C/EBP transcription factors, particularly C/EBPbeta and C/EBPdelta, are known to participate in the regulation of many genes associated with inflammation and are in fact required for the expression of many of these genes, including the proinflammatory cytokine 11-6. The overproduction of IL-6 has been associated with the pathogenesis of diseases with an inflammatory etiology, such as rheumatoid arthritis and cardiovascular disease. Therapeutic strategies for blocking the action of IL-6 represent an important facet of research directed at alleviating these diseases and detailed knowledge of the transcriptional regulation of IL-6 and other proinflammatory cytokines may provide new targets for therapy. We have developed preliminary data that lead us to hypothesize that acetylation of C/EBPbeta modulates its activity in the regulation of IL-6 and other proinflammatory cytokines. In order to test this hypothesis, the following specific aims will be addressed: 1. Directly demonstrate C/EBPbeta acetylation and its differential modulation by LPS and IL-1 beta stimulation. In vitro acetylation reactions with 14C-acetyl coenzyme A and labeling in cell culture with 3H-acetic acid will be used to detect acetylation of C/EBPbeta. Amino acid substitutions at a putative acetylation site will be used to localize the site of acetylation. Mass spectrometry will be used as a secondary approach to characterize acetylation in cultured cells and to identify acetylation sites. 2. Evaluate the role of C/EBPbeta acetylation in its binding to DNA. In vitro acetylated C/EBPbeta will be compared to """"""""mock""""""""-acetylated C/EBPbeta for its ability to dimerize, its on-rate for DNA binding and its off-rate for DNA binding. Additionally, differential binding specificity of acetylated and """"""""mock""""""""-acetylated C/EBPbeta to a variety of binding motifs will be tested. 3. Assess the functional significance of acetylation for transcriptional regulation of IL-6 expression. Transient and stable transfections of vectors expressing C/EBPbeta and a mutant form lacking acetylation sites will examine the role of acetylation in transcription of IL-6, including synergy of C/EBPbeta with NF-?B and C/EBPgamma augmentation of IL-6 expression.
