Sphingolipid Signaling in Endothelial Function

描述(申请人提供)：鞘氨醇-1-磷酸(S1P)，一种血清携带的生物活性脂质，调节血管系统的各种生物活性。S1P的大部分功能(如果不是全部的话)是由G蛋白偶联受体(GPCRs)的S1P家族介导的。已鉴定出5种S1P受体亚型(S1P1-S1P5)。此前，我们发现S1P1是一种GI偶联的GPCR，它调控内皮细胞骨架结构、趋化作用、黏附连接(AJ)和紧密连接(TJ)的形成，以及形态生成和血管生成反应。S1P/S1P1信号增强血管屏障完整性的指标--跨内皮细胞电阻(TEER)。此外，S1P1信号转导通路可抑制组胺诱导的SD大鼠血管渗漏。在机制上，S1P刺激导致两个不同的Zonula Occludens-1(ZO-1)复合体的形成，这些复合体调节内皮细胞(ECs)TJ的形成和趋化反应。这些结果表明，通过S1P1受体的S1P信号在血管功能的调节中起着重要作用。重要的是，我们最近观察到S1P1受体存在于内皮细胞的核室。此外，我们还证明了Importin？1直接与S1P1受体的第三胞内环(I3)相互作用，S1P1受体的核转位是由Importin 21-RAN核转运机制介导的。抑制S1P1的核转位对S1P介导的TEER的初始上升没有影响，但显著降低其持续上升。此外，内皮细胞核S1P-S1P1信号轴刺激Cyr61和CTGF的转录，这两个生长因子在血管生成中起重要作用。综上所述，这些结果暗示了这一提议的中心假设：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。该提案的主要目的是描述PM-和N-S1P1受体各自介导的信号级联反应和生物学反应，特别是血管完整性功能和血管生成反应的调节。本研究的目的有三个：(1)确定S1P1受体介导的内皮屏障完整性功能的机制；(2)确定核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。这项拟议研究的成功不仅将导致发现介导GPCR信号的新机制，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(S1P1)广泛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S1P1受体在调节血管屏障功能中起着关键作用；然而，核S1P1的功能仍有待阐明。本研究旨在利用体外和体内模型系统研究质膜和核S1P1受体介导的信号转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.