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J Biol Chem, Vol. 275, Issue 4, 2837-2844, January 28, 2000
From the Department of Pharmacology, New York Medical College,
Valhalla, New York 10595
Hypoxic injury provokes inflammation of many
tissues including the ocular surface. In rabbit corneal epithelial
cells, both peroxisome proliferator-activated receptor (PPAR)-inducible
cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were
increased by hypoxia. PPAR Eye closure during sleep as well as contact lens wear creates an
environment that has been described as a state of subclinical inflammation characterized by corneal swelling, conjunctival
vasodilation, and significant levels of polymorphonuclear leukocytes in
the tear film. These changes are attributed to reduced oxygen and carbon dioxide exchange, leading to corneal hypoxia and subsequent acidosis (1).
There exist a host of mediators that have been implicated in the
development and progression of corneal inflammation (2, 3). The source
of these mediators may be the injured tissue or infiltrating
inflammatory cells. Although the progression of an inflammatory
response is generally dependent on the inflammatory infiltrate, its
initiation is usually the result of inflammatory mediators released
from the damaged tissue. Among the endogenous corneal inflammatory
mediators released from the epithelium are the arachidonic acid-derived
eicosanoids, which have been implicated in the initiation, development,
and progression of an inflammatory response (4, 5). The relative
contribution of each eicosanoid to this reaction is unclear. These
eicosanoids are produced by the activity of cyclooxygenase
(COX)1 (primarily
PGE2), lipoxygenase (12(S)-HETE and
leukotrienes, e.g. LTB4) and cytochrome P450
monooxygenase (12(R)-HETE and 12(R)-HETrE) (6).
We have previously demonstrated that 12(R)-HETE and
12(R)-HETrE possess potent inflammatory properties with
12(R)-HETrE being a powerful angiogenic factor and that both
metabolites assume the role of inflammatory mediators in hypoxia- and
alkali-induced injury in the cornea, in vivo (7, 8).
Additional studies using in vitro models of corneal
epithelial injury further support a relationship between injury,
hypoxia, and the production of the CYP-derived eicosanoids (9). Studies
to identify the CYP isoform responsible for the production of
12(R)-HETE and 12(R)-HETrE revealed that a CYP4B1
isoform is readily induced in response to hypoxia in vitro
and in vivo and that antibodies against this isoform
inhibited the synthesis of these eicosanoids (10).
Hypoxia and cellular injury provoke the induction of COX-2 in many cell
types (11, 12). Moreover, induction of COX-2 mRNA and protein
together with increasing levels of prostanoids, mainly PGE2, is the hallmark of inflammation in many tissues (13,
14). Among the factors associated with the transcriptional activation of COX-2 are the PPARs. These are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and
fatty acids, including certain PGs, HETEs, as well as some of the
commonly used non-steroidal anti-inflammatory drugs (NSAIDs) (15, 16).
PPAR ligands activate transcription of COX-2 as well as the
transcription of several CYP4 genes, including CYP4B1 (17-19). Hence,
we carried out studies to determine the involvement of COX-2 in
hypoxia-induced injury of the corneal epithelium and to examine its
relation to PPAR activation and CYP4B1 expression.
RCE Cell Culture--
The RCE cell line was obtained from Dr.
Kaoru Arakai-Sasake (Osaka University, Osaka, Japan) (20). Cells were
grown to confluence in Petri dishes (60 mm) using medium containing a
mixture of Dulbecco's modified Eagle's medium/F-12 (1:1) in the
presence of 10% fetal bovine serum and 1% antibiotic/antimycotic
mixture (Fungizone, Life Technologies, Inc.); 48 h before the
experiment, the medium was changed to Dulbecco's modified Eagle's
medium/F-12 containing 0.4% fetal bovine serum and antibiotics. Cells
were then washed twice with 2 ml of sterile phosphate-buffered saline
and incubated with 2 ml of serum-free medium containing antibiotics
with and without PPAR ligands or COX inhibitors under normoxia (5%
CO2, 95% air (~20% O2)) or in a modular
tissue culture chamber (Billups-Rothenburg, Del Mar, CA) supplied
continuously with 5% CO2, 0% O2, 95%
N2 (hypoxia) and bubbled through deionized H2O
into the chamber within a 37 °C incubator for an additional 2-24 h.
In some experiments, cells were incubated with
L-buthionine-[S,R]sulfoximine (BSO, 1 mM) and maleic acid diethylester (DEM, 1 mM)
for 18 h to deplete intracellular GSH.
Western Blot Analysis--
Protein content was determined using
the DC Protein Assay (Bio-Rad) with bovine serum albumin as the
standard. Cell lysates (30 µg of protein) were mixed with Laemmli
reagent (final concentration 1%, w/v, sodium dodecyl sulfate; 10%,
v/v, glycerol; 0.5%, w/v, bromphenol blue) under reducing conditions
(4%, v/v, Measurements of PGE2 Levels--
At the end of the
incubation period, the medium was collected and stored at Measurements of Cyclooxygenase Activity--
Cells were
incubated under normoxia or hypoxia or treated with WY-14,643 (100 µM) for 12 and 24 h in a serum-free medium. Cells
were then washed twice with phosphate-buffered saline and further
incubated with arachidonic acid (10 µM) in the presence or absence of heme (2 µM), NS398 (1 µM),
indomethacin (5 µM), or GSH (10-50 mM) for
1 h in an oxygenated incubator. PGE2 levels in the
medium were measured by enzyme immunoassay as described above.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Total RNA was isolated with TRIzol reagent (Life
Technologies, Inc.) and quantified spectrophotometerically. RT reaction
was performed with 5 µg of total RNA and an oligo(dT) primer using the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech). For amplification of 28 S rRNA and PPARs ( Northern Analysis--
Total RNA (10 µg) was separated on a
1% agarose gel containing formaldehyde, visualized by ethidium bromide
staining, and transferred overnight to Hybond N plus membranes
(Amersham Pharmacia Biotech) through capillary blotting. The membranes
were probed with 32P-labeled cDNA probes in Rapid-Hyb
hybridization buffer (Amersham Pharmacia Biotech). The rabbit COX-2
cDNA probe was obtained from Dr. M. Breyer, Vanderbilt University
(22). The rabbit 28 S rRNA probe was generated by PCR as described
above. The resulting blots were subjected to autoradiography with NEF
reflection autoradiography films (NEN Life Science Products).
Densitometry analysis was done as described for the Western blot.
Statistical Analysis--
Analysis of variance and Student's
t test for unpaired data were used to evaluate differences
among control and treated samples. A p value less than or
equal to 0.05 was considered significant. Unless stated otherwise,
results are presented as means ± S.E.
COX-2 is induced in many cell types in response to injury, growth
factors and cytokines. In this respect, RCE cells exhibit a similar
response; COX-2 protein levels are markedly increased by 2-3-fold in
response to these stimuli (data not shown). Hypoxic injury is
considered an inflammatory stimulus in many tissues. In RCE cells,
hypoxia, caused a 3-fold increase in COX-2 protein levels within
24 h (Fig. 1A). The
effect of hypoxia on COX-2 protein was time-dependent and
correlated with the increasing levels of COX-2 mRNA (Fig.
1B). CYP4B1, a cytochrome P450 protein whose expression has
been correlated with the synthesis of the inflammatory eicosanoids
12(R)-HETE and 12(R)-HETrE by the corneal
epithelium (10), showed a similar time course of expression in response to hypoxia (data not shown).
The transcriptional activation of COX-2 and CYP4B1 following hypoxia
may share a common mechanism; both have been shown to be activated by
PPAR ligands. As seen in Fig.
2A, RT-PCR of RCE cell
mRNA revealed the presence of PPAR
Regulation of Cyclooxygenase-2 by Hypoxia and Peroxisome
Proliferators in the Corneal Epithelium*
,
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DISCUSSION
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and 
but not 
mRNAs were
detected in these cells. The PPAR activator, WY-14,643 increased COX-2
expression. Similarly, non-steroidal anti-inflammatory drugs with the
ability to activate PPARs induced COX-2 independently of prostaglandin
synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR
activation was not associated with a parallel increase in prostaglandin
E2 accumulation. However, the enzyme regained full
catalytic activity when: 1) hypoxic cells were re-exposed to normoxic
conditions in the presence of heme and arachidonic acid, and 2)
WY-14,643-treated cells were depleted of intracellular GSH. Consistent
with previous observations showing that the corneal production of
cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia
and inflammation, the current data suggest that hypoxic injury is a
model of inflammation in which molecules other than COX-derived
arachidonic acid metabolites play a major proinflammatory role. This
study also suggests that increased cellular GSH may be the mechanism
responsible for the characteristic dissociation of PPAR-induced COX-2
expression and activity. Moreover, we provide new insights into the
commonly observed lack of efficacy of classical non-steroidal
anti-inflammatory drugs in the treatment of hypoxia-related ocular
surface inflammation.
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-mercaptoethanol) and heated for 5 min at 85 °C.
SDS-polyacrylamide gel electrophoresis was performed using 10% (w/v)
and 3% (w/v) acrylamide for separating gel and stacking gel,
respectively. Protein transfer onto polyvinylidene difluoride membranes
(Amersham Pharmacia Biotech) was performed at 16 V for 1 h.
Membranes were saturated in blocking buffer containing 5% nonfat
dry milk and incubated for 2 h at room temperature with the
following polyclonal antibody preparations: rabbit anti-murine COX-2,
goat anti-human COX-1, rabbit anti-human HO-1, or rabbit anti-human
HO-2. After washing with buffer (Tris/HCl, 0.42%, pH 7.4, containing
0.1% Tween 20), membranes were further incubated with an anti-rabbit
or anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz
Biotechnology, Santa Cruz, CA) at dilution 1:4,000 for 45 min at room
temperature. Immunoreactive proteins were detected using
chemiluminescence substrates according to the manufacturer's
instructions and were visualized after exposure to
HyperfilmTM ECL (Amersham Pharmacia Biotech). Quantitation
was performed by scanning the blot into Photoshop and performing
densitometry with NIH Image.
80 °C.
Solid phase enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) was
performed as suggested by the manufacturer. PGE2 levels
were determined using a standard curve and a linear log-logit transformation.
, , ), a specific backward primer was used in the single reaction. The reaction mixture
was then subjected to brief incubation at 65 °C in order to
inactivate the enzyme. PCR reactions were carried out in a final volume
of 100 µl consisting of 20 mM Tris-HCl, pH 9.0, 2 mM MgSO4, 200 µM amounts of each
deoxyribonucleoside triphosphate, 4 µl of the RT first-strand
cDNA product, 0.2 µM of each forward and reverse
primer, and 2.5 units of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). The reactions were heated to 97 °C for 1 min and then immediately cycled 30 or 35 times through a 1-min
denaturing step, 1.5-min annealing step at 50 or 55 °C, and a 2-min
extension step at 72 °C. After the cycling procedure, a final 10-min
elongation step at 72 °C was performed. The following primers were
used: CYP4B1 primers, 4B1-F (5'-TCTCTGGGTTGGACAGTTCATTG) and 4B1-R
(5'-TGTCTCCTTTGCCAAACGTACAC); COX-2 primers, COX-2F (5'-GCCCTTCCTCCTGTGGCTGAT) and COX-2R (5'-TTGAGCACATCGCACACTCT); 28 S
rRNA primers, 28 S rRNA-F (5'-AAACTCTGGTGGAGGTCCGT) and 28 S rRNA-R
(5'-CTTACCAAAAGTGGCCCACTA). Primers for the different PPARs were
designed as described previously (21).
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View larger version (20K):
[in a new window]
Fig. 1.
Time-dependent expression of
COX-2 in RCE cells. Quiescent cells were incubated under normoxic
or hypoxic conditions for 8, 12, and 24 h. Total RNA was extracted
and subjected to Northern analysis using the rabbit COX-2 cDNA and
28S ribosomal RNA probes. Western analysis of COX-2 protein was carried
out in parallel and was performed as described under "Experimental
Procedures." A, a representative Western blot and
densitometry analysis of three separate experiments; B, a
representative Northern blot and densitometry analysis of three
separate experiments. Results are the mean ± S.E., *,
p < 0.05 from control, normoxia; N and 
,
normoxia; H and 
, hypoxia.
and PPAR transcripts but
no transcript for PPAR. Control experiments indicated that the
expression of PPARs , , and was readily detected in rabbit liver RNA (Fig. 2B). The specificity of these PCR products
(corneal epithelial PPAR and ) was further demonstrated by
restriction analysis based on the published sequences of the rabbit
PPARs (21).
View larger version (36K):
[in a new window]
Fig. 2.
PPAR isoform expression in RCE
cells. A, total RNA was extracted and RT reactions
performed using an oligo(dT) primer (lanes 1,
3, and 5) or specific primers (lanes
2, 4, and 6) as described under
"Experimental Procedures." B, positive controls were
amplified from rabbit liver under the same conditions as for RCE cells.
Figure shows a representative gel of three different experiments.
We further examined the effect of common peroxisome proliferators as
well as NSAIDs that have been shown to act as PPAR ligands (23) on COX
protein levels. As seen in Fig.
3A, the commonly used PPAR
ligand, WY-14,643, greatly increased COX-2 protein levels. WY-14,643
was more potent than clofibrate; its addition to the medium caused a
2-fold increase in COX-2 protein (Fig. 3A), while clofibrate
increased COX-2 protein by about 30% (data not shown). Under hypoxic
conditions, addition of WY-14,643 did not produce an additional
significant increase in COX-2 levels (Fig. 3A), suggesting
that hypoxia and the PPAR ligand may share a common mechanism for
induction of COX-2. Indomethacin as well as flurbiprofen significantly
increased COX-2 protein levels by about 2-fold. Aspirin at a
concentration of 100 µM also increased COX-2 protein levels (data not shown). Similar to WY-14,643, addition of these NSAIDs
to hypoxia-treated cells did not cause a further increase in COX-2
protein levels (data not shown). Neither hypoxia nor WY-14,643 or
NSAIDs changed the level of the constitutively expressed COX-1 protein
(Fig. 3A). Actinomycin D inhibited COX-2 expression when
used in combination with hypoxia, indomethacin, and WY-14,643 (Fig.
3B), further indicating that COX-2 is transcriptionally activated under these conditions. Indeed, Northern analysis
demonstrated that addition of WY-14,643 resulted in a 2-fold increase
in COX-2 mRNA (Fig. 4). Indomethacin
at 30 µM, while increasing COX-2 protein levels (Fig.
3A) and COX-2 mRNA (data not shown), inhibited
PGE2 levels by 60% and 80% in cells grown under normoxic
and hypoxic conditions, respectively. The effect of indomethacin on
COX-2 protein levels was not reversed by addition of exogenous
PGE2. In fact, addition of exogenous PGE2 to
normoxia- and indomethacin-treated cells had little or no effect on
COX-2 protein levels (Fig. 5), suggesting
a dissociation between COX-2 expression and PGE2 levels. Similar results were obtained in hypoxia/indomethacin-treated cells
(data not shown).
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The increase in COX-2 protein levels in response to hypoxia or
WY-14,643 was not associated with a comparable increase in PGE2 levels (Fig.
6A). In fact, marked 65% and
64% decreases in PGE2 levels were observed in cells grown
under hypoxic conditions for 12 and 24 h, respectively. In
WY-14,643-treated cells for 12 h PGE2 levels were
similar to the control levels; however, at 24 h the levels were
significantly reduced by 25% as compared with control cells (Fig.
6A). Cells grown under hypoxic conditions and treated with
WY-14,643 showed a similar decrease in PGE2 levels to that
in hypoxia, namely 701 ± 53, 448 ± 63, and 438 ± 61 pg PGE2/ml in normoxia, hypoxia, and hypoxia+WY-14,643,
respectively. These results are similar to that obtained for the
protein where no additive effect for hypoxia and WY-14,643 was observed
(Fig. 3A). There are several possibilities that could
account for the decreased PGE2 levels. One is a lack of
substrate, arachidonic acid, which has been long considered as the
rate-limiting step in prostaglandin synthesis. Addition of 10 µM arachidonic acid to hypoxia or WY-14,643-treated cells
during the culture period restored the levels of PGE2
accumulation to that seen in cells incubated under normoxic conditions
(Fig. 6B). However, the increase in PGE2 levels
in cells supplemented with arachidonic acid did not reflect the
2-4-fold increase in COX-2 protein seen in cells incubated under the
same conditions (Figs. 1 and 3).
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Another possibility is that the induced COX-2 protein is defective or
is inactive due to a lack of cofactors such as heme. In many cells, the
level of heme is regulated by the activity of heme oxygenase (HO),
specifically the inducible isoform, HO-1. Western blot analysis
revealed that only hypoxic conditions caused a marked increase in HO-1
protein levels (Fig. 7). WY-14,643 and indomethacin, while increasing COX-2 protein, had no effect on HO-1
protein level. In all experimental conditions, the protein levels of
the constitutively expressed isoform, HO-2, remained unchanged (Fig.
7). These results suggest that, at least in hypoxia, a shortage of
cellular heme may contribute to the decrease in PGE2
levels. The reduced PGE2 levels in hypoxia-treated cells may also stem from lack of oxygen. However, neither heme nor oxygen can
account for the relatively depressed PGE2 in
WY-14,643-treated cells.
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It is well documented that the catalytic activity of COX isoforms
requires the presence of hydroperoxides (24). On the other hand,
conditions of oxidative stress such as those associated with hypoxia
and inflammatory processes are associated with increased expression of
GSH-dependent peroxidase (25). Glutathione peroxidase as
well as GSH itself have been shown to inhibit COX-1 and COX-2 catalytic
activity (26). To evaluate whether changes in GSH levels underlie the
decrease in PGE2 levels, cells were incubated with BSO and
DEM to deplete endogenous GSH under hypoxia and with the addition of
WY-14,643. As seen in Fig. 8, depletion
of GSH increased PGE2 levels by 8-, 2- and 14-fold in
normoxia-, hypoxia-, and WY-14,643-treated cells, respectively.
Moreover, PGE2 accumulation in WY-14,643-treated cells was
well correlated with the increased COX-2 protein expression. In
contrast, measurement of COX activity as the conversion of arachidonic
acid to PGE2 in 1 h in the presence of exogenous GSH
demonstrated a concentration-dependent inhibition of COX
activity (30% and 40% inhibition at 10 and 50 mM GSH,
respectively).
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, p < 0.05 from normoxia treated with DEM and
BSO.
We further examined COX activity in cells grown for 12 or 24 h in
hypoxia or normoxia and in cells treated with WY-14,643 and
subsequently incubated under normoxia with 10 µM
arachidonic acid and 2 µM heme for 1 h. As seen in
Fig. 9, in 12- and 24-h hypoxia-treated
cells COX activity was 2 and 5 times higher than that of the control
cells, respectively, further indicating that the increased
immunoreactive COX-2 protein is an active enzyme and that oxygen is a
major limiting factor. However, COX activity in cells treated with
WY-14,643 remained low and was about 50% of the activity measured in
control cells (Fig. 9). Finally, addition of the selective COX-2
inhibitor, NS398, to the incubation medium revealed that about 50% of
the COX activity in cells incubated under normoxic condition is driven
by COX-2, while in hypoxia-treated cells or cells treated with
WY-14,643, this activity accounts for almost 100% (Fig.
10) suggesting that, although COX-1
protein level remains unchanged, its activity was diminished following hypoxia and WY-14643 treatment.
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Early prostaglandin research strove to establish a link between
their synthesis and ocular inflammation. It is well documented that
corneal surface injury increases production of cyclooxygenase-derived eicosanoids (PGI2, PGF2
, PGE2,
PGD2, and thromboxane A2) by the corneal
epithelium (27). There are, however, several observations that
challenge the significance of their role in the inflammatory response.
The increase in the production of these eicosanoids correlates poorly
to the inflammatory sequelae (28). Moreover, while topical applications
of micromolar concentrations of these eicosanoids are required to
elicit inflammatory effects, much lower concentrations are usually
recovered at the site of surface injury (29, 30). Furthermore, the use
of metabolic inhibitors against the cyclooxygenase pathway is not as
efficacious as corticosteroids in the treatment of ocular surface
inflammation (31, 32). These inhibitors tend to have partial efficacy
at best, indicating that other corticosteroid-sensitive mediators may
participate in the corneal inflammatory response. Studies in our
laboratory identified the formation of the CYP-derived arachidonic acid
metabolites 12(R)-HETE, a Na,K-ATPase inhibitor with
chemotactic activity, and 12(R)-HETrE, a powerful
proinflammatory mediator that has been shown to stimulate vasodilation,
increase anterior chamber proteins, chemoattract polymorphonuclear
leukocytes, and elicit angiogenesis (see Ref. 6 and references
therein). The endogenous conversion of arachidonate to these
metabolites is increased following hypoxia in vitro and
in vivo and after chemical injury in vivo. Their
production rate correlates with the inflammatory response in
vivo and inhibition of their formation attenuates the inflammation
(reviewed in Ref. 6). In all, these studies clearly place the
CYP-derived 12-hydroxyeicosanoids as corneal epithelial-derived
mediators of ocular surface inflammation. However, the discovery of
COX-2 as the inducible isoform of cyclooxygenase that is differently
affected by common NSAIDs brought back the question of the involvement
of prostaglandins in the ocular inflammatory process. The contemporary
view is that prostaglandins are mediators of numerous biological
processes including inflammation (33).
In the present study we examined the presence and inducibility of COX-2 in corneal epithelial cells following hypoxia-induced injury; such injury has been associated with a severe inflammatory response in vivo and with increased production of 12-HETE and 12-HETrE in vivo and in vitro (7). We found that COX-2, but not COX-1, mRNA and protein levels are markedly increased in RCE cells following hypoxia. Hypoxia has been shown to stimulate the expression of COX-2 in the lung and endothelial cells (11, 12, 34). While acute exposure (2 h) to hypoxia increased COX activity in endothelial cells (35), chronic exposure (24 h) in these cells and in alveolar macrophages resulted in decreased COX-2 protein and PGE2 synthesis (36, 37). In our study, 12-24 h of hypoxia markedly decreased PGE2 accumulation while increasing COX-2 expression. The use of the selective COX-2 inhibitor NS-398 further suggested that COX activity in hypoxic cells is essentially that of COX-2, whereas in control (normoxia) cells, both COX-1 and COX-2 contributed equally to the production of PGE2.
Recent studies demonstrated that a CYP4B1 isoform is induced in the
corneal epithelium in response to hypoxia and is involved in the
production of 12-HETE and 12-HETrE (10). A similar pattern of induction
was observed in RCE cells. The increased CYP4B1 and COX-2 mRNA
levels suggest a common mechanisms for regulation. Indeed, the rabbit
lung CYP4B1 has been shown to be activated by clofibrate, a PPAR
activator (15). Likewise, COX-2 expression has been shown to be induced
by numerous PPAR ligands (17). Moreover, clofibrate increased
conversion of arachidonic acid to 12-HETE and 12-HETrE in the corneal
epithelium (10). These findings indicate the possibility that PPAR
activation may underlie the increased expression of COX-2 and CYP4B1 in
hypoxia-treated cells. RT-PCR analysis clearly detected signals for
PPAR and PPAR, but not PPAR, in hypoxia-treated cells.
Treatment of cells with WY-14,643, a selective PPAR and activator, markedly increased COX-2 expression. Furthermore, NSAIDs
known to act as peroxisome proliferators such as indomethacin,
flurbiprofen, and aspirin (23) also increased COX-2 protein and
mRNA. Interestingly, several NSAIDs have been shown to inhibit
NF
B activation (38); the latter may be an important transcriptional
activator of COX-2 expression in response to hypoxia (11). The
observation that these NSAIDs did not decrease COX-2 expression under
hypoxic conditions argues against a dependence on NFB in the
hypoxia-induced expression of COX-2. In all, these findings suggest the
possibility that PPAR activation, at least in part, contributes to the
hypoxia-induced expression of COX-2 and CYP4B1. To this end, addition
of PPAR ligands to hypoxia-treated cells did not yield a further
increase in either COX-2 protein, mRNA, or PGE2
accumulation, suggesting that hypoxia and PPAR ligands may share a
similar mechanism, e.g. PPAR activation, by which they
transcriptionally activate COX-2 expression.
Noteworthy is the fact that the increased expression of COX-2 after addition of a PPAR ligand was, as in hypoxia, associated with a decrease in PGE2 accumulation. However, while COX enzymatic activity in hypoxic cells could be fully expressed, when oxygen is replenished, to reflect the 3-4-fold increase in protein levels, it was just about 50% of control in cells treated with WY-14,643. Ledwith et al. (39) have shown that PPAR ligands, including WY-14643, cause little or no increase in PGE2 levels in mouse liver cells, while markedly increasing COX-2 expression. These authors demonstrated that PPAR ligands do not inhibit COX enzymatic activity and suggested that these agents exhibit an indirect inhibitory effect on COX activity. Our findings concur with this report; however, they also offer a potential mechanism for WY-14,643. As indicated before, GSH peroxidase exerts a regulatory control on COX activity (26). Depletion of GSH in cells treated with WY-14,643 restored COX activity and increased it to levels that better reflected the increase in protein expression induced by WY-14,643. It is possible that the indirect inhibitory activity of WY-14,643 referred to in the study of Ledwith et al. (39) is an effect on GSH metabolism leading to increased GSH levels that have been shown in our study as well as others to inhibit COX activity. Indeed, there are numerous reports demonstrating the effect of peroxisome proliferators including WY-14,643 on GSH metabolism (40).
The dissociation between COX-2 expression and PGE2 levels in cells that were subjected to hypoxia could be the result of several factors operating under these conditions. One such factor is the availability of the substrate, arachidonic acid. However, experiments in which cells were prelabeled with arachidonic acid indicate that the levels of free arachidonic acid are, in fact, increased during hypoxia (data not shown). This is in accordance with reports which demonstrated that in many tissues ischemia/hypoxia leads to increased fatty acid release, including arachidonic acid, subsequent to activation of phospholipase A2 (41). One may argue that, when COX is inhibited, such increases may be shifted to other metabolic enzymes such as lipoxygenases and CYP monooxygenase. However, these cells do not express significant levels of either lipoxygenase or cytochrome P450 monooxygenase activity in culture under normoxia or hypoxia (data not shown). The small increase in PGE2 accumulation may be due to the fact that the addition of exogenous arachidonic acid resulted in increased hydroperoxide levels, an essential requirement for COX catalytic activity (42). There are several reports indicating the presence of hydroperoxides with various preparations of arachidonic acid (43). It is noteworthy that PGE2 levels in hypoxia-treated cells were essentially the same when either arachidonic acid was added or GSH was depleted. This suggests that the increased hydroperoxide level rather than substrate is the major factor contributing to the increased PGE2 levels. With regard to hypoxia, factors such as lack of oxygen and increasing heme degradation due to rapid induction of HO-1 (44) are likely to be the major factors in limiting COX activity. Indeed, reoxygenation in the presence of heme restored COX activity in hypoxia-treated cells. On the other hand, the WY-14,643 inhibitory effect on COX activity was not removed by reoxygenation and heme.
The apparent disparity between expression of COX-2 and endogenous
levels of PGE2 in the injured cells may have important
pathophysiological and therapeutic implications. It is well documented
that prolonged exposure of the corneal epithelium to hypoxia in the
form of eye closure or contact lens wear leads to a pronounced
inflammatory response (7). It is also known that NSAIDs are only
partially effective in treating ocular surface inflammation. Inasmuch
as COX-2 is defined as an inflammatory gene, in the context of this type of injury and this tissue, it seems likely that its rapid induction does not lead to increased levels of PGE2,
i.e. an increase in its expression does not yield a parallel
increase in PGE2, further indicating that the inflammatory
response in the corneal epithelium is not prostanoid-mediated under
these conditions. Moreover, under these conditions, NSAIDs may
intensify the inflammatory response by activating PPARs and
subsequently amplifying the response via induction of CYP4B1 and
production of the proinflammatory eicosanoids, 12-HETE and 12-HETrE.
The increased production of the latter in the presence of NSAIDs may
also result from shunting arachidonic acid to this pathway. To this
end, preliminary studies have demonstrated induction of PPARs and
, COX-2, and CYP4B1 expression in corneal epithelium from rabbit
eyes that were subjected to hypoxia via eye closure and contact lens
wear; in this model the inflammatory response including edema,
vasodilation, and neovascularization correlates with the duration of
contact lens wear and is associated with increased synthesis of 12-HETE
and 12-HETrE.
The events demonstrated in the cultured corneal epithelial cells may
also occur in vivo not only in the cornea but also in other
tissues where hypoxia produces an inflammatory response such as in
vascular occlusion diseases. Moreover, the results of this study
suggest that the therapeutic action of peroxisome proliferators such as
fibrates may involve an increase in GSH levels and a decrease in the
level of peroxidation. Similarly, NSAIDs may act in such a way by
activating PPARs and thus indirectly promoting antioxidant mechanisms.
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ACKNOWLEDGEMENT |
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We thank Dr. Matthew Breyer (Department of Medicine, Vanderbilt University) for the rabbit COX-2 cDNA.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant EY05613.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by a student fellowship from the Institute of
Pharmacological Sciences (Prof. Giancarlo Folco), University of Milan,
Milan, Italy.
§ To whom correspondence should be addressed. Tel.: 914-594-4153; Fax: 914-594-4303; E-mail: michal_schwartzman@nymc.edu.
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ABBREVIATIONS |
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The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; PPAR, peroxisome proliferator-activated receptors; CYP, cytochrome P450; RCE, rabbit corneal epithelium; HO, heme oxygenase; HETE, hydroxyeicosatetraenoic acid; HETrE, hydroxyeicosatrienoic acid; LT, leukotriene; NSAID, non-steroidal anti-inflammatory drug; RT, reverse transcription; PCR, polymerase chain reaction; BSO, L-buthionine-[S,R]sulfoximine; DEM, maleic acid diethylester.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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|---|
| 1. |
Sack, R. A.,
Tan, K. O.,
and Tan, A.
(1992)
Invest. Ophthalmol. Vis. Sci.
33,
626-640 |
| 2. | Bruce, A. S., and Brennan, N. A. (1990) Surv. Ophthalmol. 35, 25-58[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Smolin, G. (1989) Eye 3, 167-171 |
| 4. | Williams, K. I., and Higgs, G. A. (1988) J. Pathol. 156, 101-110[CrossRef][Medline] [Order article via Infotrieve] |
| 5. | Williams, R. N., Delamere, N. A., and Paterson, C. A. (1985) Exp. Eye Res. 41, 733-738[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Laniado Schwartzman, M. (1997) in Advances in Ocular Toxicology (Green, K. , Edelhauser, H. F. , Hackett, R. B. , Hull, D. S. , Potter, D. E. , and Tripathi, R. C., eds) , pp. 3-20, Plenum Press, New York |
| 7. | Conners, M. S., Stoltz, R. A., Webb, S. C., Rosenberg, J., Dunn, M. W., Abraham, N. G., and Schwartzman, M. L. (1995) Invest. Ophthalmol. Vis. Sci. 36, 828-840[Abstract] |
| 8. |
Conners, M. S.,
Urbano, F.,
Vafeas, C.,
Stoltz, R. A.,
Dunn, M. W.,
and Laniado Schwartzman, M.
(1997)
Invest. Ophthalmol. Vis. Sci.
38,
1963-1971 |
| 9. |
Vafeas, C.,
Mieyal, P. A.,
Urbano, F.,
Falck, J. R.,
Chauhan, K.,
Berman, M.,
and Laniado Schwartzman, M.
(1998)
J. Pharmacol. Exp. Ther.
287,
903-910 |
| 10. |
Mastyugin, V.,
Aversa, E.,
Vafeas, C.,
Mieyal, P.,
and Laniado Schwartzman, M.
(1999)
J. Pharmacol. Exp. Ther.
289,
1611-1619 |
| 11. |
Schmedtje, J. F., Jr.,
Ji, Y.-S.,
Liu, W.-L.,
DuBois, R. N.,
and Runge, M. S.
(1997)
J Biol. Chem.
272,
601-608 |
| 12. |
Chida, M.,
and Voelkel, N. F.
(1996)
Am. J. Physiol.
270,
L872-L878 |
| 13. |
DuBois, R. N.,
Abramson, S. B.,
Crofford, L.,
Gupta, R. A.,
Simon, L. S.,
Van De Putte, L. B.,
and Lipsky, P. E.
(1998)
FASEB J.
12,
1063-1073 |
| 14. | Vane, J. R., Bakhle, Y. S., and Botting, R. M. (1998) Annu. Rev. Pharmacol. Toxicol. 38, 97-120[CrossRef][Medline] [Order article via Infotrieve] |
| 15. | Willson, T. M., and Wahli, W. (1997) Curr. Opin. Chem. Biol. 1, 235-241[CrossRef][Medline] [Order article via Infotrieve] |
| 16. |
Forman, B. M.,
Chen, J.,
and Evans, R. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4312-4317 |
| 17. |
Meade, E. A.,
McIntyre, T. M.,
Zimmerman, G. A.,
and Prescott, S. M.
(1999)
J Biol. Chem.
274,
8328-8334 |
| 18. | Claire, A. E., and Simpson, M. (1997) Gen. Pharmacol. 28, 351-359[Medline] [Order article via Infotrieve] |
| 19. | Zeldin, D. C., Plitman, J. D., Kobayashi, J., Miller, R. F., Snapper, J. R., Falck, J. R., Szarek, J. L., Philpot, R. M., and Capdevila, J. H. (1995) J. Clin. Invest. 95, 2150-2160 |
| 20. |
Araki, K.,
Ohashi, Y.,
Kinoshita, S.,
Hayashi, K.,
Yang, X. Z.,
Hosaka, Y.,
Aizawa, S.,
and Handa, H.
(1993)
Invest. Ophthalmol. Vis. Sci.
34,
2665-2671 |
| 21. |
Guan, Y.,
Zhang, Y.,
Davis, L.,
and Breyer, M. D.
(1997)
Am. J. Physiol.
273,
F1013-F1022 |
| 22. | Chang, M. S., Tsai, J. C., Yang, R., DuBois, R. N., Breyer, M. D., and O'Day, D. M. (1997) Curr. Eye Res. 16, 1147-1151[CrossRef][Medline] [Order article via Infotrieve] |
| 23. |
Lehmann, J. M.,
Lenhard, J. M.,
Oliver, B. B.,
Ringold, G. M.,
and Kliewer, S. A.
(1997)
J Biol. Chem.
272,
3406-3410 |
| 24. | Kulmacz, R. J. (1998) FEBS Lett. 430, 154-157[CrossRef][Medline] [Order article via Infotrieve] |
| 25. | Mates, J. M., and Sanchez-Jimenez, F. (1999) Frontiers Biosci. 4, D339-D345[Medline] [Order article via Infotrieve] |
| 26. | Capdevila, J. H., Morrow, J. D., Belosludtsev, Y. Y., Beauchamp, D. R., DuBois, R. N., and Falck, J. R. (1995) Biochemistry 34, 3325-3337[CrossRef][Medline] [Order article via Infotrieve] |
| 27. | Bazan, H. E. P., Birkle, D. L., Beuerman, R. W., and Bazan, N. G. (1985) Curr. Eye Res. 4, 175-179[Medline] [Order article via Infotrieve] |
| 28. | Gluud, B. S., Jensen, O. L., Krogh, E., and Birgens, H. S. (1985) Acta Ophthalmol. 63, 375-379 |
| 29. | Napoli, S. A., Helm, C., Insler, M. S., Ensley, H. E., Pretus, H. A., and Feigen, L. P. (1990) Ann. Ophthalmol. 22, 30-34[Medline] [Order article via Infotrieve] |
| 30. |
Srinivasan, B. D.,
and Kulkarni, P. S.
(1980)
Invest. Ophthalmol. Vis. Sci.
19,
1087-1093 |
| 31. | Srinivasan, B. D., and Kulkarni, P. S. (1989) in The Ocular Effect of Prostaglandins and Other Eicosanoids (Bito, L. Z. , and Stjernschantz, J., eds) , pp. 229-249, Alan R. Liss, New York |
| 32. |
Haynes, W. L.,
Proia, A. D.,
and Klintworth, G. K.
(1989)
Invest. Ophthalmol. Vis. Sci.
30,
1588-1593 |
| 33. | Bazan, N. G., and Allan, G. (1997) Surv. Ophthalmol. 41 Suppl. 2, S23-S34 |
| 34. |
Ji, Y. S.,
Xu, Q.,
and Schmedtje, J. F., Jr.
(1998)
Circ. Res.
83,
295-304 |
| 35. |
Michiels, C.,
Arnould, T.,
Knott, I.,
Dieu, M.,
and Remacle, J.
(1993)
Am. J. Physiol.
264,
C866-C874 |
| 36. |
Farber, H. W.,
and Barnett, H. F.
(1991)
Circ. Res.
68,
1446-1457 |
| 37. | Hempel, S. L., Monick, M. M., and Hunninghake, G. W. (1996) Am. J. Respir. Cell Mol. Biol. 14, 170-176[Abstract] |
| 38. |
Kopp, E.,
and Ghosh, S.
(1994)
Science
265,
956-959 |
| 39. |
Ledwith, B. J.,
Pauley, C. J.,
Wagner, L. K.,
Rokos, C. L.,
Alberts, D. W.,
and Manam, S.
(1997)
J. Biol. Chem.
272,
3707-3714 |
| 40. | Cai, Y., Appelkvist, E. L., and DePierre, J. W. (1995) J. Biochem. Toxicol. 10, 87-94[CrossRef][Medline] [Order article via Infotrieve] |
| 41. | Mukherjee, A. B., Miele, L., and Pattabiraman, N. (1994) Biochem. Pharmacol. 48, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
| 42. |
Marshall, P. J.,
Kulmacz, R. J.,
and Lands, W. E.
(1987)
J. Biol. Chem.
262,
3510-3517 |
| 43. | Allen, K. G., Huang, C. J., and Morin, C. L. (1990) Anal. Biochem. 186, 108-111[CrossRef][Medline] [Order article via Infotrieve] |
| 44. |
Lee, P. J.,
Jiang, B.-H.,
Chin, B. Y.,
Iyer, N. V.,
Alam, J.,
Semenza, G. L.,
and Choi, A. M. K.
(1997)
J. Biol. Chem.
272,
5375-5381 |
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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