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...

5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. Here, we demonstrate that extracellular signal-regulated kinases (ERKs) can phosphorylate 5-LO in vitro. Efficient phosphorylation required the presence of unsaturated fatty acids and was abolished when Ser-663 was mutated to alanine. In intact HeLa cells stimulated with arachidonic acid (AA), impaired 5-LO product formation was evident in cells expressing the S663A-5-LO mutant compared with cells expressing wild-type 5-LO. For Mono Mac 6 cells, priming with phorbol myristate acetate (PMA) before stimulation with ionophore was required for ERK1/2 activation and efficient 5-LO phosphorylation, in parallel with substantial AA release and 5-LO product formation. Inhibition of PKC by GF109203x or MEK1/2 by U0126 (or PD98059) abolished the 5-LO up-regulation effects of PMA. In contrast, these inhibitors failed to suppress 5-LO product formation induced by stimuli such as AA plus ionophore, which apparently do not involve the ERK1/2 pathway. Based on inhibitor studies, ERKs are also involved in AA-stimulated 5-LO product formation in PMNL, whereas a role for ERKs is not apparent in 5-LO activation induced by ionophore or cell stress. Finally, the data suggest that ERKs and p38 MAPK-regulated MAPKAPKs can act in conjunction to stimulate 5-LO by phosphorylation.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. We show that stimulation of polymorphonuclear leukocytes (PMNL), rat basophilic leukemia (RBL)-1, or transfected HeLa cells with arachidonic acid (AA) caused prominent 5-LO product formation that coincided with the activity of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase. 5-LO product formation in AA-stimulated PMNL and RBL-1 cells was independent of Ca2+. However, in HeLa cells expressing a 5-LO mutant lacking potential 5-LO phosphorylation sites, removal of Ca2+ caused a prominent loss of 5-LO activity. For Mono Mac 6 (MM6) cells, AA failed to activate ERKs, and AA-induced 5-LO product formation was only minute. Also, activation of ERKs by phorbol esters did not lead to prominent 5-LO product synthesis. Instead, 5-LO activation in MM6 cells required Ca2+ or alternative signaling pathways induced by hyperosmotic stress. In summary, mechanisms for activation of 5-LO differ considerably between cell types.

Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca 2؉ -dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80 -100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca 2؉ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca 2؉ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca 2؉ . In summary, our data suggest that interaction of Ca 2؉ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca 2؉ , a low lipid hydroperoxide level is sufficient for 5-LO activation. 5-Lipoxygenase (5-LO)1 catalyzes the initial steps in the biosynthesis of leukotrienes (LTs) and 5(S)-hydro(pero)xyeicosatetraenoic acid (5(S)-H(P)ETE) from arachidonic acid (AA) (for review, see Ref. 1). Due to the pivotal biological functions of 5-LO metabolites (2, 3), the activity of 5-LO is tightly regulated. In intact cells, Ca 2ϩ and phosphorylation seem to be primary signals that activate 5-LO. Moreover, the membranebound 5-LO-activating protein (FLAP) (4) and the redox state (5, 6) have a strong impact on cellular 5-LO product formation.In cell-free systems Ca 2ϩ , ATP, phospholipids (membranes), lipid hydroperoxides (LOOH), and leukocyte-derived proteins have been shown to enhance 5-LO catalysis (reviewed in Ref. 1). However, the degree of stimulation by each of these components depends on the assay conditions, i.e. the source of 5-LO (isolated, in crude homogenates or cellular fractions), presence of other cofactors, the concentration of AA, etc. LOOH are of importance for the initial conversion of the active site iron from the ferrous (resting) to the ferric (active) state (7, 8). Accordingly, glutathione peroxidases (GPx) that reduce LOOH inhibit 5-LO product synthesis in vitro and in intact cells (5, 9 -15), and conditions that are associated with an increased peroxidetone promote 5-LO product formation (6,16,17). Two Ca 2ϩ ions bind to the N-terminal C2-like domain of 5-LO with a K d of 6 M (18, 19). Half-maximal activation of purified 5-LO was determined at 1-2 M Ca 2ϩ , whereas 4 -10 M Ca 2ϩ causes maximal activation of the enzyme (20,21). In intact cells lower concentrations of Ca 2ϩ (200 -300 nM) seem to be sufficient for 5-LO activation (22,23). It was shown that C...