15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells
Journal of Neuroinflammation
15d-PGJ2 induces apoptosis of mouse oligodendrocyte precursor cells
Zhongmin Xiang 0
Tong Lin 0
Steven A Reeves 0
0 Address: CNS Signaling Laboratory, MassGeneral Institute for Neurodegenerative Disease (MIND), Massachusetts General Hospital, Harvard Medical School , 114 16th Street, Charlestown, MA 02129 , USA
Background: Prostaglandin (PG) production is associated with inflammation, a major feature in multiple sclerosis (MS) that is characterized by the loss of myelinating oligodendrocytes in the CNS. While PGs have been shown to have relevance in MS, it has not been determined whether PGs have a direct effect on cells within the oligodendrocyte lineage. Methods: Undifferentiated or differentiated mouse oligodendrocyte precursor (mOP) cells were treated with PGE2, PGF2α, PGD2 or 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2). Cell growth and survival following treatment were examined using cytotoxicity assays and apoptosis criteria. The membrane receptors for PGD2 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ, as well as reactive oxygen species (ROS) in the death mechanism were examined. Results: PGE2 and PGF2α had minimal effects on the growth and survival of mOP cells. In contrast, PGD2 and 15d-PGJ2 induced apoptosis of undifferentiated mOP cells at relatively low micromolar concentrations. 15d-PGJ2 was less toxic to differentiated mOP cells. Apoptosis was independent of membrane receptors for PGD2 and the nuclear receptor PPARγ. The cytotoxicity of 15d-PGJ2 was associated with the production of ROS and was inversely related to intracellular glutathione (GSH) levels. However, the cytotoxicity of 15d-PGJ2 was not decreased by the free radical scavengers ascorbic acid or α-tocopherol. Conclusion: Taken together, these results demonstrated that 15d-PGJ2 is toxic to early stage OP cells, suggesting that 15d-PGJ2 may represent a deleterious factor in the natural remyelination process in MS.
Prostaglandin (PG)s are a group of 20-carbon fatty acids
derived from membrane lipids. By sequential enzymatic
reactions of phospholipase A2 (PLA2), housekeeping
cyclooxygenase (COX)-1 or inducible COX-2, PGH2 is
generated and then converted to PGE2, PGD2, PGF2α,
PGI2 (prostacyclin) and TXA2 (thromboxane A2) by their
respective PG isomerases . For example, PGH2 is first
converted to PGD2 by lipocalin-type PGD2 synthase
(LPGDS) or hematopoietic (H)-PGDS, which then
undergoes sequential non-enzymatic dehydration reactions to
form 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2). PGs generally act
through membrane-bound G-protein coupled PG
receptors with the exception of 15d-PGJ2, which has no
defined membrane receptor, although reported to be an
activator of the PGD2 receptor DP2 . Instead, 15d-PGJ2
is a natural ligand for the nuclear receptor peroxisome
proliferator-activated receptor (PPAR)γ , which has a
major role in the regulation of proliferation,
differentiation and lipid metabolism [4,5]. Moreover, 15d-PGJ2 has
been shown to induce apoptosis of cultured cortical
neurons [6,7], endothelial cells , hepatic myofibroblasts
, granulocytes  and cancer cells , through both
PPARγ-dependent and PPARγ-independent mechanisms
Mounting evidence suggests that PGs play important roles
in neuroinflammatory diseases such as multiple sclerosis
(MS), an autoimmune disease of the central nervous
system (CNS) in which T- and B cells attack components of
the myelin sheath leading to loss of myelin as well as
myelinating oligodendrocytes [12-14]. As a natural repair
mechanism, oligodendrocyte precursor (OP) cells
proliferate and differentiate within the demyelination sites to
replenish the lost myelinating oligodendrocytes [15,16].
In patients with MS and in the experimental autoimmune
encephalomyelitis (EAE) rodent model, the
demyelination foci are typically characterized by inflammatory
infiltrates containing myelin-specific T- and B cells, and
activated microglia and astrocytes [12,14,17-19]. These
inflammatory cells are known to secrete cytotoxic
cytokines such as TNFα and interleukin (IL)-6 [12,20], as
well as PGs such as PGE2, PGD2 and PGF2α [21-23].
Bacterial lipopolysaccharide (LPS), which is a potent
proinflammatory factor that induces abundant PGD2 or
15dPGJ2 production in microglia cultures [24,25], and in the
CSF and spinal cord following systemic administration
[26,27]. In MS demyelination foci, gene expression of PG
related enzymes such as PLA2 , COX-2  and
LPGDS  are up-regulated. Increased L-PGDS in
perineuronal oligodendrocytes and H-PGDS in microglia are
also observed in the mouse twitcher demyelination model
[31,32]. Additional evidence has shown that H-PGDS is
increased in activated T helper (Th)2 cells in vitro .
While these findings suggest that OP cells are exposed to
a PG-rich environment, little is known regarding the effect
these PGs have on OP cells.
In this study, we examined the effect of PGs on mouse OP
(mOP) cells. We found that PGD2 and its dehydration
end product 15d-PGJ2 induce apoptosis of OP cells in a
PPARγ-independent manner, while more mature OP cells
are relatively resistant. These results suggest that PGD2
and 15d-PGJ2 may contribute to MS pathology by
inducing OP cell death.
Materials and reagents
N1 supplement, insulin, biotin, staurosporine,
indomethacin, NS398, SC58125, GW9662, N-acetyl cysteine
(NAC), buthionine sulfoximine (BSO), ascorbic acid,
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and
bisbenzimide were obtained from Sigma (St. Louis, MO); High
glucose DMEM, DMEM/F12 (1:1), fetal bovine serum,
penicillin/streptomycin, Trizol, PCR reagents and
enzymes were from Invitrogen (Carlsbad, CA); SYBR
green PCR mix was from Amersham (Piscataway, NJ);
15d-PGJ2, PGD2, PGE2, PGF2α, T0070907, AH6809,
BAY-u3405 and GSH kit were from Cayman Chemicals
(Ann Arbor, MI); Cover-slips were from Bellco
Biotechnology (Vineland, NJ); LDH cytotoxicity assay kit was
from Promega (Madison, WI); TUNEL kit and cell death
ELISA kit were from Roche (Indianapolis, IN);
5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was from
Molecular Probes (Eugene, OR); Goat anti-MBP was from
Santa Cruz Biotechnology (Santa Cruz, CA); rabbit
antiNG2 was kindly provided by Dr. W. Stallcup; rabbit
antiπGST was from MBL (Woburn, MA); A2B5 hybridoma
was from ATCC (Menassas, VA); normal donkey serum
and all secondary antibodies were from Jackson
ImmunoResearch (West Grove, PA); Fluorescent mounting
medium with or without nuclear dye DAPI was from
Vector Laboratories (Burlingame, CA).
Mouse oligodendrocyte precursor (mOP) cell line
The mOP cell line developed in this lab  and the rat
oligodendrocyte cell line CG4  were used in this
study. Both cell lines were maintained in CG4
proliferation medium (PM) as described previously . CG4 PM
consists of 70% high glucose DMEM, 30% conditioned
medium from B104 neuroblastoma cell line,
supplemented with 0.5% N1 supplements, biotin 10 μg/ml,
insulin 5 μg/ml and 1% penicillin/streptomycin.
Differentiation of mOP cells was induced in
differentiation medium (DM), which is different from CG4 PM only
in that the 30% conditioned medium was from confluent
mOP cell cultures instead of B104 neuroblastoma
cultures. The use of conditioned medium from confluent
mOP cells was based on the previous report that
oligodendrocytes are self-inhibiting in proliferation  and
our observation of a differentiation-promoting effect
from medium obtained from confluent mOP cell cultures
(data not shown). Conditioned medium from confluent
cells was obtained as follows: Approximately 50%
confluent mOP cell cultures were grown for 1 wk in PM without
medium change, medium was collected, filtered, and then
used to make DM. mOP cells were cultured in DM for 3 d
Drug treatment of cell cultures
mOP cell cultures were grown to 60–70% confluency in
12- or 24-well plates and then serum-starved (CG4 PM
without conditioned medium) for 24 h before
experiments. PGs were added to the medium for 24–48 h. For
15d-PGJ2 or PGD2 preparation, the original solvent ethyl
acetate was evaporated, and PGD2 or 15d-PGJ2 was
redissolved in PBS before adding to the medium. For other
chemicals, a corresponding amount of the solvent
(DMSO or ethanol) was added to control cultures with
concentrations less than 0.2%. All experiments were
performed 3–5 times and each treatment in triplicates.
Cell growth/viability assay
Cells were assayed using
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT). MTT is
converted to a blue formazan product by mitochondria
dehydrogenases only in live cells, and can be used as a
cytotoxicity assay . In this regard, the MTT assay has
been used to specifically address 15d-PGJ2-induced cell
death in neurons and endothelial cells [7,8]. Cells were
incubated in medium with MTT (50 μg/ml) for 1 h at
37°C. The formazan product was dissolved in DMSO, and
absorbance at 600 nm was measured using a plate reader.
Additionally, lactate dehydrogenase (LDH) enzymatic
activity in the medium was measured using the
CytoTox96 kit (Promega) according to the manufacturer's
instructions. LDH is released into the medium upon cell
lysis, and the activity measured in the medium is therefore
proportional to the number of lysed cells. The amount of
cell death (percentage) was calculated as released LDH/
total LDH (value obtained by lysing all cells in the
Terminal deoxynucleotidyl transferase (TdT) dUTP nick
end labeling (TUNEL) and nuclear staining
TUNEL staining was performed using a kit from Roche
following the manufacturer's instructions. In brief, cells that
had been grown on coverslips were fixed in 4%
paraformaldehyde for 20 min and then rinsed in PBS. After
permeablization for 15 min at RT with 0.1% Triton X-I00 in
0.1% citrate buffer, the cells were incubated with TUNEL
mix (TdT enzyme and fluorescein-dUTP) for 1 h at 37°C.
After rinsing with PBS, the coverslips were mounted on
glass slides with fluorescence mounting medium and
inspected under a fluorescence microscope. Four random
areas for each coverslip (20× objective view) were
surveyed and the number of cells counted. For nuclear
staining, bisbenzimide was added to the medium at 1 μg/ml
for 20 min. After washing, mOP cells were mounted for
ELISA-based cell death assay
Apoptotic cell death was also quantified using an ELISA
kit that quantifies indirectly the histone-containing
nucleosomes after DNA fragmentation. The culture medium
was collected. Attached cells were then collected using
trypsin digestion (0.25% for 5 min), combined with the
culture supernatant, and then the mix was pelleted at
1,500 × g for 5 min. After carefully removing the
supernatant, the cells were lysed in incubation buffer for 30 min
at RT. After centrifugation at 20,000 × g for 10 min, the
supernatants (cytoplasmic fraction containing
nucleosomes) were added to the plate according to the
manufacturer's instructions. DNA fragmentation was then
examined calorimetrically using a plate reader at 405 nm.
DNA gel electrophoresis
Cells were harvested and lysed in hypotonic buffer (50
mM Tris (pH7.9) containing 1% Triton X-I00, 10 mM
EDTA and 50 μg/ml RNase A) for 5 min at RT. The lysates
were centrifuged at 10,000 × g for 10 min, and the
supernatant containing short DNA fragments was collected.
After phenol/chloroform extraction, DNA was
precipitated with sodium acetate and ethanol, resuspended in TE
buffer, separated on a 1.2% agarose gel containing
ethidium bromide and then visualized with a UV illuminator.
mOP cells grown on poly-D-lysine coated cover-slips were
fixed in 4% paraformaldehyde for 20 min and rinsed in
PBS. After permeablization for 15 min at RT with 0.2%
Triton X-I00 in PBS and 10% normal donkey serum to
block unspecific binding, mOP cells were incubated with
primary antibodies: goat anti-MBP (1: 100), rabbit
antiNG2 (1: 200), rabbit anti-GST (1: 1000), all diluted in
PBS with 1% normal donkey serum. For A2B5,
hybridoma medium was used directly without dilution. After
three washes with PBS, the cells were incubated with
appropriate Cy2- or Cy3-conjugated secondary antibodies
(1:200 in PBS with 1% donkey serum) for 1 h at RT in the
dark. After PBS washes, the cover-slips were mounted on
slides with fluorescence mounting medium containing
the nuclear dye DAPI and examined using an Olympus
BX60 microscope equipped with epifluorescence optics.
Reactive oxygen species (ROS) detection
ROS production was detected using the fluorescence
probe carboxy-H2DCFDA. mOP cells plated on
poly-Dlysine coated coverslips were washed twice with DMEM
and then incubated in loading solution (DMEM with 25
μM DCFDA) for 30 min at 37°C in the dark. Cells were
washed twice and then treated with 15d-PGJ2. Coverslips
were rinsed with DMEM before mounting on slides and
fluorescence (FITC filter) images of cells were taken
immediately using a fluorescence microscope equipped
with a digital camera (DP70, Sony). Ten fields (40×
objective) for each coverslip were sampled (>400 cells), the
mean pixel values (0–255) of individual cells were
analyzed using NIH imaging software (NIH, Bethesda, MD).
All treatments were performed in duplicate and data
expressed were averaged values of all cells counted in each
Total intracellular GSH content was measured using a kit
from Cayman according to the manufacturer's
instructions. In brief, mOP cells were scraped from 6-well plate,
pelleted by centrifugation at 700 × g for 5 min,
homogenized in 1 ml cold buffer, and then centrifuged at 10,000
× g for 15 min at 4°C. The supernatant was collected and
protein concentration was measured. The supernatant was
deproteinated by mixing with metaphosphoric acid
before GSH content measurement. GSH content was
expressed as μmol/mg protein.
Total RNA was isolated using the Trizol reagent according
to the manufacturer's instructions. First-strand cDNA was
synthesized using reverse transcriptase (Superscript) and
oligo(dT) primer. PCR reactions were performed using 1
μg cDNA and Taq polymerase. Primers for PPARγ
amplification were: 5'-TTT TCA AGG GTG CCA GTT TC-3' and
5'AAT CCT TGG CCC TCT GAG AT-3'. The expected PCR
product size is 198 bp. All reactions were carried out with
iCycler (BioRad, Hercules, CA) using SYBR green PCR
mix, which allows automated signal quantification. The
PCR parameters were 35 cycles with 94°C denaturation
for 20 sec, 60°C annealing for 30 sec, and 72°C extension
for 50 sec. Quantification was performed using the ΔΔ
method. The PCR products were confirmed by ethidium
bromide-stained agarose gel electrophoresis. cDNA
derived from a postnatal day 20 mouse brain was used as
a positive control.
Statistical analysis was performed using InStat and Prism
software (GraphPad Software, San Diego, CA). Student
ttest (two-tailed) was used to assess the difference between
two groups. One-way ANOVA was used to assess
differences among groups (more than three) with
NewmanKeuls post-test. When appropriate, two-way ANOVA and
Bonferroni posttest were used to assess differences among
groups with two independent variables. All significance
levels were set at p < 0.05.
PGD2 and 15d-PGJ2 but not PGE2 or PGF2α induced mOP
We have previously developed mouse OP (mOP) cells
 from the post-natal mouse brain that can be
sustained for long periods in culture and which display
properties similar to those of the rat CG4 oligodendrocyte cell
line . When grown in proliferation medium (PM)
mOP cells assume bipolar or tripolar morphology and
express the OP cell markers NG2 and A2B5 (Fig. 1A). We
first examined whether endogenous PG production by
oligodendrocytes has a role in mOP cell growth and survival.
In these experiments we used MTT assay as an initial assay
for cell death and chemical inhibitors of enzymes
responsible for PG production. mOP cells were treated with 10
μM of indomethacin (COX-1 and COX-2 inhibitors),
NS398 (COX-2 specific inhibitor) or SC58125 (COX-2
specific inhibitor) for 48 h. No differences in mOP cell
growth and survival were observed compared to vehicle
treated (Data not shown). We next tested whether direct
applications of PGs to the culture medium of mOP cells
affected growth and survival. PGE2 or PGF2α treatment
(0.1, 1 and 10 μM) for 24 h had no effect on mOP cell
growth (Fig. 1B). Extended treatment with PGE2 or
PGF2α (10 μM) for 48 h also had no effect (data not
shown). In contrast, PGD2 and 15d-PGJ2 induced
significant cell death in a dose-dependent manner as early as 24
h (Fig. 1C–D). mOP cells were more sensitive to 15d-PGJ2
(50% effective concentration (EC50) 1.0 μM) than to
PGD2 (EC50 16.6 μM). To confirm the cytotoxicity of
15d-PGJ2, we used a more specific cell death assay, which
measures the enzymatic activity of lactate dehydrogenase
(LDH) released in the medium by dead cells. Treatment of
15d-PGJ2 (1.0 μM) induced significant cell death at 24 h,
and more dramatically at 48 h. The effect of these PGs was
also examined on the rat oligodendrocyte cell line CG4
using MTT assay and similar results were observed (data
Apoptotic death of mOP cells induced by 15d-PGJ2
We next examined whether mOP cell death induced by
15d-PGJ2 was apoptotic. mOP cells were treated with
15d-PGJ2 for 24 h and assayed for apoptosis by staining
with the DNA-binding dye bisbenzimide, which can
demonstrate nuclear condensation characteristic of apoptosis,
and using the TUNEL staining method, which detects
apoptosis-associated DNA strand breaks . In
untreated mOP cells, a small percentage (~3.8%) of the
cells displayed condensed nuclei when stained with
bisbenzimide (Fig. 2A). However, when mOP cells were
treated with 1 μM 15d-PGJ2 for 24 h the percentage of
cells with condensed nuclei doubled to 7.5% (Fig. 2A).
TUNEL staining revealed similar results where 2.5% of
untreated and 6.0% PGJ2-treated cells were positive for
TUNEL staining (Fig. 2B). Further evidence for 15d-PGJ2
induced apoptosis of mOP cells was obtained using an
ELISA-based cell death assay, which quantifies indirectly
the histone-containing nucleosomes generated due to
DNA fragmentation. In this assay, 15d-PGJ2 induced
apoptotic DNA fragmentation ~2-fold over that observed
in untreated cells (Fig. 2C). Lastly, we assessed DNA
fragmentation (mono- and oligonucleosomes) in
15d-PGJ2treated mOP cells using agarose gel electrophoresis.
15dPGJ2 increased DNA fragmentation in mOP cells over that
gTFohigdeueenrfedfer1cotcoytfePpGrsecounrstoher (gmroOwPt)hcaenllds survival of mouse
oliThe effect of PGs on the growth and survival of
mouse oligodendrocyte precursor (mOP) cells. (A)
mOP cells express the oligodendrocyte precursor surface
markers NG2 (red) and A2B5 (Green). Scale bar, 20 μm. (B)
mOP cells were treated with PGE2 or PGF2α (0.1, 1 and 10
μM) and examined using the MTT assay after 24 h. (C-D)
mOP cells were treated with the indicated concentrations of
PGD2 or 15d-PGJ2 and examined using the MTT assay after
24 h. Data are the average of 3–4 experiments and expressed
as percentage of the control group (vehicle treated). (E)
mOP cells were treated with 15d-PGJ2 (1 μM) and examined
using the LDH assay after 24 h and 48 h. Data are expressed
as percentage of the total LDH. Asterisks indicate significant
difference versus control group (One-way ANOVA with
Dunnet posttest for C and D, Student t-test for E, *(p <
0.05), **(p < 0.01).
cFCeihgllasurraecte2rization of 15d-PGJ2-induced apoptosis in mOP
Characterization of 15d-PGJ2-induced apoptosis in
mOP cells. mOP cells were treated with 15d-PGJ2 (1 μM)
for 24 h, and then stained with the nuclear dye bisbenzimide
or labeled with the TUNEL method. Cells with condensed
nuclei (A) or that were TUNEL-positive (B) were counted,
and expressed as percentage of the total number of cells. For
apoptosis ELISA (C) and DNA fragmentation gel analysis (D),
DNA from mOP cells that had been treated with 15d-PGJ2
(5 μM) for 24 h was extracted and analyzed. Staurosporine
(STA, 100 nM) treatment was used as positive control. M,
molecular standards. Asterisks indicate significant difference
versus control group (t-test, two-tailed, *(p < 0.05), **(p <
observed in untreated cells (Fig. 2D). Staurosporine
(STA), a well described inducer of apoptosis , induced
DNA fragmentation in mOP cells similar to that of
15dPGJ2-treated cells (Fig. 2C–D).
15d-PGJ2-induced apoptosis of mOP cells occurs
independently of PPARγ or PGD2 receptors
15d-PGJ2 is a natural ligand for PPARγ and has been
shown to induce apoptosis in a variety of cell types
through a PPARγ-dependent pathway [6,8]. We therefore
investigated whether 15d-PGJ2-induced apoptosis in
mOP cells was through a PPARγ-dependent pathway. Real
time RT-PCR analysis demonstrated a small amount of
PPARγ amplification in mOP cells (data not shown). To
further examine whether PPARγ has a role in 15d-PGJ2
induced apoptosis of mOP cells we tested whether
pharmacological inhibition of PPARγ protects mOP cells from
the cytotoxic effects of 15d-PGJ2. Pre-incubation of mOP
cells with the irreversible PPARγ antagonists GW9662 (10
μM) or T0070907 (100 nM) did not block
15d-PGJ2induced apoptotic cell death (Fig. 3). These results
pro1PF5PigdAu-RPrγGe oJ32r cPyGtoDto2xmiceitmy bornanmeOrePcceepltlsorosccurs independently of
15d-PGJ2 cytotoxicity on mOP cells occurs
independently of PPARγ or PGD2 membrane receptors.
mOP cells cultured on coverslips were treated with
15dPGJ2 (1 μM), in the absence or presence of the irreversible
PPARγ antagonists GW9662 (GW, 10 μM) or T0070907 (T,
100 nM), or nonspecific PGD2 receptor (DP) antagonist
AH6809 (AH, 10 μM) or specific DP2 antagonist BAY-u3405
(BAY, 5 μM) for 24 h, and apoptotic cell death was examined
using bisbenzimide staining. Cells were counted and
expressed as percentage of control (untreated). Data shown
were average of three experiments. Asterisks indicate
significant difference versus control group (Two-way ANOVA
Bonferroni posttest, **(p < 0.01), ***(p < 0.001)).
vide evidence that the cytotoxic effect of 15d-PGJ2 is not
mediated through the PPARγ pathway.
15d-PGJ2 has been reported to be an activator of the
Gprotein-coupled receptor PGD2 DP2 . To test whether
15d-PGJ2 exerts its effect through the PGD2 DP2 receptor,
mOP cells were pretreated with the non-specific DP
antagonist AH6809 (which blocks both DP1 and DP2
receptors) or the specific PGD2 DP2 receptor antagonist
BAYu3405. Neither BAY-u3405 (5 μM) nor AH6809 (10 μM)
blocked 15d-PGJ2-induced death (Fig. 3). These results
suggest that 15d-PGJ2 induces mOP cell death
independently of known membrane G-protein-coupled receptors
15d-PGJ2 cytotoxicity and ROS
Previous reports have suggested that 15d-PGJ2 may
induce intracellular oxidative stress [38,39]. To examine
whether there is increased ROS production in
15d-PGJ2treated mOP cells we preloaded cells with the fluorescent
ROS probe DCFDA prior to 15d-PGJ2-treatment. ROS
production was significantly increased in mOP cells as
early as 45 min after treatment with 15d-PGJ2 (10 μM)
(Fig. 4A). Glutathione (GSH) is an important antioxidant
i1Fn5ifglduu-ePrnGecJe42dcbyytointotrxaicietlyluilnavrogllvuetsatfhreioenreadleicvaelsproduction and is
15d-PGJ2 cytotoxicity involves free radical
production and is influenced by intracellular glutathione
levels. (A) Time course of 15d-PGJ2-induced ROS production.
mOP cells were preloaded with the fluorescent ROS probe
DCFDA for 30 min, and then treated with 15d-PGJ2 (10 μM)
for 1 h. ROS production was expressed as DCFDA
fluorescence intensity (pixel value). (B) mOP cells were pre-treated
or not with NAC (1 mM), Ascorbic acid (1 mM) or
α-tocopherol (1 mM) for 1 h prior to treatment of 15d-PGJ2 (5 μM)
for 24 h, toxicity was examined by counting the apoptotic
cells with condensed nuclei. (C) mOP cells were treated or
not with BSO (100 μM) for 4 h and the total level of
intracellular GSH was measured. (D) mOP cells were treated with
BSO (100 μM) for 1 h and then co-treated with 15d-PGJ2 (1
μM) for 24 h. Cells treated with BSO or 15d-PGJ2 alone or
untreated were included as controls. Toxicity was examined
by counting the apoptotic cells with condensed nuclei.
Asterisks indicate significant difference (One-way ANOVA with
Newman-Keuls or Dunnet posttest, or two way ANOVA
with Bonferroni posttest, *(p < 0.05), **(p < 0.01) ***(p <
0.001); two-tailed t-test used in C).
that protects cells from oxidative damage by ROS. We
therefore tested whether manipulations of the
intracellular level of GSH could affect apoptotic cell death induced
by 15d-PGJ2. Pretreatment of mOP cells with NAC, which
is a precursor molecule for GSH synthesis and a reducing
agent for oxidized GSH , provided ~60% protection
against 15d-PGJ2 induced death, while pre-incubation
with the antioxidants ascorbic acid or α-tocopherol did
not provide protection (Fig. 4B). In contrast, application
of buthionine sulfoximine (BSO), an inhibitor for
γglutamylcysteinase synthatase  which depletes
intracellular GSH (Fig. 4C), was toxic to mOP cells by itself and
sensitized mOP cells to a lower concentration of
15dPGJ2 (Fig. 4D). These results suggest that the toxicity of
15d-PGJ2 to mOP cells is related to intracellular GSH
15d-PGJ2 cytotoxicity is dependent on the stage of
Developmental stage susceptibility to various cytotoxic
stimuli has been reported previously [42-45]. We tested
whether the effect of 15d-PGJ2 on oligodendrocytes is
stage-dependent. mOP cells were induced to differentiate
in differentiation medium (DM). In contrast to the simple
morphology that undifferentiated mOP cells display
(97.7% with 3 or less processes and 2.3% with 4 to 6
branches, n = 287 cells) (Fig. 1A), differentiated mOP cells
display complex process formation (17.9% with 3 or less
processes, 50.2% with 4 to 6 branches, and 31.9% with
more than 6 branches, n = 304 cells). While displaying
increased immunoreactivity to the late stage markers
πGST and MBP, differentiated mOP cells still showed
punctate staining to early stage OP cell marker A2B5 (Fig. 5A–
When differentiated mOP cells were treated with
15dPGJ2, higher concentrations of 15d-PGJ2 were needed for
significant cytotoxicity as judged using MTT assay (Fig.
6A), with an EC50 of 9.8 μM. Maturation stage-specific
cytoxicity was also measured using apoptosis criteria
(condensed nuclei). While 1 μM 15d-PGJ2 was sufficient
to induce significant cell death of undifferentiated mOP
cells (see Fig. 2A), a 10-fold higher concentration of
15dPGJ2 was required to induce comparable death in
differentiated mOP cells (Fig. 6B). These results demonstrated
that differentiated mOP cells are more resistant to
While PGs have been shown to display a range of activities
on various cell types , few studies have been carried out
on cells within the oligodendrocyte lineage. Our data
indicate that PGD2/15d-PGJ2 may represent another
group of factors in addition to cytotoxic cytokines
produced during inflammation that are toxic to OP cells.
Our results demonstrated that 15d-PGJ2 at ≥1 μM is toxic
to mouse OP cells. While baseline production of PGD2
from the whole mouse brain has been calculated to be
approximately 2 nM , higher concentrations may,
however, occur during inflammatory conditions. LPS
treatment can mimic inflammatory conditions and
induces PGD2/15d-PGJ2 production in mixed glial cell
cultures [24,25] and in animal models [26,27]. The
production of 15d-PGJ2 in the medium of primary microglial
cell cultures was calculated to be in the range of 10 nM
FMiigcurorgera5phs showing mOP cell differentiation
Micrographs showing mOP cell differentiation. mOP cells were induced to differentiate in DM. Differentiated mOP cells
display elaborate process extension with secondary and tertiary branching (A, Phase), and immunostain positive for the mature
oligodendrocyte markers πGST (B, Green) and MBP (D, Red). However, differentiated mOP cells still stain positive for the
early stage OP cell marker A2B5, in a punctate fashion (C, Green). Scale bar, 20 μm.
dTFeihgveueelrofefpem6cteonfta1l5sdta-PgeGoJ2f-oinldiguocdeednderoatchytiessdependent on the
The effect of 15d-PGJ2-induced death is dependent
on the developmental stage of oligodendrocytes.
mOP cells were induced to differentiate in DM and then
treated with 15d-PGJ2 for 24 h. Toxicity was examined by
MTT assay (A) and by counting cells with condensed nuclei
stained by the nuclear dye bisbenzimide (B). Asterisks
indicate significant difference (One-way ANOVA with Dunnet
post test, *(p < 0.05), **(p < 0.01).
. However, it is likely that 15d-PGJ2 concentrations in
vivo can be orders higher because of the more confined
interstitial space. Indeed this has been observed
previously for extracellular levels of glutamate. Upon
inhibition of glutamate uptake the interstitial glutamate
concentration increases to over 100–150 times (200–300
nM) the minimal value maintainable by glutamate
transporters (2 nM) . In a more recent study, 15d-PGJ2 was
found to be increased to 600 pg/mg protein  (~0.1
μM), in the ischemic cortex. These results suggest that the
toxic levels of 15d-PGJ2 we observed in our in vitro
experiments may also occur in vivo.
Our results demonstrated that 15d-PGJ2 at 1 μM
decreases MTT values at 24 h by ~50%. This relatively
large reduction likely represents a combination of cell
death, reduced cell proliferation, and compromised
mitochondrial activity. Using a more cell death specific LDH
assay, and an array of apoptotic assays, we demonstrated
that 15d-PGJ2 induces apoptotic cell death in mOP cells,
which has been observed in other cell types [6-11].
The mechanism(s) for this apoptosis has not been clearly
elucidated. While 15d-PGJ2 is a known ligand for PPARγ
and has been implicated in apoptosis in a variety of cell
types [6,8], in our studies 15d-PGJ2 induced apoptotic
death of mOP cells independently of PPARγ, since the
irreversible PPARγ antagonists GW9662 or T0070907 did
not provide protection. Consistent with our findings,
15d-PGJ2 toxicity is observed in hepatic myofibroblasts
that lack PPARγ expression .
15d-PGJ2 has been shown previously to induce free
radical production [38,39], potentially due to its unsaturated
α, β carbonyl moieties in the cyclopentanone rings. In
addition, these moieties are capable of reacting with thiol
groups by Michael addition  and thus able to modify
the functions of important proteins such as thioredoxin
. Reduced GSH is the most abundant non-protein
thiol group-containing molecule and can readily
conjugate to 15d-PGJ2 via glutathione-S-transferase (GST).
Conjugation prevents free 15d-PGJ2 from attacking other
intracellular targets. In this regard, our studies show that
depleting intracellular GSH by BSO potentiates
15d-PGJ2induced cytotoxicity and increasing intracellular levels of
reduced GSH (by NAC) provides protection. In our study,
antioxidants such as ascorbic acid or α-tocopherol, which
act as electron donors to halt free radical production,
provided no protection for the cytotoxic effect of 15d-PGJ2
on mOP cells. This has been observed previously in
neuronal cell types where ascorbic acid did not provide
protection against PGJ2-induced toxicity , or
dopamineinduced apoptosis . This lack of protection may be
due to ascorbic acid-mediated depletion of intracellular
GSH pool which would offset its beneficial effect .
Interestingly, in a study using cultured OP cells, ascorbic
acid provided no protection against cystine
deprivationinduced death, while α-tocopherol provided protection,
without blocking the depletion of intracellular GSH .
Taken together with our results these findings suggest that
ROS production may not be the only event responsible for
15d-PGJ2-induced cell death. These other events may
include reduction in mitochondrial membrane potential
, inhibition of NFκB activation , and inhibition of
transcription factor AP-1 associated  gene expression
that is involved in cell survival and apoptosis [1,55].
Of special interest, our studies demonstrate that 15d-PGJ2
is more toxic to early stage OP cells than to their more
differentiated counterpart. The higher resistance that mature
oligodendrocytes display has been reported previously in
response to lysophosphatidic acid , IFNγ ,
cysteine deprivation and hydrogen peroxide (H2O2)
treatment [42,44]. Although the death mechanisms may be
different, they all include oxidative stress and suggest that
mature oligodendrocytes have a better system to fend off
oxidative stress. While GSH may be more directly
involved in providing protection against oxidative stress,
mature oligodendrocytes do not in fact have a higher GSH
level than immature oligodendrocytes . In this regard,
maturational up-regulation of glutathione peroxidase
, π-glutathione-S-transferase  and L-PGDS (also a
GST)  in oligodendrocytes, may contribute to more
effective removal of electrophilic molecules such as
While our studies has demonstrated that 15d-PGJ2 is
cytotoxic to OP cells, we are aware that 15d-PGJ2 can have
effects on other cells that contribute to the demyelination
and remyelination process. Several previous cell culture
studies have shown that 15d-PGJ2 can inhibit activation
of microglia [25,58,59] and astrocytes , and is toxic to
microglia depending on its concentration . Moreover,
systemic application of 15d-PGJ2 has been shown to be
protective in the rodent EAE model [61-63], which is
consistent with its inhibitory effect on microglia and immune
cells. However, the beneficial effect due to microglia
inhibition and toxic effect on OP cells are not mutually
exclusive. The pathological role of 15d-PGJ2 in vivo, therefore,
remains to be better defined and likely will depend on the
level of endogenous 15d-PGJ2 production. Further
investigations, taking into consideration the interaction
between producer and recipient cells in a defined brain
area, are clearly warranted to elucidate the role of
15dPGJ2 in vivo.
In conclusion, we found that PGE2 and PGF2α have
minimal effects on the growth and survival of mOP cells,
while PGD2 and 15d-PGJ2 induce apoptosis at low
micromolar concentrations independently of membrane
receptors for PGD2 and the nuclear receptor PPARγ. The
cytotoxicity of 15d-PGJ2 on mOP cells is associated with
the production of ROS, and affected by manipulations of
intracellular glutathione level but not by the free radical
scavengers ascorbic acid or α-tocopherol. Additionally,
15d-PGJ2 is more toxic to early stage OP cells than to
differentiated OP cells. Taken together, these results suggest
that 15d-PGJ2 may represent a deleterious factor in the
natural remyelination process in MS.
ZX and TL performed the experiments. ZX and SAR
conceived the project and drafted the manuscript. All authors
have read and approved the final version.
We would like to thank Dr. W. Stallcup for NG2 antibody, Dr. I. Duncan
for the CG4 oligodendrocyte cell line and Dr. van Echten-Deckert for the
B104 neuroblastoma cell line.
Consilvio C , Vincent AM , Feldman EL : Neuroinflammation, COX2, and ALS--a dual role ? Exp Neurol 2004 , 187 ( 1 ): 1 - 10 .
Monneret G , Li H , Vasilescu J , Rokach J , Powell WS : 15 - Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils . J Immunol 2002 , 168 ( 7 ): 3563 - 3569 .
Kliewer SA , Lenhard JM , Willson TM , Patel I , Morris DC , Lehmann JM : A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation . Cell 1995 , 83 ( 5 ): 813 - 819 .
Debril MB , Renaud JP , Fajas L , Auwerx J : The pleiotropic functions of peroxisome proliferator-activated receptor gamma . J Mol Med 2001 , 79 ( 1 ): 30 - 47 .
5. Walczak R , Tontonoz P : PPARadigms and PPARadoxes: expanding roles for PPARgamma in the control of lipid metabolism . J Lipid Res 2002 , 43 ( 2 ): 177 - 186 .
6. Rohn TT , Wong SM , Cotman CW , Cribbs DH : 15 - deoxydelta12 , 14 - prostaglandin J2, a specific ligand for peroxisome proliferator-activated receptor-gamma, induces neuronal apoptosis . Neuroreport 2001 , 12 ( 4 ): 839 - 843 .
7. Yagami T , Ueda K , Asakura K , Takasu N , Sakaeda T , Itoh N , Sakaguchi G , Kishino J , Nakazato H , Katsuyama Y , Nagasaki T , Okamura N , Hori Y , Hanasaki K , Arimura A , Fujimoto M : Novel binding sites of 15- deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons . Exp Cell Res 2003 , 291 ( 1 ): 212 - 227 .
8. Bishop-Bailey D , Hla T : Endothelial cell apoptosis induced by the peroxisome proliferator-activated receptor (PPAR) ligand 15-deoxy-Delta12, 14-prostaglandin J2 . J Biol Chem 1999 , 274 ( 24 ): 17042 - 17048 .
9. Li L , Tao J , Davaille J , Feral C , Mallat A , Rieusset J , Vidal H , Lotersztajn S : 15 -deoxy-Delta 12, 14 - prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors . J Biol Chem 2001 , 276 ( 41 ): 38152 - 38158 .
10. Ward C , Dransfield I , Murray J , Farrow SN , Haslett C , Rossi AG : Prostaglandin D2 and its metabolites induce caspasedependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism . J Immunol 2002 , 168 ( 12 ): 6232 - 6243 .
11. Fukushima M , Kato T , Narumiya S , Mizushima Y , Sasaki H , Terashima Y , Nishiyama Y , Santoro MG : Prostaglandin A and J: antitumor and antiviral prostaglandins . Adv Prostaglandin Thromboxane Leukot Res 1989 , 19 : 415 - 418 .
12. Steinman L , Martin R , Bernard C , Conlon P , Oksenberg JR : Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy . Annu Rev Neurosci 2002 , 25 : 491 - 505 .
13. Wolswijk G : Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis . Brain 2000 , 123 ( Pt 1): 105 - 115 .
14. Zamvil SS , Steinman L : Diverse targets for intervention during inflammatory and neurodegenerative phases of multiple sclerosis . Neuron 2003 , 38 ( 5 ): 685 - 688 .
15. Levine JM , Reynolds R , Fawcett JW : The oligodendrocyte precursor cell in health and disease . Trends Neurosci 2001 , 24 ( 1 ): 39 - 47 .
16. Ruffini F , Kennedy TE , Antel JP : Inflammation and remyelination in the central nervous system: a tale of two systems . Am J Pathol 2004 , 164 ( 5 ): 1519 - 1522 .
17. De Keyser J , Zeinstra E , Frohman E : Are astrocytes central players in the pathophysiology of multiple sclerosis? Arch Neurol 2003 , 60 ( 1 ): 132 - 136 .
18. Holley JE , Gveric D , Newcombe J , Cuzner ML , Gutowski NJ : Astrocyte characterization in the multiple sclerosis glial scar . Neuropathol Appl Neurobiol 2003 , 29 ( 5 ): 434 - 444 .
19. Martino G , Adorini L , Rieckmann P , Hillert J , Kallmann B , Comi G , Filippi M : Inflammation in multiple sclerosis: the good, the bad, and the complex . Lancet Neurol 2002 , 1 ( 8 ): 499 - 509 .
20. Benveniste EN : Cytokine actions in the central nervous system . Cytokine Growth Factor Rev 1998 , 9 ( 3-4 ): 259 - 275 .
21. Minghetti L , Levi G : Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide . Prog Neurobiol 1998 , 54 ( 1 ): 99 - 125 .
22. Murphy S , Pearce B , Jeremy J , Dandona P : Astrocytes as eicosanoid-producing cells . Glia 1988 , 1 ( 4 ): 241 - 245 .
23. Tanaka K , Ogawa K , Sugamura K , Nakamura M , Takano S , Nagata K : Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets . J Immunol 2000 , 164 ( 5 ): 2277 - 2280 .
24. Gebicke-Haerter PJ , Bauer J , Schobert A , Northoff H : Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells . J Neurosci 1989 , 9 ( 1 ): 183 - 194 .
25. Bernardo A , Ajmone-Cat MA , Levi G , Minghetti L : 15 - deoxydelta12 , 14 - prostaglandin J2 regulates the functional state and the survival of microglial cells through multiple molecular mechanisms . J Neurochem 2003 , 87 ( 3 ): 742 - 751 .
26. Grill M , Peskar BA , Schuligoi R , Amann R : Systemic inflammation induces COX-2 mediated prostaglandin D2 biosynthesis in mice spinal cord . Neuropharmacology 2006 , 50 ( 2 ): 165 - 173 .
27. Mouihate A , Boisse L , Pittman QJ : A novel antipyretic action of 15-deoxy-Delta12,14-prostaglandin J2 in the rat brain . J Neurosci 2004 , 24 ( 6 ): 1312 - 1318 .
28. Kalyvas A , David S : Cytosolic phospholipase A2 plays a key role in the pathogenesis of multiple sclerosis-like disease . Neuron 2004 , 41 ( 3 ): 323 - 335 .
29. Rose JW , Hill KE , Watt HE , Carlson NG : Inflammatory cell expression of cyclooxygenase-2 in the multiple sclerosis lesion . J Neuroimmunol 2004 , 149 ( 1-2 ): 40 - 49 .
30. Chabas D , Baranzini SE , Mitchell D , Bernard CC , Rittling SR , Denhardt DT , Sobel RA , Lock C , Karpuj M , Pedotti R , Heller R , Oksenberg JR , Steinman L : The influence of the proinflammatory cytokine , osteopontin, on autoimmune demyelinating disease. Science 2001 , 294 ( 5547 ): 1731 - 1735 .
31. Mohri I , Taniike M , Taniguchi H , Kanekiyo T , Aritake K , Inui T , Fukumoto N , Eguchi N , Kushi A , Sasai H , Kanaoka Y , Ozono K , Narumiya S , Suzuki K , Urade Y : Prostaglandin D2-mediated microglia/ astrocyte interaction enhances astrogliosis and demyelination in twitcher . J Neurosci 2006 , 26 ( 16 ): 4383 - 4393 .
32. Taniike M , Mohri I , Eguchi N , Beuckmann CT , Suzuki K , Urade Y : Perineuronal oligodendrocytes protect against neuronal apoptosis through the production of lipocalin-type prostaglandin D synthase in a genetic demyelinating model . J Neurosci 2002 , 22 ( 12 ): 4885 - 4896 .
33. Lin T , Xiang Z , Cui L , Stallcup W , Reeves SA : New mouse oligodendrocyte precursor (mOP) cells for studies on oligodendrocyte maturation and function . J Neurosci Methods 2006 , 157 ( 2 ): 187 - 194 .
34. Louis JC , Magal E , Muir D , Manthorpe M , Varon S : CG-4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type2 astrocytes . J Neurosci Res 1992 , 31 ( 1 ): 193 - 204 .
35. Mosmann T : Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays . J Immunol Methods 1983 , 65 ( 1-2 ): 55 - 63 .
36. Gavrieli Y , Sherman Y , Ben-Sasson SA : Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation . J Cell Biol 1992 , 119 ( 3 ): 493 - 501 .
37. Couldwell WT , Hinton DR , He S , Chen TC , Sebat I , Weiss MH , Law RE : Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines . FEBS Lett 1994 , 345 ( 1 ): 43 - 46 .
38. Kondo M , Shibata T , Kumagai T , Osawa T , Shibata N , Kobayashi M , Sasaki S , Iwata M , Noguchi N , Uchida K : 15 - Deoxy-Delta ( 12 ,14)- prostaglandin J( 2 ) : the endogenous electrophile that induces neuronal apoptosis . Proc Natl Acad Sci U S A 2002 , 99 ( 11 ): 7367 - 7372 .
39. Shibata T , Yamada T , Ishii T , Kumazawa S , Nakamura H , Masutani H , Yodoi J , Uchida K : Thioredoxin as a molecular target of cyclopentenone prostaglandins . J Biol Chem 2003 , 278 ( 28 ): 26046 - 26054 .
40. Yan CY , Greene LA : Prevention of PC12 cell death by N-acetylcysteine requires activation of the Ras pathway . J Neurosci 1998 , 18 ( 11 ): 4042 - 4049 .
41. Griffith OW : Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis . J Biol Chem 1982 , 257 ( 22 ): 13704 - 13712 .
42. Back SA , Gan X , Li Y , Rosenberg PA , Volpe JJ : Maturation-dependent vulnerability of oligodendrocytes to oxidative stressinduced death caused by glutathione depletion . J Neurosci 1998 , 18 ( 16 ): 6241 - 6253 .
43. Baerwald KD , Popko B : Developing and mature oligodendrocytes respond differently to the immune cytokine interferon-gamma . J Neurosci Res 1998 , 52 ( 2 ): 230 - 239 .
44. Baud O , Greene AE , Li J , Wang H , Volpe JJ , Rosenberg PA : Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes . J Neurosci 2004 , 24 ( 7 ): 1531 - 1540 .
45. Dawson J , Hotchin N , Lax S , Rumsby M : Lysophosphatidic acid induces process retraction in CG-4 line oligodendrocytes and oligodendrocyte precursor cells but not in differentiated oligodendrocytes . J Neurochem 2003 , 87 ( 4 ): 947 - 957 .
46. Qu WM , Huang ZL , Xu XH , Aritake K , Eguchi N , Nambu F , Narumiya S , Urade Y , Hayaishi O : Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep . Proc Natl Acad Sci U S A 2006 , 103 ( 47 ): 17949 - 17954 .
47. Jabaudon D , Shimamoto K , Yasuda-Kamatani Y , Scanziani M , Gahwiler BH , Gerber U : Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin . Proc Natl Acad Sci U S A 1999 , 96 ( 15 ): 8733 - 8738 .
48. Lin TN , Cheung WM , Wu JS , Chen JJ , Lin H , Chen JJ , Liou JY , Shyue SK , Wu KK : 15d-prostaglandin J2 protects brain from ischemia-reperfusion injury . Arterioscler Thromb Vasc Biol 2006 , 26 ( 3 ): 481 - 487 .
49. Murphy RC , Zarini S : Glutathione adducts of oxyeicosanoids . Prostaglandins Other Lipid Mediat 2002 , 68 - 69 : 471 - 482 .
50. Li Z , Jansen M , Ogburn K , Salvatierra L , Hunter L , Mathew S , Figueiredo-Pereira ME : Neurotoxic prostaglandin J2 enhances cyclooxygenase-2 expression in neuronal cells through the p38MAPK pathway: A death wish ? J Neurosci Res 2004 , 78 ( 6 ): 824 - 836 .
51. Offen D , Ziv I , Sternin H , Melamed E , Hochman A : Prevention of dopamine-induced cell death by thiol antioxidants: possible implications for treatment of Parkinson's disease . Exp Neurol 1996 , 141 ( 1 ): 32 - 39 .
52. Ray DM , Bernstein SH , Phipps RP : Human multiple myeloma cells express peroxisome proliferator-activated receptor gamma and undergo apoptosis upon exposure to PPARgamma ligands . Clin Immunol 2004 , 113 ( 2 ): 203 - 213 .
53. Rossi A , Kapahi P , Natoli G , Takahashi T , Chen Y , Karin M , Santoro MG : Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase . Nature 2000 , 403 ( 6765 ): 103 - 108 .
54. Perez-Sala D , Cernuda-Morollon E , Canada FJ : Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Delta 12,14-prostaglandin J2 . J Biol Chem 2003 , 278 ( 51 ): 51251 - 51260 .
55. Shaulian E , Karin M : AP-1 as a regulator of cell life and death . Nat Cell Biol 2002 , 4 ( 5 ): E131 - 6 .
56. Tansey FA , Cammer W : A pi form of glutathione-S-transferase is a myelin- and oligodendrocyte-associated enzyme in mouse brain . J Neurochem 1991 , 57 ( 1 ): 95 - 102 .
57. Urade Y , Hayaishi O : Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase . Biochim Biophys Acta 2000 , 1482 ( 1- 2 ): 259 - 271 .
58. Kitamura Y , Kakimura J , Matsuoka Y , Nomura Y , Gebicke-Haerter PJ , Taniguchi T : Activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibit inducible nitric oxide synthase expression but increase heme oxygenase-1 expression in rat glial cells . Neurosci Lett 1999 , 262 ( 2 ): 129 - 132 .
59. Petrova TV , Akama KT , Van Eldik LJ : Cyclopentenone prostaglandins suppress activation of microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-Delta12,14-prostaglandin J2 . Proc Natl Acad Sci U S A 1999 , 96 ( 8 ): 4668 - 4673 .
60. Zhao ML , Brosnan CF , Lee SC : 15 -deoxy-delta ( 12 ,14)- PGJ2 inhibits astrocyte IL-1 signaling: inhibition of NF-kappaB and MAP kinase pathways and suppression of cytokine and chemokine expression . J Neuroimmunol 2004 , 153 ( 1-2 ): 132 - 142 .
61. Diab A , Deng C , Smith JD , Hussain RZ , Phanavanh B , Lovett-Racke AE , Drew PD , Racke MK : Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis . J Immunol 2002 , 168 ( 5 ): 2508 - 2515 .
62. Storer PD , Xu J , Chavis JA , Drew PD : Cyclopentenone prostaglandins PGA2 and 15-deoxy-delta12,14 PGJ2 suppress activation of murine microglia and astrocytes: implications for multiple sclerosis . J Neurosci Res 2005 , 80 ( 1 ): 66 - 74 .
63. Natarajan C , Bright JJ : Peroxisome proliferator-activated receptor-gamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation . Genes Immun 2002 , 3 ( 2 ): 59 - 70 .