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 nova 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 acute phase reactants. Previously, we found that a protein produced by monocytes induced CRP synthesis. Recently, we found that this protein referred to as B-cell Stimulatory Factor, Interferon-beta 2 or Interleukin-6 is one factor responsible for the induction of CRP in our cell culture system. To a lesser extent, leukemia inhibitory factor (HILDA) also has some stimulatory activity. However, we found that IL-6 alone is necessary and sufficient to initiate CRP transcription. We are also determining the cis-acting elements responsible for the regulatory control of acute phase protein 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 in the upstream promoter region. These include 2 distal enhancers an 2 proximal IL-6 responsive element (6RE) flanking a negative regulatory region. Using mobility shift and methylation interference assays we have identified the binding sites and presence of a number of trans-acting factors: C/EBP, NFIL-6, HNF-3, HNF-4, and several Octamer-like factors. Several of these factors respond to IL-6 in a positive or negative regulatory manner. We are attempting to identify and clone a modifications which may be altering their binding affinity.
