The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1–dependent manner

Abstract

<jats:title>Abstract</jats:title><jats:p>Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome by damaging renal microvascular endothelium. The pentameric B subunits of Stx types 1 and 2 (Stx1B and Stx2B) are sufficient to stimulate acute VWF secretion from endothelial cells, but Stx1B and Stx2B exert distinct effects on Ca2+ and cAMP pathways. Therefore, we investigated other signaling components in StxB-induced VWF exocytosis. Incubation of HUVECs with StxB transiently increased phospholipase D (PLD) activity. Inhibition of PLD activity or shRNA-mediated PLD1 knockdown abolished StxB-induced VWF secretion. In addition, treatment with StxB triggered actin polymerization, enhanced endothelial monolayer permeability, and activated RhoA. PLD activation and VWF secretion induced by Stx1B were abolished on protein kinase Cα (PKCα) inhibition or gene silencing but were only moderately reduced by Rho or Rho kinase inhibitors. Conversely, PLD activation and VWF exocytosis induced by Stx2B were reduced by Rho/Rho kinase inhibitors and dominant-negative RhoA, whereas attenuation of PKCα did not affect either process. Another PLD1 activator, ADP-ribosylation factor 6, was involved in VWF secretion induced by Stx1B or Stx2B, but not histamine. These data indicate that Stx1B and Stx2B induce acute VWF secretion in a PLD1-dependent manner but do so by differentially modulating PKCα, RhoA, and ADP-ribosylation factor 6.</jats:p>

Shiga Toxin-Associated Hemolytic Uremic Syndrome: A Narrative Review

“…Shiga toxins induce a profound remodeling of the gene expression repertoire of endothelial cells rather than prompting cell death, provided that vascular cells are subjected to sublethal concentrations of Shiga toxin [199,202]. The net effect is that endothelial cells adopt a prothrombogenic phenotype by expressing increased levels of tissue factor (TF) [203], releasing augmented levels of von Willebrand factor [204,205], and activating platelets [206] via the CXCR4/CXCR7/SDF-1 pathway [202]. In addition, Stx stimulates the expression of adhesion molecules [207] and inflammatory chemokines [208], thereby potentiating the cytotoxity of Stx [209] and promoting the adhesion of leucocytes to endothelial cells, which in turn exacerbate thrombosis and tissue damage.…”

Exocytosis of Weibel–Palade bodies: how to unpack a vascular emergency kit

“…Phorbol esters, such as phorbol 12‐myristate 13‐acetate (PMA), are potent (non‐physiological) stimulators of VWF secretion that directly activate PKC by mimicking the action of diacylglycerol . Histamine, vascular endothelial growth factor (VEGF) and Shiga toxin 1B (Stx1B) elevate [Ca 2+ ] i in a PLC‐dependent manner, and simultaneously either signal through the Ca 2+ ‐independent PKCδ (VEGF and histamine) or utilize the Ca 2+ ‐dependent PKCα (Stx1B and histamine) . Among its targets are components of the SNARE machinery, the assembly of which is partly regulated through PKC‐dependent phosphorylation events .…”

“…Both VEGF and histamine activate PKCδ; however, broad‐range PKC inhibition or specific depletion of PKCδ significantly reduced secretory responses after VEGF but not after histamine administration . PKCα is activated by Stx1B as well as by histamine, but is only essential for Stx1B‐induced release; inhibition of PKCα did not block histamine‐induced VWF secretion and, in some studies, even enhanced it . Paradoxically, three independent studies showed that blocking Ca 2+ signaling, which entirely abolishes VWF release induced by both histamine and PKCα‐dependent Stx1B, was not sufficient to fully inhibit VEGF‐induced VWF secretion .…”

Weibel−Palade bodies at a glance

“…One way RalA might operate is through activation of phospholipase D1 (PLD1). PLD1 is required for VWF release (Disse et al, 2009;Huang et al, 2012) and RalA can activate PLD1 in some cell types (Vitale et al, 2005) − although this has not been demonstrated in endothelial cells. Alternatively, in other cells types, RalA can interact with components of the exocyst complex, which then targets vesicles to the plasma membrane (Moskalenko et al, 2002).…”