The heterogeneity of tissue mast cells is now well recognized in the mouse, rat, and human, but little is known about how mast cells develop from bone marrow progenitors or how distinct phenotypes and attendant functions are regulated. The in vitro development of cytokine-regulated populations of mouse mast cells that are phenotypically distinct, the cloning of genes that encode the proteoglycan core and numerous mouse mast cell proteases (mMCP), and the production of antibodies that are specific to these granule constituents provide the opportunity to analyze the regulation of gene transcription, mRNA stability, protein translation, and granule targeting and maturation of proteases in different populations of cultured mouse mast cells.
In Specific Aim 1 of this Project, the molecular mechanisms by which cytokines induce and/or suppress the steady-state levels of mRNAs that encode granule proteases in mouse bone marrow-derived mast cells (mBMMC) will be studied. The methylation pattern of the chromosome 14 complex where the mast cell chymase (mMCP-1 to mMCP-5) genes reside will be analyzed in cells that do and do not express these proteases. DNase-I hypersensitivity analyses and transient transfection experiments employing human growth hormone gene/mMCP reporter constructs with deletion and site-directed mutagenesis of active sites will be used to identify the critical nucleotides in the cis-acting elements in the 5' flanking regions and/or introns of the mMCP-4 and mMCP-7 genes that regulate their transcription in different populations of mast cells. Mast cell-specific trans-acting factors that regulate transcription of these mMCPs will be identified by gel-mobility-shift assays; they then will be UV -tagged and purified. A nuclear-run-on analysis followed by a transient-transfection approach analogous to that used to identify the AU-rich destabilizing domain in cytokine transcripts will be used to identify the specific nucleotides in the mMCP transcripts that regulate their stability in mBMMC.
In Specific Aim 2, immunochemical and biochemical techniques will be used in kinetic experiments to investigate the effects of certain cytokines on the regulation of translation and granule accumulation of proteases in mBMMC Transient-transfection and site-directed mutagenesis experiments will be carried out in rat basophilic leukemia cells to identify the specific amino acid residues in mMCP-5 that control its targeting to the granule and its interaction with serglycin proteoglycans.
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