p38 Mitogen-Activated Protein Kinase Mediates Lipopolysaccharide and Tumor Necrosis Factor Alpha Induction of Shiga Toxin 2 Sensitivity in Human Umbilical Vein Endothelial Cells

Abstract

Escherichia coli O157:H7 Shiga toxin 2 (Stx2), one of the causative agents of hemolytic-uremic syndrome, is toxic to endothelial cells, including primary cultured human umbilical vein endothelial cells (HUVEC). This sensitivity of cells to Stx2 can be increased with either lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-␣). The goal of the present study was to identify the intracellular signaling pathway(s) by which LPS and TNF-␣ sensitize HUVEC to the cytotoxic effects of Stx2. To identify these pathways, specific pharmacological inhibitors and small interfering RNAs were tested with cell viability endpoints. A time course and dose response experiment for HUVEC exposure to LPS and TNF-␣ showed that a relatively short exposure to either agonist was sufficient to sensitize the cells to Stx2 and that both agonists stimulated intracellular signaling pathways within a short time. Cell viability assays indicated that the p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 and the general protein synthesis inhibitor cycloheximide inhibited both the LPS and TNF-␣ sensitization of HUVEC to Stx2, while all other inhibitors tested did not inhibit this sensitization. Additionally, SB202190 reduced the cellular globotriaosylceramide content under LPS-and TNF-␣-induced conditions. In conclusion, our results show that LPS and TNF-␣ induction of Stx2 sensitivity in HUVEC is mediated through a pathway that includes p38 MAPK. These results indicate that inhibition of p38 MAPK in endothelial cells may protect a host from the deleterious effects of Stx2.

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

“…Primary cells from both species were used as controls for the conditional immortalization. Primary HUVEC and RPTEC were sensitive, as previously reported, while primary murine aortic endothelial cells were not (62,73). The sensitivities of cells to the cytotoxic effect of Stx2 generally correlated with their level of Gb 3 expression (Table 1 and Fig.…”

“…In contrast, murine glomerular endothelial cells, podocytes, and primary cells failed to express detectable Gb 3 . When these cells were incubated with LPS for 24 h to test for cytokine-mediated Gb 3 upregulation, only HUVEC produced more Gb 3 (data not shown) (62).…”

“…Cells were treated with Stx2 at a concentration between 1 fM and 10 nM for 24 h. CCK-8 cell viability assays were performed to determine the 50% cytotoxic dose (CD 50 ) (Dojindo Molecular Technologies, Gaithersburg, MD). Stx2 coincubation with 1 g/ml LPS was tested in all cell types but enhanced only HUVEC cytotoxicity (62). Caspases were inhibited with 100 M Q-VD-OPH (MP Biochemicals, Solon, OH) suspended in dimethyl sulfoxide (DMSO) for 1 h before and after the addition of Stx2 (8).…”

Vascular endothelial cell Toll-like receptor pathways in sepsis

“…31,235 Treatment with the TLR4 agonist LPS has been reported to activate p38-MAPK and JNK in HUVECs, and p38-MAPK, JNK and ERK1/2 in HMVECs. 214,235,236 Finally, treatment with the TLR9 agonist CpG DNA has been reported to activate p38-MAPK, but not ERK1/2, in mouse lung endothelial cells. 9 Because of the importance of the conventional MAPKs in endothelial cell signaling, and because there are some apparent differences in the role of the ERKs in endothelial cells and leukocytes, each is reviewed in more detail below.…”

Fractalkine and CX3CR1 Mediate Leukocyte Capture by Endothelium in Response to Shiga Toxin

“…Apoptosis signal-regulating kinase 1 responds to inducers of cellular stress that encompass Stx1 and Stx2 due to their capacity for the ribotoxic stress response (72). The p38 MAPK also mediates LPS and TNF-␣-enhanced sensitization of HUVEC to Stx2 by increasing the expression of its receptor, Gb3/CD77 (73). Cumulatively, inhibition of NF-B-, AP1-, and p38 MAPK-mediated intracellular signaling abrogates direct and indirect cytotoxic effects of Stx2 by reducing expression of its receptor as well as membrane-bound FKN and other (MCP-1 and IL8) chemokines.…”