Activation of 5-lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes

Abstract

Abstract5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. This study showed that various forms of cell stress, such as chemical stress (sodium arsenite), osmotic stress, or heat shock lead to substantial formation of 5-LO products in freshly isolated human polymorphonuclear leukocytes (PMNLs), when exogenous arachidonic acid (10 μM) was present. In parallel, cell stress led to activation of p38 MAPK (mitogen-activated protein kinase) and mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) kinases, which can phosphorylate 5-LO in vitro. Interestingly, arsenite also caused redistribution of 5-LO from the cytosol to the nuclear membrane. Only minor activation of extracellular signal-regulated kinases and c-jun NH2-terminal kinases was observed, implying that these MAPKs are less important for 5-LO product formation in stress-stimulated PMNLs. Stimulation of 5-LO product formation by Ca++-ionophore A23187 or thapsigargin depended on Ca++; almost no 5-LO product formation was observed in freshly isolated PMNLs when Ca++ was depleted by chelating agents. Also the response toN-formylmethionyl-leucyl-phenylalanine (fMLP) was clearly diminished, but some 5-LO product formation remained. In contrast, stress-induced product formation and translocation of 5-LO, as well as activation of p38 MAPK, occurred also after Ca++ depletion. Moreover, the p38 MAPK inhibitor SB203580 blocked stress-induced 5-LO product formation efficiently, whereas ionophore- or thapsigargin-induced formation of 5-LO products was less sensitive. These data show that cell stress can activate 5-LO in isolated PMNLs by a mechanism that does not involve Ca++ mobilization. This mechanism could function independently of Ca++-mediated 5-LO activation for stimulation of leukotriene biosynthesis under physiologic conditions as well as in inflammatory diseases.