Intracellular S1P & signaling in lung endothelial cells

描述(申请人提供)：鞘氨醇-1-磷酸(S1P)是一种生物活性的脂质介质，不仅作为G蛋白偶联的SIP1-5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钙动员、增殖和抑制凋亡。早些时候，我们和其他人已经证明，血小板来源的和外源性添加的S1P可以增强内皮屏障功能和趋化作用。我们的初步实验表明，人肺内皮细胞(ECs)可以将外源性和血浆来源的S1P迅速转化为细胞内S1P；然而，调节EC关键功能的细胞内S1P信号的分子机制尚不清楚。最近，一些脂质磷酸酶(LPP)和两个鞘氨酸激酶(Sphks)已经在哺乳动物细胞中被克隆和鉴定，它们可能调节细胞外和细胞内S1P水平的动态平衡。这项提案将阐述LPP和Sphks在调节内皮细胞钙稳态、增殖和细胞骨架重组中的作用。推测LPPs和Sphks调控人肺内皮细胞内S1P的产生和信号转导。具体目标1将描述LPP及其在调节内皮细胞内S1P产生中的作用。具体目标2将确定Sphks 1和Sphks 2在催化LPP作用于外源S1P所产生的鞘氨醇磷酸化中的作用和调节。具体目标3将解决细胞内S1P的细胞靶点和功能。综上所述，这些实验将揭示LPP和Sphk调节循环血浆中细胞内S1P产生的新机制，并确定S1P参与肺内皮细胞钙稳态、黏附分子的分泌和表达、趋化和伤口愈合的细胞内靶点。更好地了解LPP和Sphks调节S1P在循环中细胞内形成的分子机制，将为了解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.