Abstract 4912: Multidrug Resistance Associated Protein-1 (MRP-1) Regulates Endothelial Cell Apoptosis
The multidrug resistance associated protein-1 (MRP-1) is the main transporter of oxidized glutathione in endothelial cells, and blockade of MRP-1 prevents endothelial cell dysfunction induced by reactive oxygen species (ROS). We therefore sought to investigate the effect of MRP-1 downregulation on TNFα-induced endothelial cell apoptosis. HAEC’s were either transfected with a siRNA against MRP-1 or a negative control siRNA and treated with TNFα/cycloheximide(CHX ) for 3h or 6h. TNFα/CHX lead to endothelial cell apoptosis as indicated by Annexin V staining (FACS analysis) and increased cleavage of caspase 3 (Western blot). Downregulation of MRP-1 via siRNA significantly diminished Annexin V staining and caspase 3 cleavage. TNFα/CHX augmented endothelial cell superoxide production which was significantly attenuated by siRNA against MRP-1. Further, pre-treatment with the glutathione precursor N-acetylcysteine prevented cleavage of caspase 3. TNFα/CHX mediated activation of caspases 7 and 8 were reduced when MRP-1 was downregulated, while no influence was found on phosphorylation levels of the Fas-Associated protein with Death Domain (FADD). TNFα/CHX induced phosphorylation of p38-, ERK- and JNK-MAP-kinases. While phosphorylation of p38 MAP kinase and ERK was not influenced by MRP-1 downregulation, TNFα/CHX induced JNK phosphorylation was significantly attenuated in HAEC’s transfected with MRP-1 siRNA. These data indicate a major role for MRP-1 in regulation of TNFα/CHX-induced endothelial cell apoptosis via modulation of ROS levels, caspase activation, and JNK phosphorylation.