Abstract
Shiga-like toxin (SLT) has been implicated in the pathogenesis of hemolytic uremic syndrome and its attendant endothelial cell (EC) injury. Key serotypes of Escherichia coli produce SLT-1 in addition to another highly pro-inflammatory molecule, lipopolysaccharide (LPS). It has previously been established that SLT-1 induces EC apoptosis and that LPS enhances this effect. LPS alone has no affect on human EC viability, and the mechanism for this enhancement remains unknown. In the present report, we demonstrate that SLT-1 sensitizes EC to LPS-induced apoptosis. Pretreatment with SLT-1 sensitized EC to LPS-induced apoptosis, whereas pretreatment with LPS did not influence SLT-1-induced apoptosis. SLT-1 exposure resulted in decreased expression of FLICE-like inhibitory protein (FLIP), an anti-apoptotic protein that has previously been shown to block LPS-induced apoptosis. This SLT-1-mediated decrease in FLIP expression preceded the onset of apoptosis elicited by SLT-1 alone or in combination with LPS. SLT-1-mediated decrements in FLIP expression correlated in a dose- and time-dependent manner with sensitization to LPS-induced apoptosis. Finally, transient or stable overexpression of FLIP protected against LPS enhancement of SLT-1-induced apoptosis, and this protection corresponded with sustained expression of FLIP. Together, these data suggest that SLT-1 sensitizes EC to LPS-induced apoptosis by inhibiting FLIP expression.
Highlights
Shiga toxin and Shiga-like toxin-1 (SLT-1),1 produced by Shigella dysenteriae serotype 1 and certain strains of Escherichia coli, respectively, have been implicated in the pathogenesis of hemolytic uremic syndrome (HUS) [1,2,3]
Consistent with previous reports [7, 20], SLT-1 directly induced endothelial cell (EC) apoptosis, whereas LPS alone had no effect on EC viability (Fig. 1)
To determine whether the enhanced EC apoptosis elicited by LPSϩSLT-1 requires LPS bioactivity, caspase activity was assayed in EC exposed to medium, LPS, SLT-1, or LPSϩSLT-1 in the presence or absence of polymyxin B (Fig. 1B)
Summary
Shiga toxin and Shiga-like toxin-1 (SLT-1), produced by Shigella dysenteriae serotype 1 and certain strains of Escherichia coli, respectively, have been implicated in the pathogenesis of hemolytic uremic syndrome (HUS) [1,2,3]. SLT-1 and Shiga toxin have been demonstrated to induce apoptosis in EC isolated from various anatomical sites [5,6,7,8,9,10] Another toxin contributing to EC injury and/or dysfunction is endotoxin or lipopolysaccharide (LPS), a component of the outer membrane of all Gram-negative bacteria including the strains of E. coli implicated in HUS [11,12,13,14]. In the presence of cycloheximide (CHX), a protein synthesis inhibitor, de novo synthesis of FLIP is inhibited, and existing FLIP molecules are rapidly degraded by the proteasome [20, 30] This decrease in FLIP expression sensitizes human EC to LPSinduced apoptosis as does specific down-regulation of FLIP using antisense oligonucleotides [20]. Because another protein synthesis inhibitor, CHX, has been previously shown to sensitize EC to LPS-induced apoptosis via inhibition of FLIP expression, we decided to investigate whether SLT-1 could influence FLIP expression and, thereby, sensitize EC to LPS-induced apoptosis
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