CD14 is a membrane receptor of mononuclear phagocytes and neutrophils that binds the lipopolysaccharide/lipopolysaccharide binding protein complex and signals the cell to begin producing tumor necrosis factor. The Applicant proposes to study the induction of CD14 biosynthesis in monocytic cell lines at a genetic level. In extensive preliminary work, she has shown: 1) that CD14 and its mRNA are both unregulated on exposure of the cells to 1,25,-dihydroxyvitamin D3; 2) by nuclear run-on, that the increase in mRNA is due at least in part to increased synthesis; and 3) that increased message synthesis requires new protein synthesis. She has cloned the CD14 gene including >4 kb of upstream sequence of which she has sequenced 500 bp, and has demonstrated by deletion analysis and DNAse I footprinting a number of regulatory sites in that 500 bp region, including a 1,25-dihydroxyvitamin D3-responsive element and an Sp1 site that works in monocytic lines but not in non-phagocyte lines. The proposals represent a continuation of this work. The stability of DC14 and RNA will be analyzed by northern blotting or pulse-chase experiments, depending on half-life. Chromatin of monocytic and non- phagocyte cell lines will be analyzed for DNAse hypersensitive sites near the CD14 gene. Promoters and enhancers farther than 500 bp upstream from the start of CD14 transcription will be identified by deletion analysis, and the DNA containing such elements will be sequenced and compared with upstream sequences from other monocyte-specific genes, and analyzed for monocyte specificity by transfection of the deletions into monocyte and non-phagocyte cell lines. Particular attention will be directed toward identifying elements responsive to 1,25-dihydroxyvitamin D3 and IL-4, looking for the latter in 3' flanking and intragenic regions if it isn't found in the 5' flanking region. The existence of monocyte-specific trans-activators for response elements upstream footprinting of nuclear proteins from monocyte and non-phagocyte lines and in vivo by site- directed mutagenesis of constructs containing the 5' flanking region of the CD14 gene in front of a luciferase reporter gene, and trans- activators for specific elements will be identified by gel shift assays. Future plans, discussed in outline only, include the purification and characterization of monocyte-specific trans-activating factors and functional studies of regulatory sequences in transgenic animals and as a means of driving arbitrary genes (e.g., c-myc) in monocyte cell lines.
