Phospholipase D1 Is Required for Regulated Secretion of VWF Induced by the B-Subunits of Shiga-Like Toxins 1 and 2

Abstract 2113

Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome (D+HUS) by damaging renal microvascular endothelium. Stx is composed of an active (A) N-glycosidase subunit that is responsible for cytotoxicity, and 5 binding (B) subunits that interact with cell surface Gb3 and promote Stx endocytosis. We have demonstrated that catalytically inactive pentameric B5 subunits of Shiga-like toxin type 1 and 2 (Stx B5) are sufficient to stimulate the acute secretion of von Willebrand factor (VWF) from human endothelial cells and can cause thrombotic microangiopathy in Adamts13–/– mice. Because our previous observations indicated that Stx1 B5 and Stx2 B5 exert distinct effects on Ca2+ and cAMP signaling pathways, we investigated the role of alternative signaling components in Stx B5-induced VWF exocytosis. Incubation of human umbilical vein endothelial cells (HUVECs) with Stx1 B5 (5 nM), Stx2 B5 (5 nM) or histamine (100 μM) caused a time-dependent increase in phospholipase D (PLD) activity that was maximal at 10 minutes after exposure to agonists. For HUVECs under static conditions, inhibition of PLD with n-butanol, or shRNA mediated PLD1 knock down, abolished Stx1 B5- or Stx2 B5-induced acute VWF secretion assayed by ELISA of conditioned media. To assess the stimulated secretion of cell-associated VWF strings under fluid shear stress, HUVECs were perfused in a flow chamber with fluorescently labeled anti-VWF antibody. When Stx1 B5 or Stx2 B5 (5 nM) was added to the perfusate, maximal induction of VWF strings was observed within 5 minutes by immunofluorescence video microscopy. However, tert-butanol, a structural analog of n-butanol that does not inhibit PLD activity, had no effect on VWF secretion or VWF string formation. In addition, treatment of HUVECs with Stx1 B5 or Stx2 B5 triggered actin stress fiber formation (assayed by fluorescence microscopy of Alexa Fluor 488-phalloidin-treated cells), enhanced monolayer permeability (assayed by the penetration of FITC-dextran in transwell chambers), and increased the level of GTP-bound RhoA (assayed by pull-down with Rhotekin-RBD protein coupled to agarose beads). These data indicate that B5 subunits of both Stx1 and Stx2 activate RhoA. Interestingly, Stx1 B5-induced PLD activation and VWF secretion were significantly reduced by pretreatment of HUVECs with the PKC-α inhibitor Go6976 (2 μM) or by shRNA mediated PKC-α knock down, but not by pretreatment with the Rho inhibitor exoenzyme C3 (1 μM) or Rho kinase inhibitor Y27632 (20 μM). Conversely, Stx2 B5-induced PLD activation and VWF secretion were reduced by these Rho/Rho kinase inhibitors, but not by inhibition of PKC-α with Go6976 or by PKC-α shRNA knock down. In addition, transfection of HUVECs with a plasmid encoding dominant negative RhoA (T19N) reduced Weibel-Palade body exocytosis induced by both Stx1 B5 and Stx2 B5. These data indicate that the B5 subunits of both Stx1 and Stx2 activate RhoA and induce acute VWF secretion in a PLD1 dependent manner. However, Stx1 B5 activates PKC-α mediated, Ca2+-dependent signaling, whereas Stx2 B5 preferentially activates RhoA mediated, Ca2+-independent signaling.