Nontransformed cells depend on growth factors for their survival, proliferation and differentiation. In addition to polypeptide growth factors, an emerging group of naturally occurring phospholipid growth factors have been discovered. Within this group, lysophosphatidic acid (LPA, 1-acyl-2-lyso-sn-glycero-3-phosphate), nature's simplest phospholipid, was found to elicit growth factor-like effects in almost every cell type spanning the phylogenetic tree, from Dictyostelium to humans. These biological effects include: 1) the mitogenic or antimitogenic regulation of the cell cycle, 2) regulation of Ca2+ homeostasis, 3) regulation of cell shape, migration, and tumor cell invasiveness, and 4) the prevention of apoptosis. LPA elicits these effects via multiple G protein-coupled receptors, which have distinct pharmacological and signal transduction properties. LPA is generated from activated and injured cells, including platelets; blood serum is consequently a very rich source. In addition to LPA, serum contains at least eight other lipids that constitute approximately 90% of the biological activity. In contrast, plasma is rich in endogenous antagonists of LPA. The first objective of this proposal is to elucidate the chemical structure of these lipid agonists and antagonists present in human serum and plasma. The structure of the chromatographically purified lipids will be elucidated by mass-, infrared-, and NMR spectrometry. The biological effects of the newly identified agonists and antagonists on the cell cycle, Ca2+ homeostasis, cell shape, and apoptosis will be determined. The second objective is to characterize the structure-activity relationship and signal transduction properties of four putative LPA receptors, and two that have already been identified. Preliminary evidence indicates that these receptors are selectively activated by three naturally occurring lipids, LPA, 1-0-cis- alk-1'-enyl-2-lyso-sn-glycero-3-phosphate, and 1-acyl-2,3-cyclic-sn- glycero-3-phosphate. The Hep G2 human hematoma cell line, which is devoid of these lipid receptors, was used to generate clonal cell lines that stably express each of these receptors. These cell lines will be used to determine the agonist/antagonist specificity and the signal transduction properties of each receptor. New information from these proposed studies will facilitate the assessment of the physiological role of these lipids, provide novel pharmacological tools for their analysis, and pave the way toward expanded therapeutic applications. The FDA has approved clinical trials of HK-Cardisol, an LPA-containing preservative for organ explants. The biotechnology industry has just begun to explore the utilization of LPA to prevent apoptosis associated with myocardial ischemia, chemotherapy, and AIDS.
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