TNFalpha-mediated endothelial cell apoptosis

TNFalpha has been found to promote endothelial cell dysfunction and apoptosis associated with a variety of human diseases. The mechanism by which endothelial cells resist or succumb to TNFalpha-induced apoptosis is poorly understood. Inhibition of the protein synthesis machinery is one of the mechanisms by which TNFalpha promotes apoptosis in endothelial cells. In this thesis, we report that induction of apoptosis in human umbilical vein endothelial cells (HUVECs) by treating them with a combination of TNFalpha and cycloheximide (CHX) [TNFalpha/CHX] led to apoptosis that is associated with increased activity of caspase-3 and activation of Bad. TNFalpha/CHX treatment led to increased phosphorylation of p38 MAPK and rapid degradation of eIF4E regulator, 4E-BP1, in a p38 MAPK-dependent manner. We show that CHX decreases the expression and activity of PP2A, a phosphatase known to dephosphorylate both p38 MAPK and 4E-BP1. Furthermore, silencing PP2A expression with siRNA transfection sensitized HUVECs to TNFalpha-mediated apoptosis, suggesting a protective role for PP2A in the apoptotic process. CHX treatment also led to decreased eIF4E phosphorylation without altering levels of eIF4E total protein level. Our data suggest that (i) HUVECs elude TNFalpha-induced apoptosis by PP2A-dependent dephosphorylation of p38 MAPK and 4E-BP1 and (ii) TNFalpha/CHX-induced inhibition of PP2A leads to increased p38 activity and a hyperphosphorylted 4E-BP1 resulting in enhanced apoptosis in HUVECs. Degradation of 4E-BP1 may be one mechanism by which eIF4E activity is modulated and TNFalpha-mediated vascular endothelial cell apoptosis is potentiated by CHX.