We show here for the first time that LPS and TNF-α use a p38 MAPK-dependent pathway to sensitize endothelial cells to Stx2-induced cell death. p38 MAPK has been identified as a signaling factor in both Stx-induced cell death (54
) and TNF-α induction of Gb3
) in other cell types, so we examined if one or both of these roles were evident in HUVEC. Since LPS and TNF-α have been shown to increase Gb3
levels over a 24-h time period (48
), p38 inhibitors were used to treat HUVEC before, during, and/or after 24 h of induction using LPS or TNF-α. We found that p38 MAPK inhibitors were effective at reducing LPS- or TNF-α-induced Stx2-mediated cell death only when given prior to the inducer, indicating that in HUVEC, p38 is principally involved in the induction of the Stx2 receptor and not in toxin-mediated cell death. This conclusion is supported by the TLC data which showed that one of the p38 inhibitors, SB202190, reduced LPS- and TNF-α-induced Gb3
We show here that both LPS and TNF-α induce Stx2-mediated cell death in a dose-dependent manner. These inflammatory mediators have been shown to increase Gb3
and/or Stx sensitivity in kidney cell types such as mesangium (53
), tubular epithelial cells (10
), and glomerular microvascular endothelial cells even though TNF-α does not affect the retrograde transport of the Stx B subunit in these cells (65
). We also show that these cells required minimal times of exposure to LPS or TNF-α in order to have increased sensitivity to Stx2.
Signal transduction of HUVEC in response to LPS and TNF-α is rapid, with TNF-α causing effects in as little as 5 min and LPS causing effects after 1 h of stimulation. This explains why LPS and TNF-α can sensitize HUVEC to the cytotoxic effects of Stx2 after only 1 h of stimulation. Our results are in concert with previous findings that showed that I-κBα degradation occurs more rapidly in the presence of TNF-α than in the presence of LPS (69
LPS and TNF-α are known to stimulate multiple pathways in HUVEC (11
), so we examined each pathway individually using various inhibition methods in order to either include or exclude a pathway's role in Stx2 sensitization. The pharmacological inhibitors that almost completely inhibited LPS and TNF-α induction of Stx2 sensitivity were SB202190 and SB203580, which are closely related inhibitors of p38 MAPK, and cycloheximide, a general inhibitor of protein synthesis. Cycloheximide has been shown to reduce the amount of TNF-α-induced Stx1 binding to human femoral vein endothelial cells and HUVEC (60
), theoretically by inhibiting translation of one or more of the biosynthetic enzymes required to make the functional Stx receptor, Gb3
. Our hypothesis included the possibility that multiple pathways could be utilized to increase Gb3
, and thereby Stx2, sensitization. If multiple pathways were involved, then inhibition of any one of the necessary pathways should partially block LPS- or TNF-α-induced sensitivity to Stx2. Indeed, a PKCζ pseudosubstrate reduced LPS-induced Stx2 sensitivity by 50% in HUVEC. No other inhibitor tested partially reduced LPS-induced Stx2 sensitivity; however, it was shown previously that myristolated PKCζ pseudosubstrate nonspecifically caused the phosphorylation of p38 MAPK, ERK1/2, Akt, and endothelial nitric oxide synthase in porcine pulmonary artery endothelial cells (32
), making interpretation of these data difficult. Inhibition of p38 MAPK did not completely reverse LPS- or TNF-α-induced Stx2 sensitivity; however, the negative results obtained with all other signal transduction inhibitors tested imply that if an additional pathway is involved, it is not one of the pathways identified in endothelial cells.
p38 MAPK has been implicated at multiple stages in Stx-mediated HUS. Upon release from the bacterium, Stx interacts with the intestinal epithelium, where p38 MAPK mediates Stx-induced release of IL-8 (58
), eIF4E phosphorylation (8
), caspase 3 cleavage, and cell death (54
). After crossing the intestinal epithelium (23
) and entering the vasculature, Stx stimulates circulating monocytes to secrete TNF-α in a p38-dependent manner (4
). Once in the vasculature, the main targets of Stx are the brain and kidneys (56
). Stricklett et al. showed that p38 MAPK is required for TNF-α induction of Gb3
and Stx1 sensitivity in human brain endothelial cells (55
); moreover, Morigi et al. implicated p38 in Stx2-mediated expression of endothelin-1, which is involved in glomerular hemodynamics (45
). It is noteworthy that in all cases, inhibiting p38 MAPK would theoretically benefit the host. Thus, p38 MAPK may represent a therapeutic target for reducing the harmful host response to Stx2 if these in vitro results can be verified in an animal model.
In conclusion, p38, a common pathway intermediate, was shown to be involved in sensitization of HUVEC to Stx2 by both LPS and TNF-α. p38 MAPK is required by both agonists to increase cellular Gb3 levels and subsequent Stx2 sensitivity but not for the Stx2-induced cytotoxicity that follows. These findings support the hypothesis that p38 MAPK is an effective target in patients exposed to Stx2 for minimizing symptoms associated with HUS.