STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a

Abstract

Key Points• Recruitment of STXBP1 by Slp4-a promotes WeibelPalade body exocytosis.• Ex vivo EIEE4 endothelial cells haploinsufficient for STXBP1 have impaired Weibel-Palade body exocytosis.Vascular endothelial cells contain unique rod-shaped secretory organelles, called WeibelPalade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormoneevoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine-and forskolin-induced VWF secretion.To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine-and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature. (Blood. 2014;123(20):3185-3194) IntroductionEndothelial cells line the lumen of all blood vessels, providing a highly dynamic barrier that plays a crucial role in maintaining vascular homeostasis. They contain specialized secretory organelles called Weibel-Palade bodies (WPBs) that allow the endothelium to store and release, in a regulated fashion, a presynthesized cocktail of hemostatic, inflammatory, and angiogenic mediators in response to endothelial activation, injury, or stress. [1][2][3] The main component of these organelles is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation and stabilizing coagulation factor VIII. In addition to VWF, several soluble chemokines (eg,, IL-8) as well as the integral membrane proteins CD63 and P-selectin are stored in these organelles. [4][5][6][7][8][9] Coordinated expression of CD63 and P-selectin on the endothelial cell surface after WPB exocytosis is crucial for leukocyte extravasation at sites of inflammation. 10 The presence of angiopoietin-2 and insulin-like growth factor-binding protein 7 in WPBs points toward a critical role for the organelle in regulation of angiogenesis. 11-13The precise composition of mediators stored in WPBs depends crucially on the physical, mechanical, and chemical signals in the local microenvironment; for example, targeting of eotaxin-3, IL-8, and IL-6 has been observed in response to pro-...

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

“…Cord blood was collected from umbilical veins within 48 hours after delivery and was processed for BOEC isolation essentially as described before . cbBOECs were cultured in EC growth medium (EGM)‐2 (Lonza, Basel, Switzerland, CC‐3162) supplemented with 18% fetal calf serum (EGM‐18; Bodinco, Alkmaar, The Netherlands) . Human embryonic kidney 293T (HEK293T) cells were cultured in Gibco Dulbecco's Modified Eagle Medium, high glucose, pyruvate (ThermoFisher, Landsmeer, The Netherlands) for regular passing and seeding and in EGM‐18 for virus production.…”

Regulation of VWF expression, and secretion in health and disease

“…In addition, Bierings et al reported the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis [42]. Using a nonbiased proteomic screen for targets of Slp4-a, they identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells [43]. Applying co-immunoprecipitations and siRNA techniques, analysis of isolated blood outgrowth endothelial cells from patient carrying mutation in STXBP1, they demonstrate that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1 [43].…”

“…Using a nonbiased proteomic screen for targets of Slp4-a, they identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells [43]. Applying co-immunoprecipitations and siRNA techniques, analysis of isolated blood outgrowth endothelial cells from patient carrying mutation in STXBP1, they demonstrate that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1 [43]. Interestingly, STXBP5 has also been shown to differentially regulate exocytosis in endothelial cells and platelets [44, 45].…”

A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel–Palade body exocytosis in endothelial cells

“…, 2011a; van Breevoort et al. , 2012, 2014). However, it is unknown how the cortically anchored WPBs are transferred to the fusion-mediating SNARE machinery after secretagogue stimulation and whether a plasma membrane–tethered WPB intermediate is formed in the course of acute WPB exocytosis.…”