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Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cieutí Ficas-Universidad Complutense de Madrid), Facultad de Farmacia, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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induces apoptosis through the synthesis of high concentrations
of NO due to the expression of NO synthase-2. In addition to NO,
activated macrophages release other molecules involved in the
inflammatory response, such as reactive oxygen intermediates and PGs.
Treatment of macrophages with cyclopentenone PGs, which are
synthesized late in the inflammatory onset, exerted a negative
regulation on cell activation by impairing the expression of genes
involved in host defense, among them NO synthase-2. However, despite
the attenuation of NO synthesis, the percentage of apoptotic cells
increased with respect to activated cells in the absence of
cyclopentenone PGs. Analysis of the mechanisms by which these PGs
enhanced apoptosis suggested a potentiation of superoxide anion
synthesis that reacted with NO, leading to the formation of higher
concentrations of peroxynitrite, a more reactive and proapoptotic
molecule than the precursors. The effect of the cyclopentenone
15-deoxy-
12,14-PGJ2 on superoxide synthesis
was dependent on p38 mitogen-activated protein kinase activity, but was
independent of the interaction with peroxisomal proliferator-activated
receptor . The potentiation of apoptosis induced by cyclopentenone
PGs involved an increase in the release of cytochrome c
from the mitochondria to the cytosol and in the nitration of this
protein. These results suggest a role for cyclopentenone PGs in the
resolution of inflammation by inducing apoptosis of activated
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Resolution of inflammation is accomplished by the presence of
anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13)
(8) and by the negative regulation of the activation
process exerted by some of the effector molecules released by activated
macrophages, in particular ROI, RNI, and cyclopentenone PGs
(15, 16, 17, 18, 19). At the end of the inflammatory response, the
cells that have participated in the process are removed by apoptosis
(12, 20, 21, 22, 23). Induction of apoptosis in activated
macrophages has been recognized as a physiological and altruistic
mechanism that helps to reduce the inflammatory stress and to avoid the
establishment of chronic persistent infection by intracellular
pathogens (3, 13, 24). The contribution of NO to trigger
apoptosis in macrophages has been well established (12, 23). The elevated synthesis of NO due to the expression of NOS-2
releases mitochondrial mediators that initiate caspase activation,
leading to a characteristic apoptotic death (25, 26, 27).
However, in several in vivo models of inflammation the synthesis of
anti-inflammatory PGs has been described, namely
15-deoxy-12,14-PGJ2
(15dPGJ2) and related cyclopentenone PGs
(17), that exert their effects through the inhibition of
I
B kinase (IKK2) and activation of the peroxisomal
proliferator-activated receptor (PPAR) (15, 19, 28, 29). According to these data, in the presence of
15dPGJ2 the synthesis of NO is significantly
reduced, and therefore, the intrinsic apoptotic capacity of NO might be
compromised. To address this apparent controversy we have investigated
in this work the action of anti-inflammatory PGs on the apoptotic
response in macrophages activated with LPS/IFN-. Under these
conditions, an important inhibition of NOS-2 expression was observed
due to the presence of 15dPGJ2. However, the
percentage of apoptotic cells increased significantly. As our data
show, the likely mechanism by which cyclopentenone PGs favor apoptosis
is through an important increase in the synthesis of ROI that react
immediately with NO, leading to the formation of peroxynitrite, a more
potent and efficient inductor of apoptosis than NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained from Roche (Mannheim, Germany), Calbiochem (Darmstadt, Germany), and Sigma (St. Louis, MO). Peptides were purchased from Bachem (Bubendorf, Switzerland). Materials and chemicals for electrophoresis were obtained from Bio-Rad (Richmond, CA). Potential-sensitive fluorescent probes were purchased from Molecular Probes (Eugene, OR). Abs were obtained from PharMingen (San Diego, CA) and New England Biolabs (Beverly, MA). Culture media were obtained from BioWhittaker (Verviers, Belgium).
Cell culture conditions
The murine macrophage cell line RAW 264.7 was cultured
(6–8 x 104
cells/cm2) in RPMI 1640 medium supplemented with
2 mM glutamine, 10% FCS, and antibiotics (50 µg/ml of penicillin,
streptomycin, and gentamicin). After 2 days, the cell layers were
washed with PBS, and the culture medium was replaced with phenol
red-free RPMI 1640 containing 0.5 mM arginine and 0.5% FCS and
stimulated (30). PGs and rosiglitazone were added 5 min
before activation with LPS and IFN-.
Flow cytometric analysis of apoptosis
Propidium iodide (PI) staining was performed after incubation of the cells with 0.005% PI, following a previously described protocol (30, 31). Cells were carefully resuspended and run in a FACScan cytometer (Becton Dickinson, San Jose, CA) equipped with a 25-mW argon laser. Quantification of the percentage of apoptotic cells was performed using a dot plot of the forward scatter against the PI fluorescence. Cell sorting and analysis of viable and apoptotic populations were performed to confirm the criteria of gating (30).
Flow cytometric analysis of mitochondrial transmembrane potential
m
Cells were incubated for 15 min at 37°C in the presence of the potential-sensitive probes chloromethyl X-rosamine, 3,3'-dihexyloxacarbocyanine iodide, tetrachlorotetraethylbenzimidazolylcarbocyanine iodide, and rhodamine 123, all at 40 nM (25), followed by analysis in a FACScan flow cytometer. The fluorescence in the presence of 10 µM of the uncoupling agent m-chlorophenylhydrazone carbonylcyanide was considered as 100%, and values were calculated as the percent change in fluorochrome fluorescence.
Measurement of ROI synthesis
Cells challenged for the indicated periods of time with different stimuli were incubated for 15 min with 10 µM 2',7'-dichlorofluorescein diacetate (DCFH) or hydroethidine (HE); and the fluorescence corresponding to the oxidized probes was followed by analysis in a flow cytometer as previously described (25, 32). Simultaneous incubation of the cells with the probes and 50 µM t-butyl hydroperoxide was used as a positive control of ROI release.
Synthesis of peroxynitrite
Peroxynitrite (OONO-) was synthesized by reaction
of hydrogen peroxide with nitrous acid as previously described
(33, 34). Briefly, a 0.6 M solution of sodium nitrite was
mixed vigorously with an equal volume of 0.7 N HCl and 0.6 M
H2O2 and immediately
stabilized with 1.2 N NaOH. Unreacted
H2O2 was removed by passing
the solution through an MnO2 column. Appropriate
controls were conducted to ensure that after decomposition of
peroxynitrite at pH 6.0, the reaction mixture did not affect cell
viability. The concentration of peroxynitrite was determined before use
by measuring the absorbance at 302 nm (
= 1670
M-1 cm-1).
Preparation of cytosolic and nuclear protein extracts
RAW 264.7 cells were washed twice with ice-cold buffer A (10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml Tosyl-L-phenylalanine chloromethyl ketone, 5 mM NaF, 1 mM NaV04, and 10 mM Na2MoO4) containing 120 mM NaCl and scraped off the plate. Lysis of the cells was performed at 4°C with 0.2 ml of buffer A supplemented with 0.5% Nonidet P-40 and under continuous shaking. After centrifugation of the cell lysate, the supernatant was stored at -80°C (cytosolic extract), and the pellet was resuspended in 50 µl of buffer A supplemented with 20% glycerol and 0.4 M KCl and gently shaken for 30 min at 4°C. Nuclear protein extracts were obtained by centrifugation at 13,000 x g for 15 min, and the supernatants were stored at -80°C. Protein content was assayed using the Bio-Rad protein reagent. All steps of cell fractionation were conducted at 4°C.
Immunoprecipitation and Western blot analysis
The presence of cytochrome c in the cytosol was
determined in cell extracts prepared as previously described
(26). To evaluate the nitration of cytochrome
c, equal amounts of supernatant protein (50 µg) were
immunoprecipitated with anti-NO-Y Ab (a gift from J. S.
Beckman, University of Alabama, Birmingham, AL) and revealed by Western
blot with anti-cytochrome c mAb (clone
7H8.2C12;PharMingen) following the instructions of the Ab supplier. The
levels of IB
, IB
, and phosphorylated or dephosphorylated
p38, c-Jun N-terminal kinase (JNK), and extracellular- regulated kinase
ERK) 1 and ERK2 were determined by Western blot using cytosolic
extracts and specific commercial Ab (Santa Cruz Biotechnology (Santa
Cruz, CA) and New England Biolabs, respectively). The proteins were
size-separated in 12% SDS-PAGE, transferred to a polyvinylidene
difluoride membrane and incubated with the indicated Ab. The bands
recognized were visualized by the ECL technique.
NF-B mobility shift assay (EMSA)
The consensus sequence of NF-B
(5'-TGCTAGGGGGATTTTCCCTCTCTCTGT-3', corresponding to nucleotides
-978 to -952 of the murine NOS-2 promoter) was used
(19). Aliquots of 50 ng of annealed oligonucleotide were
end-labeled with Klenow enzyme fragment in the presence of 50 µCi of
[-32P]dCTP and the other unlabeled dNTPs in
a final volume of 50 µl. For each binding assay 3 µg of nuclear
protein was incubated for 15 min at 4°C with the DNA (5 x
104 dpm) and 2 µg of poly(dI:dC), 5% glycerol,
1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and
10 mM Tris-HCl (pH 7.8) in a final volume of 20 µl. The DNA-protein
complexes were separated on native 6% polyacrylamide gels in 0.5%
Tris-borate-EDTA buffer. Supershift assays were conducted after
incubation of the nuclear extracts with 2 µg of Ab (anti-p50,
anti-c-Rel, and anti-p65) for 1 h at 4°C, followed by
EMSA (data not shown).
Caspase assay
The DEVDase activity (corresponding mainly to caspases 3 and 7) was determined in cell lysates using N-acetyl-DEVD-7-amino-4-methylcoumarin as fluorogenic substrate and following the instructions of the supplier (PharMingen). The corresponding peptide aldehyde and Z-VAD fluoromethyl ketone were used to inhibit caspase activity in vivo and to ensure the specificity of the reaction in the in vitro assay. The linearity of the caspase assay was determined over a 30-min reaction period.
Statistical analysis
The data shown are the mean ± SEM (n = 3–4). Statistical significance was estimated with Student’s t test for unpaired observations. p < 0.05 was considered significant. In studies using Western blot analysis, linear correlations between increasing amounts of input protein and signal intensity were observed (correlation coefficients >0.84).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of apoptosis in LPS/IFN--activated cells was
dependent on the synthesis of NO (26, 30), since the
selective NOS-2 inhibitor N-(3-aminomethylbenzyl)acetamidine
(1400W) abolished this effect (Fig. 1
A). Treatment of cells with
up to 2 µM 15dPGJ2 did not affect cell
viability, but significantly increased the apoptosis induced by
LPS/IFN- (100 ng/ml and 5 U/ml, respectively). However, the
dose-dependent apoptosis elicited by 15dPGJ2
in LPS/IFN--activated cells was in parallel with an inhibition
of NOS-2 expression and, therefore, of NO synthesis (Fig. 1, B and C). Apoptosis was measured by flow
cytometry (Fig. 1D). Moreover, this apoptosis remained
dependent on the synthesis of NO, since it was suppressed efficiently
by 1400W. These results suggest that in activated cells treated with
15dPGJ2 apoptosis was dependent on NO synthesis
and on another molecule produced in response to PG challenge. Similar
results were obtained using elicited peritoneal macrophages.
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-activated
cells. However, the percentage of apoptotic cells increased under these
conditions, suggesting a minor contribution of endogenous
15dPGJ2 to resolution of inflammation (Table I).
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plus
15dPGJ2 condition) regardless of the probe used
(Fig. 2A), confirming previous
results in these cells (26). Moreover, in cells treated
with 15dPGJ2 and LPS/IFN- a 3.7-fold increase
in cytochrome c in the cytosol was measured with respect to
cells incubated with LPS/IFN- (Fig. 2B). Analysis of NO
modifications of cytosolic cytochrome c showed the presence
of nitrotyrosine in activated cells incubated with
15dPGJ2.
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involved a 2.2-fold increase in DEVDase activity,
presumably due to caspase 3 activation, that was inhibited efficiently
after treatment with aldehyde (DEVD.CHO). Indeed, a more general
caspase inhibitor such as z-VAD prevented apoptosis to similar levels
as did DEVD.CHO (Fig. 3).
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activation
15dPGJ2 and related cyclopentenone PGs have
been described as agonists of PPAR (15, 35, 36). To
determine whether the effect of 15PGJ2 was due to
the engagement of this nuclear receptor, the actions of several PGs and
structurally unrelated PPAR ligands were investigated. As Fig. 4A shows, among the PGs
assayed, only 15dPGJ2 and
PGA2 potentiated apoptosis. Treatment of
activated RAW 264.7 cells with 10 µM of the PPAR ligand
rosiglitazone did not exert any effect on apoptosis, suggesting that
cyclopentenone PGs were acting through a mechanism distinct from
PPAR ligation. Moreover, the RAW cells used failed to express
PPAR upon challenge with LPS/IFN- as did cultured peritoneal
macrophages under similar experimental conditions (Fig. 4B).
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To further investigate the mechanisms by which
15dPGJ2 enhanced apoptosis in
LPS/IFN--activated cells, we analyzed the release of ROI and RNI by
following the fluorescence corresponding to the oxidation of HE and
DCFH. As Fig. 5A shows, the
oxidation of HE, reflecting mainly the synthesis of
O2-, increased moderately in
LPS/IFN--activated cells treated with 15dPGJ2.
When NO synthesis was transiently suppressed after inhibition of NOS-2
with 1400W, a further increase in HE oxidation was observed, indicating
an overproduction of O2-.
However, the ability O2- to
oxidize HE was lost in the presence of NO, probably due to the rapid
reaction between both reactive species producing peroxynitrite (see
Discussion). Regarding DCFH fluorescence, a time-dependent
increase was observed in cells treated with
15dPGJ2 and LPS/IFN-, with an apparent steady
state rate of oxidation after 8 h of stimulation (Fig. 5B). These kinetics paralleled the rate of NO synthesis due
to the expression of NOS-2.
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). Treatment of cells with
ebselen, which reacts with ROI and NO, abrogated DCFH oxidation and
apoptosis (37). Inhibition of NOS-2 with 1400W
significantly decreased the oxidation of DCFH and prevented apoptosis.
Incubation of cells with 2,3-dimethoxy-1,4-naphtoquinone (DMNQ), which
released O2- in solution,
enhanced the fluorescence of DCFH in LPS/IFN--activated cells, but
not in resting macrophages, suggesting that an
O2--derived metabolite was the
main molecule responsible for DCFH oxidation (Fig. 6A). When
cells were incubated with Mn3+-tetrakis(4-benzoic
acid)porphyrin, a permeant porphyrine that reduces
O2-, a significant, but not
complete, decrease in DCFH fluorescence was observed (data not shown).
These results together with the observation of an increase in
cytochrome c nitration (Fig. 2
B) as well as
previous reports on DCFH oxidation (38, 39) suggest that
peroxynitrite was the main contributor to the changes in DCFH
fluorescence in activated macrophages treated with
15dPGJ2. In agreement with these data, incubation
of RAW 264.7 cells with substances that release NO and
O2- in solution enhanced DCFH
oxidation. As Fig. 6B shows, incubation of cells with
S-nitrosogluthathione (GSNO) and DMNQ significantly
increased the oxidation of DCFH and promoted apoptosis. Indeed, when
cells were treated with the radical scavenger ebselen, the changes in
fluorescence of DCFH and apoptosis were abolished. Moreover, incubation
of cells with peroxynitrite promoted dose-dependent parallel changes in
DCFH fluorescence and apoptosis (Fig. 6C).
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Since addition of 15dPGJ2 4 h or more
later than LPS/IFN- challenge did not affect the percentage of
apoptotic cells (data not shown), we investigated potential early
signaling pathways involved in the enhancement of
O2- synthesis elicited by this
PG, among them the phosphorylation state of members of the MAPK family
and the activation of NF-B. As Table II shows, inhibition of the p38 MAPK with
SB203580 or SB202190 significantly reduced DCFH oxidation and apoptosis
(65 and 75%, respectively), whereas the inactive analogue SB202474
failed to affect these parameters. Inhibition of the MAP/ERK kinase/ERK
pathway with PD98059 (50 µM) did not modify these parameters.
Moreover, the phosphorylation state of p38, ERK1 and 2, and JNK was
investigated by Western blot. Treatment of LPS/IFN--activated cells
with 15dPGJ2 did not affect ERK1 and 2
phosphorylation, but exerted a moderate inhibition of p38 (31%) and
JNK (17%) phosphorylation (Fig. 7A). The other likely
candidate to be affected by 15dPGJ2 in
LPS/IFN- activated cells was NF-B (18, 19, 28). As
Fig. 7B shows, activated RAW 264.7 cells exhibited a
significant reduction of NF-B activity when incubated with
15dPGJ2 and a complete inhibition in the presence
of ebselen. The cytosolic levels of IB and IB inversely
paralleled the activation of NF-B as determined by EMSA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, apoptosis was very dependent on the synthesis of NO, probably
because the synergism between both stimuli resulted in efficient NOS-2
expression and a high output of NO, minimizing the contribution of
other proapoptotic pathways. Under these conditions, cyclopentenone PGs
significantly inhibited NOS-2 expression due to the inactivation of
IKK2 and the transcriptional inhibition dependent on PPAR activation
(15, 19, 28). In RAW 264.7 cells, however, the effects of
15dPGJ2 cannot be attributed to PPAR activity,
since this protein was undetectable by Western blot, and rosiglitazone,
a PPAR ligand, failed to mimic the action of cyclopentenone PGs.
Moreover, in other cell types, 15dPGJ2 and
PPAR ligands exerted apoptotic effects independent of
proinflammatory stimulation; in human monocyte-derived macrophages
activation of PPAR, but not PPAR, was sufficient to induce
apoptosis, although the activation of caspases (in particular, caspase
3) was more efficient when cells were treated with TNF- and IFN-
(41). In human vascular endothelial cells, PPAR
activation by 15dPGJ2 or ciglitizone was also
sufficient to induce apoptosis (42). Contrary to this
situation, exogenously added 15dPGJ2 induced
proliferation of COX-2-depleted colorectal cancer cells (HCA-7)
(43), indicating that the effects of these molecules are
very dependent on the cell type. Regarding the mechanism by which
15dPGJ2 enhanced apoptosis in RAW 264.7 cells, we
analyzed the changes in m and the release of cytochrome
c to the cytosol under these conditions. Our data show that
m was maintained or even increased after treatment with
15dPGJ2, indicating the involvement of a pathway
similar to that described for the apoptosis dependent on high
concentrations of NO (26). Interestingly, the amount of
nitrated cytochrome c increased upon treatment with PGs,
suggesting an improved efficiency in the synthesis of peroxynitrite.
Indeed, previous studies indicated that nitration of cytochrome
c was an early event in the release of this protein from the
mitochondria and preceded the changes in m (26, 44).
The remarkable potentiation of DCFH oxidation elicited by
15dPGJ2 in LPS/IFN- activated cells provides
an important clue to understand the observed enhancement of apoptosis.
The pathways involved in the increase in
O2- synthesis and the
mechanisms used by the cells to sense oxidative stress are a subject of
current research (4). Recently, it has been reported that
p66shc, a protein containing a Src homology 2
domain and involved in the transmission of mitogenic signals from
membrane receptors to Ras, acts as a sensor of oxidative stress,
becoming phosphorylated in Ser and initiating an as yet uncharacterized
signaling pathway that regulates oxidative stress and life span in
mammalian cells (45). In addition to this, various reports
describe a cross-talk between ROI signaling through the MAPK pathways
and activation of transcription factors implicated in early gene
regulation (NF-B and AP-1), the balance of them influencing cell
viability (4, 18, 46). The enzymes of the MAPK pathways,
p38, ERK1 and 2, and JNK, are transiently activated in LPS-stimulated
macrophages (5, 6). Using inhibitors of some of these MAPK
pathways, we identified an important contribution of p38 activity to
the synthesis of ROI that participate in the induction of apoptosis in
RAW 264.7 cells. Our data show a moderate inhibitory effect by
15dPGJ2 (<36%) on LPS/IFN--dependent
activation of p38 and JNK, but not on the ERK1 and 2 activities. In the
same vein, in cardiac myocytes deficient of MEK kinase it has been
shown that p38 constitutes an important step for apoptotic ROI
signaling (47). Alternatively, overexpression of catalase
and superoxide dismutase or decomposition of
O2- with permeant porphyrines
protected cells from ROS injury (48). Therefore, the
potential regulatory effects of
O2- on the balance between
viability and apoptosis of the macrophage are complex. Priming of RAW
264.7 cells with low concentrations (5 µM) of the
O2- donor DMNQ protected
against NO-dependent cell death when the apoptotic stimulation was
performed 15 h after DMNQ challenge. In this case, the mechanism
involved an impaired activation of NF-B and AP-1 compared with
nonprimed cells (49). The experiments reported in this
work provide the characterization of an additional mechanism of
potentiation of apoptosis in activated macrophages through the reaction
of NO and O2- to yield the
potent oxidant peroxynitrite (14, 50, 51). Regarding
NF-B activation by ROI as a sensor of oxidative stress in
macrophages (4, 18), a paradoxical situation appears when
the synthesis of O2- is
accomplished by cyclopentenone PGs; the inhibitory effect of these
lipids on IKK activity, and therefore on IB phosphorylation, results
in an impaired NF-B activation.
Moreover, in the presence of a potent radical scavenger such as
ebselen, ROI synthesis and NF-B activity were inhibited, and
apoptosis was completely abrogated. Although these data suggest that
inhibition of NF-B activity is not sufficient to promote apoptosis
in cells treated with ebselen, it cannot be excluded that the
impairment of NF-B activity exerted by 15dPGJ2
might contribute to some extent to apoptosis. To summarize our data, a
schematic representation of the concerted mechanism involving the
synthesis of peroxynitrite from NO and
O2- is shown in Fig. 8.
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The physiopathological relevance of the synthesis of cyclopentenone PGs can be deduced from models of carrageenin-induced inflammation in which a two-phase PG release has been described after expression of COX-2. PGE2 synthesis predominates during the early inflammatory step, whereas 15dPGJ2 substitutes PGE2 formation at the end of the process, coincident with the accumulation of macrophages (16, 17). Therefore, cyclopentenone PGs, in addition to their characterization as anti-inflammatory molecules, can be viewed as important factors in the resolution of the inflammatory process through the potentiation of apoptosis (24). Attempts to determine the contribution to apoptosis of the endogenous synthesis of 15dPGJ2 by activated RAW 264.7 cells treated with the COX-2 inhibitor NS398 resulted in an enhancement of cell death due to an up-regulation of NOS-2 expression. Moreover, these data reveal a moderate contribution of macrophages to the synthesis of 15dPGJ2 during the resolution of inflammation, suggesting a paracrine, rather than autocrine, regulatory loop for this PG.
In summary, our data stress the relevance of cooperative signaling leading to the synthesis of superoxide and NO to favor apoptosis. This mechanism is especially relevant at the end of the inflammatory process, when cyclopentenone PGs are produced and the synthesis of NO decreases due to the action of negative regulatory pathways (among them NO itself) and to arginine exhaustion. In this way, pharmacological modulation of the NOS-2 and COX-2 pathways can provide additional tools to regulate the balance between apoptosis and cell viability and, therefore, to control the inflammatory process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lisardo Boscá, Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain.
3 Abbreviations used in this paper: ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates; 1400W, N-(3-aminomethylbenzyl)acetamidine; COX-2, cyclooxygenase-2; 15dPGJ2, 15-deoxy-12,14-PGJ2; DCFH, 2',7'-dichlorofluorescein diacetate; DMNQ, 2,3-dimethoxy-1,4-naphtoquinone; ERK, extracellular-regulated kinase; HE, hydroethidine; IKK, IB kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NOS-2, NO synthase-2; PPAR, peroxisomal proliferator-activated receptor ; PI, propidium iodide; m, mitochondrial transmembrane potential; DEVD.CHO, aldehyde; GSNO, S-nitrosogluthathione.
Received for publication May 1, 2000. Accepted for publication September 5, 2000.
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E. S. Musiek, L. Gao, G. L. Milne, W. Han, M. B. Everhart, D. Wang, M. G. Backlund, R. N. DuBois, G. Zanoni, G. Vidari, et al. Cyclopentenone Isoprostanes Inhibit the Inflammatory Response in Macrophages J. Biol. Chem., October 21, 2005; 280(42): 35562 - 35570. [Abstract] [Full Text] [PDF]
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 E. B. E. Berry, J. A. Keelan, R. J. A. Helliwell, R. S. Gilmour, and M. D. Mitchell Nanomolar and Micromolar Effects of 15-Deoxy-{Delta}12,14-prostaglandin J2 on Amnion-Derived WISH Epithelial Cells: Differential Roles of Peroxisome Proliferator-Activated Receptors {gamma} and {delta} and Nuclear Factor {kappa}B Mol. Pharmacol., July 1, 2005; 68(1): 169 - 178. [Abstract] [Full Text] [PDF]
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 M. Pignatelli, J. Sanchez-Rodriguez, A. Santos, and A. Perez-Castillo 15-Deoxy-{Delta}-12,14-prostaglandin J2 induces programmed cell death of breast cancer cells by a pleiotropic mechanism Carcinogenesis, January 1, 2005; 26(1): 81 - 92. [Abstract] [Full Text] [PDF]
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Q. Si, M.-L. Zhao, A. C. A. Morgan, C. F. Brosnan, and S. C. Lee 15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits IFN-Inducible Protein 10/CXC Chemokine Ligand 10 Expression in Human Microglia: Mechanisms and Implications J. Immunol., September 1, 2004; 173(5): 3504 - 3513. [Abstract] [Full Text] [PDF]
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 A. Mangla, A. Khare, V. Vineeth, N. N. Panday, A. Mukhopadhyay, B. Ravindran, V. Bal, A. George, and S. Rath Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses Blood, August 15, 2004; 104(4): 1191 - 1197. [Abstract] [Full Text] [PDF]
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 R. Grau, M. A. Iniguez, and M. Fresno Inhibition of Activator Protein 1 Activation, Vascular Endothelial Growth Factor, and Cyclooxygenase-2 Expression by 15-Deoxy-{Delta}12,14-Prostaglandin J2 in Colon Carcinoma Cells: Evidence for a Redox-Sensitive Peroxisome Proliferator-Activated Receptor-{gamma}-Independent Mechanism Cancer Res., August 1, 2004; 64(15): 5162 - 5171. [Abstract] [Full Text] [PDF]
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A. Cheron, J. Peltier, J. Perez, A. Bellocq, B. Fouqueray, and L. Baud 15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits Glucocorticoid Binding and Signaling in Macrophages through a Peroxisome Proliferator-Activated Receptor {gamma}-Independent Process J. Immunol., June 15, 2004; 172(12): 7677 - 7683. [Abstract] [Full Text] [PDF]
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 J. F. Maddox, A. C. Domzalski, R. A. Roth, and P. E. Ganey 15-Deoxy Prostaglandin J2 Enhances Allyl Alcohol-Induced Toxicity in Rat Hepatocytes Toxicol. Sci., February 1, 2004; 77(2): 290 - 298. [Abstract] [Full Text] [PDF]
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A. E. Rusinol, D. Thewke, J. Liu, N. Freeman, S. R. Panini, and M. S. Sinensky AKT/Protein Kinase B Regulation of BCL Family Members during Oxysterol-induced Apoptosis J. Biol. Chem., January 9, 2004; 279(2): 1392 - 1399. [Abstract] [Full Text] [PDF]
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D. Perez-Sala, E. Cernuda-Morollon, and F. J. Canada Molecular Basis for the Direct Inhibition of AP-1 DNA Binding by 15-Deoxy-{Delta}12,14-prostaglandin J2 J. Biol. Chem., December 19, 2003; 278(51): 51251 - 51260. [Abstract] [Full Text] [PDF]
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S. Hortelano, P. G. Traves, M. Zeini, A. M. Alvarez, and L. Bosca Sustained Nitric Oxide Delivery Delays Nitric Oxide-Dependent Apoptosis in Macrophages: Contribution to the Physiological Function of Activated Macrophages J. Immunol., December 1, 2003; 171(11): 6059 - 6064. [Abstract] [Full Text] [PDF]
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A. Nencioni, K. Lauber, F. Grunebach, L. Van Parijs, C. Denzlinger, S. Wesselborg, and P. Brossart Cyclopentenone Prostaglandins Induce Lymphocyte Apoptosis by Activating the Mitochondrial Apoptosis Pathway Independent of External Death Receptor Signaling J. Immunol., November 15, 2003; 171(10): 5148 - 5156. [Abstract] [Full Text] [PDF]
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C.-W. Chen, Y.-H. Chang, C.-J. Tsi, and W.-W. Lin Inhibition of IFN-{gamma}-Mediated Inducible Nitric Oxide Synthase Induction by the Peroxisome Proliferator-Activated Receptor {gamma} Agonist, 15-Deoxy-{Delta}12,14-Prostaglandin J2, Involves Inhibition of the Upstream Janus Kinase/STAT1 Signaling Pathway J. Immunol., July 15, 2003; 171(2): 979 - 988. [Abstract] [Full Text] [PDF]
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 O. Belton and D. Fitzgerald Cyclooxygenase-2 inhibitors and atherosclerosis J. Am. Coll. Cardiol., May 21, 2003; 41(10): 1820 - 1822. [Full Text] [PDF]
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M. Cippitelli, C. Fionda, D. Di Bona, A. Lupo, M. Piccoli, L. Frati, and A. Santoni The Cyclopentenone-Type Prostaglandin 15-Deoxy-{Delta}12,14-Prostaglandin J2 Inhibits CD95 Ligand Gene Expression in T Lymphocytes: Interference with Promoter Activation Via Peroxisome Proliferator-Activated Receptor-{gamma}-Independent Mechanisms J. Immunol., May 1, 2003; 170(9): 4578 - 4592. [Abstract] [Full Text] [PDF]
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 J. L. Oliva, D. Perez-Sala, A. Castrillo, N. Martinez, F. J. Canada, L. Bosca, and J. M. Rojas The cyclopentenone 15-deoxy-Delta 12,14-prostaglandin J2 binds to and activates H-Ras PNAS, April 15, 2003; 100(8): 4772 - 4777. [Abstract] [Full Text] [PDF]
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 A. Castrillo, P. G. Traves, P. Martin-Sanz, S. Parkinson, P. J. Parker, and L. Bosca Potentiation of Protein Kinase C {zeta} Activity by 15-Deoxy-{Delta}12,14-Prostaglandin J2 Induces an Imbalance between Mitogen-Activated Protein Kinases and NF-{kappa}B That Promotes Apoptosis in Macrophages Mol. Cell. Biol., February 15, 2003; 23(4): 1196 - 1208. [Abstract] [Full Text] [PDF]
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 C. E. Clay, A. Monjazeb, J. Thorburn, F. H. Chilton, and K. P. High 15-Deoxy-{Delta}12,14-prostaglandin J2-induced apoptosis does not require PPAR{gamma} in breast cancer cells J. Lipid Res., November 1, 2002; 43(11): 1818 - 1828. [Abstract] [Full Text] [PDF]
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F. Bureau, C. Desmet, D. Melotte, F. Jaspar, C. Volanti, A. Vanderplasschen, P.-P. Pastoret, J. Piette, and P. Lekeux A Proinflammatory Role for the Cyclopentenone Prostaglandins at Low Micromolar Concentrations: Oxidative Stress-Induced Extracellular Signal-Regulated Kinase Activation Without NF-{kappa}B Inhibition J. Immunol., May 15, 2002; 168(10): 5318 - 5325. [Abstract] [Full Text] [PDF]
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G. Monneret, H. Li, J. Vasilescu, J. Rokach, and W. S. Powell 15-Deoxy-{Delta}12,1412,14-prostaglandins D2 and J2 Are Potent Activators of Human Eosinophils J. Immunol., April 1, 2002; 168(7): 3563 - 3569. [Abstract] [Full Text] [PDF]
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T. Shibata, M. Kondo, T. Osawa, N. Shibata, M. Kobayashi, and K. Uchida 15-Deoxy-Delta 12,14-prostaglandin J2. A PROSTAGLANDIN D2 METABOLITE GENERATED DURING INFLAMMATORY PROCESSES J. Biol. Chem., March 15, 2002; 277(12): 10459 - 10466. [Abstract] [Full Text] [PDF]
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R. B. Clark The role of PPARs in inflammation and immunity J. Leukoc. Biol., March 1, 2002; 71(3): 388 - 400. [Abstract] [Full Text] [PDF]
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 A.-L. Levonen, D. A. Dickinson, D. R. Moellering, R. T. Mulcahy, H. J. Forman, and V. M. Darley-Usmar Biphasic Effects of 15-Deoxy-{Delta}12,14-Prostaglandin J2 on Glutathione Induction and Apoptosis in Human Endothelial Cells Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1846 - 1851. [Abstract] [Full Text] [PDF]
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 A. Castrillo, D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, and L. Bosca Protein Kinase C{epsilon} Is Required for Macrophage Activation and Defense Against Bacterial Infection J. Exp. Med., October 29, 2001; 194(9): 1231 - 1242. [Abstract] [Full Text] [PDF]
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A. Castrillo, M. Mojena, S. Hortelano, and L. Bosca Peroxisome Proliferator-activated Receptor-gamma -independent Inhibition of Macrophage Activation by the Non-thiazolidinedione Agonist L-796,449. COMPARISON WITH THE EFFECTS OF 15-DEOXY-Delta 12,14-PROSTAGLANDIN J2 J. Biol. Chem., August 31, 2001; 276(36): 34082 - 34088. [Abstract] [Full Text] [PDF]
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E. Cernuda-Morollon, E. Pineda-Molina, F. J. Canada, and D. Perez-Sala 15-Deoxy-Delta 12,14-prostaglandin J2 Inhibition of NF-kappa B-DNA Binding through Covalent Modification of the p50 Subunit J. Biol. Chem., September 14, 2001; 276(38): 35530 - 35536. [Abstract] [Full Text] [PDF]
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