The goal of our project has been the elucidation of the molecular events involved in the regulation of the acute phase response in man. We have concentrated our attention on the prototype acute phase reactant, C-reactive protein (CRP), which is known to mediate innate immunity to infectious agents, i.e. parasites. Previously we found that Interleukin-6 (IL-6), was necessary and sufficient to initiate CRP transcription in a human hepatoma cell culture system. We have determined the cis-acting elements and the trans-acting factors responsible for the regulatory control of acute phase gene expression. We have isolated the upstream promoter region for the CRP gene and have shown that this region confers inducibility. We have identified both positive and negative regulatory elements, which include two distal enhancers and two proximal IL-6 responsive elements (IL-6REs) flanking a negative regulatory region. Using mobility shift, methylation interference, and immunodepletion assays, we have identified the binding sites and the presence of a number of trans-acting factors, including: NF-IL6a, HNF-1a, HNF-3 and several Octamer-like factors. Site-specific mutagenesis of the two IL-6REs as well as other surrounding elements indicated a synergistic effect of the two IL-6REs when bound to NFIL-6a. By domain swapping the DNA binding region of NFIL-6a with that of Gal4 or GCN4, while maintaining the NFIL-6a activation domain, we have produced a chimeric NFIL-6a factor. When this factor is co-transfected into hepatoma cell with the expression plasmid containing a Gal4 or GCN4 site, it reproduced the observed synergistic activation by IL-6. Currently, we have examined the modification of the different forms of NFIL-6a following IL-6 signal transduction. Thr-, Ser- specific phophorylation patterns which are unique to the different forms of NFIL-6a were identified and small truncated forms of NFIL-6a were confirmed to be specific for IL-6 induction. LAP* (46kd) was found both in the cytosal and in the nuclear extracts; LAP (36kd) was found mainly in the cytosal extract and LIP (20kd) & a 30kd form of NFIL-6a were found only in the nuclear extract; a 14kd & a 4kd forms of NFIL-6a were found only in the nuclear extract after stimulated by IL-6. These data suggested that the mechanism for NFIL-6a to activate transcription of the CRP gene is not only involved in the NFIL-6a protein phosphorylation, but also in the NFIL-6a protein proteolysis.
