Lipoxygenases metabolize arachidonic acid to an array of oxygenated products including leukotrienes, monohydroxyeicosatetraenoic acids (HETE), and lipoxins with broad spectrums of biological activities. Two 12- lipoxygenase isozymes categorized as """"""""platelet-type"""""""" (P-12LO) and """"""""leukocyte-type"""""""" (L-12LO) have been isolated and characterized. P-12LO is found in platelets and skin. L-12LO exhibits a widespread distribution. We propose that the two 12LO isozymes possess distinct biological functions based on their different biochemical properties and localization. Absence of good, specific 12LO inhibitors has prevented definitive studies regarding 12LO function, so far. Therefore, a targeted gene disruption strategy will be invoked to generate P-12LO and L-12LO deficient mice. Initial studies, however, will focus on refining in detail the cell specific sites of 12LO expression in normal mice, in particular, looking at P-12LO expression in skin and L-12LO expression in blood cells, epithelial cells lining the airways, and organs like pancreas and adrenal gland. 12(S)-HETE can evoke endothelial cell retraction, surface integrin expression on endothelial cells and tumor cells, and keratinocyte chemotaxis at nanomolar concentrations. P-12LO deficient mice will be tested for various platelet function parameters ex vivo and hemostatic competence in vivo. Skin function in relation to wound healing, maintenance of the permeability barrier and psoriasis will be examined. In L-12LO deficient mice, on the other hand, blood cells will be tested for chemotaxis, phagocytosis and alterations in membrane properties in response to cellular activation. Mouse models of inflammation will be induced to examine the importance of L-12LO in the inflammatory response. 12(S)-HETE has been proposed to modulate glucose-induced insulin secretion and angiotensin II-induced aldosterone synthesis. The absence of this lipoxygenase product in L-12LO-deficient mice will permit testing the importance of this pathway in these key pancreatic and adrenal functions. Experiments to delineate the relationship of the highly related L-12LO and 15-lipoxygenase (15LO) enzymes will be performed. Finally, the murine P- 12LO and L-12LO enzymes, which share 58 % sequence identity, will be studied by in vitro mutagenesis experiments to elucidate some of the molecular properties responsible for governing the specificity differences of the two 12LO isozymes. The proposed studies here should contribute significantly to our understanding of the biological importance of 12LO products.
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