12,14-prostaglandin J2 and a peroxisome proliferator-activated receptor 
agonist on macrophage activation
,
Departments of
* Microbiology and Immunology,
Medicine,
Pharmacology, and
¶ Physiology and Neuroscience, Medical University of South Carolina, Charleston; and
Department of Pharmacology and Physiology, University of South Carolina School of Medicine, Columbia
Correspondence: James A. Cook, Ph.D., Professor of Physiology/Neuroscience, Medical University of South Carolina, 167 Ashley Ave. Suite 607, Charleston, SC 29425. E-mail: cookja{at}musc.edu
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12,14-prostaglandin J2
(15-PGJ2) appears to possess anti-inflammatory properties.
Unlike other prostaglandins, it has no known plasma membrane receptor.
Its effects have been thought to occur through activation of the
nuclear peroxisome proliferator-activated receptor 
(PPAR), but
15-PGJ2 may exhibit effects independent of PPAR. We
hypothesized that 15-PGJ2 modulates macrophage (M
)
mediator production by acting on cell signaling proteins upstream of
PPAR. The effects of 15-PGJ2 on bacterial endotoxin
LPS-induced rat peritoneal M mediator production were compared with
those of a specific PPAR agonist, BRL 49653 (BRL), and to the
eicosanoids prostaglandin D2 (PGD2) and
cicaprost (CICA, a prostacyclin analogue). 15-PGJ2
inhibited LPS-induced production of NO, TNF-
, and thromboxane
B2 (TxB2). Equimolar concentrations of
PGD2 and CICA significantly inhibited LPS-stimulated
TNF- but not NO, and CICA increased TxB2 production. BRL
inhibited LPS-induced NO, but augmented LPS-induced TNF- and
TxB2. 15-PGJ2 also inhibited degradation of
LPS-induced I
B and phosphoactivation of ERK 1/2, but BRL had no
significant effect on either protein. The cyclopentenone ring
2-cyclopenten-1-one also inhibited LPS-induced ERK 1/2 activation;
however, neither 15-PGJ2 nor the cyclopentenone inhibited
PMA-induced ERK 1/2 activation. Inhibition of LPS-stimulated mediator
production by 15-PGJ2 differed from inhibition by
PGD2, CICA, and BRL. The ability of 15-PGJ2 to
inhibit LPS-induced M mediator production and cell signaling may
occur in part through reactivity of its cyclopentenone ring.
Key Words: BRL 49653 • ERK 1/2 • IB • NO • TNF- • TxB2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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s) are exposed to endotoxin, the
lipopolysaccharide (LPS) component of the cell walls of gram-negative
bacteria, they produce increased levels of proinflammatory mediators
including tumor necrosis factor alpha (TNF-) [1
],
nitric oxide (NO) [2
], and thromboxane B2
(TxB2) [3
]. This overproduction of
proinflammatory mediators leads to the deleterious effects associated
with endotoxin, including fever, hypotension, multiple organ failure,
and death [4
, 5
]. Production of
proinflammatory mediators in Ms stimulated with LPS is preceded by
an increase in activation of certain cell signaling proteins. Evidence
has accumulated that LPS activates a kinase cascade analogous to that
used by interleukin (IL)-1, including phosphorylation of the
inhibitor of B protein (IB) [6
,
7
]. Phosphorylation and subsequent degradation of
IB allows the release and nuclear translocation of nuclear
factor-B (NF-B). LPS also activates multiple mitogen-activated protein kinase cascades [8
], including
the extracellular signal-regulated receptor kinases 1 and 2 (ERK 1/2)
[9
].
Some prostaglandins appear to possess anti-inflammatory properties in
response to LPS and other stimuli. For example, prostacyclin
(PGI2), which binds to the plasma membrane IP receptor
[10
], inhibits LPS-induced TNF- production in Ms
through increases in intracellular levels of cyclic AMP
[11
]. Prostaglandin D2 (PGD2),
which binds to the plasma membrane DP receptor [12
],
inhibits NO production in vascular smooth muscle cells
[13
]. Studies involving cyclopentenone prostaglandins of
the prostaglandin J2 (PGJ2) series have
demonstrated that PGJ2 and its metabolites
inhibit proinflammatory-mediator production in response to many stimuli
[14
, 15
]. These prostaglandins are produced
when PGD2 undergoes spontaneous dehydration to form
PGJ2. Additional metabolites of PGJ2 are
12-PGJ2 (12-PGJ2) and
15-deoxy-12,14-PGJ2
(15-PGJ2) [16
]. A unique characteristic of
the PGJ2 family is that, unlike other prostaglandins, these
eicosanoids have no known plasma membrane receptors and are thought to
exert anti-inflammatory effects by activating a nuclear receptor called
the peroxisome proliferator-activated receptor (PPAR).
PPAR is a member of the superfamily of nuclear receptors for
steroid, thyroid, and retinoid hormones that require ligand binding for
activation [17
]. Upon activation with ligand, PPAR
forms a heterodimer with the retinoid X receptor. The complex binds to
the PPAR response element in the promoter region of certain genes to
modulate transcription [18
, 19
]. Although
endogenous PPAR agonists have yet to be identified, members of the
PGJ2 family of prostaglandins are ligands for PPAR. Of
the PGJ2 series, 15-PGJ2 has the highest
binding affinity for PPAR [20
, 21
].
Thiazolidinediones, a class of antihyperglycemic compounds including
BRL 49653 (BRL), troglitazone, and pioglitazone, also bind and activate
PPAR [22
]. However, there are conflicting reports as
to whether 15-PGJ2 modulates mediator production solely
through PPAR. In one study, both 15-PGJ2 and the
synthetic thiazolidinediones BRL and troglitazone inhibited inducible
NO synthase (iNOS) expression and NO production in mouse peritoneal
Ms stimulated with interferon- [15
]. Another study
demonstrated that 15-PGJ2 and synthetic PPAR agonists
inhibit the production of proinflammatory cytokines, including TNF-,
IL-6, and IL-1ß, in human monocytes stimulated with phorbol
12-myristate 13-acetate (PMA) and okadaic acid [23
]. In
contrast, other studies have demonstrated that 15-PGJ2 may
inhibit mediator production through PPAR-independent pathways.
Studies by Rossi et al. [24
] and Straus et al.
[25
] showed that PGJ2 and
15-PGJ2 inhibit NF-B translocation by blocking the
ability of IB kinases and ß (IKKß to phosphorylate
IB. Petrova et al. [26
] demonstrated that, whereas
15-PGJ2 inhibited NF-B transcription activity and iNOS
mRNA and NO production in primary microglial cells, the PPAR agonist
troglitazone had no significant effect.
In this study, we hypothesized that 15-PGJ2 modulates
LPS-induced M activation through both PPAR-independent and
-dependent mechanisms. To test this hypothesis, we compared the effects
of 15-PGJ2 on LPS-induced NO, TNF-, and TxB2
production in rat peritoneal Ms with the effects of
PGD2, the stable PGI2 analogue
cicaprost (CICA) [27
], and the synthetic PPAR agonist
BRL. The effects of 15-PGJ2 on LPS-induced rat peritoneal
M activation of the cell signaling proteins IB and ERK 1/2
were compared with the effects of BRL to determine whether
15-PGJ2 acts through PPAR-independent mechanisms.
Additionally the effects of 15-PGJ2 on ERK 1/2
activation were compared with those of 2-cyclopenten-1-one to determine
whether ERK 1/2 inhibition occurs through reactivity of the
cyclopentenone ring of 15-PGJ2.
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s were harvested by peritoneal lavage from
ether-anesthetized rats with RPMI 1640 (Mediatech Inc., Washington, DC)
and L-glutamine containing penicillin (50 IU/mL),
streptomycin (50 µg/mL), and sodium heparin (10 U/mL). These Ms
(106 cells/mL) were plated on 24-well plates (Fisher,
Pittsburgh, PA) and allowed to adhere for 2 h at 37°C in 95%
incubator air and 5% O2. The cells were washed three times
with medium before experiments. For mediator production, Ms were
stimulated with medium with or without LPS (10 µg/mL) and with or
without simultaneous treatment with a vehicle control (dimethyl
sulfoxide [DMSO] or ethanol), 15-PGJ2,
12-PGJ2, PGJ2, PGD2 (Cayman
Chemicals, Ann Arbor, MI), BRL (a kind gift from SmithKline-Beecham,
Philadelphia, PA), or 2-cyclopenten-1-one (Sigma Chemical Co., St.
Louis, MO). Cells were incubated for 24 h at 37°C in 95%
incubator air and 5% O2. After 24 h, the supernatants
were collected for TxB2, TNF-, and NO quantification.
All samples were run in duplicate in each experiment. To collect
proteins for Western blot analysis, peritoneal Ms were extracted
from rats by lavage as described above and allowed to adhere for 2 h on 6-well plates. The cells were then incubated for 30 min with
medium alone or medium plus LPS (50 µg/mL).
Assays for TxB2, TNF-,
and NO production
NO production by macrophages was assessed by measuring the
amount of nitrite, a stable metabolite product of NO, in cell culture
supernatants, using the Griess reagent as previously described
[28
]. TNF- production was measured using an
enzyme-linked immunosorbent assay. To coat the plate, 50 µL of
purified anti-rat TNF- antibody (R&D Systems, Minneapolis, MN; 4
µg/mL in 0.1 M Na2CO3 binding solution) was
loaded in each well of a 96-well enhanced-binding plate and allowed to
adhere overnight at 4°C. The plates were washed with
phosphate-buffered saline (PBS) containing 0.05% Tween-20
(PBS/Tween-20) and blocked for 2 h at room temperature with
blocking buffer (BB) containing PBS (pH 7.0) and 10% fetal bovine
serum. After washing, standards and samples were added to the plates,
and the plates were incubated overnight at 4°C. Next, the plates were
washed with PBS/Tween-20. Biotinylated anti-rat TNF antibody (R&D
Systems, 0.5 µg/mL in BB) was then loaded. The plates were incubated
for 1 h at room temperature and then washed with PBS/Tween-20, and
a 1:1000 dilution of streptavidin in BB was added. The plates were
incubated for 45 min. Finally, they were washed eight times with
PBS/Tween-20, and a 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)
substrate solution containing 10 µL of 3%
H2O2 was added. TNF- production was measured
within 1 h at an absorbance of 415 nm. TxB2 production
was measured in cell culture medium by radioimmunoassay as previously
described [29
].
Western blot analysis of cellular proteins
Cells were lysed with radioimmunoprecipitation assay lysis
buffer (pH 7.4), containing 20 mM
N-2-hydroxypiperazine-N'-2-ethanesulfonic acid,
1% Triton X-100, 50 mM NaCl, 1 mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid, 5 mM ß-glycerophosphate, 30 mM sodium pyrophosphate, 100 mM
sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/mL
of leupeptin, and 0.2 µg/mL of pepstatin A). The lysates were
sonicated, and proteins were concentrated using a Millipore (Bedford,
MA)) filtering system. The proteins were then collected, 10-µL
aliquots were taken to determine protein concentrations, using the
Bio-Rad (Richmond, CA) DC protein assay for microplates, and the
remaining samples were mixed with an equal volume of 2x Laemmli sample
buffer for Western blot analysis [30
]. Immunoblot assays
were performed as previously described [31
]. Briefly
summarized, one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide
electrophoresis was performed according to the method of Laemmli
[30
]. Samples were resuspended through a 1.5-mm, 4–12%
acrylamide resolving gel. Proteins were then electrophoretically
transferred to polyvinylidene difluoride membranes, blocked for 1 h in 7.5% powdered-milk solution in 0.1% Tris-buffered saline-Tween
(TBS-T [0.1% Tween-20, 20 mM Tris, and 5 mM NaCl, pH 7.5]), washed
with TBS-T, and incubated overnight with anti-phosphoactivated ERK 1/2
antibody (1:10,000; New England Biolabs, Beverly, MA) or with
anti-IB antibody (1:1,000; Rockland, Gilbertsville, PA). After
washing with TBS-T, membranes were incubated with anti-AP-linked rabbit
anti-immunoglobulin G (IgG) antibody (1:4,000; Amersham, Arlington
Heights, IL) for 1 h, washed with TBS-T (0.15% Tween 20) five
times for 20 min per time, and incubated with enhanced
chemiluminescence detection reagents. The bands were detected after
exposure to enhanced chemiluminescence hyperfilm.
Statistical analysis
Values are expressed as means ± SE.
Statistical analyses of data were performed by unpaired Student’s
t-test, for studies comparing the effects of the
eicosanoids at equimolar concentrations, or by analysis of variance
followed by Fisher’s probable least-squares difference test for
comparing dose-response studies. Statview software (SAS Institute,
Cary, NC) was used to calculate statistical significance. The 50%
effective concentration (EC50) and 50% inhibitory
concentration (IC50) values were calculated using GraphPad
Prism software (San Diego, CA). The null hypothesis was rejected when
the P value was P 
0.05.
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s were stimulated for 24 h with LPS
(10 µg/mL) either alone or with 3 µM concentrations of
PGJ2, 12-PGJ2, or 15-PGJ2. LPS
significantly (n = 3–4, P 0.05)
stimulated NO production, measured by nitrite, to 1,808 ± 225
ng/mL compared with basal levels of 303 ± 140 ng/mL (Fig. 1
). The PGJ2 metabolite 15-PGJ2 (3 µM)
significantly inhibited NO production by 96 ± 5%.
12-PGJ2 also significantly inhibited NO production to a
lesser degree by 70 ± 16% (P 0.05). However,
the parent PGJ2 compound did not significantly
alter LPS-induced NO.
 View larger version (23K): [in a new window] |
Figure 1. Comparison of the effects of 15-PGJ2 with those of
12-PGJ2 and PGJ2 on
LPS-induced NO production. Rat peritoneal Ms were
stimulated with medium alone, LPS alone, or LPS with each compound, and
NO production, indicated by the levels of nitrite, was measured.
*P 0.05 compared to BAS; #, P 0.05 compared
to LPS.
|
, and
TxB2 productions were stimulated for 24 h with LPS (10 µg/mL)
with or without 10 µM concentrations of 15-PGJ2,
PGD2, or CICA, and their effects on NO, TNF-, and
TxB2 production were measured. 15-PGJ2
significantly inhibited (n = 3, P0.05)
LPS-stimulated NO production by 80 ± 6% relative to NO levels
(2,260 ± 172 ng/mL) induced by LPS alone (Fig. 2a
). In contrast, PGD2 and CICA had no significant
effects on LPS-induced NO production. 15-PGJ2 significantly
inhibited (n = 3, P 0.05) TNF-
production by 97 ± 1% compared with LPS control levels (710 ± 26 pg/mL) (Fig. 2b)
. PGD2 and CICA also inhibited
(n = 3, P0.05) TNF- production by
50 ± 12% and 61 ± 35%, respectively. LPS-induced
TxB2 production was inhibited (n = 3,
P 0.05) 60 ± 8% by 15-PGJ2 (Fig. 2c)
compared with the LPS control (18.5 ± 3 ng/mL).
PGD2 had no effect on TxB2 production, but CICA
significantly increased (n = 3, P
0.05) TxB2 production by 77 ± 37% (Fig. 2c)
over
control LPS levels.
 View larger version (28K): [in a new window] |
Figure 2. Comparison of the effects of 15-PGJ2 with those of other
prostaglandins on LPS-induced NO (A), TNF- (B), and
TxB2 (C) production. Rat peritoneal Ms were stimulated
with medium alone, LPS alone, or LPS with each compound. TNF- and
TxB2 are expressed as a percent difference of LPS set at
100%. n = 3–4; *, P 0.05 compared
with BAS; #, P < 0.05 compared with
LPS.
|
agonist BRL on
LPS-induced NO, TNF-,
and TxB2 productions were stimulated for 24 h with LPS (10
µg/mL) either alone or with BRL (0.1–100 µM), and production of
NO, TNF-, and TxB2 was measured. The effects of various
concentrations of 15-PGJ2 and BRL were compared (Fig. 3a
). 15-PGJ2 significantly inhibited (n = 4, P 0.05) LPS-induced NO production at
concentrations ranging from 1 to 10 µM. In contrast, BRL had no
effect on LPS-induced NO production at concentrations ranging from 0.1
to 10 µM. However, at 100 µM concentrations, BRL significantly
inhibited (n = 6, P 0.05)
LPS-induced NO production by 41 ± 8%. In contrast to the
inhibitory effect on production of NO by the PPAR- agonist, BRL (100
µM) significantly augmented (n = 6, P 0.05) TNF- production by 134 ± 19% (Fig. 3b)
. Treatment
with BRL (10 and 100 µM) also augmented (P 0.05)
LPS-induced TxB2 production by 212 ± 15%
(n = 3) and 365 ± 22% (n = 6),
respectively, over control LPS levels (Fig. 3c)
.
 View larger version (15K): [in a new window] |
Figure 3. Concentration response of the effects of BRL on LPS-induced
NO, TNF-, and TxB2 production. Rat
peritoneal Ms were stimulated with medium alone, LPS alone, or LPS
with increasing concentrations of 15-PGJ2 or BRL, and (A)
Nitrite production, (B) TNF- production, and (C) TxB2
production were measured (n = 2–6, P <
0.05 compared with BAS; #, P 0.05 compared with
LPS.
|
B degradation and ERK 1/2
phosphoactivationB degradation in cell lines. To confirm that
15-PGJ2 inhibits degradation in rat peritoneal
Ms, the effects of 15-PGJ2 on LPS-induced
IB were examined. Rat peritoneal Ms were pretreated for 1 h with 15-PGJ2 (0.05–10 µM) and then stimulated for 30
min with medium alone or with LPS (50 µg/mL). Stimulation of
Ms with LPS resulted in 79 ± 9% degradation
(n = 4, P 0.05) of IB protein
compared with degradation of control basal IB (Fig. 4b
). Pretreatment with 15-PGJ2 produced a
concentration-dependent augmentation 326 ± 60% of basal IB
expression , with an EC50 of 1.12 µM. LPS stimulation
shifted the EC50 for
15-PGJ2-induced IB protein expression to 2.4 µM.
 View larger version (19K): [in a new window] |
Figure 4. Concentration response of the effects of 15-PGJ2 on
LPS-induced IB degradation and ERK 1/2
phosphoactivation. Rat peritoneal Ms were pretreated for 1 h
with 15-PGJ2 (0.05–10 µM) and then stimulated for 30 min
with LPS (50 µg/mL). Cellular lysate was prepared and subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). IB degradation
and ERK 1/2 phosphoactivation were determined by Western blot analyses
using IB-specific antibodies and phosphospecific ERK 1/2
antibodies, respectively. (A) Representative gel of IB
degradation and ERK 1/2 phosphoactivation from one of three independent
experiments. Mean ± SE of scanning densitometry data
for IB degradation (B) and ERK 1/2 phosphoactivation (C) obtained
from three independent experiments. Data represent the percent change
compared with a control set at 100%. n = 3–4; *,
P 0.05 compared with BAS.
|
0.05) ERK 1/2
phosphoactivation relative to basal levels (Fig. 4a) . Pretreatment with
15-PGJ2 completely inhibited LPS-induced ERK 1/2
phosphoactivation (n = 4, P 0.05) at
5 and 10 µM concentrations. The 15-PGJ2 IC50
value for inhibition of LPS-induced ERK 1/2 phosphoactivation was 0.93
µM. Ms were also pretreated with BRL (0.1–100 µM) to determine
effects on signaling. In contrast to the inhibitory effects of
15-PGJ2 observed with pretreatment, BRL had no
significant effect on LPS-induced IB degradation (Fig. 5a
), nor did it affect LPS-induced ERK 1/2 phosphoactivation
(Fig. 5b)
.
 View larger version (18K): [in a new window] |
Figure 5. Concentration response of the effects of a PPAR agonist on
LPS-induced IB degradation and ERK 1/2
phosphoactivation. Rat peritoneal Ms were pretreated for 1 h
with 15-PGJ2 (0.05–10 µM) and then stimulated for 30 min
with LPS (50 µg/mL). Cellular lysate was prepared and subjected to
SDS-PAGE. IB degradation and ERK 1/2 phosphoactivation were
determined by Western blot analyses using IB-specific antibodies
and phosphospecific ERK 1/2 antibodies, respectively. (A)
Representative gels of IB degradation and ERK 1/2
phosphoactivation from one of three independent experiments. Mean ± SE of scanning densitometry data for IB
degradation (B) and ERK 1/2 phosphoactivation (C) obtained from three
independent experiments. Data represent the percent change compared
with a control set at 100%. n = 1–3; *,
P 0.05 compared with BAS; #, P
0.05 compared with LPS.
|
0.05) ERK 1/2 activation by LPS. To
examine whether the inhibition of ERK 1/2 was direct or a result of
inhibition of a signaling protein upstream of ERK 1/2, the effects of
15-PGJ2 and 2-cyclopenten-1-one on PMA-induced ERK 1/2
activation were also examined. In contrast to LPS-induced ERK 1/2
inhibition, neither 15-PGJ2 nor 2-cyclopenten-1-one
inhibited PMA-induced ERK 1/2 activation.
 View larger version (27K): [in a new window] |
Figure 6. Effects of 15-PGJ2 and 2-cyclopenten-1-one on
LPS-induced ERK 1/2 phosphoactivation. Rat peritoneal Ms
were pretreated for 1 h with 15-PGJ2 (5 µM) or
2-cyclopenten-1-one (100 and 300 µM) and then stimulated for 30 min
with LPS (50 µg/mL) or PMA (6 µg/mL). A crude membrane fraction was
prepared and subjected to SDS-PAGE. ERK 1/2 phosphoactivation was
determined by Western blot analyses of phosphospecific ERK 1/2
antibody. (A) Representative gel of ERK 1/2 phosphoactivation from one
of three independent experiments. Mean ± SE of
scanning densitometry data for ERK 1/2 phosphoactivation (B) obtained
from three independent experiments. Data represent the percent change
compared with a control set at 100%. n = 3–4; *,
P < 0.05 compared with BAS.
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agonist BRL had differential effects on LPS-induced mediator
production. Both 15-PGJ2 and BRL inhibited LPS-induced NO
production in rat peritoneal Ms stimulated with LPS. However,
15-PGJ2 inhibited LPS-induced TNF- and TxB2
production, whereas BRL potentiated LPS-induced TNF- and
TxB2 production. The differential effects of
15-PGJ2 and BRL on TNF- and TxB2 production
suggest that 15-PGJ2 may modulate the production of these
metabolites through mechanisms that are independent of PPAR
activation. The latter effects of 15-PGJ2 do not appear to
be a result of cross-reactivity with receptors of other eicosanoids.
Stimulation of Ms with PGD2 and the PGI2
analogue CICA, which activate DP and IP receptors, respectively, did
not inhibit LPS-induced NO production, whereas CICA augmented
LPS-induced TxB2 production. Thus, the ability of
15-PGJ2 to inhibit all three mediators suggests that it may
affect LPS-induced proximal signaling events. Experiments conducted to
address this phenomenon led to a second major finding of this
study—that 15-PGJ2 and the PPAR agonist BRL have
differential effects on LPS-induced cell signaling events.
Specifically, 15-PGJ2 inhibited, in a
concentration-dependent manner, both LPS-induced IB degradation
and ERK 1/2 phosphoactivation, whereas BRL had no significant effect on
either protein. In addition, the cyclopentenone also inhibited
LPS-induced ERK 1/2 activation.
Our data suggest that 15-PGJ2 acts independently of PPAR
to modulate M activation by inhibiting LPS-induced signaling
proteins. Cellular degradation of IB with subsequent NF-B
nuclear translocation is also linked to induction of
proinflammatory genes. Previous studies by Rossi et al.
[32
] have suggested that 15-PGJ2 inhibits
phorbol ester-induced NF-B translocation through inhibition of
phosphorylation and degradation of IB in Jurkat cells, HeLa
cells, and human monocytes. The inhibition of IB phosphorylation
by 15-PGJ2 was attributed to inhibition of IKK/ß
activity by covalent modifications of critical cysteine residues in the
IKK/ß proteins and in the DNA-binding domain of NF-B
[24
, 25
]. Activation of CD14 receptors by
LPS involves a Toll-like receptor 4-coupled kinase cascade in which
IKK/ß is activated through NF-B-inducing kinase
[7
]. Therefore, 15-PGJ2 may similarly
inhibit LPS-induced IB. In most mammalian cells, IB protein
is degraded within 10 min of stimulation and resynthesized within 60
min [33
]. In this study, 60-min pretreatment of
15-PGJ2 markedly augmented IB expression
in basal and LPS-induced Ms. The latter finding suggests that
15-PGJ2 may affect de novo synthesis of IB and/or
alter IB degradation. The effect of 15-PGJ2 on
up-regulation of the constitutive expression of cellular IB has
not been previously reported.
Inhibition of ERK 1/2 by 15-PGJ2 has not been
previously observed. Because LPS-induced ERK 1/2 activation is
essential for induction of TNF- and TxB2 formation in
rat peritoneal Ms [34
], this may constitute an
important anti-inflammatory function of 15-PGJ2. Since
inhibition of IKK/ß was attributed to reactivity of cyclopentenone
ring of 15-PGJ2, it is possible that
15-PGJ2 may inhibit LPS-induced ERK 1/2
phosphoactivation through a similar mechanism. To examine this
possibility, the effects 15-PGJ2 and 2-cyclopenten-1-one on
ERK 1/2 activation were compared. Both 15-PGJ2 and
2-cyclopenten-1-one inhibited LPS-induced ERK 1/2 activation,
suggesting that this inhibition by 15-PGJ2 is also a result
of reactivity of its cyclopentenone ring. Since neither compound
inhibited PMA-induced ERK 1/2 activation, the inhibition by
15-PGJ2 is not direct. PMA activates ERK 1/2 through
induction of protein kinase C. In turn, protein kinase C activates ERK
at the level of ras, raf, or MEK [35
]. Thus,
15-PGJ2 appears to inhibit ERK 1/2 by inhibiting a
signaling protein that is upstream of ras, raf, and MEK.
15-PGJ2 may also affect other signaling pathways as
suggested from 15-PGJ2 inhibition of JNK
phosphoactivation in a rat insulinoma cell line [36
].
The latter study also demonstrated that pretreatment with
15-PGJ2 and the PPAR agonist troglitazone resulted in an
increase in the expression of heat shock protein 70 (hsp70), which
inhibits NF-B activation and iNOS expression in rat microglial cells
[37
, 38
]. Induction of hsp70 also leads to
inhibition of in vivo LPS-induced TxB2
production in porcine models [39
] and to decreased
TNF- production in rats [40
]. Whether
15-PGJ2-induced induction of hsp70 may be, in part, a
mechanism for altering LPS signaling pathways in Ms remains to be
determined.
Other studies suggest that 15-PGJ2 affects mediator
production independently of PPAR activation. In a study comparing
the effects of 15-PGJ2 and several synthetic PPAR
agonists on LPS-induced TNF- and IL-6 production in murine Ms,
only 15-PGJ2 significantly inhibited the production of the
two cytokines [41
]. Also, studies have demonstrated
that, whereas 15-PGJ2 inhibited NO production in a
microglial cell line, the PPAR agonist troglitazone had no effect on
NO production [26
]. In the current study, BRL augmented
LPS-induced TxB2 production. Although this phenomenon has
not been previously reported with PPAR agonists, studies have
demonstrated that ligand activation of another PPAR receptor subtype,
PPAR, increases cyclooxygenase-2 expression in murine Ms
[36
]. On the other hand, studies have demonstrated that
15-PGJ2 inhibits cyclooxygenase-2 induction in
LPS-stimulated fetal hepatocytes [42
] and in
LPS-activated RAW264.7 M cell lines. The latter data are consistent
with our data showing inhibition of LPS-induced
TxB2 by 15-PGJ2.
It is not known whether 15-PGJ2 is an endogenous mediator
of inflammatory events. Although PGJ2 has been measured in
murine mesangial cells [43
] and rat livers
[44
] and 15-PGJ2 has been measured in rat
inflammatory exudate [45
], the endogenous concentrations
measured ranged from picograms to nanograms per milliliter. These
endogenous concentrations were far below the pharmacological
concentrations (in micromolars) of 15-PGJ2 used in the
current study to elicit anti-inflammatory effects. Nagoshi et al.
[13
] suggested that PGD2 inhibits NO
production in vascular smooth muscle cells through metabolism of
PGD2 to the parent PGJ2 compound and to the
PGJ2 metabolites 12-PGJ2 and
15-PGJ2. The lack of an effect of PGD2 or
PGJ2 on LPS-induced NO production in our study may suggest
cellular differences in M and smooth muscle with regard to
PGD2 and PGJ2 metabolism.
In conclusion, our data demonstrate that 15-PGJ2 suppresses
NO, TNF-, and TxB2 production induced by
LPS. Although the eicosanoids possessing membrane receptors,
PGD2 and CICA, inhibited LPS-induced TNF- production, NO
production and TxB2 production were not inhibited. Although
BRL inhibited LPS-induced NO production, it had no effect on TNF-
production and augmented LPS-induced TxB2 production. Thus,
15-PGJ2 had effects that were distinct from those of
PGD2, a PGI2 analogue, and a
PPAR agonist. The ability of 15-PGJ to alter LPS-induced cell
signaling pathways may be a potential mechanism for its
anti-inflammatory effects. Since 2-cyclopenten-1-one inhibited
LPS-induced ERK 1/2 activation similarly to 15-PGJ2,
15-PGJ2 may modulate M activation through mechanisms
that involve reactivity of its cyclopentenone ring.
Received June 19, 2000; revised November 25, 2000; accepted November 27, 2000.
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