Intracellular S1P & signaling in lung endothelial cells

描述（由申请人提供）：鞘氨醇-1-磷酸（S1P）是一种生物活性脂质介质，不仅作为g蛋白偶联的SIP 1-5受体（以前称为内皮分化基因受体）的细胞外配体，而且还作为细胞内第二信使参与Ca2+动员，增殖和抑制凋亡。早些时候，我们和其他人已经证明血小板衍生和外源性添加S1P增强内皮屏障功能和趋化性。我们的初步实验表明，人肺内皮细胞（ECs）迅速将外源性和血浆来源的S1P转化为细胞内的S1P；然而，调控关键EC功能的细胞内S1P信号的分子机制尚不清楚。近年来，在哺乳动物细胞中克隆并鉴定了几种脂质磷酸磷酸酶（LPPs）和两种鞘氨醇激酶（Sphks），它们可能调节细胞外和细胞内S1P水平的动态平衡。本研究将探讨LPPs和Sphks在调节内皮钙稳态、增殖和细胞骨架重组中的作用。假设LPPs和Sphks调节人肺内皮细胞中S1P的细胞内生成和信号传导。特异性Aim 1将描述LPPs及其在调节内皮细胞内S1P生成中的作用。特异性Aim 2将确定Sphks 1和Sphks 2在催化由LPPs作用于外源S1P产生的鞘氨醇磷酸化中的作用和调控。特异性目标3将讨论细胞内S1P的细胞靶点和功能。综上所述，这些实验将揭示LPPs和Sphks调节循环血浆中细胞内S1P产生的新机制，并确定S1P在肺内皮细胞中参与钙稳态、分泌、粘附分子表达、趋化性和伤口愈合的细胞内靶点。更好地理解LPPs和Sphks在循环中介导细胞内S1P形成的调控分子机制，将为S1P在正常和病理条件下（如动脉粥样硬化和哮喘）作为肺内皮细胞内细胞内第二信使的可能生理作用提供新的见解。

DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, not only acts as an extra-cellular ligand for the G-protein coupled SIP 1-5 receptors (formerly known as endothelial differentiation gene receptors), but also functions as an intra-cellular second messenger involved in Ca2+ mobilization, proliferation and suppression of apoptosis. Earlier, we and others have demonstrated that platelet derived and exogenously added S1P enhance endothelial barrier function and chemotaxis. Our preliminary experiments suggest that human lung endothelial cells (ECs) rapidly convert exogenous and plasma-derived S1P to intra-cellular S1P; however, it is unclear the molecular mechanisms of intra-cellular S1P signaling that regulate key EC functions. Recently, several lipid phosphate phosphatases (LPPs) and two sphingosine kinases (Sphks) have been cloned and characterized in mammalian cells which may regulate the dynamic balance between extra- and intra-cellular S1P levels. This proposal will address the role of LPPs and Sphks in regulating endothelial calcium homeostasis, proliferation and cytoskeletal reorganization. It is postulated that "LPPs and Sphks regulate intra-cellular generation and signaling of S1P in human lung ECs. Specific Aim 1 will characterize the LPPs and their role in regulating intra-cellular S1P production in the endothelium. Specific Aim 2 will determine the role and regulation of Sphks 1 and 2 in catalyzing the phosphorylation of sphingosine derived by the action of the LPPs on exogenous S1P. Specific Aim 3 will address the cellular targets and functions of intra-cellular S1P. Taken together, these experiments will uncover novel mechanisms by which LPPs and Sphks regulate production of intra-cellular S1P from circulating plasma and also identify intra-cellular targets of S1P involved in calcium homeostasis, secretion, and expression of adhesion molecules, chemotaxis, and wound healing in lung endothelial cells. A better understanding of molecular mechanisms of regulation of LPPs and Sphks in mediating intra-cellular formation of S1P from circulation will provide new insights into possible physiological role of S1P as an intra-cellular second messenger in lung endothelial cells under normal and pathological conditions such as atherosclerosis and asthma.