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). Our development of an in vitro cell culture system to monitor the induction of acute phase protein synthesis has made it possible not only to determine the extent of de novo CRP synthesis, the type of processing of the protein and the transcriptional level of regulation, but also to determine the types of biological factors responsible for the induction of the acute phase reactants. Previously, we found that a protein produced by monocytes induced CRP synthesis. Subsequently, we found that this protein, referred to as Interleukin-6 (IL-6), was necessary and sufficient to initiate CRP transcription in our cell culture system. Currently, we are determining 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 activation and DNA binding regions of NFIL-6a with those of Gal4 and GCN4, we are attempting to determine the mechanism of cooperativity between the factors which results in synergistic activation. In addition, we are examining the modification of the different forms of NFIL-6a following IL-6 signal transduction, and the role these different forms play in induction or repression of the acute phase response.
