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
Leukotrienes (LTs) are potent lipid mediators most
strongly implicated in asthma and allergic diseases. A
substantial capacity for the complete biosynthesis of
LTs from arachidonic acid (AA) is largely confined to
leukocytes. This biosynthetic pathway can be triggered
by a variety of stimuli, including antigens, microbes,
cytokines, complement, oxidants, immune complexes,
and toxins. These stimuli activate signal transduction
cascades that in turn activate LT-forming enzymes.
Briefly, a phospholipase A2 (PLA2), probably the
cytosolic (cPLA2) or a secretory (sPLA2) isoform,
initiates the pathway by catalyzing the hydrolysis of
AA from membrane phospholipids. The liberated free
AA is then oxygenated at C-5 and subsequently
dehydrated by the actions of the 5-lipoxygenase (5-LO)
enzyme, yielding the epoxide intermediate LTA4. The
efficient utilization of endogenous arachidonate by 5-LO
requires a helper protein termed ‘‘5-LO activating
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
be modified extracellularly by sequential amino acid
$
Supported by the National Institutes of Health through R01-
HL58897 and R01-AI43574.
*Corresponding author. Tel.: +1-734-763-9077; fax: +1-734-764-
4556.
E-mail address: petersm@umich.edu (M. Peters-Golden).
removal to yield LTD4 and then LTE4. LTs C4, D4 and
E4 are known as cysteinyl LTs (cysLTs) and comprise the
myotropic and edemagenic activities long known as
‘‘slow reacting substance of anaphylaxis.’’ LTB4 is best
recognized for its potent chemotactic and leukocyte-
activating effects.
5-LO is a member of the family of arachidonate
lipoxygenases, but differs in certain critical respects
from other members of the family. Its requirement for
interaction with FLAP in order to initiate the 5-
lipoxygenation of AA in intact cells is one such
distinction. Although steps in LT synthesis both
proximal and distal to the actions of 5-LO/FLAP are
subject to regulatory control, modulation of the expres-
sion and function of these two proteins is a critical
determinant of cellular capacity for LT biosynthesis.
Knowledge of these processes has expanded enormously
in the last decade. In this review, we provide an overview
of the molecular, biochemical, and cellular aspects of 5-
LO and FLAP expression and action.
2. Biochemical properties of 5-LO
All of the plant, fungal and animal lipoxygenases are
capable of inserting oxygen on the appropriate sub-
strate, which is typically a polyunsaturated fatty acid
containing a series of cis double bonds. Early studies of
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
 ARTICLE IN PRESS
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
oxygenation as well as a dehydration step that produces
LTA4. All lipoxygenases also contain a non-heme iron
atom that is involved in catalysis. The form of the iron
atom is critical to determining the catalytic potential of
the enzyme, with oxidation to the ferric (Fe3+) form
conferring activity. As a result, low levels of peroxides
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
activation by calcium. In the absence of calcium, 5-LO
has minimal catalytic activity [1–3]. The addition of
calcium stimulates both the oxygenation and dehydra-
tion reactions of 5-LO. Maximal activation is obtained
with 4–10 mM calcium [4]. Also, early biochemical
studies found that 5-LO activity from unstimulated
leukocytes was in the soluble fraction of crude cell
lysates. The addition of calcium to cell lysates led to
membrane association of 5-LO [5]. Taken together,
these results indicated that cell stimulation causing a
transient rise in intracellular calcium would induce 5-LO
to move from its soluble site to a membrane, leading to
catalysis. This ‘‘translocation’’ event is discussed in
depth below.
It was found that ATP, and other nucleotides to a
lesser extent, could significantly enhance the activity of
5-LO in cell lysates [6]. Likewise, LT synthetic capacity
in intact cells was reduced under conditions of ATP
depletion [7]. However, stimulation of 5-LO activity did
not involve hydrolysis of ATP or the generation of
inorganic phosphate as an energy source. Instead, ATP
directly bound to the 5-LO enzyme. Analysis of ATP
binding to 5-LO suggested two potential binding
sequences [8]. Interestingly, the enhancement of 5-LO
activity by ATP only occurs in the presence of calcium
[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
oxygen species can alter the oxidative state of the iron in
5-LO and in this way affect activity. It has also been
shown that nitric oxide (NO) can be a potent inhibitor
of LT synthesis [16,17]. NO can directly inhibit the
activity of recombinant 5-LO [17], presumably through
its effects on the critical iron atom. NO can also interact
with reactive oxygen species to generate peroxynitrite,
which can also inhibit 5-LO activity. Although nitrotyr-
osination of 5-LO has been observed at high concentra-
tions of peroxynitrite [18], it does not appear that this
mechanism accounts for inhibition seen at lower
concentrations.
3. Structural features of 5-LO
The cloning of the cDNA for human, rat, mouse and
hamster 5-LO indicated that these genes have been
highly conserved across species. Furthermore, compar-
ison of the primary amino acid sequences of the 5-LO
proteins from different species with other plant and
mammalian lipoxygenases revealed a high level of
similarity, particularly in their carboxy terminal resi-
dues. This similarity suggested that common structural
characteristics might underlie their common functions.
To date, four lipoxygenase proteins have been crystal-
lized and resolved structurally; three are plant lipox-
ygenases [19–21] and the fourth is rabbit reticulocyte 15-
lipoxygenase [22]. All have two domains: an amino
terminal b-barrel structure and a carboxy terminal
structure consisting predominantly of a-helices (Fig. 1).
The carboxy terminal region is highly conserved across
all plant and animal lipoxygenases. In contrast, the
amino terminal domain varies greatly, even between
mammalian lipoxygenases.
Within the carboxy terminal regions of all lipoxy-
genases are two clustered patterns of amino acids,
termed lipoxygenase iron-binding signatures, which
contain conserved histidine residues that serve to hold
iron inside the molecule. An isoleucine residue at the
Fig. 1. Secondary structure of rabbit reticulocyte 15-lipoxygenase. All
known lipoxygenases have two distinct domains, the b-barrel domain
and the catalytic domain. In 5-LO, the b-barrel can bind calcium and
direct movement to cellular membranes. The catalytic domain contains
the non-heme iron (Fe) essential to catalysis, as well as regulatory sites
for nuclear import, ATP binding and phosphorylation by MAPKAP
kinase 2.
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extreme carboxy terminus also appears to be necessary
for retaining the iron atom. The presence of iron
indicates that this larger segment of the 5-LO protein
contains the site of catalysis and, as a result, is
commonly referred to as the ‘‘catalytic domain’’.
Surprisingly, there is no consensus on how substrate
enters this catalytic domain and interacts with the metal
center of any lipoxygenase [23]. Presumably, specific
residues and pocket size would control the positioning
of the substrate relative to iron, determining the site of
oxygenation, as has been proposed [22]. However, the
available crystal structures from the different lipoxy-
genases suggest a variety of potential sites for substrate
access. It has been noted that each of these structures
is based on the ferrous, or inactive, form of the
enzyme and that conformational alterations associated
with oxidation of the active site might reveal the access
site [23].
Given the similarity of different lipoxygenase catalytic
domains, it has been possible to model the 5-LO
catalytic domain after the known rabbit reticulocyte
15-LO structure [24]. This model suggested that the
catalytic domain of 5-LO involves 15 a-helices organized
around a central helix that contains the iron-binding
patterns described above. Several additional structural
features are predicted to be found in the catalytic
domains of all lipoxygenases, including 5-LO. Most
notable among these is a region described as an SH3-
binding site [25]. This domain, which has been shown to
bind actin in vitro, is highly conserved across mamma-
lian 5-, 12- and 15-LOs. The relevance of this region to
lipoxygenase function in vivo is unknown.
There are some regions within the catalytic domain
that are unique to 5-LO. One such region is a putative
ATP-binding site near Trp201 [8]. This region is highly
conserved among 5-LOs from different species and is
poorly conserved in other lipoxygenases. Another such
region involves a four amino acid insertion in the
general lipoxygenase sequence, producing a site for
phosphorylation by MAPKAP kinase 2 at Ser271 [26].
Like the ATP-binding site, this site is found in 5-LO
sequences from different species but is absent from all
other lipoxygenases.
The amino terminal sequences of the different
lipoxygenases, although dissimilar in their primary
sequences, have consistently been found to form a b-
barrel structure. In other proteins such as cPLA2 and
protein kinase C, b-barrel structures can bind calcium
and mediate calcium-dependent membrane association.
Consistent with this, the amino terminus of 5-LO has
been shown both to bind calcium [27] and to be both
necessary and sufficient for membrane association [28].
This indicates that calcium, rising in concentration
during cell stimulation, would bind to the b-barrel
region of 5-LO and drive translocation, thus placing the
enzyme close to its substrate. It is of interest that one of
101
the ATP-binding sites on 5-LO, at TRP75, also happens
to be close to the calcium-binding site on the b-barrel
[24], providing a possible molecular basis for coopera-
tion between these two cofactors; this possibility has not
as yet been tested.
4. Inhibitors of 5-LO
There has been great interest in identifying inhibitors
of 5-LO activity, because of the importance of 5-LO-
derived LTs in the pathogenesis of asthma and other
inflammatory diseases. Several competitive inhibitors of
AA metabolism, including zileuton, AA-861, ABT-761,
ZD-2138 and L-739010, are very specific for 5-LO over
other lipoxygenases. It should be noted that these
inhibitors may have biochemical actions in addition to
their effects on 5-LO [18,29].
Some other commonly used inhibitors of 5-LO, such
as nordihydroguaiaretic acid (NDGA), are general
lipoxygenase inhibitors, blocking catalysis by altering
the redox state of the iron necessary for all lipoxy-
genases. Such inhibitors may reduce LT generation, but,
because of their lack of specificity of action, their
efficacy cannot be attributed solely to their effects on the
5-LO pathway.
A novel compound, MK-886, was found to be a very
effective inhibitor of LT synthesis [30] in intact
leukocytes but not in studies with leukocyte homo-
genates or purified 5-LO. This indicated that MK-886
was inhibiting 5-LO indirectly, perhaps by associating
with an essential cofactor. Subsequent experiments
isolated a membrane-associated protein that could bind
MK-886 [31], and because this protein appeared to be
essential in determining 5-LO activation, it was termed
5-lipoxygenase activating protein, or FLAP.
5. Properties and structural features of FLAP
The subsequent cloning of FLAP allowed the expres-
sion of FLAP, with or without 5-LO, in cells that
normally lack both enzymes. Results from these studies
indicated that FLAP was required for ionophore-
induced LT synthesis from endogenous substrate
[32,33]. Interestingly, FLAP can enhance, but is not
necessary for, the processing of exogenous AA by 5-LO.
In cells that express both FLAP and 5-LO, changes in
FLAP expression greatly influence cellular LT synthetic
capacity [34,35]. Taken together, these findings suggest
that FLAP may more accurately be viewed as a
5-lipoxygenase facilitating protein.
The mechanism of FLAP action is discussed below.
However, it has been demonstrated that FLAP is also an
AA-binding protein [36]. Furthermore, AA competes
with MK-886 for binding with FLAP [37]. These
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102 M. Peters-Golden, T.G. Brock / Prostaglandins, Leukotrienes and Essential Fatty Acids 69 (2003) 99–109
observations suggest that FLAP facilitates 5-LO action
by enhancing the delivery of AA to 5-LO, and that MK-
886 inhibits LT synthesis by directly interfering with this
process.
FLAP is now recognized as a member of a protein
superfamily designated as membrane-associated pro-
teins in eicosanoid and glutathione metabolism, or
MAPEG [38]. Hydropathy plots predicted from the
primary sequences of member proteins are similar. This
similarity in hydropathy likely reflects common patterns
within the primary sequences and should result in
similar positioning of the proteins in membranes.
Furthermore, these similarities might suggest additional
parallels in protein function.
Three proteins involved in eicosanoid metabolism
have been assigned to the MAPEG superfamily: FLAP,
LTC4 synthase and membrane-associated PGE synthase
(mPGES). The primary sequence of FLAP is approxi-
mately 50% similar to that of LTC4 synthase [39,40] and
less so (33% similarity with two gaps) to mPGES [41].
Significantly, all three proteins share the pattern ER-
x(3)-A-x(2)-N-x(2)-D/E that has been shown to be
important for binding MK-886 in FLAP [42]. Presum-
ably for this reason, MK-886 is able to inhibit all three
proteins [41].
There are points of difference between FLAP and
these other MAPEG members. No enzymatic activity
has been identified for FLAP, unlike other members of
the MAPEG superfamily. Also, FLAP activity is not
modulated by glutathione, unlike PGE synthase, which
is glutathione dependent, and LTC4 synthase, which
utilizes glutathione during catalysis. Finally, LTC4
synthase appears to function as a homodimer [43], while
FLAP does not.
6. Inhibitors of FLAP
As noted above, FLAP was first identified because of
its ability to bind MK-886, a potent inhibitor of LT
synthesis in intact cells (IC50=100 nM). The effective-
ness of MK-886 as a FLAP inhibitor results from its
capacity to fill the binding site for its ‘‘substrate’’ AA.
As noted above, similarities in the lipid-binding sites of
FLAP, LTC4 synthase and mPGES allows MK-886 to
inhibit each of these enzymes; differences between the
sites may explain differences in inhibitory concentra-
tions (IC50=11 mM MK-886 for LTC4 synthase and
3.2 mM for PGE synthase-1). It is important to note that
MK-886 has been shown to have effects independent of
its actions on FLAP, LTC4 synthase and mPGES. Thus,
MK-886 alters gene expression in cancer cells, ultimately
leading to cell death [44], a result that may reflect non-
specific, FLAP-independent actions. Other inhibitors
that also inhibit AA binding to FLAP have also been
developed (see, for example, reference [45]). To our
knowledge, no FLAP inhibitor is currently in clinical
development.
7. Intracellular sites of LT biosynthesis
The first decade of research into LT biosynthesis was
dominated by efforts to characterize the relevant
enzymes, their cofactors, and their cellular and tissue
distribution. Little was known about the intracellular
sites at which these enzymes functioned. A foundation
for our current understanding of such matters derives
from studies conducted in the late 1980s and early 1990s,
in which crude fractionation methods were used to study
the locale of 5-LO and FLAP in resting and activated
cells. This work demonstrated that FLAP was found in a
particulate fraction in both states [31]. By contrast, 5-LO
was found in the soluble fraction of resting leukocytes,
but rapidly (i.e., within 0.5–15 min) redistributed in a
calcium-dependent manner to a particulate fraction
upon activation [5]. cPLA2 exhibited properties similar
to 5-LO [46]. From these early studies, two assumptions
were generally made. The first was that cell activation
colocalized cPLA2 and 5-LO with FLAP at the same
membrane site. The second was that this site was the
plasma membrane—a logical assumption inasmuch as
LTs are known to be predominantly secreted mediators.
However, the crude cellular disruption and fractionation
methods employed in these early studies were not
capable of rigorously supporting such assumptions.
In the early to mid-1990s, these issues were more
definitively addressed by several laboratories using more
careful disruption/fractionation as well as immunomi-
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
cPLA2 and FLAP in activated cells. Surprisingly,
however, the site at which these proteins were found
upon cell activation proved to be not the plasma
membrane, but the nuclear envelope (NE) and contig-
uous perinuclear endoplasmic reticulum (PER) [47–54].
Functional studies demonstrated in parallel that most
AA hydrolyzed by cPLA2 upon cell activation derived
from phospholipids of the NE/PER [52]. Moreover, the
enzyme LTC4 synthase has also been localized to this
same site [40,55]. Therefore, all four key enzymes
necessary for synthesis of the proximate cysLT, LTC4,
are assembled at the NE/PER.
In addition to a calcium-dependent mechanism for
5-LO translocation, a calcium-independent mechanism
that involves phosphorylation of 5-LO by mitogen-
activated protein kinases, in response to agents such
as cellular stress or unsaturated fatty acids, has recently
been described [56]. Although available data
are consistent with the possibility that this latter
pathway likewise results in association with the NE/
 ARTICLE IN PRESS
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PER, this remains to be confirmed by immunomicro-
scopic techniques.
FLAP was originally thought to be a membrane
docking protein for 5-LO [57], and selective localization
of FLAP to the NE/PER could have provided an
explanation for selective translocation of 5-LO to this
site. However, initial attempts failed to reveal evidence
of a direct interaction between FLAP and 5-LO.
Subsequent studies demonstrated membrane association
of 5-LO in transfected cells even in the absence of
FLAP, although LT synthesis could not be initiated [58].
Finally, immunofluorescence [59] and immunoelectron
microscopic [49] analysis revealed that the FLAP
inhibitor MK-886 caused a near-complete inhibition of
LT synthesis without any effect on NE/PER transloca-
tion of 5-LO. These data cast doubt on its role as a
docking protein, favoring the alternative role in
substrate presentation discussed above. An alternative
explanation for selective targeting of 5-LO (and cPLA2)
to the NE might relate to the preferential capacity of the
C2 domains of both proteins to associate with phos-
phatidylcholine, together with the fact that the NE is
enriched in phosphatidylcholine, as compared to the
inner plasma membrane [60]. The molecular mechan-
isms responsible for targeting the integral membrane
proteins FLAP and LTC4 synthase selectively to this
intracellular membrane are not known.
Agents such as lipopolysaccharide and phorbol esters
are not themselves capable of triggering LT synthesis, but
are well known to ‘‘prime’’ cells for enhanced LT
production in response to a triggering agonist. It has
been demonstrated that pretreatment with each of these
agents enhances the translocation of 5-LO to the NE/PER
in response to triggering agonists [61,62]. Likewise,
substances that increase intracellular levels of cyclic
AMP, including prostaglandin E2, are recognized to
inhibit cellular LT biosynthesis. This inhibitory action
has recently been linked to impaired translocation of 5-
LO to the NE/PER [10]. Other studies have demonstrated
that inhibitors of p38 mitogen-activated protein kinase
[63] and tyrosine kinases [14] including Janus kinase 3 [64]
inhibit LT biosynthesis in parallel with inhibition of 5-LO
translocation, although the precise role of 5-LO phoshor-
ylation in translocation has not been defined. These
studies further underscore the importance of translocation
to the NE for 5-LO activation.
Prolonged stimulation of LT biosynthesis in macro-
phages and mast cells results in persistent NE/PER
association and inactivation of 5-LO. However, when
stimulation is transient, membrane association is rever-
sible. Under these circumstances, re-stimulation can
result in another round of rapid translocation and LT
synthesis [59]. This capacity for rapid and repeatable
responses distinguishes LT generation from that of
other classes of inflammatory mediators, and may be an
important property for long-lived cells.
103
An alternative to the NE/PER as a site for assembly
of enzymes in the LT biosynthetic cascade has been
suggested. These enzymes have also been identified by
immunomicroscopic techniques in lipid bodies, non-
membrane-delimited cytoplasmic droplets which can
increase in number in response to inflammatory stimuli
(see accompanying article by Bandeira-Melo and Well-
er). On the basis of a novel immunolocalization
technique for detecting intracellular LTC4, it has been
suggested that lipid bodies are the site of eosinophil
LTC4 formation in response to chemokines and
cytokines, whereas the NE/PER is relevant for stimuli
that raise intracellular calcium [65,66]. NE/PER accu-
mulation of 5-LO has also been observed in macro-
phages stimulated with zymosan or formyl peptide and
in mast cells stimulated with immune complexes [59].
Moreover, NE localization has been demonstrated in
situ in alveolar macrophages from lung biopsy speci-
mens obtained from patients with idiopathic pulmonary
fibrosis. In the latter circumstance, NE immunostaining
correlated with increased LT levels in lung tissue as well
as with overproduction of LTs by alveolar macrophages
ex vivo [67]. In the absence of a discrete membrane, it is
not clear how the enzyme assembly necessary for
activation of cPLA2 or 5-LO might be mediated in an
organelle such as the lipid body. Nonetheless, it is
certainly plausible that sites other than the NE/PER
may contribute to LT synthesis, and that these alter-
native sites may assume prominence under certain
situations in certain cell types.
A macromolecular complex of sequential enzymes in
a metabolic pathway has been termed a ‘‘metabolon’’
[68]. Such a complex would be expected to result in a
cellular microenvironment that allows efficient channel-
ing of metabolic intermediates. It is still not known if the
components of the LT biosynthetic metabolon directly
associate with each other, or if their proximity merely
increases the likelihood of metabolic coupling. We also
know little about the capacity of intermediates such as
AA or LTA4 to cross relevant membranes; however,
their capacity to do so would seem to be a relevant
variable in understanding the significance of compart-
mentalization data for LT-forming proteins. In any
case, the role of the NE/PER as a site for this metabolon
is entirely counter-intuitive, given that LTs are known to
be secreted extracellularly. This fact strongly suggests
that LTs subserve important functions within or near
the cell nucleus (see below).
8. Regulation of 5-LO compartmentalization in resting
cells
As noted above, the NE/PER is both the site at which
FLAP is localized and the site to which 5-LO
translocates in activated leukocytes. However, the
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intracellular compartmentalization of 5-LO in resting
cells has proven to be a complex matter. First, it varies
in a cell-specific manner: 5-LO is largely cytosolic in
resting peritoneal macrophages [47] as well as mono-
cytes [48], neutrophils [69], and eosinophils [70] isolated
from peripheral blood. By contrast, mast cells [69,71]
and alveolar macrophages [49,53] contain both cytosolic
and intranuclear pools of enzyme. The intranuclear 5-
LO is strictly soluble in the alveolar macrophage,
whereas soluble as well as bound pools exist within the
nucleus of the mast cell [53]. The presence of 5-LO
within the nucleus is the consequence of nuclear import
through the nuclear pore complex. Import of a protein
of the size of 5-LO requires a nuclear localization signal
(NLS), and several candidate motifs have been proposed
[72–74]. It now seems likely that 5-LO contains at least
two distinct NLSs [74] as well as a nuclear export signal
[75] that together determine compartmentalization. The
fact that alveolar macrophages appear to be unique
among cells of the macrophage lineage in the fact that
they contain an intranuclear pool of 5-LO suggests that
the process of nuclear import is triggered by factors
unique to the pulmonary alveolar milieu [76]. It is
important to recognize that 5-LO localization within the
nucleus is not itself indicative of activation of LT
biosynthesis. Regardless of its site of origin in a resting
cell, LT biosynthesis generally appears to require
translocation of 5-LO to the NE/PER. It is assumed
that 5-LO originating from the cytosol would associate
with the outer membrane of the NE/PER, while enzyme
originating from within the nucleus would associate with
the inner nuclear membrane.
A further degree of complexity involves the fact that,
within a given cell type, 5-LO can be rapidly (i.e., within
15–60 min) imported into the nucleus in response to
in vivo or experimental conditions. Again, such import
is unassociated with enzyme activation and LT synth-
esis, and is to be distinguished from the calcium-
dependent association with NE that accompanies LT
synthesis. Import of 5-LO from the cytosol into the
nucleus has been observed when peripheral blood
neutrophils [77,78] or eosinophils [70] are recruited to
inflammatory sites in vivo, or adhered to various
surfaces in vitro. It has also been described when
eosinophils [79] or human in vitro culture-derived mast
cells [80] are primed with interleukin (IL)-5.
9. Implications of 5-LO localization in resting and
activated cells
As mentioned above, the fact that LT biosynthesis
occurs at the NE suggests the likelihood that these
mediators act at the nucleus. Although classical G
protein-coupled receptors for prostaglandin E2 have
been identified on the NE [81] in addition to their
expected locale on the plasma membrane, such has not
as yet been observed for LT receptors. LTB4 has been
reported by some investigators [82] but not all [83] to be
a ligand for the intranuclear receptor/transcription
factor, peroxisome proliferator activated receptor-a; in
any case, it seems entirely likely that other intranuclear
‘‘receptors’’ for LTs will be recognized. Because LTA4 is
a highly reactive electrophile, it might react readily with
diverse intranuclear targets with resulting consequences
for nuclear function; the possibility that DNA might be
such a target is suggested by the demonstration that it
can interact with nucleosides and nucleotides in vitro
[84]. It should also be recognized that reactive oxygen
species are byproducts of the 5-LO reaction [85], and
their generation near or within the nucleus could
influence redox tone and thereby modulate, for example,
the activity of oxidant-sensitive transcription factors
such as nuclear factor (NF)-kB. Finally, because free
AA might itself exert intranuclear effects, 5-LO action
could be important by virtue of its ability to consume
substrate.
The resting locale of 5-LO may influence LT
biosynthetic capacity in several ways. First, it has been
observed that within a given cell type, different 5-LO
pools exhibit different capacities for translocation to the
NE upon stimulation. Thus, while the alveolar macro-
phage contains both intranuclear and cytosolic pools, it
appears that only the intranuclear pool is capable of
translocating; by contrast, both pools are capable of
translocating in mast cells [53]. Nuclear import that
occurs when neutrophils are adhered appears to increase
LT synthetic capacity [77]. Nuclear import observed in
eosinophils upon adherence appears to reduce capacity
for translocation and LT biosynthesis with activation
[70]; by contrast, that observed with IL-5 treatment
increased LT synthesis [79]. The cellular mechanisms for
such differences in ability to translocate are not known,
but could reflect differences in distribution of critical
protein kinases, import proteins, or anchors.
Second, compartmentalization could influence cata-
lytic activity. This could occur on the basis of variations
in the local concentrations of cofactors or factors
required for optimal enzyme activity, such as calcium,
hydroperoxides, ATP, or protein kinases. Alternatively,
a cytosolic inhibitor of 5-LO has been described in
macrophages [86,87], and nuclear import, as observed in
alveolar macrophages, may represent a means of escape
from this metabolic brake.
Third, compartmentalization could alter the degree to
which 5-LO couples with upstream or downstream
enzymes. As with 5-LO, both cytosolic and intranuclear
pools of cPLA2 and LTA4 hydrolase have been
described. Nuclear import of LTA4 hydrolase appears
to accompany that of 5-LO seen during differentiation
of alveolar macrophages, but not that observed with
adherence of peripheral blood neutrophils [88]. The
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colocalization of both enzymes within the nucleus of the
alveolar macrophage may increase their degree of
metabolic coupling and thereby explain why this cell
type is a poor donor of LTA4 for transcellular reactions,
in contrast to the neutrophil [89].
Although conventional thinking has focused on the
metabolic function of 5-LO, it is important to consider
potential non-metabolic roles for this protein in the
nucleus. For example, 5-LO has been observed in a yeast
two-hybrid assay to interact with other nuclear and non-
nuclear proteins [90]. Moreover, even in resting cells not
synthesizing LTs, 5-LO has been identified in the
euchromatin region of the nucleus [49], the site of active
gene transcription. These findings raise the possibility
that 5-LO might regulate transcriptional events in the
nucleus.
Although many questions remain about the biological
significance of 5-LO compartmentalization within the
cell, alterations in its locale, owing to either mutations in
the protein or abnormalities in cellular machinery,
represent a potential mechanism for disease.
10. Regulation of 5-LO and FLAP expression
Although enzymes in the LT biosynthetic cascade
both proximal (PLA2s) and distal (LT synthases) are
ubiquitously expressed among various cell types, sig-
nificant expression of 5-LO and FLAP is confined to
bone-marrow-derived cells. These include neutrophils,
eosinophils, monocytes/macrophages, mast cells, certain
lymphocyte populations, and dendritic cells. Lower level
expression has been variably documented in other cells
and tissues, such as epithelial cells, fibroblasts, and in
various parts of the brain. In leukemic cell lines of
granulocytic or monocytic origin, baseline expression of
both proteins is very low, but it can be enhanced by
treatment with various agents that induce phenotypic
differentiation (e.g., phorbol esters, retinoic acid,
DMSO, and vitamin D3) [35,91,92]. Tissue-specific
differences in expression levels of 5-LO and FLAP have
also been described in mature macrophages. Thus,
human alveolar macrophages contain B8-fold more 5-
LO and B35-fold more FLAP protein (per unit of
cellular protein) than do their precursors, the peripheral
blood monocyte [34]. Increased alveolar macrophage
expression of FLAP, in particular, appears to be related
to factors in the alveolar milieu [93]. Prominent among
such factors are cytokines. Indeed, cytokines and related
substances have been the most extensively investigated
mediators capable of upregulating expression of 5-LO
and/or FLAP. 5-LO expression has been reported to be
increased by colony-stimulating factors [94,95], IL-1
[96], IL-3 [97], and transforming growth factor-b (TGF-
b) [98]. Increased FLAP expression has been induced by
colony-stimulating factors [94,99,100], IL-3 [101], vita-
105
min D3 [92] and oxidized low-density lipoprotein [102].
Cell specificity in such regulation has also been
observed. For example, granulocyte macrophage col-
ony-stimulating factor increased both 5-LO [95] and
FLAP [99] expression in neutrophils, but not in
macrophages (in which it only increased expression of
cPLA2) [103]. Increased expression of 5-LO and/or
FLAP has been observed in various leukocyte popula-
tions in asthmatic subjects [104] or in allergic animal
models of asthma [105]; enhanced expression in these
situations very likely reflects the influence of cytokines.
Finally, it is interesting to note that glucocorticoids
augment expression of 5-LO and FLAP in various cell
types [106–108], a fact that might serve to oppose the
broad anti-inflammatory actions of these agents.
As would be expected, expression of 5-LO and FLAP
can also be reduced below normal baseline levels. This
has been observed under conditions of deprivation of
some of the inducing factors noted above. For example,
dietary deficiency of vitamin D3 reduces FLAP expres-
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
antibody in animals [110] or to naturally occurring CD4
depletion in patients with advanced HIV infection [111].
In all of these instances, impaired LT synthetic capacity
accompanies the decline in steady-state levels of 5-LO
and/or FLAP. There are few examples of substances
that actively suppress expression of these proteins; this
has, however, been described for IL-4 and IL-13
treatment of monocytes [96].
Although precedent exists for post-transcriptional
regulatory mechanisms (e.g., [95,98]), transcriptional
regulation appears to be the dominant means by which
expression of 5-LO and FLAP is controlled. Analysis of
the promoter regions of the genes encoding these
proteins has revealed some of the known cis elements
that may mediate transcription factor interactions. For
example, the 5-LO promoter contains consensus binding
sites for Spl, Sp3, Egr-1, Egr-2, NF-kB, GATA, Myb,
and AP family members [112–114]. FLAP, by contrast,
has a TATA box and potential AP-2 and glucocorticoid
receptor binding sites [115]. The functional significance
of some of these sites is unknown. In addition, these cis
elements do not necessarily explain many of the known
regulatory influences discussed above. Examples include
the restricted expression in myeloid cells, tissue-specific
differences among macrophage populations, and the
effects of differentiating agents. Likewise, it is not
understood why 5-LO and FLAP often exhibit parallel
changes in expression despite substantial differences in
their promoter regions. A genetic polymorphism in the
number of Spl/Egr-1 binding sites in the 5-LO promoter
that modestly reduces its transcriptional activity has
been described [116]. Although the functional impact of
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
not been established, it has been associated with a
reduced likelihood of therapeutic response to a 5-LO
inhibitor among asthmatic patients [117]. Likewise, a
FLAP promoter polymorphism that is somewhat more
frequent in asthmatics than in control subjects has been
reported [118], but its functional significance is also
uncertain. A more complete understanding of the role of
genetic variation in expression of these proteins under
different circumstances will be an important objective of
future studies.
11. Concluding remarks
Although asthma represents the historical roots of LT
biology, our appreciation of the biological significance
of these lipid mediators has expanded considerably and
will undoubtedly continue to do so. 5-LO and FLAP
carry out the first committed steps in LT synthesis from
AA. Therefore, understanding how these proteins are
expressed, how they work, and how they interact with
each other as well as with upstream and downstream
metabolon components will remain a central focus for
those seeking to comprehend LT generation and its
regulation in health and disease. Over the last quarter
century, a rudimentary appreciation of the biochemis-
try, pharmacology, and molecular and cellular biology
of 5-LO/FLAP has been gained. However, many
uncertainties remain. Only by continued integration of
all of these perspectives and by incorporating insights
from genetics will the desired progress be achieved. It
will also be essential that therapeutic opportunities
arising from a better understanding of these proteins be
capitalized upon. The last decade has witnessed many
surprises, particularly related to the cell biology of
5-LO. Further surprises almost certainly await us.
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