Activation of phospholipase A2 and MAP kinases by oxidized low density lipoproteins in immortalized GP8.39 endothelial cells

In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A(2) (cPLA(2)). A putative role for MAPKs in this process has emerged. Here, we studied the contribution of Ca2+-independent phospholipase A(2) (iPLA(2)), and the role of the MAP kinase family as well as both cPLA(2) and iPLA(2) mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH2-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA, activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 mu M hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA(2) protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA2 protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC-ERK-p38-JNK pathways and PLA(2) activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.