Abstract
Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.
Highlights
Sphingosine 1-phosphate (S1P) is a naturally occurring lysophospholipid that plays a crucial role in maintaining vascular homeostasis [1,2]
This receptor family consists of 5 members of which S1P receptor 1 (S1PR1) and S1PR3 have been shown to be expressed in endothelial cells [10]
A timecourse revealed that the kinetics of von Willebrand factor (VWF) release were not affected by down-regulation of endogenous S1PR1 (Figure 1D)
Summary
Sphingosine 1-phosphate (S1P) is a naturally occurring lysophospholipid that plays a crucial role in maintaining vascular homeostasis [1,2]. S1PR1, S1PR2 and S1PR3 are the major S1P receptors in the cardiovascular system [9] It is well-established that S1P promotes the barrier function of endothelial cells [10]. WPBs are rod-shaped organelles that store von Willebrand factor (VWF), a multimeric adhesive glycoprotein crucial for platelet plug formation as well as the leukocyte receptor P-selectin [15,16]. CD63 a co-receptor for P-selectin is stored within these organelles firmly linking WPB exocytosis to leukocyte extravasation [17,18]. Vaso-active components like endothelin and calcitonin-gene related peptide as well as the angiogenic mediators angiopoietin-2 and insulin-like growth factor binding protein 7 (IGFBP7) are stored in WPBs [19,20,21,22]. A number of other bioactive compounds that include chemoattractants like interleukin 6 and 8 and eotaxin-3 have been localized to these organelles [23,24,25]
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