Vibrio vulnificus Cytolysin Induces Superoxide Anion-initiated Apoptotic Signaling Pathway in Human ECV304 Cells

Kang-Beom Kwon,Jeong-Yeh Yang,Do-Gon Ryu,Hye-Won Rho,Jong-Suk Kim,Jin-Woo Park,Hyung-Rho Kim,Byung-Hyun Park

doi:10.1074/jbc.m108645200

Kang-Beom Kwon, Jeong-Yeh Yang + Show 6 more

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https://doi.org/10.1074/jbc.m108645200

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Abstract

Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia.

Highlights

Vibrio vulnificus, previously designated as a lactose-positive vibrio, is a halophilic bacterium which inhabits marine or estuarine areas
Caspase-1 inhibitor, Ac-YVADCHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where V. vulnificus cytolysin (VVC)-induced apoptosis is triggered by the generation of O2., release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation
A kinetic analysis of O2. generation upon exposure to VVC revealed a significant increase in the intracellular concentration of O2. with the maximum attained at 5 min following VVC treatment, which dropped to base-line level by 30 min induced (Fig. 2A)

Summary

Introduction

Previously designated as a lactose-positive vibrio, is a halophilic bacterium which inhabits marine or estuarine areas. We already reported that hemolysis caused by VVC is colloid-osmotic in nature and those cytolysins, after binding to membranes, were oligomerized to form small pores on cell membranes (11, 12). Apoptosis can be initiated by oxidative stress mediated by the generation of reactive oxygen intermediates (14). This hypothesis arose from several observations that linked oxidative stress to apoptosis. These include the induction of oxidative stress by apoptotic stimuli such as tumor necrosis factor-␣ (15); apoptosis inhibition by antioxidants such as N-acetylcysteine (16), catalase (17), and 3,3,5,5-tetramethylpyrroline N-oxide (TEMPO) (18); and the direct induction of apoptosis by hydrogen peroxide (19)

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