MEGAKARYOCYTE AND PLATELET PROTEOGLYCANS

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This is the third revision of a competitive renewal proposal. The long-range goal of this proposal is to understand the structure, function and regulation of expression of the serglycin proteoglycan. It is known to be made by megakaryocytes and myeloid cells. It is contained in platelet alpha granules and mast cell secretory granules, and is released upon stimulation. It is constitutively secreted from lymphocytes and upregulated during activation. This laboratory has recently found that the molecule is also expressed in human endothelial cells, in murine yolk sac, and in embryonic stem cells. Serglycin binds to several matrix molecules and cytokines, and could be involved in modulating cell activation and adhesion. Serglycin may be important for early fetal development. The glycosaminoglycan (GAG) chain length, number and composition differ amongst cell types and are important determinants of the binding characteristics of molecules made by different cells. The proposal will attempt to establish the in vivo functional roles of serglycin. In Specific Aim 1, the investigators will characterize the GAG chain content of serglycins from different cell sources in order to determine their propensity for binding to different molecules. In specific Aim 2, they will perform functional analyses of serglycin by analyzing the ability of native and modified serglycins from different sources to bind to matrix molecules and cytokines. These experiments may thus elucidate the structural basis for the mechanism of action of the serglycin family of proteoglycans, and possibly lead to the development of the therapeutic agents. They will prepare homozygous knockouts of the gene in embryonic stem cells and monitor the effects on formation of embryoid bodies and hematopoietic cells. In Specific Aim 3, they will localize serglycin protein and mRNA in fetal and adult tissues in situ to determine sites of synthesis and deposition in vivo. This will enable them to better understand the function f this molecule. In Specific Aim 4, they will prepare promoter constructs of murine serglycin and compare regulation in several cell types with their data on cell-specific expression of the human gene. Promoter construct which have been found to be active in vitro will be used to generate transgenic mice in order to analyze the cell specificity of promoter regulation in vivo. Further studies could generate unique methods for cell-specific knockouts of the gene in mice to establish the function of this molecule in different cells.