Sphingolipid Signaling in Endothelial Function

性状（由申请人提供）：鞘氨醇-1-磷酸（S1 P）是一种血清携带的生物活性脂质，可调节血管系统的各种生物活性。大多数（如果不是全部的话）S1 P功能由G蛋白偶联受体（GPCR）的S1 P家族介导。已鉴定出5种S1 P受体亚型（S1 P1-S1 P5）。以前，我们表明，S1 P1，一个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的反应。S1 P/S1 P1信号增强跨内皮电阻（TEER），这是血管屏障完整性的指标。此外，S1 P1转导的信号抑制组胺诱导的血管渗漏在SD大鼠。从机制上讲，S1 P刺激导致两种不同的闭锁小带-1（ZO-1）复合物的形成，其调节内皮细胞（EC）中TJ的形成和趋化反应。这些结果表明，通过S1 P1受体的S1 P信号是重要的血管功能的调节。重要的是，我们最近观察到S1 P1受体存在于EC的核区室中。此外，我们证明importin <$1直接与S1 P1受体的第三胞内环（i3）相互作用，并且S1 P1受体的核转位是由importin 21-Ran核转运机制介导的。抑制S1 P1的核转位对S1 P介导的TEER的初始升高没有影响，但显著降低了其持续升高。此外，内皮细胞核S1 P-S1 P1信号轴刺激Cyr 61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，这些结果表明了这一提议的中心假设：“质膜（PM-）和核（N）-S1 P1受体在调节内皮功能，特别是在血管完整性和血管生成中起关键作用”。该提案的主要目标是表征PM-和N-S1 P1受体介导的相应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。本研究的主要目的是：（1）阐明S1 P1受体介导的内皮屏障完整性功能的机制;（2）确定内皮细胞核S1 P1受体的功能;（3）利用动物模型阐明S1 P1受体在体内的生理功能。这项研究的成功不仅将导致发现介导GPCR信号传导的新机制，而且还可能发展为未来的治疗用途。公共卫生相关性：鞘氨醇-1-磷酸受体亚型1（S1 P1）大量存在于血管内皮细胞的质膜和核室中。最近的证据有力地表明，质膜S1 P1受体在调节血管屏障功能中起着至关重要的作用，然而，核S1 P1的功能仍有待阐明。拟议的研究旨在通过使用体外和体内模型系统来表征质膜和核S1 P1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.