Tumor Necrosis Factor Alpha and Lipopolysaccharide Induce Shiga Toxin Sensitivity in Endothelial Cells Through p38 Mitrogen Activated Protein Kinase

Stone, Matthew Kevin, Department of Microbiology, University of Virginia
Obrig, Tom, Department of Microbiology, University of Virginia
Brautigan, David, Department of Microbiology, University of Virginia
Lynch, Kevin, Department of Pharmacology, University of Virginia
Pizarro, Theresa
Erickson, Loren, Department of Microbiology, University of Virginia
Pemberton, Lucy, Department of Microbiology, University of Virginia

Hemolytic Uremic Syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is the leading cause of acute renal failure in children, and is characterized by thrombocytopenia, hemolytic anemia, and kidney failure where glomerular endothelial damage is frequently observed. Escherichia coli O157:H7 Shiga toxin 2 (Stx2), one of the causative agents of HUS, is toxic to endothelial cells including primary cultured human umbilical vein endothelial cells (HUVEC). This sensitivity of the 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 siRNAs were tested with cell viability endpoints. A time course and dose response of LPS and TNF exposure to HUVEC showed that a relatively short exposure to either agonist was sufficient to sensitize the cells to Stx2, and that both agonists stimulate intracellular signaling pathways within a short time. Cell viability assays indicate p38 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. Additionally, SB202190 reduced the cellular Gb 3 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 iii that includes p38 MAPK. These results indicate that inhibition of p38 MAPK in endothelial cells may protect the host from the deleterious effects of Stx2.

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PHD (Doctor of Philosophy)
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