines, viruses, and second-messenger agonists. There is an overall functional similarity between IL-6 and c-fos promoters, since transfection of excess amounts of either promoter DNA into intact HeLa cells modulates the function of the heterologous promoter construct. Furthermore, the transcription regulatory factor Fos transrepresses both the IL-6 and c-fos promoters. The 115-base pair (bp) region from -225 to -111 in the IL-6 5'-flanking region, which shares nucleotide sequence similarity with the c-fos serum response (SRE) and adjacent AP-1-like (the CGTCA motif) elements, confers responsiveness to several reagents, including serum, forskolin, and phorbol ester, upon the heterologous herpesvirus thymidine kinase (TK) promoter. In gel shift assays using nuclear extracts from HeLa cells, the 115-bp IL-6 enhancer formed several complexes that (i) were increased when extracts from induced HeLa cells were used and (ii) were inhibited most efficiently by the fos E DNA fragment (-700 to -100) and by c-fos oligonucleotides containing an intact AP-1-like site (the CGTCA motif). The 23-bp oligonucleotide designated AR1 from within the IL-6 enhancer region (-173 to -151) contains a CGTCA motif and bound nuclear proteins that also associated with c-fos oligonucleotides containing either an intact SRE or AP-1-like site. A single copy of AR1 inserted upstream of the herpesvirus TK promoter rendered this heterologous promoter inducible by IL-1 alpha, tumor necrosis factor, and serum as well as by activators of the protein kinase A (forskolin) and protein kinase C (phorbol ester) signal transduction pathways. Mutations in the AP-1-like site within AR1 (CGTCA----GTTCA) decreased inducibility of the chimeric IL-6/TK/chloramphenicol acetyltransferase gene by phorbol ester and by forskolin but not by serum, IL-1 alpha, or tumor necrosis factor. These data not only show that the AR1 segment from within the IL-6 enhancer binds nuclear proteins that also bind to c-fos regulatory elements but also demonstrate that a single copy of this 23-bp element is functionally sufficient to confer responsiveness to a variety of inducers and thus define a multiple-response element.