The Molecular Biology and Regulation of 5-Lipoxygenase

OLOF P. RÅDMARK

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

	INTRODUCTION

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5-Lipoxygenase (5LO) catalyzes the conversion of arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), and further to the allylic epoxide 5(S)-trans-7,9-trans-11,14-cis-eicosatetrenoic acid (leukotriene A₄, LTA₄). Thus, 5LO has a central role in leukotriene biosynthesis. This article covers some basic properties of 5LO. Studies regarding the 5LO gene and targeted gene disruption, and 5LO intracellular compartmentalization, are described in other parts of this supplement.

	PURIFICATION, CLONING OF cDNA, STRUCTURAL PROPERTIES

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5LO has been purified from different types of leukocytes, as monomeric enzymes with estimated molecular weights between 72,000 and 80,000 (1). Mammalian 5LO cDNAs have been cloned from human, rat, mouse, and hamster (6). The deduced sequences contain 672 or 673 amino acids, with more than 90% identity. A probable cloning artifact in the C terminus of rat 5LO cDNA has been noted (11).

Recombinant human 5LO and porcine leukocyte 5LO were shown to contain iron, and iron as well as enzyme activity were lost when 5LO was exposed to oxygen (12). Electron paramagnetic resonance (EPR) studies of the ferric enzyme showed a multicomponent g6 signal in the EPR spectrum, which indicated that the metal center in its oxidized state existed in more than one (but related) forms (13). The role of iron in the generally accepted scheme for the lipoxygenase reaction is to act as electron acceptor and donor, during hydrogen abstraction and peroxide formation.

In almost all lipoxygenases, six conserved histidine residues are present. Also, the C termini are similar among the lipoxygenases. On the basis of mutagenesis studies and comparisons with the crystal structures of soybean LO-1 three of the conserved histidines and the C-terminal isoleucine appear to function as ligands to the prosthetic iron in 5LO. It was suggested that one of the histidine ligands in 5LO could function as a so-called replaceable ligand (see Reference 11 for review).

	STIMULATION OF 5-LIPOXYGENASE ACTIVITY

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Calcium

Calcium stimulates both the oxygenase and the LTA₄ synthase activities, as well as the pseudoperoxidase activity of 5LO (14). It was originally observed that calcium ionophore activated biosynthesis of SRS-A (slow-reacting substance of anaphylaxis) and other 5LO products in intact cells (see Reference 15 for review). In homogenates of RBL-1 cells, calcium stimulated synthesis of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-DHETE) (16), and subsequently, calcium has been used as an activator in assays of purified 5LO. However, it should be observed that 5LO has some activity also without addition of calcium (1, 5, 17). The concentration of calcium giving half-maximal activation of purified 5LO is quite low (1-2 µM) and full activation is reached at 4-10 µM (18, 19). We found that 5LO actually binds calcium in a reversible manner, with a K_d of 6 µM, and an apparent maximum binding of two calcium per 5LO (20). Calcium increases the hydrophobicity of 5LO, and promotes membrane association (compare below). Also, other cations (Ba²⁺, Sr²⁺, and Mn²⁺) can activate, while Zn²⁺, Cu²⁺, and Co²⁺ inhibit, 5LO (4, 18, 19).

ATP

ATP was first shown to stimulate crude 5LO (21). In the presence of calcium K_a values for ATP were 30-100 µM, but ATP alone was inefficient (4, 17, 22). The costimulatory effect has been confirmed with purified enzymes, and also other nucleotides can stimulate 5LO (21, 23, 24). However, ATP had a stimulatory effect on purified 5LO also when [Ca²⁺] was 20 nM, far below the efficient concentration of calcium. Thus, ~ 0.1 mM ATP gave maximum activation (24). The affinity for ATP has been used to purify 5LO.

Lipid Hydroperoxide

For lipoxygenase catalysis, the ferrous iron of the resting form should be oxidized to the ferric form, by lipid hydroperoxide. Leukocyte 5LO in crude homogenates was stimulated by 15-, 12-, or 5-HPETE, or by 13-hydroperoxy-octadecadienoic acid (13-HPODE) but not by cumene hydroperoxide, tert-butyl hydroperoxide, or hydrogen peroxide (25). Reducing agent (dithiothreitol, DTT) counteracted 5LO activation in crude homogenates (25, 26). Also, glutathione peroxidase added to in vitro assays of 5LO inhibited product formation (27, 28). Thus, reduction of lipid hydroperoxide in the cell by glutathione peroxidases should be important for control of 5LO activity (29), and for the lymphocyte cell line BL41-E95-A, no 5LO activity could be detected unless preincubation with Dnp-Cl or 1-chloro-2,4-dinitrobenzene, or diamide was performed (30). Studies using selenium-deficient RBL cells indicated that phospholipid hydroperoxide glutathione peroxidase was most important for control of the lipid hydroperoxide level, and thus 5LO activity (31). It is of interest that the upregulation of 5LO activity observed after culture of HL-60 cells with transforming growth factor a and vitamin D₃ was related to the appearance of a peroxidase-insensitive 5LO activity (32).

Membrane Structures

Microsomal membranes stimulate 5LO enzyme activity (see, e.g., Reference 33). Also, phosphatidylcholine (PC) stabilized 5LO during purifications (2, 3), and PC vesicles could replace cellular membranes as a stimulatory factor (34). It was subsequently found that the ratio of arachidonic acid to phospholipids affected the activity of purified 5LO, suggesting that the concentration of substrate at a lipid-water interphase is important (35). From another study it was concluded that 5LO performs an interfacial reaction, in the same manner as phospholipase A₂ (36), and calcium causes association of 5LO with synthetic PC liposomes (19). 5LO can also bind to cellular membrane fractions, as first found when polymorphonuclear leukocytes (PMNLs) or RBL cells were homogenized in the presence of calcium (see, e.g., Reference 37). However, it was pointed out that 5LO can sediment in a 100,000 g pellet in the absence of membrane, and thus apparent membrane binding in vitro may not always reflect a physiological process (38). Ionophore A23187 has been used as a stimulus for membrane binding of 5LO in intact cells (see, e.g., Reference 37). Also, more physiological stimuli resulted in membrane association, for example, N-formyl-methionyl-leucyl-phenylalanine (fMLP) in studies of dimethyl sulfoxide (DMSO)-differentiated HL-60 cells (39), and IgE/antigen in studies with RBL cells and mast cells (40, 41).

	5-LIPOXYGENASE-ACTIVATING PROTEIN

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The drug MK-886 was found to inhibit 5LO in intact human leukocytes, but not in broken cells or after purification of 5LO. Also, MK-886 was reported to block membrane association of 5LO in human leukocytes (38). A radiolabeled analog to MK-886, carrying a photoactivatable azido group, was prepared. In elegant experiments, this compound was used to mark an 18-kD protein in neutrophils, which could then be purified (42). Complementary DNAs, isolated from rat and human, encoded novel proteins (5LO-activating protein, FLAP) with three transmembrane-spanning regions and two hydrophilic loops. Human osteosarcoma cells were transfected with cDNA for 5LO and/or FLAP. Both proteins were required for leukotriene production in osteosarcoma cells stimulated by A23187 (no exogenous arachidonic acid) (43). In subsequent studies regarding the subcellular localization, it was demonstrated that most of the FLAP is associated with the nuclear membrane, and some with endoplasmic reticulum (44).

It is often assumed that FLAP functions as a membrane anchor for 5LO, but a direct association between the two proteins has not been shown. The finding that MK-886 counteracted 5LO membrane association in intact human leukocytes and in RBL cells was thought to explain the mechanism for the inhibitory effect of MK-886. However, several reports state that MK-886 inhibits LT biosynthesis, without an effect on membrane association (46, 49). It was thus suggested that LT biosynthesis can be a two-step process consisting of FLAP-independent binding of 5LO to the membrane of the nuclear envelope, followed by FLAP-dependent activation of the enzyme. In addition to its still possible role in membrane association of 5LO, FLAP appears to function as a substrate transfer protein. Thus, FLAP binds to an ¹²⁵I-labeled photoaffinity analog of arachidonic acid (53). Also, it was presented that arachidonic acid and other cis-unsaturated fatty acids compete with inhibitors (BAY X1005, MK-886) regarding binding to FLAP (54, 55). FLAP also has an effect when exogenous substrate is presented to the cells. In studies of transfected Spodoptera frugiperda (Sf9) cells, FLAP was thus shown to stimulate 5LOs use of exogenous arachidonic acid as substrate, and the conversion of 5-HPETE to LTA₄ was promoted, but FLAP was not obligatory for 5LO activity in the intact cell (56). It has been shown that FLAP can also promote the use of other 5LO substrates [12(S)-HETE, 15(S)-HETE], and the effects with arachidonic acid were confirmed (57).

	BINDING OF 5-LIPOXYGENASE TO CELLULAR MEMBRANES, SUBCELLULAR LOCALIZATION, THE SH3 MOTIF

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For some time it was thought that 5LO in a resting cell resided in the cytosol, and simply became membrane bound in connection with activation to produce LTs. Findings regarding the distribution of 5LO in rat alveolar macrophages were the first not to fit with this model (49). At some stage in the life span of the inflammatory cell, 5LO can be imported into the nucleus (44, 58). Different patterns have been observed, particularly for peripheral blood leukocytes as compared with alveolar macrophages (47). In blood PMNLs, 5LO resides in the cytosol until the cell is activated to produce leukotrienes, and then 5LO binds to the nuclear membrane. In alveolar macrophages a large part of 5LO is found inside the nucleus already when cells are isolated, in a location (somehow associated with euchromatin) that after subcellular fractionation was described as nuclear soluble. On activation to produce leukotrienes, the intranuclear 5LO binds to the nuclear membrane (46, 47). A study regarding glycogen-elicited rabbit peritoneal neutrophils showed that nuclear import of 5LO could be disparate from LT biosynthesis (59). Thus, the elicitation procedure led to import of 5LO, from the cytosol to a nuclear-soluble compartment. Subsequent stimulation with ionophore gave binding of 5LO to the nuclear envelope and LT biosynthesis. As alveolar macrophages, the elicited PMNLs had higher activation thresholds (for A23187) as compared with nonelicited cells; however these cells also had an increased capacity for LT biosynthesis (59). This may be linked to the intranuclear localization of 5LO, and it could be that alveolar macrophages have actually been elicited in vivo, before isolation from the tissue.

It would appear that binding of 5LO to the perinuclear membrane always occurs in connection with LT biosynthesis. However, there may not be an obligatory coupling between these events, as demonstrated in experiments where 5LO was bound to membrane fractions in the presence of zileuton (52, 60). Also, membrane binding can be reversible, particularly when cells are stimulated in a less vigorous fashion (IgE/antigen, fMLP, zymozan, ionophore for short time). Furthermore, membrane binding does not necessarily lead to inactivation of 5LO, although this may occur when cells are activated with ionophore for a longer time (which also gives persistent membrane association) (41, 52). Association of 5LO with the nucleus has been observed also for other cell types (9, 30, 61), and several studies thus support the idea that 5LO associates with nuclear membranes when cells are activated, and that this is the locale for LT biosynthesis. However, another organelle, i.e., lipid bodies, has also been suggested (62).

5LO contains an Src homology 3 (SH3) binding motif, which allows 5LO to bind to the SH3 domain of growth factor-bound receptor protein 2 (Grb2) (63). A competitor peptide containing the 5LO SH3 binding motif inhibited binding to Grb2, and also interfered with translocation of 5LO from cytosol to membrane in intact human neutrophils. It thus appears possible that translocation of 5LO (nuclear import and/ or membrane binding) can involve the SH3 motif. It was also presented that 5LO could bind to cytoskeletal proteins (-actinin and actin), and that 5LO activity was inhibited by guanine nucleotides. Kinase activity was important for cellular 5LO activity and membrane association, as indicated by the effect of inhibitors of tyrosine kinase and mitogen-activated protein kinase kinase 1 in neutrophils and HL-60 cells, and a model for the interaction of 5LO with SH3SH2 domain proteins was depicted (64, 65). The 5LO sequence contains basic nuclear localization sequences, but none of these appeared important for nuclear import of 5LO. Instead, a part (amino acids 1-80) of the putative -barrel of 5LO efficiently directed import of a green fluorescent protein (GFP) fusion protein to the nucleus, as demonstrated by immunofluorescence microscopy of transfected HEK 293 cells (66).

It is possible that 5LO could have different roles in different subcellular locales. It was suggested that 5LO in the nucleus could have functions independent of its enzyme activity, or that it could be relevant for formation of LTs with autocrine functions. On the other hand, 5LO in the cytosol or in lipid bodies could have a role in the generation of paracrine LTs (59, 62, 63).

	UPREGULATION OF 5-LIPOXYGENASE DURING DIFFERENTIATION: EFFECTS OF CYTOKINES

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An extensively used model for myeloid cell maturation is in vitro differentiation of the human leukemic cell line HL-60. Differentiation of this cell line leads to upregulation of 5-LO mRNA, protein, and activity (see, e.g., Reference 67). A factor in human serum was found to augment the upregulation of 5LO activity (by ninefold) in HL-60 cells during differentiation. This upregulation was not primarily due to increased expression of 5LO, as there was only twofold more 5LO protein (68). The factor was identified as transforming growth factor  (TGF-), and the effect of TGF- was increased by granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor  (TNF-) (69). However, a cellular lipid was also required for TGF- to be effective, and this was found to be vitamin D₃ (VD3) (70). When HL-60 cells were treated with TGF- and a low concentration of VD3 the same effect as with serum was obtained, i.e., a more prominent increase in enzyme activity than in the amount of 5LO protein. At higher concentrations of VD3, there was also an increased expression of 5LO. Thus, TGF- and VD3 have two effects: increased activity and increased 5LO expression. Regarding the effect on 5LO activity, it was shown that after culture of HL-60 cells with TGF- and VD3, the 5LO activity of cell homogenates was no longer inhibited by DTT or glutathione (GSH). Such peroxidase-insensitive 5LO activity was also present in homogenates of PMNLs, and it was suggested that the differentiation process induced components of importance for conversion of cellular 5LO to the active ferric form (32).

TGF- and VD3 had a prominent effect on 5LO in the human monocytic cell line Mono Mac 6 (71). In nondifferentiated Mono Mac cells, no 5LO activity or protein could be detected, but after culture with TGF- and VD3, activity increased more than 500-fold, and expression about 100-fold. The increased expression was due both to upregulated transcription, and to improved transcript elongation and maturation (72). Also, VD3 upregulated 5LO metabolism in differentiating blood monocytes, and ionophore-induced formation of LTB₄ and 5-HETE was reduced in alveolar macrophages from VD3-deficient rats (73, 74). It is of interest that for the promyelocytic cell line U937, expression of 5LO and FLAP was not sufficient for 5LO activity; it was required that the cells were also differentiated (with DMSO) (75). Synergistic effects of TGF- and VD3 on leukemic cell lines have been described (76). These agents together stimulate the terminal differentiation of monocytic cells, and it appears reasonable that the effects on 5-LO are part of such a differentiation process during maturation of myeloid cells in the bone marrow. Also, when the keratinocyte cell line HaCaT, or normal human keratinocytes, were cultured under conditions promoting differentiation, increased expression of 5LO was observed (77).

The effects of TGF- and VD3 thus seem related to cellular differentiation. However, GM-CSF can prime mature human leukocytes for increased leukotriene biosynthesis. Mechanisms have been presented, and concern increased availability of endogenous fatty acid substrate, and intracellular calcium levels. That GM-CSF could also upregulate 5LO expression was first indicated by the effects of cycloheximide or actinomycin D, which inhibited the effect of GM-CSF on priming of 5LO activity (78). Subsequently it was found that 3 nM GM-CSF (60 min) gave an approximately threefold increase in the amount of 5LO protein in human PMNLs (79). GM-CSF did not affect the level of 5LO mRNA, or its stability, indicating that the effect was posttranslational. However, another study claimed that a longer exposure to GM-CSF (more than 6 hr) caused increased 5LO gene transcription in human PMNLs (80). GM-CSF also upregulated 5LO expression and activity in monocytes. Thus, monocytes were cultured in the presence of lectin-stimulated lymphocyte supernatants, resulting in increased amounts of 5LO (and FLAP) mRNA and protein. The effect of the lymphocyte conditioned medium could be mimicked by GM-CSF or interleukin 3 (IL-3) (81). Similar results were obtained with the monocyte-like cell line THP-1 (82), and the same group also found that dexamethasone could increase expression of 5LO and FLAP in these cells (83). IL-3 can stimulate expression of 5LO also in mouse mast cells (84). When cells were cultured with Kit ligand and IL-10, the simultaneous addition also of IL-3 (time period, 2-5 wk) caused a substantial increase in the amount of 5LO protein.

	Footnotes

Correspondence and requests for reprints should be addressed to Olof P. Radmark, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden. E-mail: Olof.Radmark{at}mbb.ki.se.

Acknowledgments: Supported by grants from the Swedish Medical Research Council (03X-217), from the European Union (BMH4-CT96-0229), from the Verum Foundation, and from the Vårdal Foundation (A95 067).

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