These studies propose to identify and characterize genetic elements regulating the transcription of genes encoding tryptases, which are the most abundant proteins of human mast ells. They are serine proteases with trypsin-like substrate specificity and are stored in secretory granules. With heparin and histamine, they are released extracellularly after mast cell activation by antigen-bound IgE, neuropeptides and other stimuli. Except for small amounts expressed in a minority of basophils, tryptases are exclusive to mast cells. They are of special interest to pulmonologists because their concentration is higher in the lung than in any other organ, their levels in lung bronchoalveolar lining fluid increase in several forms of lung disease, and their in vitro properties suggest involvement in bronchoconstriction, lung and airway fibrosis, and tissue remodeling. The first generation of selective tryptase inhibitors is now in clinical trials for treatment of asthma. Nevertheless, much about the expression of these biologically important enzymes remains poorly understood. In prior work, the laboratory explored biological properties of tryptases and cloned and sequenced human tryptase cDNAs and genes. The studies proposed here explore the regulation of these unique, highly expressed mast cell products at the level of the gene. Cis-acting elements flanking the human tryptase I gene and embedded within the gene's unusual first intron will be defined by transfection of tryptase- transcribing cell lines with plasmids containing protease regulatory elements linked to reporter genes. Homologous regions in the vicinity of related human tryptase genes will be sequenced and compared to those found to regulate tryptase I gene expression. DNA sequences involved in binding to mast cell-specific nuclear regulatory proteins will be defined by DNA footprinting, gel mobility shift, and related approaches. These studies will help us to understand the genetic basis of the exceptional levels of tryptase gene expression in humans. Because tryptases are specific to mast cells and are its most abundant products, the study of tryptase gene regulation may eventually lead to the identification of """"""""master"""""""" regulatory proteins controlling the expression of the mast cell's unique repertoire of proteins and mediators.
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