Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways

Abstract

IntroductionIngestion of food contaminated with Shiga toxin (Stx)-producing Escherichia coli, such as strain O157:H7, can cause bloody diarrhea that develops into diarrhea-associated hemolytic uremic syndrome (D ϩ HUS) that is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. D ϩ HUS is the most common cause of acute renal failure in children and is fatal in ϳ 3%-5% of cases. 1 Several mechanisms seem to influence the course of tissue injury by Stx. Stx-producing E coli make 2 major types of Stx, each of which is composed of 1 catalytically active A subunit (ϳ 33 kDa) and 5 B subunits (ϳ 7.7 kDa) that form a homopentameric ring. Stx1 is identical to the major toxin produced by Shigella dysenteriae serotype 1. Stx2 is ϳ 50%-60% identical in sequence to Stx1 and occurs in several variants. Stx1 binds to globotriaosylceramide (Gb3) more tightly than Stx2, but Stx2 is more toxic in animal models and is more often associated with D ϩ HUS in humans than Stx1. [2][3][4] The Stx B subunits bind to Gb3 on cell surfaces and facilitate internalization of the Stx holotoxin that undergoes retrograde transport to the endoplasmic reticulum. The Stx A subunit is then translocated into the cytoplasm where it cleaves the adenine base at position 4324 of 28S ribosomal RNA, thereby blocking protein synthesis and initiating a series of responses that culminate in cell death. 5 In addition, treatment of human umbilical vein endothelial cells (HUVECs) or glomerular microvascular endothelial cells (HGMECs) with nanomolar concentrations of Stx1 or Stx2 induces rapid VWF secretion and the formation of long cellassociated VWF strings. 6 Administration of Stx2 to Adamts13 Ϫ/Ϫ mice also causes fatal microvascular thrombosis that resembles thrombotic thrombocytopenic purpura, and Adamts13 Ϫ/Ϫ mice are protected from the lethal effects of Stx if they also lack VWF. 7 We recently found that the B subunits from Stx1 or Stx2, in the absence of the A subunits, are sufficient to stimulate VWF secretion by HUVECs or HGMECs and that Stx2B subunits can induce thrombotic microangiopathy in Adamts13 Ϫ/Ϫ mice. 8 These responses indicate the existence of Stx B-induced signaling pathways that may be responsible for some of the biologic effects attributed previously to the cytotoxic Stx A subunit.At least 3 signaling pathways can contribute to agonist-induced secretion of VWF. Histamine and thrombin activate phospholipase C (PLC)-␤ that generates inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. IP 3 increases the level of intracellular Ca 2ϩ that is required for VWF secretion induced by these agonists. In contrast, epinephrine and vasopressin induce VWF secretion by increasing intracellular cAMP and activating protein kinase A (PKA). 9 Alternatively, vascular endothelial growth factor (VEGF) stimulates VWF secretion through a pathway that depends on protein kinase C (PKC)-␦ but not on Ca 2ϩ or cAMP. 10 Whether any of these pathways contribute to Stx B-induced VWF secretion has not been determined previous...

Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion

“…Stxbp5 mRNA was expressed in murine tissues, including lung, spleen, and aorta, as measured by quantitative real-time PCR (qPCR; Figure 1B). Stxbp5 mRNA was also found ECs through discrete signaling pathways (37)(38)(39)(40)(41)(42)(43)(44). However, the detailed mechanisms controlling the endothelial SNARE machinery are not fully known.…”

The B Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi Up-Regulates Chemokines, Cytokines, and Adhesion Molecules in Human Macrophage, Colonic Epithelial, and Brain Microvascular Endothelial Cell Lines

“…However, purified B subunits of Stx1 and Stx2 have been reported to induce secretion of von Willebrand factor by human endothelial cells and to contribute to thrombotic microangiopathy in mice (35). Interestingly, this appeared to involve activation of distinct signaling pathways by Stx1B and Stx2B, dependent upon protein kinase C␣ and protein kinase A, respectively (36). Thus, Stx B subunits may play a direct role in the endothelial injury that is the hallmark of the systemic complications of STEC disease in humans.…”

cAMP-induced secretion of endothelial von Willebrand factor is regulated by a phosphorylation/dephosphorylation switch in annexin A2

“…by guest www.bloodjournal.org From more importantly, we identify for the first time a phosphorylation/ dephosphorylation target activated in the course of cAMP-triggered VWF secretion. Although evidence for a role of PKA in cAMPdependent VWF secretion has been obtained through inhibitor experiments, 16,17,40,41 PKA substrates in this pathway are not known. Although counterintuitive at a first glance, we show that PKA (indirectly) triggers a dephosphorylation of AnxA2, thereby promoting AnxA2-S100A10 complex formation that in turn is required for efficient WPB exocytosis.…”

“…Future experiments are needed to reveal whether this pathway also operates in the recently described PKA-dependent release of VWF that is triggered by Shiga toxin 2B and occurs without elevation of intracellular cAMP levels. 17 Previous in vitro approaches identified PKC phosphorylation sites in the N-terminal domain of AnxA2 that affect the stability of the AnxA2-S100A10 complex, 32 and a recent study revealed a PKCdependent serine phosphorylation of AnxA2 in endothelial cells that induced a dissociation from S100A10. 24 Thus, PKC seems to be primarily responsible for complex-destabilizing serine phosphorylation in AnxA2.…”

“…[12][13][14][15] The point of this convergence and more general aspects of a cross-talk between the Ca 21 -and cAMP-dependent pathways initiating WPB exocytosis are not known. Furthermore, although PKA activity is required downstream of cAMP in the regulation of WPB exocytosis, 16,17 targets of its activity that are controlled by a phosphorylation/ dephosphorylation switch in the course of WPB exocytosis remain to be identified.…”