Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H2O2) formation, we analysed H2O2 release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H2O2 release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H2O2 release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A, inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H2O2 release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B-4, but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C-4, restored LPA-stimulated H2O2 release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B-4 are required for LPA-stimulated H2O2 release in HaCaT cells.