INDUCTION OF ACUTE PHASE PROTEIN BIOSYNTHESIS

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The acute phase response consists of a large number of defensive and adaptive mechanisms that represent a phenomenon of great biologic importance. Our long-term goals have to delineate the mechanisms that mediate induction of the major human acute phase protein C-reactive protein (CRP) in hepatocytes. IL-6 induces transcription of CRP in the human hepatoma cell line Hep 3B through C/EBP and STAT3 transcription factors. We recently found that the re1 protein p50 participates in IL-6induced CRP expression in two unexpected ways: 1) p50 binds to a non-consensus kappaB site overlapping the proximal C/EBP binding site of the CRP promoter to form ternary complexes with C/EBP species, which are required for CRP induction by IL-6. 2) Constitutive binding of p50 to a non-consensus kappaB site adjacent to the STAT3 response element was displaced by STAT3 binding in the presence of IL-6. These findings did not reflect classic NFkappaB activity; 1) CRP expression was inhibited by over-expressed pp65 and 2) p65 was not found in EMSA complexes. These observations give rise to our working hypotheses, that non-classic re1 family members, including p50, affect IL-6 activated transcription factors at two sites in the proximal promoter: at the C/EBP binding site by stabilizing binding of C/EBP proteins and at the STAT3 binding site, by affecting binding of this factor to its cognate site. Our specific aims, which are designed to delineate the precise molecular mechanisms that mediate transcription factor interactions in CRP induction, are: I. To define the mechanisms by which p50 influences CRP expression at the critical C/EBP binding site centered at -53. We will determine whether binding of p50 to this region affects affinity and stability of C/EBPbeta for binding at this site and will determine whether these factors physically interact. We will determine if p50 stimulates expression of the endogenous CRP gene and will define the domains of p50 and p65 that mediate their activities. II. To define the mechanisms by which p50 influences CRP expression at the STAT3 binding site. II. To define the To define the mechanisms by which p50 influence CRP expression at the STAT3 binding site and whether increasing p50 affinity for its site alters CRP expression. III. To delineate the specific roles played by IL-6 and IL-1beta in regulating expression on the proximal CRP promoter. IV. To investigate the cytokine response elements in the major response region between -256 and -157 of the CRP promoter. These studies have the potential of contributing substantially to an area of great current interest delineation of the mechanisms by which transcription factors, particularly NFkappaB proteins, interact to influence natural promoters and gene expression.