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J Biol Chem, Vol. 273, Issue 16, 9886-9893, April 17, 1998
From the Department of Biochemistry, Michigan State University,
East Lansing, Michigan 48824 and the Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory
drugs like aspirin and ibuprofen. These enzymes catalyze the committed step in the formation of prostanoids from arachidonic acid. Although PGHS-1 and -2 are similar biochemically, a number of studies suggest that PGHS-1 and PGHS-2 function independently to form prostanoids that
subserve different cellular functions. We have hypothesized that these
isozymes may reside, at least in part, in different subcellular
compartments and that their compartmentation may affect their access to
arachidonic acid and serve to separate the functions of the enzymes. To
obtain high resolution data on the subcellular locations of PGHS-1 and
-2, we employed immunoelectron microscopy with multiple antibodies
specific to each isozyme. Both PGHS-1 and -2 were found on the lumenal
surfaces of the endoplasmic reticulum (ER) and nuclear envelope of
human monocytes, murine NIH 3T3 cells, and human umbilical vein
endothelial cells. Within the nuclear envelope, PGHS-1 and -2 were
present on both the inner and outer nuclear membranes and in similar
proportions. Western blotting data showed a similar distribution of
PGHS-1 and -2 in subcellular fractions, and product analysis using
isozyme-specific inhibitors suggested that both enzymes generate the
same products in NIH 3T3 cells. Thus, we are unable to attribute the
independent functioning of PGHS-1 and PGHS-2 to differences in their
subcellular locations. Instead, the independent operation of these
isozymes may be attributable to subtle kinetic differences
(e.g. negative allosteric regulation of PGHS-1 at low
concentrations of arachidonate (500-1000 nM)). A further
conclusion of importance from a cell biological perspective is that
membrane proteins such as PGHS-1 and -2, which are located on the
lumenal surface of the ER, are able to diffuse freely among the ER and
the inner and outer membranes of the nuclear envelope.
Two biochemically similar enzymes with markedly different patterns
of expression catalyze the committed step in the formation of
prostaglandins from arachidonic acid. The prostaglandin
endoperoxide H synthases (PGHS-1 and
PGHS-2)1 each form
PGH2 from arachidonic acid through sequential
cyclooxygenase and peroxidase activities (1). Biologically active
prostanoids including PGE2, PGF2,
PGI2, PGD2, and thromboxane A2 are
then formed by specific prostaglandin synthases. PGHS-1 is expressed constitutively in nearly all mammalian tissues and forms prostanoids central to several housekeeping functions including water reabsorption in the kidney, vascular homeostasis, and platelet aggregation. PGHS-2,
although absent from most cells, can be rapidly and dramatically induced in many cell types upon treatment with inflammatory cytokines, growth factors, and tumor promoters (2). PGHS-1 and -2 share 60%
primary sequence identity (3, 4), and their x-ray crystal structures
are virtually superimposable (5-7). Kinetic profiles suggest similar
if not identical reaction mechanisms (8, 9). There are, however,
sequence differences near the active site resulting in subtle
differences in substrate specificities and differential sensitivities
to various nonsteroidal anti-inflammatory drugs (10, 11). These
differences have been exploited in preparing a new generation of
selective inhibitors of PGHS-2 that may inhibit pain, inflammation, and
tumorigenesis while preserving the capacity of PGHS-1 to synthesize
housekeeping prostanoids.
Despite their many biochemical similarities, results of several
experiments make it difficult to argue that PGHS-2 exists primarily to
augment the biosynthetic capacity of PGHS-1 (see "Discussion" in
Ref. 12). PGHS expression patterns and apparently differential access
to cellular pools of arachidonate (13-15) have led to the hypothesis
that PGHS-1 and -2 represent two independent prostanoid biosynthetic
systems. The inducible expression of PGHS-2, which is encoded by an
immediate early gene, occurs in conjunction with nuclear events such as
cell differentiation and replication (2). Consequently, the inducible
enzyme, especially the subset of enzyme molecules localized to the
nuclear envelope, has been hypothesized to be the major source of
prostanoids involved in a putative peroxisome proliferator activated
receptor-mediated nuclear signaling system (16-18).
PGHS-1 and PGHS-2 are both integral membrane proteins of the
endoplasmic reticulum (ER) and nuclear envelope (12). Based on previous
confocal immunolocalization work from our laboratory, we suggested that
differential compartmentation of the two isozymes may serve, at least
in part, to separate the activities of PGHS-1 and -2 within cells (12).
Although both enzymes are present in the ER and nuclear envelope, the
conclusion of the earlier work was that PGHS-2 was preferentially
associated with the nuclear envelope (12), whereas PGHS-1 was equally
distributed between the ER and nuclear envelope. To test this model, we
used immunoelectron microscopy and Western blotting of PGHS-1 and -2 in
subcellular fractions. In addition, to test for isozyme-specific
channeling of PGH2 to downstream PG synthases, we analyzed
the prostanoid products formed by PGHS-1 and -2. Immunogold
labeling experiments revealed that both enzymes are on the outer
nuclear membrane; this was expected due to the continuity of this
membrane with the ER (19). Somewhat to our surprise, both PGHS-1 and
PGHS-2 were also present on the inner nuclear membrane. Overall, our studies have indicated that the locations of the isozymes and the
nature of the products are the same for both PGHS-1 and -2.
All materials were purchased from Sigma unless otherwise
noted.
Isolation and Preparation of Human Monocytes for Electron
Microscopy--
Peripheral blood mononuclear cells (PBMCs) were
isolated from several healthy human volunteers. Heparinized blood was
diluted 1:1 in Hanks' balanced salts solution (HBSS, Life
Technologies, Inc.) and spun against a Ficol-Hypaque (LSM-Organon
Teknika) gradient for 20 min at 500 × g. PBMCs were
isolated as a band from the interface and washed 2 × with HBSS
before resuspension in 10 ml of RPMI medium (Life Technologies, Inc.)
containing 5% autologous serum and penicillin/streptomycin (100 units/ml). Cells (106/ml) were incubated in the presence or
absence of 1 ng/ml lipopolysaccharide (LPS; from Jayne Chen at Merck)
and incubated with gentle agitation in a water-saturated 5%
CO2 atmosphere at 37 °C for 24 h. Fixation was
performed after LPS- or vehicle-stimulated cells were collected by
centrifugation and resuspended in appropriate media (see below). Monocytes comprise 5-8% of PBMCs. Most of the other cells in PBMC preparations are T and B lymphocytes that do not normally express PGHSs
and, therefore, provide a convenient internal negative control against
PGHS immunostaining. Because ultrathin sectioning makes possible the
observation of more than one section from a given cell, care was taken
to use several different samples in the interest of gathering data from
many different cells. PBMCs from each volunteer were separated into
between 5 and 10 separate pellets per experiment. Between every few
grids of thin sections taken from a single pellet, several thick (1 µm) sections were cut to maximize the number of different cells
observed per experiment.
Preparation of NIH 3T3 Cells and HUVEC Cells for Electron
Microscopy--
Murine NIH 3T3 cells express PGHS-1 constitutively
(20). Serum-starved, quiescent 3T3 cells were cultured and then
stimulated by the addition of 10% fetal calf serum (HyClone
Laboratories, Inc., Logan, UT) for 3 h to induce PGHS-2 as
described previously (20). After serum stimulation, cells from five
100-mm culture dishes were removed from the growing surface with a
rubber policeman, resuspended in DMEM (Life Technologies, Inc.), and
immediately fixed (see below). HUVECs (Cell Systems, Seattle, WA) were
thawed from stocks frozen at passage 1 and expanded through passage
3-4 as suggested by the manufacturer. After stimulation for 20 h
with 10 ng/ml IL-1 Fixation and Cryoprotection for Electron Microscopy--
Cells
(PBMCs, NIH 3T3, and HUVEC) were obtained in a 2.5-ml suspension of an
appropriate medium and immediately fixed by the addition of 2.5 ml of
2× fixative. Two different fixation methods were employed to allow for
differences in the sensitivity to fixation of PGHS-1 and -2 antigens.
Microwave/glutaraldehyde-fixed cells were fixed exactly as described
previously (21). Cells fixed with Nakane fixative (22) alone were
treated by adding 2× Nakane fixative (1× Nakane = 0.1 M NaIO4, 0.75 M lysine, 0.0375 M phosphate buffer, 2% paraformaldehyde (Fisher)) to an
equal volume of cell suspension and incubated for 2 h at room
temperature. These cells were then washed twice with 1× Nakane
fixative and resuspended in fresh 1× Nakane before an overnight (or
shorter) incubation at 4 °C. NIH 3T3 cells immunolabeled for PGHS-2
were only fixed at 4 °C for an additional 2 h after the 2-h
room temperature fixation. After the overnight fixation step, both
microwave/glutaraldehyde- and Nakane-fixed cells were collected by
centrifugation, resuspended in 0.1 M sucrose/PBS, and
pelleted in 2% low gelling temperature agarose, 0.1 M
sucrose, PBS. Cryoprotection was performed for 2 h (25 °C) or
overnight (4 °C) in polyvinylpyrrolidone, 2.3 M sucrose/phosphate buffer (pH 7.2) (polyvinylpyrrolidone-sucrose) (21).
Cell pellets were mounted on bull's-eye specimen pins (Ted Pella,
Inc.), frozen by plunging into liquid propane, and stored under liquid
nitrogen until use.
Immunogold Labeling--
Immunogold labeling was performed
essentially as described previously (21). Briefly, ultrathin
(75-80-nm) sections of cells were cut at Antibodies Specific for PGHS-1 and PGHS-2--
All primary
antibodies were raised in rabbits as described previously (24). For
PGHS-2 staining of human monocytes and HUVECs, a polyclonal antibody
raised against ovine PGHS-2 was used. This antibody (from Dr. Jilly
Evans, Merck Frosst) cross-reacts with human PGHS-2 but not with ovine
or human PGHS-1 (25). PGHS-1 immunostaining of NIH 3T3 mouse
fibroblasts was performed with an affinity purified anti-peptide
antibody directed against amino acids
Leu274-Arg288 of murine PGHS-1 (26). This
antibody does not cross-react with PGHS-2 in Western blotting
experiments. Another antibody, an IgG fraction from a polyclonal
antibody raised against PGHS-1 (27), recognizes only PGHS-1 and was
used for immunolabeling of NIH 3T3 cells and HUVECs. PGHS-2 labeling of
NIH 3T3 cells was performed using an affinity purified anti-peptide
antibody directed against an 18-amino acid cassette near the C terminus
of PGHS-2 (12, 28); this antibody does not cross-react with any known
PGHS-1 but recognizes human and murine PGHS-2 on Western blots.
Distribution of PGHS-1 and PGHS-2 between Inner and Outer
Membranes of the Nuclear Envelope--
We determined the distribution
of PGHS-1 and PGHS-2 between the inner and outer nuclear membranes
essentially as described previously (21). Gold particles lying on or
within one 5-nm particle diameter of the inner nuclear membrane were
designated as being on the inner nuclear membrane. Particles lying on
or within one 5-nm particle diameter of the outer nuclear membrane were
designated as being on the outer nuclear membrane. When a gold particle
was observed in the nuclear envelope and not within one particle
diameter of either membrane, it was designated as lumenal. Analysis of
the distribution of PGHS-1 and PGHS-2 was performed on a total of 24 and 32 cells, respectively. Distribution analysis was limited to
well-preserved sections of nuclear envelope. Only regions of cells in
which the inner and outer nuclear envelope were clearly distinguishable
were used for our analyses.
Statistical Analysis--
The distribution analysis described
above was performed using at least three experimental groups of well
preserved cells for both PGHS-1 in NIH 3T3 cells and PGHS-2 in
monocytes. The analysis of PGHS-2 in NIH 3T3 cells was performed on
seven cells taken from two separate experiments. Analysis of the mean
distribution percentages using Student's t test showed that
there was no significant difference between the inner membrane/outer
membrane distribution of PGHS-1 and -2 in the cell types analyzed. It
is possible for a protein of the nuclear envelope to be present in
significantly different amounts on the inner and outer nuclear
membranes, as evidenced by our previous work (21) on 5-lipoxygenase and
FLAP.
Subcellular Fractionation and Western Analysis--
NIH 3T3
cells were prepared as whole cell lysates, microsomes, or isolated
nuclei. For whole cells, harvests of three 100-mm culture dishes of
serum-stimulated NIH 3T3 cells in PBS were collected by centrifugation
and resuspended in Hanks' balanced salts solution (HBSS). The
resuspended cells were then disrupted by sonication and Dounce
homogenization to produce whole cell lysates. Microsomes were prepared
as described previously (29-31) from 10 plates of cells except that
the 200,000 × g pellet was resuspended in HBSS. Isolation of cell nuclei (32, 33) began with the harvest of 20 dishes
of 3T3 cells in PBS followed by centrifugation at 1000 × g. Nuclei were isolated in the presence of 0.2 or 1%
saponin as described below. Isolation of nuclei in the absence of
detergent was begun by resuspending the nuclei in 10 ml of cold Buffer
A (10 mM Tris, 10 mM NaCl, 1 mM
EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg leupeptin/ml, and 3 mM MgCl2
(pH 8.0)) and incubation on ice for 5 min, followed by 5 s of
gentle vortexing. After passage 10 times through a 20-gauge needle, the
crude nuclei were again collected by centrifugation at 1000 × g. The pellet was resuspended in 6 ml of cold Buffer A and
incubated on ice for 5 min. After an additional 10 passages through a
20-gauge needle, the suspension was subjected to gentle homogenization
in a Teflon homogenizer. Half of the suspension was placed in each of
two Beckman 5-ml UltraClear ultracentrifuge tubes. A nuclear spin
cushion was prepared by dissolving 6.16 g of sucrose in 10 ml of
Buffer B (60 mM KCl, 15 mM NaCl, 15 mM Tris, 0.15 mM spermine, 0.5 mM
spermidine, and 0.5 mM dithiothreitol) to give a 1.8 M sucrose solution. After underlaying 2 ml of the sucrose
solution beneath the crude nuclear suspension, the tubes were
centrifuged for 19 min at 4 °C at 13,500 rpm in a Beckman SW50.1
rotor. Isolated nuclei were visible at the bottom of the tubes. The
liquid was removed from the tubes by aspiration, and the nuclear pellet
was resuspended in 400 µl of HBSS. Nuclei were washed 4 times with
HBSS and collected by centrifugation at 1800 rpm for 3 min in a
microcentrifuge. Purified nuclei and whole cell lysates were disrupted
by sonication before protein concentrations were determined using a
Bio-Rad Protein Assay solution. Aliquots of broken cells, microsomes,
or nuclei (20 µg) were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by Western blotting as described
previously (34). Densitometric quantitation of immunoreactive PGHS-1 or
-2 was performed using a Bio-Rad GS-505 Molecular Imaging System and Molecular Analyst software. For a given isozyme, densities are expressed as the ratio of nuclear to microsomal immunoreactivity. Student's t test was utilized to determine if these ratios
for PGHS-1 and PGHS-2 were significantly different for each
experimental condition.
Prostanoid Product Formation by Murine NIH 3T3 Cells--
Murine
NIH 3T3 cells were cultured in DMEM with 10% fetal calf serum in a
water-saturated 7% CO2 incubator. At approximately 60%
confluence, the cells were starved for 48 h by incubation in DMEM
containing 0.2% fetal calf serum. Quiescent cells were stimulated by
the addition of fetal bovine serum (final concentration of 16%) for
3 h (20). In some experiments various PGHS-1 and/or PGHS-2
inhibitors (valerylsalicylate (500 µM), NS398 (15 µM), flurbiprofen (100 µM), or aspirin (500 µM)) were added 30 min before the addition of serum (10,
35), and when used, these agents were also present in the medium during
the stimulation.
Subcellular Localization of Prostaglandin Endoperoxide H
Synthases-1 and -2 by Immunoelectron Microscopy*
,, and Department of
Biochemical and Molecular Pathology, Merck Research Laboratories,
Rahway, New Jersey 07065
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
, cells were removed from culture dishes with a rubber policeman, immediately resuspended in CS-C media (Cell Systems,
Seattle, WA), and fixed (see below). Previous PGHS immunolocalization experiments on both adherant and detached cells gave similar results, suggesting that removal of cells from the growing surface does not
affect the localization of PGHS-1 or PGHS-2 (12, 40).
106 °C on a Reichert
Ultracut S ultramicrotome fitted with a Reichert FCS cryoattachment.
Sections were collected on drops of 2.3 M sucrose and
placed on glow discharged, Formvar-coated nickel grids (Ted Pella,
Inc.). After a minimum of 1 h in blocking solution (5% milk, 1%
bovine serum albumin, PBS, 0.02% sodium azide) and washing, grids were
placed section-side down on 25-µl drops of primary antibody solutions
of various concentrations and incubated for various times depending on
the primary antibody. All antibody solutions were cleaned by filtration
through 0.2-µm filters prior to use. To control for antibody
specificity, primary antibodies were incubated with a 50-fold molar
excess of either cognate peptide (for anti-PGHS-1 or anti-PGHS-2
peptide directed antibodies) or an approximately 10-fold molar excess
of purified ovine PGHS-1 or -2 (for antibodies prepared against either
whole protein). Preadsorption of whole protein antibodies with purified PGHS-1 or -2 was performed with constant agitation at 4 °C for 24 h. The samples were centrifuged at 12,000 rpm in a Beckman Microfuge for 1 h at 4 °C, the supernatants were removed,
filtered through a 0.2-µm syringe filter, and used as primary
antibody solutions in immunogold labeling experiments. Purified ovine
PGHS-1 was from R. M. Garavito at Michigan State University;
purified ovine PGHS-2 was from Cayman Chemical Co., Ann Arbor, MI.
After incubation with primary antibody, each grid was washed eight
times for 3 min on drops of 1% bovine serum albumin, PBS, 0.02%
sodium azide before a 1-h incubation at room temperature on drops of gold-conjugated secondary antibody (Amersham Pharmacia Biotech GAR-G5
diluted 1:75 in 1% bovine serum albumin, PBS, 0.02% sodium azide).
After a washing step, sections were post-fixed and stained for contrast
by floating the grids sequentially on drops of 2% glutaraldehyde, 2%
osmium tetroxide, and 2% uranyl acetate. Polyvinyl alcohol (2%) was
used to embed the grids before observation by transmission electron
microscopy (23).
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RESULTS |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Immunogold Labeling of PGHS-2 in Human Monocytes and HUVECs-- Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human volunteers, treated for 20 h with bacterial lipopolysaccharide (LPS), and processed for electron microscopy. Western blotting of samples of these cells prior to fixation established that, as expected, PGHS-2 expression was induced by LPS but that vehicle-treated cells lacked detectable levels of this isozyme (37). Monocytes in LPS-treated samples, when incubated with an antibody raised against ovine PGHS-2, exhibited perinuclear staining for PGHS-2 (Fig. 1A). PGHS-2 labeling was completely eliminated by preadsorption of the anti-PGHS-2 antibody with purified ovine PGHS-2 (Fig. 1B). No comparable gold label was seen in vehicle-treated cells (data not shown). PGHS-2 labeling was observed on both the inner and outer membranes of the nuclear envelope in monocytes present in LPS-treated PBMC preparations (Fig. 1C). The distribution of PGHS-2 within the nuclear envelope of 26 LPS-treated monocytes was determined by counting the gold particles associated with well preserved regions of the inner and outer nuclear membranes (Table I). In performing these distribution analyses, only those segments of the nuclear envelope in which both the inner and outer nuclear membranes were clearly distinguishable were used. PGHS-2 labeling was approximately equally distributed between the inner and outer membranes of the nuclear envelope.
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-treated human umbilical vein endothelial cells (HUVEC) both of
which are known to express PGHS-2 (20, 38). An anti-peptide antibody raised against the C-terminal 18-amino acid cassette of PGHS-2 was used
to label PGHS-2 in serum-treated NIH 3T3 cells. This antibody labeled
serum-treated NIH 3T3 cells along the nuclear membrane and in the ER
(not shown). PGHS-2 immunoreactivity was distributed equally between
the inner and outer nuclear membranes in NIH 3T3 cells (Table I).
PGHS-2 labeling of serum-treated NIH 3T3 cells was eliminated by
preincubation of the antibody with its cognate peptide. Finally, PGHS-2
staining in IL-1-treated HUVECs was present on the inner and outer
nuclear membranes and in the ER, a pattern similar to that seen in
LPS-treated monocytes and serum-treated NIH 3T3 cells (data not
shown).
Immunogold Labeling of PGHS-1 in Murine NIH 3T3 Cells-- Serum-starved or serum-treated NIH 3T3 cells were fixed and processed for immunoelectron microscopy. The expectation that PGHS-1 would be expressed at similar levels in both starved and treated cells (20, 39) was confirmed by Western blotting (not shown). Serum-treated (Fig. 2A) or serum-starved (not shown) NIH 3T3 cells exhibited perinuclear and ER staining for PGHS-1. PGHS-1 labeling was eliminated by preincubation of the anti-PGHS-1 antibody with its cognate peptide (Fig. 2B). The immunostaining experiments depicted in Fig. 2 were performed using an anti-PGHS-1 antibody raised against a peptide corresponding to amino acids Leu274-Arg288 of murine PGHS-1. Identical experiments performed with another anti-PGHS-1 antibody, this one raised against whole ovine PGHS-1 (27), resulted in a pattern of staining indistinguishable from that seen with the anti-peptide antibody (not shown). PGHS-1 staining by the antibody prepared against the whole protein was eliminated by preadsorption of the antibody with purified ovine PGHS-1.
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Western Blotting of PGHS-1 and PGHS-2 in Subcellular Fractions-- Earlier immunofluorescence studies from our laboratory indicated that PGHS-1 and PGHS-2 are in the ER and nuclear envelope but that the concentration of PGHS-2 in the nuclear envelope is roughly twice that of the ER, whereas the concentration of PGHS-1 is the same in the nuclear envelope and the ER (12). In contrast, our quantitative immunoelectron microscopy indicated that PGHS-1 and -2 are present in the same subcellular locations in approximately equal proportions. There are significant differences between the fixation and staining protocols used for immunofluorescence and electron microscopy, any one of which could conceivably cause subtle differences in the patterns of staining. One difference involves the use of detergents to permeabilize the cells for immunofluorescence staining. Accordingly, we determined the effect of saponin, the detergent used in our earlier immunofluorescence work (12), on the distribution of PGHS-1 and PGHS-2 in nuclear and ER fractions from NIH 3T3 cells. When nuclei were isolated in the presence of 1% saponin from murine NIH 3T3 cells, immunoreactive PGHS-2 was relatively more abundant in nuclear membranes than in microsomal membranes, whereas PGHS-1 immunoreactivity was equally distributed between nuclear and microsomal membranes in the same experiments (Fig. 3A).3 Qualitatively similar results were obtained when nuclei were isolated in the presence of 0.2% saponin, although the difference between PGHS-1 and PGHS-2 distribution were not statistically significant (Fig. 3A). However, when Western blotting experiments were conducted on subcellular fractions isolated from serum-stimulated murine NIH 3T3 cells in the absence of detergent (Fig. 3A), PGHS-1 and PGHS-2 were similarly distributed between nuclear and microsomal membranes. We measured the ratios of immunoreactive PGHS-1 and -2 in nuclei versus microsomes using densitometry (Fig. 3B). In three experiments in the presence of 1% saponin, the ratios of nuclear to microsomal immunoreactive PGHS-1 and -2 were 1.1 ± 0.1 and 1.9 ± 0.2, respectively. In the presence of 0.2% saponin, the same ratios for PGHS-1 and -2 were 1.14 ± 0.1 and 1.26 ± 0.1. Exclusion of detergent resulted in nuclear to microsomal immunoreactivity ratios for PGHS-1 and -2 of 1.08 ± 0.1 and 1.10 ± 0.2, respectively. These data suggest that PGHS-1 and PGHS-2 can be differentially solubilized from nuclear membranes by detergents such as saponin. This may account for the differential patterns of PGHS-1 and PGHS-2 staining that have been observed in immunofluorescence studies (12).
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Products Formed via PGHS-1 and -2 in NIH 3T3 Cells-- As shown in Fig. 4A, quiescent murine NIH 3T3 cells express PGHS-1 but not PGHS-2, whereas after serum stimulation, PGHS-2 is detectable, and there is no significant change in the level of immunoreactive PGHS-1. To determine if a different set of products are produced via PGHS-1 versus PGHS-2, serum stimulated NIH 3T3 cells were treated with various PGHS-1 and PGHS-2 inhibitors, and the products formed from [1-14C]arachidonic acid were separated and quantified. The main prostanoid product derived from arachidonic acid by PGHS-1 and -2 in NIH 3T3 cells was PGE2 in all cases (Fig. 4B). Following serum stimulation, the amount of product increased by about 20%. We presume that this results from the increase in PGHS-2 (Fig. 4B). In support of this presumption are the following findings. Following treatment of serum-starved 3T3 cells with valerylsalicylate (500 µM), an inhibitor which is relatively specific for PGHS-1 (35), prostanoid production was almost completely inhibited, whereas valerylsalicylate caused incomplete (~80%) inhibition of prostanoid formation by serum-stimulated 3T3 cells. NS398 is a PGHS-2-specific inhibitor (41), and NS398 (15 µM) caused a 15-20% inhibition of prostanoid formation by serum-stimulated cells but had little effect on synthesis by serum-starved cells. Flurbiprofen inhibits both PGHS-1 and PGHS-2 (10), and flurbiprofen (100 µM) completely inhibited synthesis by both serum-starved and serum-stimulated 3T3 cells.
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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PGHS-1 and PGHS-2 have structurally homologous membrane binding domains containing four amphipathic helices which anchor the proteins to one leaflet of the lipid bilayer (5-7, 40, 42, 43). Previous work has established that both isozymes are located on the lumenal surfaces of the ER and nuclear envelope (24, 40). However, immunocytofluorescence studies suggested that PGHS-2 was more concentrated in the nuclear envelope than the ER, whereas PGHS-1 was equally distributed in both compartments (12). One possibility to account for these latter findings was that PGHS-2 was uniquely localized on the inner membrane of the nuclear envelope. The major aim of the present studies was to examine this possibility using immunoelectron microscopy. The issue of PGHS-1 versus PGHS-2 localization is of particular interest because of the potential relationship between PGHS-2-derived products generated at the nuclear envelope and nuclear signaling associated with cell replication or differentiation.
The major finding of our EM study is that in NIH 3T3 cells both PGHS-1 and PGHS-2 are present in equal proportions on both the inner and outer membranes of the nuclear envelope. Human monocytes and umbilical vein endothelial cells stained for PGHS-1 or PGHS-2 also exhibited identical distribution patterns. Our results make it clear that both isozymes are present in the same subcellular compartments and at comparable concentrations. Of course, we cannot rule out the possibility that PGHS-1 and PGHS-2 are associated with different microdomains within these compartments.
Earlier results from immunofluorescence studies which employed low concentrations of saponin to permeabilize cell membranes and had suggested that PGHS-2 is preferentially localized to the nuclear envelope (12) can be explained on the basis of differential solubilization of the two proteins from the nuclear envelope. When ER and nuclear membranes were prepared in the presence of 1% saponin, PGHS-2 was more concentrated in the nuclear fraction, whereas PGHS-1 was found in equal abundance in the ER and nuclear membranes; in the absence of saponin, PGHS-2 was present at the same concentration in the ER and nuclear envelope. Presumably these differences in affinities of the two proteins for the nuclear envelope are a result of the significant differences in the amino acid composition of the membrane binding domains of the two isozymes (1, 44).
Studies of prostaglandin product formation by whole cell, ER, and nuclear fractions from murine 3T3 cells indicated that all fractions produced the same set of products in the presence and absence of inhibitors specific for PGHS-1 or PGHS-2. These results indicate that at least in 3T3 cells, there is no isozyme-specific channeling of the endoperoxide intermediate to specific PGH2 metabolizing synthases (e.g. PGE synthase).
A model has emerged suggesting that PGHS-1 and PGHS-2 act independently and that, at least in part, the inducible isozyme, PGHS-2, provides prostaglandins for a nuclear eicosanoid signaling system (1, 17, 45). Although this model may still be correct, our results imply that any specific connection between PGHS-2 and the generation of products that function in the nucleus would have to result from differences in the expression of the activities of PGHS-1 and PGHS-2 and not from gross differences in the subcellular distributions of PGHS-2 versus PGHS-1. That is because both PGHS-1 and PGHS-2 appear to be present in the same membranes, factors other than compartmentation account for the separation of their activities into two independent systems. The most likely factors are differences in interactions with different phospholipases and/or differences in enzyme kinetics. Arm, Austen, Herschmann, and co-workers (13, 15, 46-48) have demonstrated that two separate phases of PGD2 synthesis in mast cells are independently coupled to PGHS-1 (early phase) and PGHS-2 (late phase) by different phospholipases A2. Kinetic mechanisms for separating the actions of PGHS-1 and PGHS-2 have also been described. For example, PGHS-2 has a significantly lower threshold for hydroperoxide activation than PGHS-1 thereby enabling PGHS-2 to oxygenate arachidonic acid in the presence of lower peroxide concentrations (9, 49). In addition, negative allosteric regulation of PGHS-1 by arachidonic acid, at concentrations between 0.5 nM and 1 µM, has the overall effect of causing a 2-4-fold greater rate of PGHS-2-mediated prostanoid formation (50). These kinetic differences between PGHS-1 and PGHS-2 have been identified with purified or partially purified enzyme preparations. However, it may be possible to test for kinetic differences between the two isozymes in intact cells using histochemical assays of enzyme activity (12).
Another more speculative possibility to account for the independent operation of PGHS-1 and PGHS-2 in cells where both isozymes are expressed is the existence of accessory proteins which differentially affect the rate of prostaglandin endoperoxide formation by PGHS-1 versus PGHS-2. Although no such protein(s) has been identified in the prostanoid biosynthetic system, there is a precedent for an accessory protein in the leukotriene pathway. Leukotrienes synthesized through 5-lipoxygenase arise from arachidonic acid apparently delivered to the 5-lipoxygenase by an activating protein, FLAP (51-55).
The observation that both PGHS-1 and PGHS-2 are located on the inner nuclear membrane is of interest from a cell biology perspective. To our knowledge, no other endogenous integral membrane proteins of the ER have been demonstrated to be present on the inner nuclear membrane. FLAP, another integral membrane protein involved in eicosanoid biosynthesis, has been localized to the inner and outer membranes of the NE (21, 56). However, in contrast to PGHS-1 and -2, FLAP is predominantly localized to the nuclear envelope; the orientation of FLAP in the membrane is not well characterized. One model of how integral membrane proteins synthesized in the ER reach the inner nuclear membrane involves lateral diffusion through the membrane bilayers of the NE (57-59). According to this model, membrane proteins are subject to a size constraint imposed by the lateral channel diameter of the nuclear pore complex, which serves to separate the inner and outer nuclear membranes. Both PGHS-1 and PGHS-2 are targeted initially to the ER by a KDEL-like C-terminal targeting signal (60). We propose that both isozymes are then able to bypass the nuclear pore complex and reach the inner nuclear membrane by lateral diffusion. Our reasoning is based on the nature of their interaction of PGHS-1 and PGHS-2 with membranes (i.e. via a monotopic membrane binding domain) and the fact that the proteins are on the lumenal surface of the membrane. Consistent with this concept are studies with the lamin B receptor, an integral membrane protein that is targeted exclusively to the inner nuclear membrane via specific targeting signals (58). When the nucleoplasmic/cytoplasmic oriented extramembrane domain of lamin B receptor is artificially enlarged, the protein is retained in the ER (and outer nuclear membrane). Other ER membrane proteins with large extramembrane domains oriented toward the cytoplasm are also thought to be restricted to the outer membrane of the nuclear envelope and the ER (58, 61). P450s such as thromboxane synthase and prostacyclin synthase are integral membrane proteins of the ER and have relatively large cytoplasmic domains. Thus, these proteins are not likely to be present on the nucleoplasmic face of the inner nuclear membrane and would be unable to metabolize efficiently PGH2 generated by PGHS-1 or PGHS-2 present on this membrane.
We conclude that PGHS-1 and PGHS-2 are present in similar proportions on the endoplasmic reticulum, outer nuclear membrane, and inner nuclear membrane of NIH 3T3 cells, human monocytes, and HUVECs. Fatty acid substrates for PGHSs appear to be supplied via both an sPLA2 that functions on the phospholipids on the cell surface and a cPLA2 that undergoes a Ca2+-dependent translocation to the cytosolic face of the ER and outer membrane of the nuclear envelope (62-66) and perhaps the inner membrane of the nuclear envelope (67). However, the issue of whether there is preferential coupling of different phospholipases A2 to PGHS-1 versus PGHS-2 is currently unresolved. PGH2 formed through both PGHS-1 and PGHS-2 can apparently diffuse readily through membranes (68). Any PGH2 that diffuses from the ER lumen into the cytoplasm is likely to be metabolized by enzymes such as thromboxane A2 synthase or PGI2 synthase located on the cytoplasmic surface of the ER and nuclear envelope (69). The fate of the PGH2 that diffuses into the nucleoplasm is presently unknown.
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ACKNOWLEDGEMENTS |
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We thank Jeanne Barker, Sol Scott, Jamie Flanagan, Doug Kawka, and Jayne Chen at Merck for helpful suggestions and Dr. John Heckman at the Michigan State University Center for Electron Optics for discussions and the use of equipment. We also thank Dr. Howard Worman for advice relating to integral membrane protein targeting.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK22042.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.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824. Tel.: 517-353-8680; Fax: 517-353-9334; E-mail: smithww{at}pilot.msu.edu.
1
The abbreviations used are: PGHS-1 and PGHS-2,
prostaglandin endoperoxide H synthases-1 and -2; LPS, bacterial
lipopolysaccharide; ER, endoplasmic reticulum; NE, nuclear envelope;
IL-1, the -isoform of interleukin-1; PBMC, peripheral blood
mononuclear cells; HUVEC, human umbilical vein endothelial cells; PG,
prostaglandin; DMEM, Dulbecco's modified Eagle's medium; FLAP,
5-lipoxygenase activating protein; HBSS, Hanks' balanced salt
solution; PBS, phosphate-buffered saline.
2 No immunogold labeling of PGHS-1 in vehicle- or LPS-treated monocytes in PBMC preparations was observed using several different anti-PGHS-1 antibodies, presumably due to the low level of PGHS-1 expression in these cells (37).
3 Similar results were observed in the mouse macrophage line RAW 264.7. Although PGHS-1 was always approximately equally distributed between microsomal and nuclear membranes, PGHS-2 was more concentrated in nuclear membranes 70% of the time in more than 20 experiments.
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 R. Rao, M.-Z. Zhang, M. Zhao, H. Cai, R. C. Harris, M. D. Breyer, and C.-M. Hao Lithium treatment inhibits renal GSK-3 activity and promotes cyclooxygenase 2-dependent polyuria Am J Physiol Renal Physiol, April 1, 2005; 288(4): F642 - F649. [Abstract] [Full Text] [PDF]
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 K. M. Harnett, W. Cao, and P. Biancani Signal-Transduction Pathways that Regulate Smooth Muscle Function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G407 - G416. [Abstract] [Full Text] [PDF]
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G. Giannico, M. Mendez, and M. C. LaPointe Regulation of the membrane-localized prostaglandin E synthases mPGES-1 and mPGES-2 in cardiac myocytes and fibroblasts Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H165 - H174. [Abstract] [Full Text] [PDF]
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H. Jiang, A. S. Weyrich, G. A. Zimmerman, and T. M. McIntyre Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility J. Biol. Chem., December 31, 2004; 279(53): 55905 - 55913. [Abstract] [Full Text] [PDF]
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 A. Vazquez-Tello, L. Fan, X. Hou, J.-S. Joyal, J. A. Mancini, C. Quiniou, R. I. Clyman, F. Gobeil Jr., D. R. Varma, and S. Chemtob Intracellular-specific colocalization of prostaglandin E2 synthases and cyclooxygenases in the brain Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1155 - R1163. [Abstract] [Full Text] [PDF]
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 A. K. Goff Steroid Hormone Modulation of Prostaglandin Secretion in the Ruminant Endometrium During the Estrous Cycle Biol Reprod, July 1, 2004; 71(1): 11 - 16. [Abstract] [Full Text] [PDF]
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M. Girotti, J. H. Evans, D. Burke, and C. C. Leslie Cytosolic Phospholipase A2 Translocates to Forming Phagosomes during Phagocytosis of Zymosan in Macrophages J. Biol. Chem., April 30, 2004; 279(18): 19113 - 19121. [Abstract] [Full Text] [PDF]
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 J. R. Ghilardi, C. I. Svensson, S. D. Rogers, T. L. Yaksh, and P. W. Mantyh Constitutive Spinal Cyclooxygenase-2 Participates in the Initiation of Tissue Injury-Induced Hyperalgesia J. Neurosci., March 17, 2004; 24(11): 2727 - 2732. [Abstract] [Full Text] [PDF]
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M. A. Balboa, Y. Shirai, G. Gaietta, M. H. Ellisman, J. Balsinde, and E. A. Dennis Localization of Group V Phospholipase A2 in Caveolin-enriched Granules in Activated P388D1 Macrophage-like Cells J. Biol. Chem., November 28, 2003; 278(48): 48059 - 48065. [Abstract] [Full Text] [PDF]
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I. Dey, K. Keller, A. Belley, and K. Chadee Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica PNAS, November 11, 2003; 100(23): 13561 - 13566. [Abstract] [Full Text] [PDF]
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M. ROMANO and J. CLARIA Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and tumor angiogenesis: implications for cancer therapy FASEB J, November 1, 2003; 17(14): 1986 - 1995. [Abstract] [Full Text] [PDF]
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P. Teismann, K. Tieu, D.-K. Choi, D.-C. Wu, A. Naini, S. Hunot, M. Vila, V. Jackson-Lewis, and S. Przedborski Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration PNAS, April 29, 2003; 100(9): 5473 - 5478. [Abstract] [Full Text] [PDF]
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C. W. Houchen, M. A. Sturmoski, S. Anant, R. M. Breyer, and W. F. Stenson Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine Am J Physiol Gastrointest Liver Physiol, March 1, 2003; 284(3): G490 - G498. [Abstract] [Full Text] [PDF]
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K. Asai, T. Hirabayashi, T. Houjou, N. Uozumi, R. Taguchi, and T. Shimizu Human Group IVC Phospholipase A2 (cPLA2gamma ). ROLES IN THE MEMBRANE REMODELING AND ACTIVATION INDUCED BY OXIDATIVE STRESS J. Biol. Chem., February 28, 2003; 278(10): 8809 - 8814. [Abstract] [Full Text] [PDF]
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M.-H. Wu, H.S. Sun, C.-C. Lin, K.-Y. Hsiao, P.-C. Chuang, H.-A. Pan, and S.-J. Tsai Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis Mol. Hum. Reprod., December 1, 2002; 8(12): 1103 - 1110. [Abstract] [Full Text] [PDF]
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 H. J. Lin, K. M. Lakkides, T. O. Keku, S. T. Reddy, A. D. Louie, I. H. Kau, H. Zhou, J. S. Y. Gim, H. L. Ma, C. F. Matthies, et al. Prostaglandin H Synthase 2 Variant (Val511Ala) in African Americans May Reduce the Risk for Colorectal Neoplasia Cancer Epidemiol. Biomarkers Prev., November 1, 2002; 11(11): 1305 - 1315. [Abstract] [Full Text] [PDF]
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 H. Lim and S. K. Dey Minireview: A Novel Pathway of Prostacyclin Signaling--Hanging Out with Nuclear Receptors Endocrinology, September 1, 2002; 143(9): 3207 - 3210. [Abstract] [Full Text] [PDF]
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P. Christmas, B. M. Weber, M. McKee, D. Brown, and R. J. Soberman Membrane Localization and Topology of Leukotriene C4 Synthase J. Biol. Chem., August 2, 2002; 277(32): 28902 - 28908. [Abstract] [Full Text] [PDF]
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S. M. Prescott and H. J. Yost The COXes of Danio: From mechanistic model to experimental therapeutics PNAS, July 9, 2002; 99(14): 9084 - 9086. [Full Text] [PDF]
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 F. Gobeil Jr, I. Dumont, A. M. Marrache, A. Vazquez-Tello, S. G. Bernier, D. Abran, X. Hou, M. H. Beauchamp, C. Quiniou, A. Bouayad, et al. Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors Circ. Res., April 5, 2002; 90(6): 682 - 689. [Abstract] [Full Text] [PDF]
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G. Zahner, G. Wolf, M. Ayoub, R. Reinking, U. Panzer, S. J. Shankland, and R. A. K. Stahl Cyclooxygenase-2 Overexpression Inhibits Platelet-derived Growth Factor-induced Mesangial Cell Proliferation through Induction of the Tumor Suppressor Gene p53 and the Cyclin-dependent Kinase Inhibitors p21waf-1/cip-1 and p27kip-1 J. Biol. Chem., March 15, 2002; 277(12): 9763 - 9771. [Abstract] [Full Text] [PDF]
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 M. Mendez and M. C. LaPointe Trophic Effects of the Cyclooxygenase-2 Product Prostaglandin E2 in Cardiac Myocytes Hypertension, February 1, 2002; 39(2): 382 - 388. [Abstract] [Full Text] [PDF]
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D. H. Wright, D. Abran, M. Bhattacharya, X. Hou, S. G. Bernier, A. Bouayad, J.-C. Fouron, A. Vazquez-Tello, M. H. Beauchamp, R. I. Clyman, et al. Prostanoid receptors: ontogeny and implications in vascular physiology Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2001; 281(5): R1343 - R1360. [Abstract] [Full Text] [PDF]
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T. Takano, A. V. Cybulsky, X. Yang, and L. Aoudjit Complement C5b-9 induces cyclooxygenase-2 gene transcription in glomerular epithelial cells Am J Physiol Renal Physiol, November 1, 2001; 281(5): F841 - F850. [Abstract] [Full Text] [PDF]
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S. T. Davidge Prostaglandin H Synthase and Vascular Function Circ. Res., October 12, 2001; 89(8): 650 - 660. [Abstract] [Full Text] [PDF]
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B. Poligone and A. S. Baldwin Positive and Negative Regulation of NF-kappa B by COX-2. ROLES OF DIFFERENT PROSTAGLANDINS J. Biol. Chem., October 12, 2001; 276(42): 38658 - 38664. [Abstract] [Full Text] [PDF]
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 S. J. Sawyer, S. M. Norvell, S. M. Ponik, and F. M. Pavalko Regulation of PGE2 and PGI2 release from human umbilical vein endothelial cells by actin cytoskeleton Am J Physiol Cell Physiol, September 1, 2001; 281(3): C1038 - C1045. [Abstract] [Full Text] [PDF]
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G. E. Caughey, L. G. Cleland, P. S. Penglis, J. R. Gamble, and M. J. James Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2 J. Immunol., September 1, 2001; 167(5): 2831 - 2838. [Abstract] [Full Text] [PDF]
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H. Parfenova, V. N. Parfenov, B. V. Shlopov, V. Levine, S. Falkos, M. Pourcyrous, and C. W. Leffler Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells Am J Physiol Cell Physiol, July 1, 2001; 281(1): C166 - C178. [Abstract] [Full Text] [PDF]
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 J. N. Fain, C. W. Leffler, and S. W. Bahouth Eicosanoids as endogenous regulators of leptin release and lipolysis by mouse adipose tissue in primary culture J. Lipid Res., October 1, 2000; 41(10): 1689 - 1694. [Abstract] [Full Text]
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 P. Hardy, I. Dumont, M. Bhattacharya, X. Hou, P. Lachapelle, D. R. Varma, and S. Chemtob Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy Cardiovasc Res, August 18, 2000; 47(3): 489 - 509. [Abstract] [Full Text] [PDF]
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 A Tatsuguchi, C Sakamoto, K Wada, T Akamatsu, T Tsukui, K Miyake, S Futagami, T Kishida, Y Fukuda, N Yamanaka, et al. Localisation of cyclooxygenase 1 and cyclooxygenase 2 in Helicobacter pylori related gastritis and gastric ulcer tissues in humans Gut, June 1, 2000; 46(6): 782 - 789. [Abstract] [Full Text] [PDF]
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J.-Y. Liou, S.-K. Shyue, M.-J. Tsai, C.-L. Chung, K.-Y. Chu, and K. K. Wu Colocalization of Prostacyclin Synthase with Prostaglandin H Synthase-1 (PGHS-1) but Not Phorbol Ester-induced PGHS-2 in Cultured Endothelial Cells J. Biol. Chem., May 12, 2000; 275(20): 15314 - 15320. [Abstract] [Full Text] [PDF]
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S. J. Hewett, T. F. Uliasz, A. S. Vidwans, and J. A. Hewett Cyclooxygenase-2 Contributes to N-Methyl-D-aspartate-Mediated Neuronal Cell Death in Primary Cortical Cell Culture J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 417 - 425. [Abstract] [Full Text]
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E. D. Thuresson, K. M. Lakkides, and W. L. Smith Different Catalytically Competent Arrangements of Arachidonic Acid within the Cyclooxygenase Active Site of Prostaglandin Endoperoxide H Synthase-1 Lead to the Formation of Different Oxygenated Products J. Biol. Chem., March 17, 2000; 275(12): 8501 - 8507. [Abstract] [Full Text] [PDF]
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Y. Nakatani, T. Tanioka, S. Sunaga, M. Murakami, and I. Kudo Identification of a Cellular Protein That Functionally Interacts with the C2 Domain of Cytosolic Phospholipase A2alpha J. Biol. Chem., January 14, 2000; 275(2): 1161 - 1168. [Abstract] [Full Text] [PDF]
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O. C. Trifan, R. M. Smith, B. D. Thompson, and T. Hla Overexpression of Cyclooxygenase-2 Induces Cell Cycle Arrest. EVIDENCE FOR A PROSTAGLANDIN-INDEPENDENT MECHANISM J. Biol. Chem., November 26, 1999; 274(48): 34141 - 34147. [Abstract] [Full Text] [PDF]
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A. G. Spencer, E. Thuresson, J. C. Otto, I. Song, T. Smith, D. L. DeWitt, R. M. Garavito, and W. L. Smith The Membrane Binding Domains of Prostaglandin Endoperoxide H Synthases 1 and 2. PEPTIDE MAPPING AND MUTATIONAL ANALYSIS J. Biol. Chem., November 12, 1999; 274(46): 32936 - 32942. [Abstract] [Full Text] [PDF]
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C. O. Bingham III, R. J. A. Fijneman, D. S. Friend, R. P. Goddeau, R. A. Rogers, K. F. Austen, and J. P. Arm Low Molecular Weight Group IIA and Group V Phospholipase A2 Enzymes Have Different Intracellular Locations in Mouse Bone Marrow-derived Mast Cells J. Biol. Chem., October 29, 1999; 274(44): 31476 - 31484. [Abstract] [Full Text] [PDF]
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M. Murakami, T. Kambe, S. Shimbara, S. Yamamoto, H. Kuwata, and I. Kudo Functional Association of Type IIA Secretory Phospholipase A2 with the Glycosylphosphatidylinositol-anchored Heparan Sulfate Proteoglycan in the Cyclooxygenase-2-mediated Delayed Prostanoid-biosynthetic Pathway J. Biol. Chem., October 15, 1999; 274(42): 29927 - 29936. [Abstract] [Full Text] [PDF]
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 M. M. Johnson, B. Vaughn, M. Triggiani, D. D. Swan, A. N. Fonteh, and F. H. Chilton Role of Arachidonyl Triglycerides within Lipid Bodies in Eicosanoid Formation by Human Polymorphonuclear Cells Am. J. Respir. Cell Mol. Biol., August 1, 1999; 21(2): 253 - 258. [Abstract] [Full Text]
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 H. Lim, R. A. Gupta, W.-g. Ma, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta Genes & Dev., June 15, 1999; 13(12): 1561 - 1574. [Abstract] [Full Text]
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M. Bhattacharya, K. Peri, A. Ribeiro-da-Silva, G. Almazan, H. Shichi, X. Hou, D. R. Varma, and S. Chemtob Localization of Functional Prostaglandin E2 Receptors EP3 and EP4 in the Nuclear Envelope J. Biol. Chem., May 28, 1999; 274(22): 15719 - 15724. [Abstract] [Full Text] [PDF]
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H. Shinohara, M. A. Balboa, C. A. Johnson, J. Balsinde, and E. A. Dennis Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s J. Biol. Chem., April 30, 1999; 274(18): 12263 - 12268. [Abstract] [Full Text] [PDF]
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T. G. Brock, R. W. McNish, and M. Peters-Golden Arachidonic Acid Is Preferentially Metabolized by Cyclooxygenase-2 to Prostacyclin and Prostaglandin E2 J. Biol. Chem., April 23, 1999; 274(17): 11660 - 11666. [Abstract] [Full Text] [PDF]
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 A. F. de Arriba, F. Cavalcanti, A. Miralles, Y. Bayón, A. Alonso, M. Merlos, J. García-Rafanell, and J. Forn Inhibition of Cyclooxygenase-2 Expression by 4-Trifluoromethyl Derivatives of Salicylate, Triflusal, and Its Deacetylated Metabolite, 2-Hydroxy-4-trifluoromethylbenzoic Acid Mol. Pharmacol., April 1, 1999; 55(4): 753 - 760. [Abstract] [Full Text]
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R. Patel, M. G. Attur, M. Dave, S. B. Abramson, and A. R. Amin Regulation of Cytosolic COX-2 and Prostaglandin E2 Production by Nitric Oxide in Activated Murine Macrophages J. Immunol., April 1, 1999; 162(7): 4191 - 4197. [Abstract] [Full Text] [PDF]
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R. I. Clyman, P. Hardy, N. Waleh, Y. Q. Chen, F. Mauray, J.-C. Fouron, and S. Chemtob Cyclooxygenase-2 plays a significant role in regulating the tone of the fetal lamb ductus arteriosus Am J Physiol Regulatory Integrative Comp Physiol, March 1, 1999; 276(3): R913 - R921. [Abstract] [Full Text] [PDF]
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T. Hirabayashi, K. Kume, K. Hirose, T. Yokomizo, M. Iino, H. Itoh, and T. Shimizu Critical Duration of Intracellular Ca2+ Response Required for Continuous Translocation and Activation of Cytosolic Phospholipase A2 J. Biol. Chem., February 19, 1999; 274(8): 5163 - 5169. [Abstract] [Full Text] [PDF]
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M. Murakami, T. Kambe, S. Shimbara, and I. Kudo Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways J. Biol. Chem., January 29, 1999; 274(5): 3103 - 3115. [Abstract] [Full Text] [PDF]
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K. Tada, M. Murakami, T. Kambe, and I. Kudo Induction of Cyclooxygenase-2 by Secretory Phospholipases A2 in Nerve Growth Factor-Stimulated Rat Serosal Mast Cells Is Facilitated by Interaction with Fibroblasts and Mediated by a Mechanism Independent of Their Enzymatic Functions J. Immunol., November 1, 1998; 161(9): 5008 - 5015. [Abstract] [Full Text] [PDF]
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H. Sakamoto, H. Imai, and Y. Nakagawa Involvement of Phospholipid Hydroperoxide Glutathione Peroxidase in the Modulation of Prostaglandin D2 Synthesis J. Biol. Chem., December 15, 2000; 275(51): 40028 - 40035. [Abstract] [Full Text] [PDF]
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T. Tanioka, Y. Nakatani, N. Semmyo, M. Murakami, and I. Kudo Molecular Identification of Cytosolic Prostaglandin E2 Synthase That Is Functionally Coupled with Cyclooxygenase-1 in Immediate Prostaglandin E2 Biosynthesis J. Biol. Chem., October 13, 2000; 275(42): 32775 - 32782. [Abstract] [Full Text] [PDF]
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M. Murakami, R. S. Koduri, A. Enomoto, S. Shimbara, M. Seki, K. Yoshihara, A. Singer, E. Valentin, F. Ghomashchi, G. Lambeau, et al. Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms J. Biol. Chem., March 23, 2001; 276(13): 10083 - 10096. [Abstract] [Full Text] [PDF]
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R. Koljak, I. Jarving, R. Kurg, W. E. Boeglin, K. Varvas, K. Valmsen, M. Ustav, A. R. Brash, and N. Samel The Basis of Prostaglandin Synthesis in Coral. MOLECULAR CLONING AND EXPRESSION OF A CYCLOOXYGENASE FROM THE ARCTIC SOFT CORAL GERSEMIA FRUTICOSA J. Biol. Chem., March 2, 2001; 276(10): 7033 - 7040. [Abstract] [Full Text] [PDF]
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N. Ueno, M. Murakami, T. Tanioka, K. Fujimori, T. Tanabe, Y. Urade, and I. Kudo Coupling between Cyclooxygenase, Terminal Prostanoid Synthase, and Phospholipase A2 J. Biol. Chem., September 7, 2001; 276(37): 34918 - 34927. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, M. Phoofolo, and P. F. Weller Extranuclear Lipid Bodies, Elicited by CCR3-mediated Signaling Pathways, Are the Sites of Chemokine-enhanced Leukotriene C4 Production in Eosinophils and Basophils J. Biol. Chem., June 15, 2001; 276(25): 22779 - 22787. [Abstract] [Full Text] [PDF]
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J.-Y. Liou, W.-G. Deng, D. W. Gilroy, S.-K. Shyue, and K. K. Wu Colocalization and Interaction of Cyclooxygenase-2 with Caveolin-1 in Human Fibroblasts J. Biol. Chem., September 7, 2001; 276(37): 34975 - 34982. [Abstract] [Full Text] [PDF]
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T. Yang, Y. Huang, L. E. Heasley, T. Berl, J. B. Schnermann, and J. P. Briggs MAPK Mediation of Hypertonicity-stimulated Cyclooxygenase-2 Expression in Renal Medullary Collecting Duct Cells J. Biol. Chem., July 21, 2000; 275(30): 23281 - 23286. [Abstract] [Full Text] [PDF]
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F. Gobeil Jr, I. Dumont, A. M. Marrache, A. Vazquez-Tello, S. G. Bernier, D. Abran, X. Hou, M. H. Beauchamp, C. Quiniou, A. Bouayad, et al. Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors Circ. Res., April 5, 2002; 90(6): 682 - 689. [Abstract] [Full Text] [PDF]
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