Sphingolipid Signaling in Endothelial Function

描述（由申请人提供）：鞘氨醇-1-磷酸（S1P）是一种血清性生物活性脂质，调节血管系统的各种生物活性。大多数（如果不是全部的话）S1P功能是由g蛋白偶联受体（gpcr）的S1P家族介导的。已鉴定出5种S1P受体亚型（S1P1-S1P5）。先前，我们发现S1P1，一个gi偶联的GPCR，调节内皮细胞骨架结构，趋化性，粘附连接（AJs）和紧密连接（TJs）的形成，以及形态发生和血管生成反应。S1P/ S1P1信号可增强血管屏障完整性指标-跨内皮电阻（TEER）。此外，s1p1转导的信号传导抑制组胺诱导的SD大鼠血管渗漏。从机制上讲，S1P刺激导致两种不同的封闭带-1 （ZO-1）复合物的形成，这些复合物调节内皮细胞（ECs）中TJ的形成和趋化反应。这些结果表明，通过S1P1受体传递的S1P信号在血管功能调节中起重要作用。重要的是，我们最近观察到S1P1受体存在于ECs的核室中。此外，我们证明了输入蛋白1直接与S1P1受体的第三胞内环（i3）相互作用，并且S1P1受体的核易位是由输入蛋白21-Ran核转运机制介导的。抑制S1P1的核易位对S1P1介导的TEER初始升高没有影响，但显著降低其持续升高。此外，内皮核S1P-S1P1信号轴刺激Cyr61和CTGF的转录，这两种生长因子在血管生成中具有重要的功能。总之，这些结果表明了该提案的中心假设：“质膜（PM-）和核(N)- s1p1受体在调节内皮功能，特别是在血管完整性和血管生成方面发挥关键作用”。本提案的主要目标是表征PM-和N-S1P1受体介导的各自的信号级联和生物反应，特别关注血管完整性功能和血管生成反应的调节。本研究计划有三个具体目标：(1)表征S1P1受体介导的内皮屏障完整性功能的机制；(2)确定细胞核S1P1受体在内皮细胞中的功能；(3)利用动物模型阐明S1P1受体在体内的生理功能。这项研究的成功不仅会导致发现介导GPCR信号传导的新机制，而且可能会发展到未来的治疗用途。公共卫生相关性：鞘氨醇-1-磷酸受体亚型1 （S1P1）大量存在于血管内皮细胞的质膜和核室中。最近的证据强烈表明，质膜S1P1受体在血管屏障功能的调节中起关键作用；然而，核S1P1的功能仍有待阐明。本研究旨在通过体外和体内模型系统表征质膜和核S1P1受体介导的信号传导的分子细节和生理相关性。

DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, regulates various biological activities of vasculature. Most, if not all, S1P functions are mediated by the S1P family of G-protein-coupled receptors (GPCRs). Five S1P receptor subtypes (S1P1-S1P5) have been identified. Previously, we showed that S1P1, a Gi-coupled GPCR, regulates endothelial cytoskeletal architectures, chemotaxis, formation of adherens junctions (AJs) and tight junctions (TJs), as well as morphogenic and angiogenic responses. The S1P/ S1P1 signaling enhances the transendothelial electrical resistance (TEER), an indicator of vascular barrier integrity. Moreover, the S1P1-transduced signaling inhibits the histamine-induced vessel leakage in the Sprague-Dawley (SD) rat. Mechanistically, S1P stimulation results in the formation of two distinct Zonula Occludens-1 (ZO-1) complexes which regulate the TJ formation and chemotactic response in endothelial cells (ECs). These results suggest that S1P signaling via the S1P1 receptor is important in the regulation of vascular functions. Importantly, we recently observed that S1P1 receptor is present in the nuclear compartment of ECs. Furthermore, we demonstrate that importin¿1 directly interacts with the third intracellular loop (i3) of S1P1 receptor and the nuclear translocation of S1P1 receptor is mediated by the importin 21-Ran nuclear transport machinery. Inhibition of nuclear translocation of S1P1 has no effect on the initial S1P-mediated TEER rise, yet markedly diminishes its sustained rise. Furthermore, endothelial nuclear S1P-S1P1 signaling axis stimulates the transcription of Cyr61 and CTGF, two growth factors which are functionally important in angiogenesis. Together, these results suggest the central hypothesis of this proposal: "both the plasma membrane (PM-) and nuclear (N)-S1P1 receptors play critical roles in regulating endothelial functions, particularly in vascular integrity and angiogenesis". The main goal of this proposal is to characterize the respective signaling cascades and biological responses mediated by the PM- and N-S1P1 receptors with particular focus on the regulation of vessel integrity function and angiogenic response. Three specific aims are planned in this proposal, and they are: (1) characterize the mechanisms of S1P1 receptor-mediated endothelial barrier integrity function, (2) determine the functions of nuclear S1P1 receptor in ECs, and (3) utilizing animal models to elucidate the physiological functions of S1P1 receptor in vivo. The success of this proposed research will not only lead to the discovery of novel mechanisms mediating GPCR signaling, but may also develop into future therapeutic usages. PUBLIC HEALTH RELEVANCE: Sphingosine-1-phosphate receptor subtype 1 (S1P1) is abundantly present in both plasma membrane and nuclear compartments of vascular endothelial cells. Recent evidence strongly suggests that plasma membrane S1P1 receptors play a critical role in the regulation of vascular barrier function; however, the function of nuclear S1P1 remains to be elucidated. The proposed research aims to characterize the molecular details and physiological relevance of plasma membrane and nuclear S1P1 receptor mediated signaling by using both in vitro and in vivo model systems.