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

Abstract

5-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 M) 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 cjun NH 2 -terminal kinases was observed, implying that these MAPKs are less important for 5-LO product formation in stressstimulated 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 to N-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 IntroductionMetabolism of arachidonic acid (AA) by 5-lipoxygenase (5-LO) initializes the biosynthesis of biologically active leukotrienes (LTs), which are potent mediators in inflammatory and allergic reactions. 1 Whereas LTB 4 is considered as a potent chemotactic and chemokinetic agent for phagocytes, the cysteinyl-LTs C 4 , D 4 , and E 4 cause smooth muscle contraction and increase vascular permeability. Formation of LTs in leukocytes depends on the availability of free AA from either endogenous pools liberated by activated cytosolic phospholipase A 2 (cPLA 2 ) or from transcellular migration of AA, released from surrounding cells like platelets or endothelial cells. cPLA 2 is regulated by phosphorylation on serine residues and by Ca ϩϩ , which binds to the C2 domain of the enzyme and induces its translocation and binding to the nuclear membrane (for a review, see Gijon and Leslie 2 ).In resting cells, depending on the cell type, 5-LO can occur in different soluble loci. On cell stimulation, 5-LO translocates to the nuclear membrane where it colocalizes with 5-lipoxygenaseactivating protein (FLAP) and cPLA 2 , and an orchestrated interplay between these enzymes is of importance for efficient LT formation (for reviews, see Peters-Golden and Brock 3 and Rådmark 4 ). The activation of cellular 5-LO can be induced by ligation of specific receptor...

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