Shiga Toxin 1(Stx1) Stimulates Endothelial Cell VWF Secretion through a Ca2+/PKC Signaling Pathway That Does Not Require the Active Toxin Stx1A Subunit

Abstract

Shiga toxins (Stx) consist of 5 B (binding) subunits that interact with cell surface globotriaosylceramide (Gb3), and a single A subunit that is retrotranslocated into the cytoplasm where it enzymatically inactivates ribosomal RNA. E. coli O157:H7 can express several variants of Shiga toxin (Stx) that cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. The Stx A subunit is required for cytopathic effects on endothelium. In addition, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice, but not in wild-type mice, and this effect requires the presence of von Willebrand factor (VWF). When added to cultured human endothelial cells under conditions of laminar flow, Stx rapidly induces the acute secretion of long strings of VWF that attach to the cell surface and bind platelets with high affinity, which suggests that VWF-induced platelet aggregation might contribute to the pathogenesis of HUS. Whether VWF secretion depends on ribotoxic stress is unknown, and the mechanism of Stx-induced VWF secretion has not been characterized. To address these questions, we investigated VWF secretion by human umbilical vein endothelial cells (HUVECs) treated with Stx1 holotoxin (AB5) or binding subunits (B5). Recombinant Stx1 B5 was expressed in E. coli and purified to homogeneity. The pentameric composition and purity of B5 preparations were demonstrated by gel filtration chromatography and Western blotting. Endotoxin was removed from Stx AB5 and B5 preparations by affinity chromatography. HUVECs were perfused in a parallel plate flow chamber with fluorescently labeled anti-VWF and Stx preparations, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Unexpectedly, we found that Stx1 B5 and Stx1 AB5 were equally potent in stimulating the secretion of VWF strings. String formation was maximal after 5 min and was blocked by soluble analogs of Gb3 or anti-Stx1 B subunit antibodies. Pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min), or a protein kinase C inhibitor (50 nM staurosporine, 30 min) decreased the secretion of VWF strings by 95%, 82%, or 90%, respectively. Treatment with a protein kinase A inhibitor (5 μM H89, 30 min) did not affect VWF string formation. To more directly assess Stx1-induced PLC activation, HUVECs were transfected with a plasmid expressing a PLC-delta PH domain-GFP construct, which binds membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). When HUVECs were observed by confocal microscopy, both Stx1 AB5 and Stx1 B5 caused a rapid (<60 s) redistribution of fluorescent signal from plasma membrane to cytosol, indicating the acute activation of PLC and hydrolysis of PIP2. In addition, Stx1 AB5 or B5 induced a transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min. Stx1-induced Ca2+ transients were comparable to those induced by 0.1 mM histamine or 1 U/ml thrombin. Stx-induced Ca2+ responses were inhibited by BAPTA-AM or U73122, but not by staurosporine or H89. Treatment with Stx1 AB5 or B5 had no effect on intracellular levels of cAMP. These results indicate that Stx1 B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3, PIP2 hydrolysis by PLC, increased intracellular Ca2+, and activation of PKC. Therefore, Stx1 secreted by enterohemorrhagic E. coli may contribute to the pathogenesis of HUS through at least two mechanisms that affect microvascular endothelium: cell death caused by A subunit-induced ribotoxic stress, and VWF secretion caused by an independent B subunit-induced cell signaling pathway.