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ARTICLE IN PRESS

Prostaglandins, Leukotrienes and Essential Fatty Acids 69 (2003) 99–109

5-Lipoxygenase and FLAP$


M. Peters-Golden*, T.G. Brock
Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, 1150 W. Medical Center
Drive, Ann Arbor, MI 48109-0642, USA
Accepted 1 April 2003

Abstract

The initial steps in the biosynthesis of leukotrienes from arachidonic acid are carried out by the enzyme 5-lipoxygenase (5-LO). In
intact cells, the helper protein 5-LO activating protein (FLAP) is necessary for efficient enzyme utilization of endogenous substrate.
The last decade has witnessed remarkable progress in our understanding of these two proteins. Here we review the molecular and
cellular aspects of the expression, function, and regulation of 5-LO and FLAP.
r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Leukotrienes; Compartmentalization; Nucleus; Catalysis; Metabolon; Calcium; Kinases; Inhibitors; Expression

1. Introduction removal to yield LTD4 and then LTE4. LTs C4, D4 and
E4 are known as cysteinyl LTs (cysLTs) and comprise the
Leukotrienes (LTs) are potent lipid mediators most myotropic and edemagenic activities long known as
strongly implicated in asthma and allergic diseases. A ‘‘slow reacting substance of anaphylaxis.’’ LTB4 is best
substantial capacity for the complete biosynthesis of recognized for its potent chemotactic and leukocyte-
LTs from arachidonic acid (AA) is largely confined to activating effects.
leukocytes. This biosynthetic pathway can be triggered 5-LO is a member of the family of arachidonate
by a variety of stimuli, including antigens, microbes, lipoxygenases, but differs in certain critical respects
cytokines, complement, oxidants, immune complexes, from other members of the family. Its requirement for
and toxins. These stimuli activate signal transduction interaction with FLAP in order to initiate the 5-
cascades that in turn activate LT-forming enzymes. lipoxygenation of AA in intact cells is one such
Briefly, a phospholipase A2 (PLA2), probably the distinction. Although steps in LT synthesis both
cytosolic (cPLA2) or a secretory (sPLA2) isoform, proximal and distal to the actions of 5-LO/FLAP are
initiates the pathway by catalyzing the hydrolysis of subject to regulatory control, modulation of the expres-
AA from membrane phospholipids. The liberated free sion and function of these two proteins is a critical
AA is then oxygenated at C-5 and subsequently determinant of cellular capacity for LT biosynthesis.
dehydrated by the actions of the 5-lipoxygenase (5-LO) Knowledge of these processes has expanded enormously
enzyme, yielding the epoxide intermediate LTA4. The in the last decade. In this review, we provide an overview
efficient utilization of endogenous arachidonate by 5-LO of the molecular, biochemical, and cellular aspects of 5-
requires a helper protein termed ‘‘5-LO activating LO and FLAP expression and action.
protein’’ (FLAP). LTA4 can then be hydrolyzed by
LTA4 hydrolase to LTB4, or conjugated with reduced
glutathione by LTC4 synthase to yield LTC4. LTC4 can 2. Biochemical properties of 5-LO
be modified extracellularly by sequential amino acid
$
All of the plant, fungal and animal lipoxygenases are
Supported by the National Institutes of Health through R01-
capable of inserting oxygen on the appropriate sub-
HL58897 and R01-AI43574.
*Corresponding author. Tel.: +1-734-763-9077; fax: +1-734-764- strate, which is typically a polyunsaturated fatty acid
4556. containing a series of cis double bonds. Early studies of
E-mail address: petersm@umich.edu (M. Peters-Golden). soluble fractions of leukocytes demonstrated that a

0952-3278/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0952-3278(03)00070-X
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100 M. Peters-Golden, T.G. Brock / Prostaglandins, Leukotrienes and Essential Fatty Acids 69 (2003) 99–109

single enzyme, 5-LO, can catalyze both the C-5 its effects on the critical iron atom. NO can also interact
oxygenation as well as a dehydration step that produces with reactive oxygen species to generate peroxynitrite,
LTA4. All lipoxygenases also contain a non-heme iron which can also inhibit 5-LO activity. Although nitrotyr-
atom that is involved in catalysis. The form of the iron osination of 5-LO has been observed at high concentra-
atom is critical to determining the catalytic potential of tions of peroxynitrite [18], it does not appear that this
the enzyme, with oxidation to the ferric (Fe3+) form mechanism accounts for inhibition seen at lower
conferring activity. As a result, low levels of peroxides concentrations.
are necessary for 5-LO to be functional. However,
higher levels will render the enzyme inactive.
An important determinant of 5-LO action is its 3. Structural features of 5-LO
activation by calcium. In the absence of calcium, 5-LO
has minimal catalytic activity [1–3]. The addition of The cloning of the cDNA for human, rat, mouse and
calcium stimulates both the oxygenation and dehydra- hamster 5-LO indicated that these genes have been
tion reactions of 5-LO. Maximal activation is obtained highly conserved across species. Furthermore, compar-
with 4–10 mM calcium [4]. Also, early biochemical ison of the primary amino acid sequences of the 5-LO
studies found that 5-LO activity from unstimulated proteins from different species with other plant and
leukocytes was in the soluble fraction of crude cell mammalian lipoxygenases revealed a high level of
lysates. The addition of calcium to cell lysates led to similarity, particularly in their carboxy terminal resi-
membrane association of 5-LO [5]. Taken together, dues. This similarity suggested that common structural
these results indicated that cell stimulation causing a characteristics might underlie their common functions.
transient rise in intracellular calcium would induce 5-LO To date, four lipoxygenase proteins have been crystal-
to move from its soluble site to a membrane, leading to lized and resolved structurally; three are plant lipox-
catalysis. This ‘‘translocation’’ event is discussed in ygenases [19–21] and the fourth is rabbit reticulocyte 15-
depth below. lipoxygenase [22]. All have two domains: an amino
It was found that ATP, and other nucleotides to a terminal b-barrel structure and a carboxy terminal
lesser extent, could significantly enhance the activity of structure consisting predominantly of a-helices (Fig. 1).
5-LO in cell lysates [6]. Likewise, LT synthetic capacity The carboxy terminal region is highly conserved across
in intact cells was reduced under conditions of ATP all plant and animal lipoxygenases. In contrast, the
depletion [7]. However, stimulation of 5-LO activity did amino terminal domain varies greatly, even between
not involve hydrolysis of ATP or the generation of mammalian lipoxygenases.
inorganic phosphate as an energy source. Instead, ATP Within the carboxy terminal regions of all lipoxy-
directly bound to the 5-LO enzyme. Analysis of ATP genases are two clustered patterns of amino acids,
binding to 5-LO suggested two potential binding termed lipoxygenase iron-binding signatures, which
sequences [8]. Interestingly, the enhancement of 5-LO contain conserved histidine residues that serve to hold
activity by ATP only occurs in the presence of calcium iron inside the molecule. An isoleucine residue at the
[9]. The mechanism of ATP action remains unclear.
Kinase-mediated phosphorylation can also affect
5-LO activity. There is evidence in intact cells that 5-
LO activity can be modulated by protein kinases A [10]
and C [11], protein tyrosine kinases [12], and by
mitogen-activated protein kinase (MAPK) kinase [13],
but these studies have not shown direct phosphorylation
of 5-LO. More recently, two groups have been able to
show direct phosphorylation of 5-LO by a tyrosine
kinase [14] and by MAPK-activated protein kinase 2
[15]. In both cases, inhibitors of phosphorylation
reduced leukotriene synthetic capacity, suggesting that
this post-translational modification of 5-LO stimulates
activity.
There are few examples of endogenous substances
that inhibit 5-LO activity. As noted above, reactive Fig. 1. Secondary structure of rabbit reticulocyte 15-lipoxygenase. All
oxygen species can alter the oxidative state of the iron in known lipoxygenases have two distinct domains, the b-barrel domain
and the catalytic domain. In 5-LO, the b-barrel can bind calcium and
5-LO and in this way affect activity. It has also been
direct movement to cellular membranes. The catalytic domain contains
shown that nitric oxide (NO) can be a potent inhibitor the non-heme iron (Fe) essential to catalysis, as well as regulatory sites
of LT synthesis [16,17]. NO can directly inhibit the for nuclear import, ATP binding and phosphorylation by MAPKAP
activity of recombinant 5-LO [17], presumably through kinase 2.
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extreme carboxy terminus also appears to be necessary the ATP-binding sites on 5-LO, at TRP75, also happens
for retaining the iron atom. The presence of iron to be close to the calcium-binding site on the b-barrel
indicates that this larger segment of the 5-LO protein [24], providing a possible molecular basis for coopera-
contains the site of catalysis and, as a result, is tion between these two cofactors; this possibility has not
commonly referred to as the ‘‘catalytic domain’’. as yet been tested.
Surprisingly, there is no consensus on how substrate
enters this catalytic domain and interacts with the metal
center of any lipoxygenase [23]. Presumably, specific 4. Inhibitors of 5-LO
residues and pocket size would control the positioning
of the substrate relative to iron, determining the site of There has been great interest in identifying inhibitors
oxygenation, as has been proposed [22]. However, the of 5-LO activity, because of the importance of 5-LO-
available crystal structures from the different lipoxy- derived LTs in the pathogenesis of asthma and other
genases suggest a variety of potential sites for substrate inflammatory diseases. Several competitive inhibitors of
access. It has been noted that each of these structures AA metabolism, including zileuton, AA-861, ABT-761,
is based on the ferrous, or inactive, form of the ZD-2138 and L-739010, are very specific for 5-LO over
enzyme and that conformational alterations associated other lipoxygenases. It should be noted that these
with oxidation of the active site might reveal the access inhibitors may have biochemical actions in addition to
site [23]. their effects on 5-LO [18,29].
Given the similarity of different lipoxygenase catalytic Some other commonly used inhibitors of 5-LO, such
domains, it has been possible to model the 5-LO as nordihydroguaiaretic acid (NDGA), are general
catalytic domain after the known rabbit reticulocyte lipoxygenase inhibitors, blocking catalysis by altering
15-LO structure [24]. This model suggested that the the redox state of the iron necessary for all lipoxy-
catalytic domain of 5-LO involves 15 a-helices organized genases. Such inhibitors may reduce LT generation, but,
around a central helix that contains the iron-binding because of their lack of specificity of action, their
patterns described above. Several additional structural efficacy cannot be attributed solely to their effects on the
features are predicted to be found in the catalytic 5-LO pathway.
domains of all lipoxygenases, including 5-LO. Most A novel compound, MK-886, was found to be a very
notable among these is a region described as an SH3- effective inhibitor of LT synthesis [30] in intact
binding site [25]. This domain, which has been shown to leukocytes but not in studies with leukocyte homo-
bind actin in vitro, is highly conserved across mamma- genates or purified 5-LO. This indicated that MK-886
lian 5-, 12- and 15-LOs. The relevance of this region to was inhibiting 5-LO indirectly, perhaps by associating
lipoxygenase function in vivo is unknown. with an essential cofactor. Subsequent experiments
There are some regions within the catalytic domain isolated a membrane-associated protein that could bind
that are unique to 5-LO. One such region is a putative MK-886 [31], and because this protein appeared to be
ATP-binding site near Trp201 [8]. This region is highly essential in determining 5-LO activation, it was termed
conserved among 5-LOs from different species and is 5-lipoxygenase activating protein, or FLAP.
poorly conserved in other lipoxygenases. Another such
region involves a four amino acid insertion in the
general lipoxygenase sequence, producing a site for 5. Properties and structural features of FLAP
phosphorylation by MAPKAP kinase 2 at Ser271 [26].
Like the ATP-binding site, this site is found in 5-LO The subsequent cloning of FLAP allowed the expres-
sequences from different species but is absent from all sion of FLAP, with or without 5-LO, in cells that
other lipoxygenases. normally lack both enzymes. Results from these studies
The amino terminal sequences of the different indicated that FLAP was required for ionophore-
lipoxygenases, although dissimilar in their primary induced LT synthesis from endogenous substrate
sequences, have consistently been found to form a b- [32,33]. Interestingly, FLAP can enhance, but is not
barrel structure. In other proteins such as cPLA2 and necessary for, the processing of exogenous AA by 5-LO.
protein kinase C, b-barrel structures can bind calcium In cells that express both FLAP and 5-LO, changes in
and mediate calcium-dependent membrane association. FLAP expression greatly influence cellular LT synthetic
Consistent with this, the amino terminus of 5-LO has capacity [34,35]. Taken together, these findings suggest
been shown both to bind calcium [27] and to be both that FLAP may more accurately be viewed as a
necessary and sufficient for membrane association [28]. 5-lipoxygenase facilitating protein.
This indicates that calcium, rising in concentration The mechanism of FLAP action is discussed below.
during cell stimulation, would bind to the b-barrel However, it has been demonstrated that FLAP is also an
region of 5-LO and drive translocation, thus placing the AA-binding protein [36]. Furthermore, AA competes
enzyme close to its substrate. It is of interest that one of with MK-886 for binding with FLAP [37]. These
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observations suggest that FLAP facilitates 5-LO action knowledge, no FLAP inhibitor is currently in clinical
by enhancing the delivery of AA to 5-LO, and that MK- development.
886 inhibits LT synthesis by directly interfering with this
process.
FLAP is now recognized as a member of a protein 7. Intracellular sites of LT biosynthesis
superfamily designated as membrane-associated pro-
teins in eicosanoid and glutathione metabolism, or The first decade of research into LT biosynthesis was
MAPEG [38]. Hydropathy plots predicted from the dominated by efforts to characterize the relevant
primary sequences of member proteins are similar. This enzymes, their cofactors, and their cellular and tissue
similarity in hydropathy likely reflects common patterns distribution. Little was known about the intracellular
within the primary sequences and should result in sites at which these enzymes functioned. A foundation
similar positioning of the proteins in membranes. for our current understanding of such matters derives
Furthermore, these similarities might suggest additional from studies conducted in the late 1980s and early 1990s,
parallels in protein function. in which crude fractionation methods were used to study
Three proteins involved in eicosanoid metabolism the locale of 5-LO and FLAP in resting and activated
have been assigned to the MAPEG superfamily: FLAP, cells. This work demonstrated that FLAP was found in a
LTC4 synthase and membrane-associated PGE synthase particulate fraction in both states [31]. By contrast, 5-LO
(mPGES). The primary sequence of FLAP is approxi- was found in the soluble fraction of resting leukocytes,
mately 50% similar to that of LTC4 synthase [39,40] and but rapidly (i.e., within 0.5–15 min) redistributed in a
less so (33% similarity with two gaps) to mPGES [41]. calcium-dependent manner to a particulate fraction
Significantly, all three proteins share the pattern ER- upon activation [5]. cPLA2 exhibited properties similar
x(3)-A-x(2)-N-x(2)-D/E that has been shown to be to 5-LO [46]. From these early studies, two assumptions
important for binding MK-886 in FLAP [42]. Presum- were generally made. The first was that cell activation
ably for this reason, MK-886 is able to inhibit all three colocalized cPLA2 and 5-LO with FLAP at the same
proteins [41]. membrane site. The second was that this site was the
There are points of difference between FLAP and plasma membrane—a logical assumption inasmuch as
these other MAPEG members. No enzymatic activity LTs are known to be predominantly secreted mediators.
has been identified for FLAP, unlike other members of However, the crude cellular disruption and fractionation
the MAPEG superfamily. Also, FLAP activity is not methods employed in these early studies were not
modulated by glutathione, unlike PGE synthase, which capable of rigorously supporting such assumptions.
is glutathione dependent, and LTC4 synthase, which In the early to mid-1990s, these issues were more
utilizes glutathione during catalysis. Finally, LTC4 definitively addressed by several laboratories using more
synthase appears to function as a homodimer [43], while careful disruption/fractionation as well as immunomi-
FLAP does not. croscopic techniques in a variety of leukocyte popula-
tions. These subsequent studies supported the first of the
above assumptions, i.e., that 5-LO colocalized with
6. Inhibitors of FLAP cPLA2 and FLAP in activated cells. Surprisingly,
however, the site at which these proteins were found
As noted above, FLAP was first identified because of upon cell activation proved to be not the plasma
its ability to bind MK-886, a potent inhibitor of LT membrane, but the nuclear envelope (NE) and contig-
synthesis in intact cells (IC50=100 nM). The effective- uous perinuclear endoplasmic reticulum (PER) [47–54].
ness of MK-886 as a FLAP inhibitor results from its Functional studies demonstrated in parallel that most
capacity to fill the binding site for its ‘‘substrate’’ AA. AA hydrolyzed by cPLA2 upon cell activation derived
As noted above, similarities in the lipid-binding sites of from phospholipids of the NE/PER [52]. Moreover, the
FLAP, LTC4 synthase and mPGES allows MK-886 to enzyme LTC4 synthase has also been localized to this
inhibit each of these enzymes; differences between the same site [40,55]. Therefore, all four key enzymes
sites may explain differences in inhibitory concentra- necessary for synthesis of the proximate cysLT, LTC4,
tions (IC50=11 mM MK-886 for LTC4 synthase and are assembled at the NE/PER.
3.2 mM for PGE synthase-1). It is important to note that In addition to a calcium-dependent mechanism for
MK-886 has been shown to have effects independent of 5-LO translocation, a calcium-independent mechanism
its actions on FLAP, LTC4 synthase and mPGES. Thus, that involves phosphorylation of 5-LO by mitogen-
MK-886 alters gene expression in cancer cells, ultimately activated protein kinases, in response to agents such
leading to cell death [44], a result that may reflect non- as cellular stress or unsaturated fatty acids, has recently
specific, FLAP-independent actions. Other inhibitors been described [56]. Although available data
that also inhibit AA binding to FLAP have also been are consistent with the possibility that this latter
developed (see, for example, reference [45]). To our pathway likewise results in association with the NE/
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PER, this remains to be confirmed by immunomicro- An alternative to the NE/PER as a site for assembly
scopic techniques. of enzymes in the LT biosynthetic cascade has been
FLAP was originally thought to be a membrane suggested. These enzymes have also been identified by
docking protein for 5-LO [57], and selective localization immunomicroscopic techniques in lipid bodies, non-
of FLAP to the NE/PER could have provided an membrane-delimited cytoplasmic droplets which can
explanation for selective translocation of 5-LO to this increase in number in response to inflammatory stimuli
site. However, initial attempts failed to reveal evidence (see accompanying article by Bandeira-Melo and Well-
of a direct interaction between FLAP and 5-LO. er). On the basis of a novel immunolocalization
Subsequent studies demonstrated membrane association technique for detecting intracellular LTC4, it has been
of 5-LO in transfected cells even in the absence of suggested that lipid bodies are the site of eosinophil
FLAP, although LT synthesis could not be initiated [58]. LTC4 formation in response to chemokines and
Finally, immunofluorescence [59] and immunoelectron cytokines, whereas the NE/PER is relevant for stimuli
microscopic [49] analysis revealed that the FLAP that raise intracellular calcium [65,66]. NE/PER accu-
inhibitor MK-886 caused a near-complete inhibition of mulation of 5-LO has also been observed in macro-
LT synthesis without any effect on NE/PER transloca- phages stimulated with zymosan or formyl peptide and
tion of 5-LO. These data cast doubt on its role as a in mast cells stimulated with immune complexes [59].
docking protein, favoring the alternative role in Moreover, NE localization has been demonstrated in
substrate presentation discussed above. An alternative situ in alveolar macrophages from lung biopsy speci-
explanation for selective targeting of 5-LO (and cPLA2) mens obtained from patients with idiopathic pulmonary
to the NE might relate to the preferential capacity of the fibrosis. In the latter circumstance, NE immunostaining
C2 domains of both proteins to associate with phos- correlated with increased LT levels in lung tissue as well
phatidylcholine, together with the fact that the NE is as with overproduction of LTs by alveolar macrophages
enriched in phosphatidylcholine, as compared to the ex vivo [67]. In the absence of a discrete membrane, it is
inner plasma membrane [60]. The molecular mechan- not clear how the enzyme assembly necessary for
isms responsible for targeting the integral membrane activation of cPLA2 or 5-LO might be mediated in an
proteins FLAP and LTC4 synthase selectively to this organelle such as the lipid body. Nonetheless, it is
intracellular membrane are not known. certainly plausible that sites other than the NE/PER
Agents such as lipopolysaccharide and phorbol esters may contribute to LT synthesis, and that these alter-
are not themselves capable of triggering LT synthesis, but native sites may assume prominence under certain
are well known to ‘‘prime’’ cells for enhanced LT situations in certain cell types.
production in response to a triggering agonist. It has A macromolecular complex of sequential enzymes in
been demonstrated that pretreatment with each of these a metabolic pathway has been termed a ‘‘metabolon’’
agents enhances the translocation of 5-LO to the NE/PER [68]. Such a complex would be expected to result in a
in response to triggering agonists [61,62]. Likewise, cellular microenvironment that allows efficient channel-
substances that increase intracellular levels of cyclic ing of metabolic intermediates. It is still not known if the
AMP, including prostaglandin E2, are recognized to components of the LT biosynthetic metabolon directly
inhibit cellular LT biosynthesis. This inhibitory action associate with each other, or if their proximity merely
has recently been linked to impaired translocation of 5- increases the likelihood of metabolic coupling. We also
LO to the NE/PER [10]. Other studies have demonstrated know little about the capacity of intermediates such as
that inhibitors of p38 mitogen-activated protein kinase AA or LTA4 to cross relevant membranes; however,
[63] and tyrosine kinases [14] including Janus kinase 3 [64] their capacity to do so would seem to be a relevant
inhibit LT biosynthesis in parallel with inhibition of 5-LO variable in understanding the significance of compart-
translocation, although the precise role of 5-LO phoshor- mentalization data for LT-forming proteins. In any
ylation in translocation has not been defined. These case, the role of the NE/PER as a site for this metabolon
studies further underscore the importance of translocation is entirely counter-intuitive, given that LTs are known to
to the NE for 5-LO activation. be secreted extracellularly. This fact strongly suggests
Prolonged stimulation of LT biosynthesis in macro- that LTs subserve important functions within or near
phages and mast cells results in persistent NE/PER the cell nucleus (see below).
association and inactivation of 5-LO. However, when
stimulation is transient, membrane association is rever-
sible. Under these circumstances, re-stimulation can 8. Regulation of 5-LO compartmentalization in resting
result in another round of rapid translocation and LT cells
synthesis [59]. This capacity for rapid and repeatable
responses distinguishes LT generation from that of As noted above, the NE/PER is both the site at which
other classes of inflammatory mediators, and may be an FLAP is localized and the site to which 5-LO
important property for long-lived cells. translocates in activated leukocytes. However, the
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intracellular compartmentalization of 5-LO in resting expected locale on the plasma membrane, such has not
cells has proven to be a complex matter. First, it varies as yet been observed for LT receptors. LTB4 has been
in a cell-specific manner: 5-LO is largely cytosolic in reported by some investigators [82] but not all [83] to be
resting peritoneal macrophages [47] as well as mono- a ligand for the intranuclear receptor/transcription
cytes [48], neutrophils [69], and eosinophils [70] isolated factor, peroxisome proliferator activated receptor-a; in
from peripheral blood. By contrast, mast cells [69,71] any case, it seems entirely likely that other intranuclear
and alveolar macrophages [49,53] contain both cytosolic ‘‘receptors’’ for LTs will be recognized. Because LTA4 is
and intranuclear pools of enzyme. The intranuclear 5- a highly reactive electrophile, it might react readily with
LO is strictly soluble in the alveolar macrophage, diverse intranuclear targets with resulting consequences
whereas soluble as well as bound pools exist within the for nuclear function; the possibility that DNA might be
nucleus of the mast cell [53]. The presence of 5-LO such a target is suggested by the demonstration that it
within the nucleus is the consequence of nuclear import can interact with nucleosides and nucleotides in vitro
through the nuclear pore complex. Import of a protein [84]. It should also be recognized that reactive oxygen
of the size of 5-LO requires a nuclear localization signal species are byproducts of the 5-LO reaction [85], and
(NLS), and several candidate motifs have been proposed their generation near or within the nucleus could
[72–74]. It now seems likely that 5-LO contains at least influence redox tone and thereby modulate, for example,
two distinct NLSs [74] as well as a nuclear export signal the activity of oxidant-sensitive transcription factors
[75] that together determine compartmentalization. The such as nuclear factor (NF)-kB. Finally, because free
fact that alveolar macrophages appear to be unique AA might itself exert intranuclear effects, 5-LO action
among cells of the macrophage lineage in the fact that could be important by virtue of its ability to consume
they contain an intranuclear pool of 5-LO suggests that substrate.
the process of nuclear import is triggered by factors The resting locale of 5-LO may influence LT
unique to the pulmonary alveolar milieu [76]. It is biosynthetic capacity in several ways. First, it has been
important to recognize that 5-LO localization within the observed that within a given cell type, different 5-LO
nucleus is not itself indicative of activation of LT pools exhibit different capacities for translocation to the
biosynthesis. Regardless of its site of origin in a resting NE upon stimulation. Thus, while the alveolar macro-
cell, LT biosynthesis generally appears to require phage contains both intranuclear and cytosolic pools, it
translocation of 5-LO to the NE/PER. It is assumed appears that only the intranuclear pool is capable of
that 5-LO originating from the cytosol would associate translocating; by contrast, both pools are capable of
with the outer membrane of the NE/PER, while enzyme translocating in mast cells [53]. Nuclear import that
originating from within the nucleus would associate with occurs when neutrophils are adhered appears to increase
the inner nuclear membrane. LT synthetic capacity [77]. Nuclear import observed in
A further degree of complexity involves the fact that, eosinophils upon adherence appears to reduce capacity
within a given cell type, 5-LO can be rapidly (i.e., within for translocation and LT biosynthesis with activation
15–60 min) imported into the nucleus in response to [70]; by contrast, that observed with IL-5 treatment
in vivo or experimental conditions. Again, such import increased LT synthesis [79]. The cellular mechanisms for
is unassociated with enzyme activation and LT synth- such differences in ability to translocate are not known,
esis, and is to be distinguished from the calcium- but could reflect differences in distribution of critical
dependent association with NE that accompanies LT protein kinases, import proteins, or anchors.
synthesis. Import of 5-LO from the cytosol into the Second, compartmentalization could influence cata-
nucleus has been observed when peripheral blood lytic activity. This could occur on the basis of variations
neutrophils [77,78] or eosinophils [70] are recruited to in the local concentrations of cofactors or factors
inflammatory sites in vivo, or adhered to various required for optimal enzyme activity, such as calcium,
surfaces in vitro. It has also been described when hydroperoxides, ATP, or protein kinases. Alternatively,
eosinophils [79] or human in vitro culture-derived mast a cytosolic inhibitor of 5-LO has been described in
cells [80] are primed with interleukin (IL)-5. macrophages [86,87], and nuclear import, as observed in
alveolar macrophages, may represent a means of escape
from this metabolic brake.
9. Implications of 5-LO localization in resting and Third, compartmentalization could alter the degree to
activated cells which 5-LO couples with upstream or downstream
enzymes. As with 5-LO, both cytosolic and intranuclear
As mentioned above, the fact that LT biosynthesis pools of cPLA2 and LTA4 hydrolase have been
occurs at the NE suggests the likelihood that these described. Nuclear import of LTA4 hydrolase appears
mediators act at the nucleus. Although classical G to accompany that of 5-LO seen during differentiation
protein-coupled receptors for prostaglandin E2 have of alveolar macrophages, but not that observed with
been identified on the NE [81] in addition to their adherence of peripheral blood neutrophils [88]. The
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colocalization of both enzymes within the nucleus of the min D3 [92] and oxidized low-density lipoprotein [102].
alveolar macrophage may increase their degree of Cell specificity in such regulation has also been
metabolic coupling and thereby explain why this cell observed. For example, granulocyte macrophage col-
type is a poor donor of LTA4 for transcellular reactions, ony-stimulating factor increased both 5-LO [95] and
in contrast to the neutrophil [89]. FLAP [99] expression in neutrophils, but not in
Although conventional thinking has focused on the macrophages (in which it only increased expression of
metabolic function of 5-LO, it is important to consider cPLA2) [103]. Increased expression of 5-LO and/or
potential non-metabolic roles for this protein in the FLAP has been observed in various leukocyte popula-
nucleus. For example, 5-LO has been observed in a yeast tions in asthmatic subjects [104] or in allergic animal
two-hybrid assay to interact with other nuclear and non- models of asthma [105]; enhanced expression in these
nuclear proteins [90]. Moreover, even in resting cells not situations very likely reflects the influence of cytokines.
synthesizing LTs, 5-LO has been identified in the Finally, it is interesting to note that glucocorticoids
euchromatin region of the nucleus [49], the site of active augment expression of 5-LO and FLAP in various cell
gene transcription. These findings raise the possibility types [106–108], a fact that might serve to oppose the
that 5-LO might regulate transcriptional events in the broad anti-inflammatory actions of these agents.
nucleus. As would be expected, expression of 5-LO and FLAP
Although many questions remain about the biological can also be reduced below normal baseline levels. This
significance of 5-LO compartmentalization within the has been observed under conditions of deprivation of
cell, alterations in its locale, owing to either mutations in some of the inducing factors noted above. For example,
the protein or abnormalities in cellular machinery, dietary deficiency of vitamin D3 reduces FLAP expres-
represent a potential mechanism for disease. sion in macrophages [109]. This has also been observed
in the setting of deficiency of CD4-derived lymphokines,
due either to CD4 cell depletion using a monoclonal
10. Regulation of 5-LO and FLAP expression antibody in animals [110] or to naturally occurring CD4
depletion in patients with advanced HIV infection [111].
Although enzymes in the LT biosynthetic cascade In all of these instances, impaired LT synthetic capacity
both proximal (PLA2s) and distal (LT synthases) are accompanies the decline in steady-state levels of 5-LO
ubiquitously expressed among various cell types, sig- and/or FLAP. There are few examples of substances
nificant expression of 5-LO and FLAP is confined to that actively suppress expression of these proteins; this
bone-marrow-derived cells. These include neutrophils, has, however, been described for IL-4 and IL-13
eosinophils, monocytes/macrophages, mast cells, certain treatment of monocytes [96].
lymphocyte populations, and dendritic cells. Lower level Although precedent exists for post-transcriptional
expression has been variably documented in other cells regulatory mechanisms (e.g., [95,98]), transcriptional
and tissues, such as epithelial cells, fibroblasts, and in regulation appears to be the dominant means by which
various parts of the brain. In leukemic cell lines of expression of 5-LO and FLAP is controlled. Analysis of
granulocytic or monocytic origin, baseline expression of the promoter regions of the genes encoding these
both proteins is very low, but it can be enhanced by proteins has revealed some of the known cis elements
treatment with various agents that induce phenotypic that may mediate transcription factor interactions. For
differentiation (e.g., phorbol esters, retinoic acid, example, the 5-LO promoter contains consensus binding
DMSO, and vitamin D3) [35,91,92]. Tissue-specific sites for Spl, Sp3, Egr-1, Egr-2, NF-kB, GATA, Myb,
differences in expression levels of 5-LO and FLAP have and AP family members [112–114]. FLAP, by contrast,
also been described in mature macrophages. Thus, has a TATA box and potential AP-2 and glucocorticoid
human alveolar macrophages contain B8-fold more 5- receptor binding sites [115]. The functional significance
LO and B35-fold more FLAP protein (per unit of of some of these sites is unknown. In addition, these cis
cellular protein) than do their precursors, the peripheral elements do not necessarily explain many of the known
blood monocyte [34]. Increased alveolar macrophage regulatory influences discussed above. Examples include
expression of FLAP, in particular, appears to be related the restricted expression in myeloid cells, tissue-specific
to factors in the alveolar milieu [93]. Prominent among differences among macrophage populations, and the
such factors are cytokines. Indeed, cytokines and related effects of differentiating agents. Likewise, it is not
substances have been the most extensively investigated understood why 5-LO and FLAP often exhibit parallel
mediators capable of upregulating expression of 5-LO changes in expression despite substantial differences in
and/or FLAP. 5-LO expression has been reported to be their promoter regions. A genetic polymorphism in the
increased by colony-stimulating factors [94,95], IL-1 number of Spl/Egr-1 binding sites in the 5-LO promoter
[96], IL-3 [97], and transforming growth factor-b (TGF- that modestly reduces its transcriptional activity has
b) [98]. Increased FLAP expression has been induced by been described [116]. Although the functional impact of
colony-stimulating factors [94,99,100], IL-3 [101], vita- this mutation as regards LT biosynthetic capacity has
ARTICLE IN PRESS
106 M. Peters-Golden, T.G. Brock / Prostaglandins, Leukotrienes and Essential Fatty Acids 69 (2003) 99–109

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