Objective: Tumor necrosis factor-α (TNFα) may change from a stimulator of reversible activation of endothelial cells (ECs) to a killer when combined with cycloheximide (CHX). The means by which endothelial cells are destined to either the survival pathway or the apoptotic pathway are not fully understood. We investigated the role of p38 mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) activation and their regulation of 4E-BP1 stability in ECs to determine whether this pathway contributes to apoptosis induced by TNFα and CHX.
Methods and results: Apoptosis was induced in human umbilical vein ECs (HUVECs) by treating them with a combination of TNFα and CHX (TNFα/CHX). Activation of p38 MAPK was increased in HUVECs undergoing apoptosis, which was associated with degradation of eukaryotic initiation factor 4A regulator 4E-BP1 in a p38 MAPK-dependent manner. CHX attenuated a TNFα-stimulated increase in the expression and activity of PP2A. Silencing PP2A expression with small interfering RNA transfection mimicked CHX sensitization, increasing HUVEC apoptosis with TNFα stimulation and suggesting a protective role for PP2A in the apoptotic process.
Conclusion: Our data suggest that (1) TNFα stimulates PP2A and HUVECs elude apoptosis by PP2A-dependent dephosphorylation of p38 MAPK, and (2) CHX-induced inhibition of PP2A leads to maintenance of p38 activity and degradation of 4E-BP1, resulting in enhanced TNFα-induced apoptosis.