Clinical Immunology (2006) 119, 229 — 240

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SHORT ANALYTICAL REVIEW

Prostaglandin E2 synthesis and secretion:

The role of PGE2 synthases
Jean Y. Park a,b,c,*,1, Michael H. Pillinger a,b,c,*,1, Steven B. Abramson a,b

a
The Division of Rheumatology, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA
b
The Department of Rheumatology, The Hospital for Joint Diseases, New York, NY 10003, USA
c
The Division of Rheumatology, Department of Medicine, New York Harbor Healthcare System New York Campus of the
Veterans Administration, New York, NY 10010, USA

Received 20 December 2005; accepted with revision 25 January 2006

Available online 15 March 2006

KEYWORDS Abstract Prostaglandin E2 (PGE2) is a principal mediator of inflammation in diseases such as

Prostaglandin; rheumatoid arthritis and osteoarthritis. Nonsteroidal anti-inflammatory medications (NSAIDs)
PGE2; and selective cyclooxygenase-2 (COX-2) inhibitors reduce PGE2 production to diminish the
COX; inflammation seen in these diseases, but have toxicities that may include both gastrointestinal
PGES; bleeding and prothrombotic tendencies. In cells, arachidonic acid is transformed into PGE2 via
mPGES-1; cyclooxygenase (COX) enzymes and terminal prostaglandin E synthases (PGES). Accumulating
mPGES-2; data suggest that the interaction of various enzymes in the PGE2 synthetic pathway is complex
cPGES; and tightly regulated. In this review, we summarize the synthesis and secretion of PGE2. In
Inflammation; particular, we focus on the three isoforms of the terminal PGES, and discuss the potential of
Arthritis targeting PGES as a more precise strategy for inhibiting PGE2 production.
D 2006 Elsevier Inc. All rights reserved.

Introduction
Prostaglandins (PGs) are members of the eicosanoid family
(oxygenated C20 fatty acids) and are produced by nearly all
cells within the body [01]. Prostaglandins are lipid mediators
* Corresponding author. Division of Rheumatology, Hospital for
Joint Diseases, NYU School of Medicine, 301 East 17th Street, New
York, NY 10003, USA. Fax: +212 951 3329.
E-mail address: jean.park@med.nyu.edu (J.Y. Park).
1
These authors contributed equally to the manuscript.
that are not stored by cells; rather, they are synthesized
from arachidonic acid via the actions of cyclooxygenase
(COX) enzymes, either constitutively or in response to cell-
specific trauma, stimuli, or signaling molecules [1—3]. The
most abundant prostanoid in the human body is PGE2 [4].
Depending upon context, PGE2 exerts homeostatic [1,5],
inflammatory [6], or in some cases anti-inflammatory [7]
effects. Inhibition of PGE2 synthesis has been an important
anti-inflammatory strategy for more than 100 years [8]. In
this article, we review the synthesis and cellular secretion
of PGE2, including the characteristics of the enzymes
involved in this process. Because PGE2 terminal synthases

1521-6616/$ — see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.clim.2006.01.016
230
represent a relatively recent discovery, and because PGE2
synthase inhibition has the potential to be a safer anti-
inflammatory strategy than either nonselective, or COX-2-
selective COX inhibition, we pay particular attention to PGE2
terminal synthases.
Phospholipase A2
Arachidonic acid is a polyunsaturated fatty acid derived
from dietary sources that resides in the cell membrane. It is
first liberated from cell membrane phospholipids via the
hydrolysis of sn-2 bond by phospholipase A2 enzymes (PLA2)
[3]. Arachidonic acid is then oxygenated by a COX to form
PGG2 and subsequently reduced by the same COX to yield
the unstable intermediate, PGH 2 [9]. The release of
arachidonic acid from cell membrane phospholipids deter-
mines the amount of eicosanoid production that occurs.
Therefore, PLA2 determines eicosanoid levels [10,11].
Fifteen genes are responsible for encoding the diverse
PLA2 enzymes that exist in mammals [12]. There are three
main classes of phospholipase A2 enzymes: (1) secreted PLA2
(sPLA2), (2) intracellular group VI calcium-independent PLA2
(GVI iPLA2), and (3) group IV cytosolic PLA2 (GIV cPLA2)
(Table 1).
Ten distinct mammalian sPLA2 enzymes of low molecular
weight (14—19 kDa) have been identified to date [12]. sPLA2
enzymes require calcium to hydrolyze the sn-2 position of
phospholipids and utilize a His—Asp dyad in its catalytic
mechanism. However, it has been shown that sPLA2 enzymes
have no strict fatty acid selectivity and induce arachidonic
acid release in a stimulus-independent manner [13]. iPLA2s
have a higher molecular weight (85 kDa) than sPLA2 and
retain full enzymatic activity even in the absence of
calcium. iPLA2 enzymes have long been thought of as
housekeeping enzymes as they play a role in phospholipid
acyl-chain remodeling [14]. Even though iPLA2 does not
absolutely require calcium for its enzymatic actions, it has
been reported to be upregulated by calcium or calcium-
dependent factors in some cell models [12]. Its exact
regulation is still under investigation. While each class of
PLA2 can release arachidonic acid from cell membrane
phospholipids, only cPLA2a appears to have as its primary
function the release of arachidonic acid for eicosanoid
production [12,15].
Table 1 PLA2 enzyme families
Enzyme Molecular Calcium requirement Number of
family weight for enzyme activation isoforms
cPLA2 85—110 kDa bAM Ca2+ 3 Ser/Asp dyad
sPLA2 14—19 kDa mM Ca2+ 10
iPLA2 85—90 kDa None 2 (several splice
variants exist)
J.Y. Park et al.
cPLA2a is found in most cells and tissues. Its high
specificity for the sn-2 position of arachidonic acid is
responsible for its specific role in the release of arachidonic
acid. The translocation of cPLA2a from the cytosol to the
Golgi, endoplasmic reticulum, and nuclear envelope is
upregulated in the presence of increased intracellular
calcium which binds the N-terminal C2 domain of cPLA2a
[16] (Figs. 1 and 2). Binding of the C2 domain with calcium
allows the catalytic domain of this enzyme interact with
arachidonic acid [17]. cPLA2a has also been shown to be
upregulated by the phosphorylation of Ser505, Ser727, or
Ser515 in its catalytic domain by mitogen-activating protein
kinases (MAPK), MAPK-interacting kinase, and calcium-
calmodulin kinase II [18—20]. Evaluation of cPLA2a-deficient
mice revealed decreased eicosanoid production and subse-
quent reduction in airway reactivity [21], defective female
reproductivity [21,22], and decreased platelet aggregation
[23]. In addition, cPLA2a knockout mice showed a decreased
incidence and severity of collagen-induced arthritis [24].
Therefore, cPLA2a is an integral component in the produc-
tion of PGE2, a known mediator of inflammation in arthritis.
Cyclooxygenases and the synthesis of PGE2
precursors
COX-1 and-2
In 1971, Sir John Vane reported that aspirin, salicylate, and
indomethacin inhibited prostaglandin synthesis in a dose-
dependent manner. It was initially presumed that a single
cyclooxygenase (COX) enzyme (also termed PG G/H synthase
(PGHS), PG endoperoxidase synthase, or PG synthase) was
responsible for producing prostaglandins, which in turn were
responsible for a variety of effects including pain, inflam-
mation, fever, platelet aggregation, and GI cytoprotection.
Vane proposed that all NSAIDs inhibit COX and therefore
decrease prostaglandin synthesis [25]. This COX enzyme was
purified from sheep seminal vesicles in 1976 and cloned in
1988 by several groups [26—29]. COX was found to be a
membrane-bound heme-containing glycoprotein. It was
most abundant in the endoplasmic reticulum of cells that
produce prostanoids. Its major actions are: (1) cyclization of
arachidonic acid in which a 15-hydroperoxy group is added
to form PGG2 (hence cyclooxygenase), and (2) reduction of
Catalytic site Tissue expression
Ubiquitous in most cells and tissues
(primary function of cPLA2a is to
release arachidonic acid for
eicosanoid production)
His/Asp dyad Human immune tissues
(spleen, tonsils, thymus, bone
marrow); GI organs
(colon, liver, pancreas)
Ser/His/Asp Generally ubiquitous in most cells
triad and tissues
Prostaglandin E2 synthesis and secretion 231

Figure 1 Coordinate production of PGE2 by cPLA2a, COX-1, and cPGES. (A) Unstimulated cell. Prior to cellular activation by
inflammatory stimuli, cPLA2a and cPGES are present in the cytoplasm of cells whereas COX-1 is constitutively expressed in the
nuclear envelope and endoplasmic reticulum. (B) Stimulated cell. Activation by inflammatory stimuli results in calcium influx,
leading to translocation of cPLA2a to the nuclear membrane where it enzymatically hydrolyzes membrane phospholipids to release
arachidonic acid. The enzymatic activity of COX-1 on arachidonic acid results in an unstable intermediate (PGG2) which is
subsequently converted by COX-1 to PGH2. Constitutively expressed cPGES may be stimulated to translocate from the cytosolic to the
nuclear fraction, where it preferentially coordinates with COX-1 to convert PGH2 to PGE2. PGE2 may exit the cell by simple diffusion,
or by active transport via the MRP4 transporter.

the nascent hydroperoxy group of PGG2 to form the
hydroxylated product, PGH2 [30] (Fig. 1). It has also been
noted that COX can produce prostaglandin E1 (PGE1) and
other monoenoic prostaglandins when dihomo-g-linolenic
acid (DHLA) is present as a substrate instead of arachidonic
acid [31].
By 1991, a second isoform of COX was discovered and
named COX-2. Though the original COX enzyme (COX-1) and
COX-2 are similar in structure and catalytic activity, they
were found to be genetically distinct as COX-1 mapped to
chromosome 9q32—q33.3 [32] and COX-2 to chromosome
1q25.2—q25.3 [33] (Table 2). COX-2, like COX-1, catalyzes
two sequential enzymatic reactions (oxygenation and re-
duction of arachidonic acid). The two functions of the COX
enzymes occur at distinct but interrelated sites. The
oxygenation step occurs in a channel within the COX

Figure 2 Coordinate production of PGE2 by cPLA2a, COX-2, and mPGES-1. (A) Unstimulated cell. As noted in Fig. 1A, cPLA2a is
constitutively present in the cytoplasm. In unstimulated cells, COX-2 and mPGES-1 are not expressed. (B), Stimulated cell.
Inflammatory stimulation results in calcium influx which leads to the translocation of cPLA2a from the cytosol to the nuclear
membrane where it enzymatically hydrolyzes membrane phospholipids to release arachidonic acid. Inflammatory stimuli also induce
the transcription and protein expression of both COX-2 and mPGES-1 at the nuclear membrane and endoplasmic reticulum. COX-2
transforms arachidonic acid to PGG2 which is subsequently converted to PGH2. mPGES-1 may then act on PGH2 to generate PGE2.
PGE2 may exit the cell by simple diffusion, or by active transport via the MRP4 transporter.
232 J.Y. Park et al.

molecule, while enzymatic reduction occurs at a heme-

stimulus-induced

cortex and aorta

Human cerebral
in many organs
containing site on the surface.

Not normally
Both COX-1 and COX-2 have molecular weights of 72 kDa

distribution

Ubiquitous

present in
cells, but
and share a 61% amino acid sequence homology. An

Tissue
important difference in the amino acid sequences of these
two molecules is located in their promoter regions. The
promoter region of the human COX-1 gene lacks a TATA or
CAAT box [34]. These features lead COX-1 to be a

Typically b2.5 AM;

[Arachidonic acid]

mainly exogenous
Typically N10 AM;

endogenous and
constitutive enzyme in the majority of cells (however,

Same as COX-1
COX-1 can be induced in some cell lines by the binding of

exogenous
Sp1 in its promoter region [34]). In contrast, COX-2 has
utilized

multiple transcriptional regulatory sequences in its promot-

er region, including a TATA box, an NF-IL6 motif, two AP-2
sites, three Sp1 sites, two NF-nB sites, a CRE motif, and an
E-box. COX-2 gene expression can be induced by multiple
arachidonic

cytokines and growth factors, via activation of transcrip-

0.54 AM tional regulatory proteins that act on these promoter sites
K m for

~5 AM

~5 AM

[35] (Fig. 2). Thus, COX-2 appears to be the primary COX

acid

controlling PGE2 synthesis in response to inflammation. COX

effects are widespread and extremely complex; however,
membrane N ER

studies in knockout mice for COX-1 vs. COX-2 reveal

sometimes overlapping, not altogether predictable roles
ER; nuclear

ER; nuclear
Subcellular

membrane

membrane

for these two enzymes (Table 3).

location

Nuclear

The overall crystal structures of COX-1 and COX-2 are

nearly identical; both the murine [36] and human [37] forms
of COX-2 are superimposable on the 3-dimensional structure
of COX-1. Each COX isozyme contains 3 major domains: (1)
molecular

an N-terminal epidermal growth factor (EGF) domain, (2) a

Protein

weight
72 kDa

72 kDa

65 kDa

helical membrane binding domain (MBD), and (3) a large

catalytic domain at the C-terminus [37]. The MBD contains 4
helices, which surround the opening where fatty acids (i.e.,
arachidonic acid) and NSAIDs enter the cyclooxygenase
elements in promoter region

TATA box, NF-IL6 motif, two

two NF-nB sites, CRE motif,

AP-2 sites, three Sp1 sites,
2 Sp1 motifs, 2 AP-2 sites,

active site. The upper portion of the catalytic domain at

Lacks TATA and CAAT box;
Transcriptional regulatory

the C-terminus makes up the cyclooxygenase active site that

binds these fatty acids and NSAIDs.
NF-IL6 motif, GATA

Although aspirin and NSAIDs inhibit the biosynthetic

Same as COX-1

activity of both COX enzymes, their actions are nonidenti-

cal. Aspirin irreversibly inhibits the cyclooxygenase active
site of PGHS, but does not affect the peroxidase portion of
the enzyme [38]. Interestingly, aspirin does not simply
E-box

inhibit COX-2, but rather diverts its enzymatic activity

toward the synthesis of precursors of lipoxin A4, a potent
antiinflammatory lipid [39]. In contrast, NSAIDs compete
Transcriptional

with arachidonic acid for the active sites of both COX

Constitutive

Constitutive
regulation

enzymes. While the active sites of COX-1 and COX-2 are

Inducible

similar in structure, they differ in size. The active site of

COX-2 is larger, owing to a replacement of isoleucine-434 of
COX-1 with valine-434 [40]. The identification of the
COX enzyme characteristics

difference between the active sites permitted the develop-

4—4.5 kb

ment of COX-2 specific inhibitors [41], which fit COX-2 but

5.2 kb
mRNA

are excluded from COX-1. Despite the difference in size of

3 kb
size

the active sites, the K m of COX-1 and COX-2 for arachidonic

acid remains similar [9].
Chromosome

1q25.2—25.3

Shitashige et al. showed that COX-1 and COX-2 prefer-

9q32—q33.3

entially utilize different pools of arachidonic acid to

Same as

synthesize prostaglandins. COX-2 acts on arachidonic acid

COX-1

when it is present in concentrations V2.5 AM. Endogenously

produced arachidonic acid is released at these low concen-
trations. COX-1 preferentially oxygenates arachidonic acid
Enzyme
Table 2

COX-1

COX-2

COX-3

over COX-2 when arachidonic acid is at concentrations N10

AM. These higher concentrations occur when arachidonic
acid is derived from an exogenous source, or released during
Prostaglandin E2 synthesis and secretion
Table 3 Phenotypic changes in COX knockout mice
Enzyme Phenotypic changes in KO mice
COX-1 Reduced platelet aggregation
Decreased AA-induced inflammation
Sensitive to radiation injury
Resistant to indomethacin-induced gastric
ulceration
COX-2 Defective ovulation, fertilization,
implantation, decidualization
Decreased brain injury induced by ischemia
Suppression of tumorigenesis
Renal nephropathy
Cardiac fibrosis
Peritonitis
Failure of patent ductus arteriosus closure
acute inflammation or cell injury [42]. The constitutive
presence of COX-1 may therefore be responsible for
generation of PGE2 during early phases of inflammation,
prior to COX-2 upregulation. Thus, the functions of COX-1
and 2 may be differentiated, not only by their cellular and
tissue distribution and responses to stimuli, but also by their
intrinsic kinetic properties.
Morita et al. compared the subcellular locations of COX-1
and COX-2. As detected immunocytofluorescence, COX-1 and
COX-2 both localize the endoplasmic reticulum and nuclear
envelope. While COX-1 was distributed equally between
these two compartments, COX-2 was found to be concen-
trated by a two-fold increase in the nuclear envelope
compared to the endoplasmic reticulum [43]. In contrast,
however, Spencer et al. employed immunoelectron micros-
copy as well as Western blotting of COX-1 and COX-2 in
subcellular fractions. They concluded that COX-1 and COX-2
are present in similar proportions in the endoplasmic
reticulum, as well as the inner and outer membranes of the
nuclear envelope. Therefore, the independent functions of
these two isozymes could not be attributed to a difference in
subcellular localization of COX-1 and COX-2 [44].
COX-3 and others: splice variants of COX-1
While acetaminophen is widely used as an analgesic and
antipyretic medication, its exact mechanism of action has
not been clarified. Whereas acetaminophen inhibits neither
COX-1 nor COX-2 in peripheral tissues, Flower et al.
postulated that acetaminophen inhibits an unknown COX
molecule in the brain [45]. Chandrasekharan et al. isolated
and cloned a splice variant of canine COX-1 in 2002. This
molecule, which was designated COX-3, differed from COX-1
in that a previously designated intron (intron 1) was
included in message and protein expression. The presence
of COX-3 mRNA transcript, with a size of approximately 5.2
kb, was subsequently confirmed in human cells; COX-3 was
in highest concentrations in the cerebral cortex and heart
tissue [46]. The regulation of COX-3 transcription appears to
be identical to that of COX-1 (D. Simmons, personal
communication).
COX-3 is similar to COX-1 and COX-2 in terms of structure
and enzymatic function. However, the retention of intron 1
in COX-3 seems to slow its enzymatic activity in comparison
233
to COX-1 and COX-2. Interestingly, therapeutic doses of
acetaminophen inhibit COX-3 in vitro. Thus, COX-3 repre-
sents a candidate target for acetaminophen’s mechanism of
action in the CNS [46]. Indeed, Botting et al. have
demonstrated that acetaminophen produces an analgesic
and antipyretic effect in mice by inhibiting COX-3 in the
brain and thereby decreasing levels of brain PGE2 [47]. Two
smaller splice variants of COX-1 were also isolated from
cerebral canine COX-1 and termed partial COX-1 (PCOX-1
proteins). One of these PCOX-1 proteins, PCOX-1a, also
contains intron 1 but lacks exons 5—8 of COX-1 mRNA. PCOX-
1a lacks the cyclooxygenase activity of COX-1 [46]. Splice
variants of COX-2 have also been reported, but have failed
to show enzymatic activity [48].
Prostaglandin E synthases
PGH2 itself does not play a significant role as an inflamma-
tory mediator. Rather, it serves as a substrate for various
specific enzymes that produce more stable prostanoids.
These prostanoids include PGE2, PGI2 (prostacyclin), PGD2,
PGF2A, and thromboxane A2 (TXA2), and the enzymes that
produce them from PGH2 are PGE2, PGI2, PGD2, PGF2A, and
TXA2 synthases, respectively. Because PG synthases typically
catalyze the generation of the final active products, they
are also referred to as terminal synthases [49]. The
mechanisms through which PGH2 is preferentially converted
to one product or another are not well understood. Even for
a single product, several different PG synthases may
interact with one or another COX to produce the target
prostaglandin. In the case of PGE2, studies to date suggest
the presence of at least three distinct PGE synthases (PGES)
(Table 4). Evidence suggests that COX/PG synthase interac-
tions may involve preferential functional coupling between
particular enzyme isoforms.
Microsomal prostaglandin E synthase-1 (mPGES-1)
The seminal vesicle is an organ known to contain high
concentrations of PGE2, so it is not surprising that there
were many initial attempts to isolate PGES from this organ.
Ogino et al. first reported the isolation of PGES from bovine
seminal vesicles in 1977. Moonen et al. similarly reported
the isolation of a bPGH-PGE isomerase enzymeQ from sheep
vesicular glands in 1982. Unfortunately, this enzyme could
not be functionally purified fully by either group, as it
proved to be unstable, losing activity after only 30 min even
at 258C. Both the degree of PGES activity, and the enzyme’s
stability, could be increased by the addition of glutathione.
Therefore, it was concluded that glutathione was a neces-
sary cofactor for PGES [50—52].
In 1999, Jakobsson et al. reported the cloning and
characterization of a human PGES. In particular, they
demonstrated that microsomal glutathione S-transferase 1-
like 1 (MGST1-L1), a member of the MAPEG (membrane-
associated proteins involved in eicosanoid and glutathione
metabolism) superfamily, had the ability to convert PGH2 to
PGE2. They were then able to isolate this PGES by virtue of
its 38% amino acid sequence homology with MGST1-L1. PGES
was expressed in a bacterial system (E. coli) and subsequent
membrane and cytosolic fractions were prepared. Western
234
Table 4 PGES isoforms
PGES isoform Chromosome mRNA Transcriptional Protein
size regulation molecular
weight
mPGES-1 9q34.4 14.8 kb Inducible 15—16 kDa
cPGES 12q13.13 1.9 kb Constitutive 26 kDa
mPGES-2 9q33—q34 2 kb Constitutive 33 kDa
a
Denotes best available data in tissues from normal patients.
blot data revealed a 15—16 kDa protein band in the
membrane fraction. The membrane fraction was isolated
and incubated with PGH2 and glutathione; high PGE synthase
activity (0.25 Amol/min/mg) was observed. The prostate,
testis, placenta, mammary gland, and bladder showed
intermediate levels of PGES, while high levels of PGES were
expressed in two cancer cell lines, HeLa cells and A549
pulmonary fibroblasts [53]. Enzymes in the MAPEG super-
family have two highly conserved amino acids, Arg100 and
Tyr117 [54]. Mutation of Arg100, but not of Tyr117, results in
the cessation of catalytic activity from PGES; therefore,
Arg100 is an essential amino acid for the enzymatic function
of PGES [55].
COX-2 and PGES protein expression are concordantly
induced by IL-1h, consistent with the hypothesis that PGES
and COX-2 are coregulated and that stimulated PGE2
synthesis may depend on upregulation of both of these
enzymes [53,56] (Fig. 2). To elucidate the relationship
between PGES and COX-2, the gene structure, localization,
and regulation of PGES were studied further by Forsberg et
al. [57]. The gene for PGES was cloned and isolated. PGES
was localized to chromosome 9q34.4 and was found to
comprise three exons that span approximately 14.8 kb. The
promoter region of PGES contains numerous transcription
factor binding sites, including two GC-rich boxes, an aryl
hydrocarbon response element (AHR), and two tandem
barbie boxes. Again, it was found that IL-1h upregulated
PGES and COX-2 mRNA expression as well as COX-2 promoter
activity [57]. Shortly after the membrane fraction-associat-
ed PGES was identified, another group reported the
purification and characterization of a cytosolic prostaglan-
din E synthase [58]. The membrane-bound form of PGES was
redesignated mPGES-1 (membrane-associated or microsom-
al PGES-1), and the cytosolic form of PGES became known as
cPGES (cytosolic PGES; see below).
Thoren et al. analyzed the structure of mPGES-1 by
electron crystallography. They reported a 10 angstrom
projection structure showing that mPGES-1 forms a trimer
in the crystal, similar to MGST1-L1. Hydrodynamic studies
J.Y. Park et al.
Subcellular Km for Tissue
location PGH2 distributiona
Nuclear 40 AM Prostate, testis,
membrane placenta,
mammary gland,
bladder (also
found in oncogenic
pulmonary
fibroblasts and
cervical epithelial
cells)
Cytoplasm Y Nuclear 14 AM Ubiquitous
membrane
Golgi Y Cytoplasm 28 AM Brain, heart,
skeletal muscle,
kidney, liver
done at 208C revealed that an mPGES-1/Triton X-100
detergent complex had a molecular weight of 215,000, of
which 53,700 represented the weight of mPGES-1, confirm-
ing the trimeric structure [59].
Cotransfection of human mPGES-1 and COX-2 into HEK293
cells and subsequent stimulation of these cells with either
A23187 (which invokes an immediate inflammatory response)
or IL-1h (delayed response) resulted in a significant increase
in PGE2 production. When cells were cotransfected with COX-
1 and mPGES-1 and stimulated with A23187 or IL-1h, a smaller
increase in PGE2 production resulted. It was concluded that
mPGES-1 preferentially couples with COX-2 activity to
increase the delayed production of PGE2 which is seen with
inflammation, fever, osteogenesis, and cancer. mPGES-1 also
couples with COX-2 activity to increase immediate PGE2
production when COX-2 is already present in cells. mPGES-1
does act in concert with COX-1, but typically when arachi-
donic acid concentrations are high and/or supplied exoge-
nously. Since mPGES-1, COX-1, and COX-2 are all localized to
the perinuclear region, the subcellular location of these
enzymes cannot account for the preferential interaction of
mPGES-1 with either COX enzyme [55].
Regulators of mPGES-1 expression
IL-1h and other cytokines stimulate the Erk and p38
members of the mitogen-activated protein kinase (MAPK)
family in OA chondrocytes, as well as in other cell types.
Members of the third MAPK family, Jnk, are absent from
both unstimulated and IL-1h-stimulated chondrocytes [60].
Pharmacologic inhibition of Erk (using PD98059) and
nonselective inhibition of p38a, h, and g isoforms (using
SB203580) abrogated IL-1h-stimulated expression of chon-
drocyte mPGES-1, as well as subsequent PGE2 production.
SC906, a specific inhibitor of the a isoform of p38, did not
affect mPGES-1 expression. Therefore, IL-1h regulates
chondrocyte mPGES-1 through ERK and a non-a p38
isoform [60].
Prostaglandin E2 synthesis and secretion
Early Growth Response-1 (Egr-1), an inducible zinc finger
protein that recognizes GC-rich sequences in DNA, binds a
GC box in the proximal promoter region, and subsequently
upregulates transcription of mPGES-1 [61]. Egr-1 expression
is inhibited by peroxisome proliferator-activated receptor g
(PPARg), a ligand-activated nuclear transcription factor that
is a member of the nuclear hormone receptor superfamily
[62—64]. In contrast, Egr-1 expression is positively regulated
by NF-nB, a proinflammatory transcription factor with
protean effects. Transfection of rat chondrocytes with the
NF-nB inhibitor protein InBaDN blocked both mPGES-1
expression and PGE2 synthesis. Since NF-nB additionally
upregulates COX-2 by an Egr-1-independent mechanism, NF-
nB activation may coordinately regulate the expression of
COX-2 and mPGES-1 [52,65].
15-deoxy-D12,14prostaglandin J2 (15d-PGJ2) is a prosta-
glandin with largely anti-inflammatory effects [62], that
suppresses pannus formation and inflammatory infiltrates in
rat adjuvant-induced arthritis [66]. 15d-PGJ2 acts in part by
engaging PPARg, suggesting a possible mechanism of 15d-
PDJ2 action on mPGES-1 expression via Egr-1[67]. Consistent
with this model, irreversible inhibition of PPARg by GW9662
reverses 15d-PGJ 2 inhibition of mPGES-1 expression.
[61,67,68]. 15d-PGJ2 may also regulate mPGES-1 expression
via inhibition of NF-nB [52,65]. Therefore, 15d-PGJ2 may
regulate mPGES-1 expression by a variety of mechanisms.
Cytosolic prostaglandin E synthase (cPGES)
The identification of a functional, cytosolic form of PGES
was first reported in 2000 by Tanioka et al. The molecular
mass of cPGES was determined to be 26 kDa on SDS-PAGE.
Peptide mapping of this 26 kDa protein revealed that cPGES
is identical to p23, a chaperone that binds the ATP-
dependent conformation of heat shock protein-90 (Hsp-
90). The K m of cPGES for PGH2 was determined to be 14 AM.
cPGES activity was upregulated in the presence of glutathi-
one, and inhibited by 1-chloro-2,4-dinitrobenzene, p-nitro-
phenylethyl bromide, and ethacrynic acid [58].
RNA blot analysis revealed that cPGES is most abundant
in the testis, but also is expressed in many other organs,
including heart, brain, and stomach. Immunostaining using
confocal microscopy indicates that cPGES is localized to the
cytosol. cPGES expression is largely constitutive and unaf-
fected by inflammatory stimuli. However, cPGES is upregu-
lated in the brains of rats exposed to lipopolysaccharide
[58].
The role of cPGES in the production of PGE2 in human
cells has been studied by cotransfecting the gene for cPGES,
together with the COX-1 or COX-2 gene into HEK293 cells.
PGE2 production increased significantly when cPGES was
cotransfected with COX-1 (at all arachidonic acid doses), but
was only minimally increased when cPGES was cotransfected
with COX-2. Therefore, cPGES may preferentially couple
with COX-1 to maintain PGE2 production required for
cellular homeostasis [58]. However, as mPGES-1-deficient
mice have been shown to develop normally, some authors
have questioned whether the coupling between COX-2/
mPGES-1 and COX-1/cPGES is exclusive. In porcine and rat
brains, for example, cPGES colocalizes with both COX-1 and
COX-2 in both brain parenchyma and cerebral microvascular
235
microsomes. The coordination of COX-2 with either mPGES-1
or cPGES in the brain seems to depend upon cell compart-
ment and chronologic age of species from which the brain
specimen was obtained [69]. Although PGE2 production by
COX-1 is generally considered to be constitutive, ongoing
studies in our own laboratory suggest that cPGES may
undergo a translocation from the cytosol to the nuclear
membrane to form an assemblage with COX-1 to upregulate
PGE2 production rapidly after cells are stimulated [70] (Fig.
1). Thus, although inflammatory cytokines tend not to affect
cPGES levels, it is possible that inflammation does affect
cPGES activity via effect on protein localization and possibly
activity.
Coregulators of cPGES activity
That cPGES is identical to p23 has suggested to some
investigators the possibility that heat shock protein 90
(Hsp90), which interacts with p23, may regulate cPGES
activity. Tanioka et al. reported that cPGES activity was
upregulated in vitro in the presence of Hsp90. Optimal
cPGES activation occurred when Hsp90 was added in a 1:1
ratio with cPGES. The induction of cPGES activity by Hsp90
was accompanied by a concomitant increase in cPGES/Hsp90
complex formation. Immediate increase in PGE2 production
as a consequence of cPGES activity was also seen in vivo
when rat fibroblast 3Y1 cells were stimulated with A23187 or
a physiological stimulus (bradykinin). Addition of Hsp90
inhibitors, geldanamycin and novobiocin, resulted in disso-
ciation of the cPGES/Hsp90 complex with a concurrent
decrease in stimulus-induced PGE2 generation [71].
Further evaluation of cPGES regulation has revealed that
casein kinase II (CK-II), a client protein for Hsp90, regulates
cPGES by a phosphorylation event. Phosphorylation of cPGES
occurs in parallel with increased cPGES activity and PGE2
production. In vitro, direct cPGES phosphorylation by CK-II
increases the affinity of cPGES for PGH2 (K m = 66.6 AM for
cPGES alone compared to K m = 35.7 AM for cPGES plus CK-II)
and so upregulates the enzymatic activity of cPGES. Indeed,
CK-II inhibitors decrease cPGES phosphorylation, stimulus-
induced cPGES activity, and PGE2 synthesis. In contrast,
dexamethasone and a p38 mitogen-activated protein kinase
(MAPK) inhibitor indirectly suppress cPGES activation,
apparently by inhibiting CK-II mediated phosphorylation
[72]. In vitro coincubation of Hsp90 CK-II and cPGES results
in maximal activity cPGES activity (K m = 14.9 AM for cPGES
plus CK-II with Hsp90), leading to the suggestion that Hsp90
may bmodulate the conformation of cPGES, and allow cPGES
to be phosphorylated further by CK-IIQ [72].
Microsomal prostaglandin E synthase-2 (mPGES-2)
Watanabe et al. first reported the existence of two separate
mPGES enzymes in rat tissues. They stated that glutathione
(GSH)-dependent mPGES activity localized mainly to the
genital organs and kidney. Since PGE2 also functions in the
heart, spleen, and uterus, they also searched these organs
for mPGES activity. mPGES was indeed localized to these
organs, but at a much lower level than that seen in the
genital organs and kidney. It was within the heart, spleen,
and uterus that this group discovered a novel GSH-indepen-
236
dent form of mPGES [73]. In 1999, Watanabe et al. purified
and identified a protein that possessed this GSH-indepen-
dent PGES activity from bovine heart microsomes [73]. Since
this membrane-associated PGES differed from that reported
by Jakobsson et al. in 1999, it was named microsomal
prostaglandin E synthase-2 (mPGES-2) [53]. mPGES-2 was
purified by Tanikawa et al. and reported to have a molecular
weight of 33 kDa. The gene for mPGES-2 localized to
chromosome 9q33—q34, where the genes for COX-1 and
mPGES-1 are also located. The V max and K m of mPGES-2 for
PGH2 respectively are 3.3 Amol/min mg and 28 AM. Catalysis
of PGH2 to PGE2 by mPGES-2 does not require the presence
of glutathione, as mPGES-1 does. However, mPGES-2 enzy-
matic activity is upregulated by the addition of dithiothrei-
tol (DTT-a double thiol reagent), and also is stimulated, but
to a lesser extent, by the addition of single SH reagents,
glutathione, and 2-mercaptoethanol [74,75].
Dot blot and Northern blot analyses confirmed that
mPGES-2 was present mainly in the heart and brain, but
not in the genital organs. The amino acid sequence of
mPGES-2 was conserved highly among monkey, bovine, and
human cDNA. As mPGES-1 and cPGES require glutathione for
their enzymatic activity, it was originally assumed that
mPGES-2 also would have an absolute requirement of GSH
for its activity. However, further evaluation of the amino
acid sequence of mPGES-2 did not reveal any homology with
any GSH S-transferase, so it was concluded that mPGES-2 did
not belong to this family of enzymes [75]. Detailed analysis
of the 377 amino acid sequence of mPGES-2 showed the
inclusion of a consensus region, 110Cys-x-x-Cys113, which is
also found in the active sites of thioredoxin and glutar-
edoxin. Mutation of 110Cys to serine abrogated the enzy-
matic activity of mPGES-2; mutation of 113Cys did not affect
mPGES-2 activity. Therefore, it was concluded that 110Cys
was critical for the activity of mPGES-2 [76]. mPGES-2 has
been complexed with the nonsteroidal anti-inflammatory
medication indomethacin and its subsequent crystal struc-
ture analyzed [75].
mPGES-2 is synthesized in the Golgi membrane, then
undergoes a proteolytic event where its N-terminal hydro-
phobic domain is removed. This truncated enzyme is
subsequently released into the cytoplasm. The exact
mechanism of these events still has yet to be elucidated.
mPGES-2 is a constitutively expressed enzyme in most cells
and tissues, but has been shown to be induced to high levels
in colorectal adenocarcinoma cells, suggesting that mPGES-
2 expression is also subject to upregulation under certain
conditions. While mPGES-2 can couple with both COX-1 and
COX-2 to produce PGE2 in response to acute and chronic
inflammation, respectively, it appears to demonstrate a
modest preference for coordination with COX-2 [77].
PGES in RA and OA
PGE2 is a key mediator of inflammation and pain in both RA
[78] and OA [79], so it is not surprising that the role of the
PGES in arthritis inflammation and/or progression is of great
interest. Synovial biopsy specimens from RA patients
undergoing arthroscopy or orthopedic surgery stain positive-
ly for intracellular mPGES-1 in RA synovial membranes
(pannus). mPGES-1 is particularly abundant in the lining
(intima) of RA synovial biopsy specimens. Within the
J.Y. Park et al.
subintima, mPGES-1 is present, but in fewer cells and with
less intense staining. cPGES is also found in RA synoviocyte
biopsy specimens, but fewer synovial lining cells stained for
cPGES compared to the number that stained positive for
mPGES-1 [80]. Immunohistochemical analyses of rheumatoid
arthritis (RA) synovial intimal cells reveal that mPGES-1 is
most abundant in patients with active RA. In contrast,
mPGES-2 is present in synovial lining cells from both active
and quiescent RA patients, suggesting that mPGES-2 parti-
cipates in homeostatic, rather than inflammatory PGE2
generation [77]. Westman et al. utilized immunohistologic
markers to determine which cells produce mPGES-1 in RA
synovial membranes, and reported that mPGES-1 is pro-
duced by synovial macrophages and fibroblasts, but not by T
lymphocytes or B lymphocytes. Endothelial cells have also
been shown to express mPGES-1 [80].
A small number of studies to date have examined the role
of mPGES-1 ex vivo, in cells and/or tissues derived from
arthritic joints. Korotkova et al. reported that synovial fluid
mononuclear cells (SFMC) from RA patients contained low
levels of mPGES-1 staining at baseline. Treatment with LPS
resulted in increases in mPGES-1 expression and PGE2
production, that were inhibited by either dexamethasone
or anti-TNF-a antibodies [81]. Stimulation with IL-1h con-
cordantly increases mPGES-1 and COX-2 mRNA and protein
expression, as well as PGE2 production, in RA synoviocytes
[82,83]. Therefore, mPGES-1 may be responsible for the
upregulation of PGE2 production in response to inflammatory
stimuli in RA synovial tissues. IL-1h-induced increases in
mPGES-1 and COX-2 were inhibited by dexamethasone [82].
Accumulating evidence also implicates mPGES-1 in the
pathogenesis of OA. mPGES-1 localizes to the superficial
layers of human OA cartilage, areas where OA damage first
appears [67]. IL-1h (implicated in OA pathogenesis [67]) also
was increased in these areas of cartilage, consistent with a
role for IL-1h in stimulating chondrocyte mPGES-1 and
therefore PGE2 production in OA [67]. Ex vivo, OA chon-
drocytes respond to both IL-1h and TNF-a with induction of
mPGES-1 and COX-2 mRNA and protein expression in a time-
and dose-dependent manner [60,84]. PGE2 secretion from
OA chondrocytes correlates well with mPGES-1 concentra-
tions following stimulation [60]. mPGES-1 and COX-2 were
both localized to the perinuclear region of IL-1h stimulated
chondrocytes [84], consistent with previously reported
localization in other cell types.
PGES in animal models of arthritis
Studies from animal models of arthritis support the impor-
tance of PGES, and particularly mPGES-1, in inflammatory
joint disease. Several groups have studied PGES expression
in the setting of adjuvant-induced arthritis (AIA). Kojima et
al. reported that rats whose paws are injected with
adjuvant demonstrated a significant increase in mPGES-1
mRNA in joint tissues, concordant with the anticipated paw
inflammation [84]. Claveau et al. similarly found that both
mPGES-1 and COX-2 mRNA and protein were upregulated at
early time points, followed by an increase in PGE2 in
adjuvant-injected paws. COX-1 and cPGES mRNA and
protein were only slightly induced, while mPGES-2 mRNA
was slightly decreased in these studies [85]. As mPGES-1 is a
member of the MAPEG family (which also includes 5-
Prostaglandin E2 synthesis and secretion
lipoxygenase-activating protein (FLAP) and LTC4 synthase),
these groups further assessed MK-866, a FLAP inhibitor, as a
potential inhibitor of mPGES-1 and modulator of AIA. MK-866
inhibited mPGES-1 in AIA with an approximate IC50 value of
2 AM [85,86].
As chronic inflammatory arthritis is accompanied by
central nervous system symptoms (i.e., hyperalgia, fever,
fatigue, malaise), PGE2 production as well as mPGES-1 and
COX-2 expression in the brains of AIA rats have also been
examined. mPGES-1 and COX-2 were expressed in IL-1h
receptor bearing endothelial cells along the blood—brain
barrier; mPGES-1 was also seen in the parenchyma of the
paraventricular hypothalamic nucleus. Thus, mPGES-1 may
contribute to CNS symptoms in chronic inflammation by
bupregulating the production of PGE2 along the blood brain
barrier and in the parenchyma of the hypothalamic para-
ventricular nucleusQ [87].
Another model of animal arthritis, carrageenan-induced
arthritis, showed an early, sequential induction of COX-2 and
mPGES-1 mRNA in the paws of rats. An increase in PGE2
production in the paw followed this induction. Induction of
COX-2 in the CNS of rats in carrageenan-induced arthritis
model led to the early increase of prostaglandins, throm-
boxane and prostacyclin; PGE2 was found to be increased
along with mPGES-1 in these studies. These data support
that mPGES-1 mediates increased peripheral and central
PGE2 production in inflammatory arthritis [88].
The role of mPGES-1 has been further studied in collagen-
induced arthritis (CIA), utilizing mPGES-1-deficient mice.
mPGES-1-deficient mice develop normally, and do not differ
from wild-type mice with respect to general appearance,
behavior, longevity, and hematologic parameters [89—91].
Nonetheless, CIA in mPGES-1-deficient mice was significantly
less severe and less common compared to wild-type mice.
Histopathological analysis of the mPGES-1 knockout mice
revealed reduced joint damage and a lack of proteoglycan
loss at articular surfaces following collagen-antibody injec-
tion [90,91]. In a nonarthritis model, mPGES-1-deficient
mice demonstrated less PGE2 production than wild-type mice
in response to treatment with LPS [89—91]. Macrophages are
probably among the cells dependent upon mPGES-1 for their
ability to produce PGE2, since peritoneal macrophages from
mPGES-1-deficient mice produce less PGE2 than wild-type
macrophages when incubated with arachidonic acid [91].
mPGES-1-deficient mice have also been used to study
pain, a key component of inflammation that is mediated
both peripherally and centrally via PGE2 [90,91]. Injection of
diluted acetic acid to wild-type mice produces an acute
painful reaction manifested as writhing. Writhing in mPGES-
1-deficient mice following acetic acid injection was de-
creased compared to the reaction seen in wild-type mice,
but was comparable to that seen in NSAID pretreated wild-
type mice [90,91]. Interestingly, mPGES-1 knockout mice
demonstrated reduced synthesis of prostaglandin I2, another
known mediator of pain, in response to LPS [90]. Since
mPGES-1 is not responsible for PGI2 synthesis, these data
suggest that PGE2 may indirectly regulate PGI2 production.
Prostaglandin E2 transport
As noted earlier, COX-1 and COX-2 are localized to the nuclear
envelope and endoplasmic reticulum, and it is primarily
237
within the endoplasmic reticulum that prostaglandins are
synthesized [35]. However, in order for prostaglandins to
exert their extracellular effects, they need to exit from the
cell in which they have been synthesized. Originally, the
prevailing notion was that newly synthesized prostaglandins
exited cells by passive diffusion. At physiologic pH, prosta-
glandins are anions; the electronegative cell interior favors
the diffusion of PG out of the cell [92]. However, it would
appear that the kinetics of the PG effects following stimu-
lated synthesis cannot be explained fully by this slow process
of simple diffusion [93]. Kanai et al. identified a prostaglan-
din transporter (PGT) in HeLa cells and Xenopus oocytes that
was associated with the rapid transport of PGE1, PGE2, and
PGF2a into the cell [94]. This group further studied this PGT
and found that an bobligatory, electrogenic anion exchangeQ
involving prostaglandin and lactate was responsible for the
carrier-mediated PG influx [93]. These observations, howev-
er, do not explain how prostaglandins might be transported
out of the cell after synthesis. Reid et al. studied the
multidrug resistance proteins (MRPs) and their relationship
to prostaglandin transport. They reported that MRP4 was
used specifically for the transport of PGE1 and PGE2 out of
cells. Interestingly, MRP4-dependent PGE efflux was inhib-
ited by both non-E prostaglandins and NSAIDs. Thus, NSAIDs
may reduce extracellular PGE2 levels, both by inhibiting PG
synthesis, and by inhibiting the secretion of PGE2 out of cells
from which they are synthesized [95].
Conclusion
PGE2, synthesized by many cells and tissues throughout the
body, has long been considered the principal prostaglandin
in acute inflammation, as well as in arthritic diseases such as
rheumatoid [78] and osteoarthritis [96]. Pharmacologic PGE2
blockade with aspirin and later NSAIDS has been a useful
antiinflammatory strategy for more than a century, but the
degree and severity of gastrotoxicity with chronic NSAID use
gradually became apparent. Excitement surrounded the
advent of the selective COX-2 inhibitors that match the
efficacy of traditional NSAIDs but, by sparing constitutively
active COX-1, are less toxic to the stomach. Unfortunately,
controversy has surrounded these selective COX-2 inhibitors
recently, owing to an apparent increase in the risk of
cardiovascular disease and stroke. Several investigators
have postulated that these procoagulant effects may result,
in whole or part, from the fact that selective COX-2
inhibitors reduce endothelial production of prostacyclin
(antithrombotic) while permitting platelet production of
thromboxane A2 (prothrombotic), thus disrupting the ho-
meostatic mechanisms of platelet regulation [97].
PGES have been of increasing interest since the contro-
versy surrounding selective COX-2 inhibitors emerged. As
described in detail throughout this review, mPGES-1 is
inducible, is increased in RA and OA cells, and is correlated
with PGE2 production. Theoretically, pharmacologic block-
ade of mPGES-1 (and perhaps other PGES) could decrease
proinflammatory PGE2 production while sparing other, non-E
prostanoids including prostacyclin and thromboxane A2.
Since no specific pharmacologic inhibitors of mPGES-1 or
other PGES are currently available, however, the benefits of
such a strategy remain theoretical. Further investigation into
the basic biology, regulation, and role of PGES may illumi-
238
nate the processes of inflammation, as well as the potential
utility of clinically targeting these important enzymes.
Acknowledgments
Dr. Park is supported by an NIH T32 training grant (AR007176)
to the Division of Rheumatology of New York University School
of Medicine (Steven B. Abramson, PI). Dr. Pillinger is
supported by a grant from the Arthritis Foundation New York
Chapter. The authors thank Nada Marjanovic for providing
experimental data and helpful suggestions and to Drs. Robert
Zurier and Daniel Simmons for helpful discussions.
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