Activation of cytosolic phospholipase A2 and 15-lipoxygenase by oxidized low-density lipoproteins in cultured human lung fibroblasts

In cell cultures of human lung fibroblasts, we found that oxidized LDL (oxLDL), after 24-h treatment, stimulated arachidonic acid release. Aputative role for phospholipases A2 and MAPK activities in this process was postulated. Consequently, we studied the contribution of either Ca2+-dependent, cytosolic phospholipase A2 (cPLA2) or Ca2+-independent phospholipase A2 (iPLA2), and the role of the MAP kinase family in oxLDLtoxicity to fibroblastic cells in vitro. Activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH2-terminal kinase (JNK) wasalso assessed with Western blotting. Compared with cellular samples untreated or treated with native LDL, treatment with oxLDL (50-100 μMhydroperoxides) for 24 h significantly increased the levels of either cPLA2 protein expression or constitutively phosphorylated cPLA2 protein; inaddition we observed enzyme translocation to membranes. iPLA2 activity was not stimulated by oxLDL. Arachidonic acid release appeared to beassociated with phosphorylation of ERK1/2 which was significantly enhanced in a dose-dependent manner whereas no activation of p38 and JNKswas found, indicating that these MAPKs are not involved in mediating the maximal oxLDL response. Western blotting on subcellular fractionsand confocal microscopy analyses confirmed an increase in 15-lipoxygenase (15-LO) protein expression and translocation upon activation. Asignificant increase of cyclooxygenase-2 expression into membrane fraction was also found. Collectively, the data presented link the stimulation ofERK–cPLA2–15-LO pathway by oxLDL to the prooxidant mechanism of the lipoprotein complex. It may initially stimulate the fibroblast reactionagainst the oxidation challenge as well as metabolic repair, such as during lung inflammation and pulmonary fibrosis.