The formation of prostaglandins is inhibited by anti-inflammatory steroids in intact cells and isolated organs by specifically hindering release of polyunsaturated fatty acid precursors, namely arachidonic acid. The inhibitory action of steroids is a consequence of RNA and protein synthesis. This acute anti- inflammatory activity has been correlated to steroid- dependent induction and release of phospholipase inhibitory proteins, which directly inhibit arachidonic acid release. Independently isolated proteins have been shown to be closely related and designated lipocortin. Full length cDNAs coding for human lipocortin I and II (also called calpactin) have recently been isolated. The purpose of this research is to undertake a high resolution analysis of the normal and glucocorticoid-mediated mechanisms which control the regulation of lipocortin I gene expression in vivo in the mouse, which offers established genetics, the direct analysis of animal tissues, and a wide variety of appropriate cell lines. The primary strategy of the proposal is as follows: 1) The initial goal is the isolation and total characterization of a complete murine cDNA and genomic clone by restriction and DNA sequence analysis; 2) The evaluation of tissue-specific transcription and the level of glucocorticoid-mediated transcriptional control; 3) The delineation of the promoter boundaries via the generation of deletion mutants of the lipocortin 5' flanking sequence, fusion to a reporter gene coding sequence, and assaying by transient expression and/or in stable transformants; 4) The investigation of lipocortin chromatin structure using DNase I hypersensitive studies; 5) The study of lipocortin gene regulation at nucleotide resolution by directly scanning relevant sequences in vivo to detect protein-DNA interactions using the laboratories expertise in genomic sequencing. These studies are also of medical relevance because lipocortin expression has been correlated to rheumatic diseases such as arthritis and systemic lupus erythematosus and through its steroid-mediated control of prostaglandin synthesis, a vital aspect in the inflammatory response.
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