Phosphatidylinositol‐3,4,5‐triphosphate‐dependent Rac exchange factor 1 regulates epinephrine‐induced exocytosis of Weibel–Palade bodies

Abstract

, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Objective: Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Methods: Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Results: Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory b-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Conclusion: Our findings show that PREX1 regulates epinephrine-induced release of WPBs.

Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

“…The exocytotic roles of several previously identified genes, including Rac1 (ref. 20) and myosin IIA14, were confirmed (Fig. 1a).…”

“…4b), suggesting that S142/143 phosphorylation is essential for zyxin-mediated VWF secretion. Protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) are both effectors of cAMP signalling20. S142/143 phosphorylation was inhibited by treatment with a PKA-specific inhibitor but not an EPAC inhibitor (Brefeldin A), suggesting that zyxin functions downstream of PKA (Fig.…”

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

“…Twenty‐four hours before the secretion assay, media were replaced for fresh EGM‐18 supplemented with 10 ng/mL of IL‐1β (I9401, Sigma‐Aldrich, St Louis, MO) or vehicle. Twenty‐four‐hour conditioned media were harvested, cells were pretreated for 15 minutes with serum‐free M199 medium (Gibco 22340, ThermoFisher) supplemented with 0.2% (w/v) bovine serum albumin (BSA; Serva, Heidelberg, Germany) and were stimulated with 100 μmol/L of histamine or vehicle for 30 minutes as described previously . Releasates were collected, and stimulated and unstimulated ECs were lysed in M199 with 0.2% BSA and 1% Triton X‐100 (Sigma‐Aldrich).…”

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

“…37,38 Interestingly, the Rac1 exchange factor PREX-1, which we recently identified as a regulator of cAMP-mediated WPB release, was also pulled down. 39 Annotated spectra of Rab8A and Rab27A are shown in supplemental Figures 1 and 2.…”

“…37,38 Interestingly, the Rac1 exchange factor PREX-1, which we recently identified as a regulator of cAMP-mediated WPB release, was also pulled down. 39 Annotated spectra of Rab8A and Rab27A are shown in supplemental Figures 1 and 2.Because we determined that the exocytosis-promoting capacity of Slp4-a is localized carboxyl-terminal of the SHD, we hypothesized that among the interactome of Slp4-a, DSHD effectors of Slp4-a involved in exocytosis can be found. A prominent binding partner of Slp4-a-DSHD was STXBP1 ( Figure 3A).…”