Localization of Functional Prostaglandin E2 Receptors
EP3 and EP4 in the Nuclear Envelope*
Mousumi
Bhattacharya
§,
Krishna
Peri¶,
Alfredo
Ribeiro-da-Silva
,
Guillermina
Almazan
,
Hitoshi
Shichi
,
Xin
Hou¶,
Daya R.
Varma
, and
Sylvain
Chemtob¶**
From the
Department of Pharmacology & Therapeutics,
McGill University, Montreal, PQ, Canada, H3G 1Y6, the ** Departments of
Pediatrics, Ophthalmology and Pharmacology, and the ¶ Research
Center of Hôpital Ste. Justine, University of Montreal, Montreal,
PQ, Canada H3T 1C5, and the
Department of Ophthalmology, Wayne
State University School of Medicine, Detroit, Michigan 48201
 |
ABSTRACT |
The effects of prostaglandin E2
are thought to be mediated via G protein-coupled plasma membrane
receptors, termed EP. However recent data implied that prostanoids may
also act intracellularly. We investigated if the ubiquitous
EP3 and the EP4 receptors are localized in
nuclear membranes. Radioligand binding studies on isolated nuclear
membrane fractions of neonatal porcine brain and adult rat liver
revealed the presence of EP3 and EP4. A
perinuclear localization of EP3
and EP4
receptors was visualized by indirect immunocytofluorescence and
confocal microscopy in porcine cerebral microvascular endothelial cells
and in transfected HEK 293 cells that stably overexpress these
receptors. Immunoelectron microscopy clearly revealed
EP3
and EP4 receptors localization in the nuclear envelope of endothelial cells; this is the first demonstration of the nuclear localization of these receptors. Data also reveal that
nuclear EP receptors are functional as they affect transcription of
genes such as inducible nitric-oxide synthase and intranuclear calcium
transients; this appears to involve pertussis toxin-sensitive G
proteins. These results define a possible molecular mechanism of action
of nuclear EP3 receptors.
 |
INTRODUCTION |
Prostaglandin E2
(PGE2)1 is one of
the most abundant prostanoids in the brain (1) and plays an important
role in many cerebral functions, particularly in the newborn (2).
PGE2 also influences mitogenesis (3), promotes growth and
metastasis of tumors (4), and stimulates gene transcription (5). To
date, the biological actions of PGE2 have been attributed
to result from its interaction with plasma membrane G protein-coupled
receptors termed EP, which include EP1, EP2,
EP3, and EP4 subtypes (6). Recent studies have
shown that the nuclear membrane contains high levels of
cyclooxygenase-1 and -2 and of PGE2 (7). Possible
intracellular sites of action for prostanoids are also suggested by
other data. For example, a transporter that mediates the influx of
prostanoid has been identified (8). Cytosolic phospholipase
A2 undergoes a calcium-dependent translocation
to the nuclear envelope (9), and cyclooxygenase-2 has been shown to
translocate to the nucleus in response to certain growth factors (10).
It is thus possible that prostanoids may exert some of their effects
via intracellular EP receptors, to have a direct nuclear action as
recently proposed by Goetzl et al. (11), and Morita et
al. (12).
It has generally been assumed that the signal transduction cascades are
initiated at the plasma membrane and not the nuclear membranes.
However, recent studies have disclosed that the nuclear envelope plays
a major role in signal transduction cascades (13, 14). In fact, a novel
nuclear lipid metabolism that is a part of unique nuclear signaling
cascade termed NEST (nuclear envelope signal transduction) has been
hypothesized (15). Both heterotrimeric and low molecular weight G
proteins (15, 16), phospholipase C (13), phospholipase D (15), and
adenylate cyclase (17) have shown to be localized at the nucleus. The
nuclear membranes also have distinct inositide cycles (18) and
receptors for 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate
(13). Altogether these data raise the possibility of the presence of
nuclear prostanoid receptors. This inference has recently been
suggested for EP1 (19), but whether or not this single
observation is specific for this receptor or applies to other
prostanoid receptors, especially of the widely distributed
EP3 and EP4 subtypes, is unknown.
In the present study, we investigated the possible expression of
nuclear EP3 and EP4 receptors using human
embryonic kidney (HEK) 293 cells, porcine microvascular endothelial
cells, newborn pig brain, and adult rat liver. We selected these
tissues because many high affinity PGE2 binding sites have
been reported in the plasma membranes of pig brain (20) and rat liver
(21). We focused on EP3 receptors that are most ubiquitous
of the four EP subtypes (6) and also examined localization of
EP4 receptors. Our data provide novel evidence for the
existence of EP3 and EP4 receptors in the
nuclear envelope and reveal that these receptors are functional, and
their actions appear to involve pertussis toxin (PTX)-sensitive G proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
AH23848B was a gift from Glaxo-Wellcome, UK and
M&B 28,767 from Rhone-Poulenc Rorer, UK. The following products were
purchased: PGE2, 17-phenyl trinor PGE2 (Cayman
Chemicals, Ann Arbor, MI); Dulbecco's modified Eagle's medium,
Geneticin (Life Technologies, Inc., Burlington, ON);
[3H]PGE2 (182 Ci/mmol),
45Ca2+ (2 mCi/49.5 µg of Ca), and
[
-32P] (3000 Ci/mmol) (Amersham Pharmacia Biotech,
Mississauga, ON); all other chemicals were from Sigma.
Animals--
Newborn pigs (1-3 days old) were killed with
intracardiac pentobarbital under halothane anesthesia, and tissues of
interest were removed. Adult Sprague-Dawley male rats (250-300 g) were decapitated and had livers removed.
Cell Culture--
HEK 293 cells were cultured in Dulbecco's
modified Eagle's medium with 10% fetal calf serum. Primary cultures
of porcine cerebral endothelial cells from brain microvessels (20) were
established as described previously (22).
Preparation of Subcellular Fractions--
All steps were
performed at 4 °C using solutions containing 1.1 mM
acetylsalicylic acid, 1 mM benzamidine, 0.2 mM
phenylmethylsulfonyl fluoride and 100 µg/ml soybean trypsin
inhibitor. Nuclei were isolated from adult rat liver (23) and newborn
porcine brain cortex (24). Endoplasmic reticulum (ER) was isolated as
described (25). The purity of cellular fractions was ascertained by
determining 5'-nucleotidase using a Sigma diagnostic kit as a marker
for plasma membrane (23), and glucose-6-phosphatase was assayed as a
marker for ER (26). Proteins were determined by the Bio-Rad assay. 5'-Nucleotidase activity (units/mg protein) was 240 ± 15.4 and 5.3 ± 1.3 in plasma and nuclear membrane fractions, respectively, suggesting that the nuclear membranes were relatively free of contamination by plasma membranes. Glucose-6-phosphatase (an ER marker)
specific activity (mmol PO4 released/mg of protein) was 25.2 ± 2.3 in ER and 22.8 ± 3.1 in nuclear membrane
fractions since the outer nuclear membrane is contiguous with the ER
(27).
Radioligand Binding--
Saturation isotherms of specific
binding of [3H]PGE2 to membrane fractions
from newborn porcine brain cortex and displacement of
[3H]PGE2 by receptor isoform-specific ligands
on brain cortex and rat liver was performed essentially as previously
reported (20). Receptor densities (Bmax) and
affinity constants (KD) were determined using
Prism Graphpad program (San Diego, CA).
Immunoblotting of EP Receptors--
Western blotting of
EP3
and EP4 receptors was conducted as
described (28) on newborn brain nuclear and plasma membrane fractions.
After immunoblotting using EP3
- or
EP4-specific polyclonal rabbit antibodies (29) (1:1000),
immunoreactive bands were visualized by chemiluminescence (Amersham
Pharmacia Biotech) as per manufacturer instructions.
EP3 and EP4 Receptor Expression in HEK
293 Cells--
The full-length cDNA fragments corresponding to
human EP3
(30) and EP4 (31) were cloned
separately into the mammalian expression vector pRC-CMV (Invitrogen).
HEK 293 cells (2 × 105) were transfected with 2 µg
of plasmid DNA and 8 µl of LipofectAMINE in Opti-MEM (Life
Technologies, Inc.) according to the manufacturer instructions;
Geneticin (1 mg/ml) -resistant clones were selected and maintained in
Dulbecco's modified Eagle's medium medium containing Geneticin (0.2 mg/ml).
Immunocytochemical Detection of EP Receptors--
The
immunolocalization of EP receptors in HEK 293 and porcine
cerebrovascular endothelial cells was performed by indirect immunofluorescence (32). Briefly, cells were washed in
phosphate-buffered saline (PBS), fixed in acetone-methanol (1:1) for 10 min at
20 °C and incubated for 1 h with specific rabbit
anti-EP3
or anti-EP4 receptor antibodies
(29) diluted 1:50 in PBS containing 5% goat serum, 5% fetal calf
serum, and 0.1% Triton X-100. After washing, samples were incubated
for 1 h with Texas Red-conjugated IgG (BioCan, Mississauga, ON)
diluted 1:50 in the above buffer. To detect plasma membrane
immunolocalization, permeabilization of cells with 0.1% Triton X-100
was limited to 15 min (to improve preservation of membranes) before
incubation with primary antibodies and subsequent steps. As a negative
control, either the primary antibody was omitted or primary antibody
with its cognate peptide (29) was added. Intracellular membranes
(predominantly ER) were stained using 3,3'-dihexyloxacarbocyanine
iodide (DiOC6 (3)) and nuclei were stained with DAPI, or
Sytox Green according to the instructions of the manufacturer
(Molecular Probes, Eugene, OR).
Immunoelectron Microscopy of EP Receptors--
Pre-embedding
immunogold staining was done as described in detail previously (33,
34). Porcine brain endothelial cells were fixed for 30 min at room
temperature in 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer, washed with 0.2% Triton X-100 in PBS
for 15 min at room temperature, and incubated with
anti-EP3
or anti-EP4 receptor antibodies
(1:10) overnight at 4 °C in PBS; this limited permeabilization of
cells is required for pre-embedding immunoelectron microscopy for
adequate ultrastructural preservation. The immunogold reaction (33) was
performed overnight at 4 °C with goat anti-rabbit IgG coupled to 1 nm of gold (1:50) (Amersham Pharmacia Biotech), the reaction was
intensified using an Intense Silver Enhancement kit (Amersham Pharmacia
Biotech) according to the instructions of the manufacturer. After
osmification in 1% osmium tetroxide and Epon embedding, ultrathin
sections were observed using a transmission electron microscope
(Philips 410 LS, Netherlands).
RNA Hybridization Studies--
Nuclei were isolated from
endothelial cells (35), and aliquots (100 µg of protein) were
incubated in the presence or absence of 0.1 µM
EP3 agonist, M&B 28,767, for 60 min at 37 °C in a total volume of 40 µl (per reaction tube) of 10 mM Tris-HCl
buffer (pH 8.0) containing 5 mM MgCl2, 300 mM KCl, 0.5 mM each of ATP, CTP, GTP, and UTP,
RNase guard (111 units), and DNase (10 units). RNA was extracted as
described previously (28). For the isolation of total cytoplasmic RNA,
cells were incubated in the presence or absence of test agents for
1 h and washed with ice-cold PBS. Nuclear and total RNA were
applied to a nylon membrane using a vacuum filtration apparatus (36).
32P-Labeled cDNA probes for porcine iNOS (37) and mouse
-actin (Ambion) were prepared using an oligolabeling kit (Amersham
Pharmacia Biotech); unincorporated nucleotides were removed by G-25
column chromatography. Membranes were hybridized to the radiolabeled probes and washed (36). The bands were visualized and quantified by
Phosphorimaging (Molecular Dynamics).
Nuclear Calcium Signals and Uptake--
The uptake of
45Ca2+ in isolated nuclei was determined as
described previously (14). Briefly, nuclei were resuspended in buffer A
(125 mM KCl, 2 mM
K2HPO4, 25 mM Hepes, 4 mM MgCl2, and 400 nM CaCl2, pH 7.0). 45Ca2+ (2 µCi/ml)
was added, and samples were incubated in the presence or absence of
test agents at 37 °C for different time periods. The reaction was
terminated with ice-cold buffer containing 50 mM Tris-HCl
and 150 mM KCl (pH 7.0), filtered under vacuum on glass
fiber filters (GF/B, Whatman). The radioactivity on filters was counted
on a beta-counter (Beckman LS 7500). The 45Ca2+
transient was defined as the radioactivity at a given time minus the
radioactivity at time zero.
Effects of test agents on calcium transients were measured by fura-2/AM
fluorometry as described (14, 22) with some modifications. Isolated
liver nuclei were resuspended in buffer A and preloaded with 7.5 µM fura-2/AM for 45 min at 4 °C. The nuclei were
washed and stimulated (~2 × 106 nuclei/ml) with
various test agents. The intranuclear calcium concentration was
measured with a spectrofluorometer (LS 50, Perkin Elmer, Beaconsfield,
UK) and fluorescent signal calibrated (22).
To assess the role of PTX-sensitive G proteins in nuclear
Ca2+ transients, isolated rat liver nuclei were treated
with PTX as described (38). Prior to treatment, the toxin was
preactivated by incubating at 37 °C for 10 min in 50 mM
Tris-HCl (pH 7.5) containing 100 mM dithiothreitol and 0.1 mM ATP. The isolated nuclei were incubated with
preactivated PTX (20 µg/ml) at 25 °C for 20 min in buffer A
containing 1 mM NAD and 50 µM GDP. The
treated nuclei were washed with buffer A and then the effects of
prostaglandin analogs on intranuclear calcium levels were measured by
fura-2/AM fluorometry as above.
 |
RESULTS AND DISCUSSION |
[3H]PGE2 Binding to Subcellular
Fractions--
The maximal specific binding of
[3H]PGE2 to newborn pig brain plasma
membrane, ER, and nuclear membrane fractions was comparable (Table
I); in adult rat liver, the
Bmax for [3H]PGE2 on
plasma and nuclear membranes was also similar. The affinity constant
(KD) of PGE2 binding did not
significantly differ between tissues and membrane fractions. Subtypes
of PGE2 receptors were studied by displacement of bound
[3H]PGE2 with PGE2, AH6809
(EP1 receptor antagonist), butaprost (EP2
agonist), M&B 28,767 (EP3 subtype agonist), and AH23848B (EP4 antagonist) (6). Neonatal porcine brain plasma and
nuclear membranes contained mostly EP3 receptors (nearly
100 and 45%, respectively); on nuclear membranes, the balance of EP
receptors was equally divided among EP1, EP2,
and EP4 (Fig. 1, a
and b). In adult liver plasma membranes, all four EP
receptors were detected in equal proportions (25%); in liver nuclear
membranes, EP3 was most abundant (40%), and
EP1, EP2, and EP4 consisted 15, 20, and 30% of EP receptors, respectively (Fig. 1, c and
d). [3H]PGE2 displacement data and
the availability of specific anti-EP3
and
anti-EP4 receptor antibodies (29) led us to focus our
investigation on the cellular localization of EP3
and
EP4 receptors, and the remaining studies concentrated on
these two receptor subtypes.
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Table I
Maximum specific binding of [3H]PGE2 (Bmax)
on cell fractions
Values (Bmax in fmol/mg protein,
KD in nM) are mean ± S.E. of three
experiments, in duplicate. ND, not determined.
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Fig. 1.
Competitive displacement of
[3H]PGE2 binding to
plasma and nuclear membrane from newborn pig brain and adult rat liver
by prostaglandins and analogs. Shown are brain plasma membrane
(a), nuclear membrane (b), liver plasma membrane
(c), and nuclear membrane (d). Membranes were
incubated with 10 nM [3H]PGE2 at
37 °C for 30 min in the presence or absence of 25 µM
unlabeled PGE2 to determine 100% specific binding. ,
PGE2; , AH6809 (EP1 antagonist); ,
butaprost (EP2 agonist); , M&B 28,767 (EP3
agonist); , AH2384B (EP4 antagonist). Each data point is
the mean ± S.E. of four experiments performed in duplicate.
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Expression of EP3
and EP4 Immunoreactive
Protein in Newborn Brain Subcellular Fractions--
Immunoblot
analysis revealed immunoreactive bands in plasma and nuclear fractions
of similar molecular masses (EP3
, 60 kDa;
EP4, 63 kDa) (Fig.
2).

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Fig. 2.
Immunoblot of EP3
and EP4 receptor proteins (see
arrows) in plasma membrane (P) and
nuclear membrane (N) fractions from newborn pig
brain. Top and bottom arrows point to
EP4 and EP3 bands, respectively; only one
band was detected in the range of interest (50-65 kDa).
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Indirect Immunofluorescence of EP3
and
EP4 Receptors in Porcine Cerebral Vessel Endothelial
Cells--
Because cerebral microvessels contain a number of high
affinity PGE2 binding sites (20), primary cultures of
newborn pig brain microvascular endothelial cells were used to study
the intracellular distribution of EP3
and
EP4 receptors by confocal microscopy. No fluorescence was
detected in the absence of the primary antibodies (Fig.
3a). Immunoreactivity for both
receptor subtypes was detected in the plasma membrane (Figs.
3b and 4a), in the cytoplasm, and at the nucleus
(Figs. 3c and 4b). EP3
specific
fluorescence in the nuclear envelope appeared as a perinuclear halo
(Fig. 3c); the latter was more prominent than that of
EP4 receptors (Fig. 4b). The cells were stained
with DiOC6 (3) to identify intracellular membranes, mainly
ER (Figs. 3d and 4c). Merging the images from EP3
or EP4 specific red immunofluorescence
with DiOC6 (3) green staining revealed that EP receptors
colocalized on intracellular membranes as indicated by the bright
yellow-orange fluorescence (Fig. 3e and
4d); however, the stains did not fully converge, suggesting
distinct sites particularly evident in the perinuclear region. Cells
were also stained with a nuclear stain, Sytox Green (Fig.
3g). A transverse section (Z-section) of this image
superimposed with that of the EP3
immunoreative staining
(Fig. 3h) in the same cell revealed that the
immunoreactivity was perinuclear and not intranuclear (Fig.
3i); similar results were obtained using the EP4
antibody (data not shown). No immunofluorescence was detected when the
antibodies were preincubated with their cognate peptides (Figs.
3f and 4e). EP3
and
EP4 immunoreactivity in co-localization with
DiOC6 (3) staining apparently in the nucleoli of a few
endothelial cells (Figs. 3, c-e and 4, b-d) was
not consistently observed (Fig. 3h) and remains unexplained at this point.

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Fig. 3.
Confocal microscopy of porcine cerebral
microvascular endothelial cells immunofluorescently stained for
EP3 receptors. Cells were subjected to
indirect immunofluorescent staining using affinity purified rabbit
anti-peptide EP3 antibodies followed by Texas
Red-conjugated anti-rabbit IgG. a, Texas Red-conjugated IgG
alone; note absence of immunofluorescence. EP3
immunoreactivity on plasma membrane (b) and nuclear membrane
and cytoplasm (c); note perinuclear halo in more extensively
permeabilized cells (see "Experimental Procedures"). d,
DiOC6 (3), intracellular membranes (mostly endoplasmic
reticulum) stain; e, superimposed images of panels
c and d; note red (EP3
immunoreactivity) perinuclear halo. f,
anti-EP3 in the presence of cognate peptide, (10 µg/ml), note lack of immunofluorescence; g, Sytox Green
nucleus stain; h, EP3 immunoreactivity;
i, Z section of superimposed images of panels g
and h.
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Fig. 4.
Confocal microscopy of porcine cerebral
microvascular endothelial cells immunofluorescently stained for
EP4 receptors. Cells were subjected to
indirect immunofluorescent staining using affinity purified rabbit
anti-peptide EP4 antibodies followed by Texas
Red-conjugated anti-rabbit IgG. EP4 immunoreactivity on
plasma membrane (a), and nuclear membrane and cytoplasm
(b); perinuclear halo is noted in more extensively
permeabilized cells (see "Experimental Procedures"). c,
DiOC6 (3), intracellular membranes (mostly
endoplasmic reticulum) stain; d, superimposed
images of panels b and c, note
red (EP4 immunoreactivity) perinuclear halo;
e, anti-EP4 in the presence of cognate
peptide (10 µg/ml), note absent immunofluorescence.
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Indirect Immunofluorescence of EP3
and
EP4 Receptors in HEK 293 Cells--
To assess whether this
perinuclear distribution of EP3
and EP4
receptors applies generally to cells, the localization of these
receptors was studied after transfection of cDNA for EP3
and EP4 into HEK 293 cells that do not
normally express prostanoid receptors (39); ectopically expressed EP
receptors in HEK 293 cells bind PGE2 and are functional
(39, 40). Immunoreactivity for EP3
and EP4
receptors was seen on the plasma membrane (Fig.
5, c and g) and
perinuclear area, which are in proximity to each other in the
transfected HEK 293 cells (Fig. 5d, h), which are relatively small and
contain limited cytoplasm compared with endothelial cells. As expected,
no immunofluorescence was detected in the wild-type cells (Fig.
5a) or after preincubation of the antibodies with their
cognate peptide epitopes (Fig. 5 f and j).

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Fig. 5.
EP3 and
EP4 immunofluorescent staining of overexpressing
clones of human EP3 or human
EP4 receptors in HEK 293 cells.
a, cells transfected with vector alone, note absence of
immunofluorescence; b, nuclear stain (DAPI) of cells from
panel a. EP3 immunoreactivity on plasma
membrane (c) and nuclear membrane (d); note the
perinuclear halo in more permeabilized cells (see "Experimental
Procedures"). e, nuclear stain (DAPI) of cells from
panel d. f, anti-EP3 in the
presence of cognate peptide (10 µg/ml), note the lack of
immunofluorescence. EP4 immunoreactivity on plasma membrane
(g) and nuclear membrane (h), note the
perinuclear halo in more permeabilized cells (see "Experimental
Procedures"). i, nuclear stain (DAPI) of cells from
panel h; j, anti-EP4 in the presence
of cognate peptide (10 µg/ml).
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Immunogold Labeling of EP3
and EP4
Receptors--
Thus far, indirect immunofluorescence studies revealed
a perinuclear localization of EP3
and EP4
receptors. To distinguish the nuclear envelope, immunoelectron
microscopy of porcine cerebrovascular endothelial cells was performed
and confirmed that EP3
and EP4
immunoreactivity was indeed at the nuclear envelope (Fig. 6, c and f). As
expected, these receptors were detected on plasma membranes (Fig. 6
b and e) and Golgi vesicles (Fig. 6d).
No immunogold staining was observed in the absence of the primary
antibodies (Fig. 6a) or in the nucleoli of cells (data not
shown). EP3
and EP4 nuclear envelope
immunogold staining was detected in the majority of cells observed
(95% of cells, over 100 cells observed in each case).

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Fig. 6.
Immunogold localization of
EP3 and EP4 receptors
in porcine cerebrovascular endothelial cells by electron microscopy
(see arrows). a, anti-rabbit
gold-conjugated IgG alone; note absence of immunostaining when primary
antibody is omitted. EP3 immunoreactivity on plasma
membrane (b) and nuclear membrane (c).
EP4 immunoreactivity on Golgi vesicles (d),
plasma membrane (e), and nuclear membrane (f).
Scale bar in each represents 0.5 µm.
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Effects of Stimulation of Porcine Cerebrovascular Endothelial Cells
Nuclear EP Receptors on iNOS Gene Transcription--
Recent studies
have shown that endogenous PGE2 has a stimulatory effect on
inducible nitric-oxide synthase (iNOS) (41, 42). We tested whether the
stimulation of nuclear EP receptors by prostaglandin analogs may affect
iNOS transcription, as determined by dot hybridization of RNA studies.
Stimulation of intact nuclei isolated from primary cultures of porcine
brain endothelial cells with EP3 receptor agonist M&B
28,767 (0.1 µM) increased transcription of iNOS (Fig. 7a) to a greater extent than
after stimulation of whole cells.

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Fig. 7.
Effects of nuclear EP receptor stimulation on
iNOS gene transcription and nuclear calcium transients.
a, effects of EP3 agonist M&B 28,767 (0.1 µM) on iNOS transcription in neonatal porcine cerebral
microvascular endothelial cells (primary culture from newborn brain) as
determined by dot blot hybridization of RNA; MB and
C refer to M&B 28,767 and control (unstimulated),
respectively. -Actin dot blot indicates equal loading. One
representative dot blot of three is shown. b, effect of M&B
28,767 (1 µM) on 45Ca2+ uptake by
isolated liver nuclei; control refers to absence of drug. The free
calcium concentration was 400 nM. The movement of
45Ca2+ transient after a given time was defined
as radioactivity at a given time minus the radioactivity at time 0. c, typical tracings showing effects of PGE2
analog 16,16-dimethyl PGE2 (1 µM) and
EP3 agonist M&B 28,767 (0.01-1 µM) on
intranuclear calcium concentrations
([Ca2+]n) in isolated liver nuclei
loaded with fura-2/AM; arrow shows the time of application
of test agents. d, peak increases in isolated liver
intranuclear calcium ([Ca2+]n)
after addition of M&B 28,767 (1 µM) in the presence or
absence of PTX preincubation (20 µg/ml, 20 min at 25 °C).
Experiments (b-d) were carried out on three independent
isolations of intact nuclei, each one performed in duplicate.
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Effects of Stimulation of Rat Liver Nuclear EP Receptors on
Ca2+ Transients--
The nuclear envelope contains
distinct nuclear calcium pools that play crucial roles in major nuclear
functions including gene transcription (13). The amplitude and duration
of calcium signals have been shown to control differential activation
of transcription factors (43). In addition, Ca2+ can
activate iNOS independent of protein kinases C and A (44). We tested
whether stimulation of nuclear EP3 receptors with
prostaglandin analogs could affect calcium concentrations in isolated
nuclei of liver; stimulation of EP4 receptors could not be
performed because of lack of availability of specific agonists.
Application of M&B 28,767 (1 µM, an
EP3-selective agonist) to intact isolated nuclei caused
rapid nuclear uptake of 45Ca2+ (Fig.
7b). In addition, this EP3 agonist produced a
dose-dependent increase in rat liver nuclear calcium as
determined by fura-2/AM, a fluorescent dye which localizes in the
nuclear envelope space (45) (Fig. 7c); M&B 28,767 (1 µM) was nearly as effective as the nonselective EP
agonist 16,16-dimethyl PGE2 (1 µM).
We determined whether the nuclear calcium uptake evoked by the
EP3 agonist M&B 28,767 was dependent on G proteins.
EP3 couples to Gi or Go (46), which
are known to affect Ca2+ mobilization (46, 47); such G
proteins are detected in rat liver nuclei (38). Because PTX causes
these G proteins to lose their ability to associate with receptors, we
tested the effects of PTX on M&B 28,767-induced Ca2+
transients. Pretreatment of isolated nuclei with PTX markedly attenuated the stimulatory effect of M&B 28,767 (1 µM) on
intranuclear calcium levels, suggesting the involvement of a
PTX-sensitive G protein in mediating the effects of nuclear
EP3 receptors (Fig. 7d). In contrast, M&B 28,767 did not inhibit forskolin-stimulated cAMP formation (data not shown).
These findings are consistent with coupling of nuclear EP3
receptors to G proteins which may directly control Ca2+
channels independently of second messengers, as mostly reported for
Gi (47, 48).
In conclusion, the data presented provides the first clear evidence for
the presence of the G protein-coupled receptors, EP3
and
EP4 at nuclear membranes of native tissues as well as
primary and transfected cells. Furthermore, these receptors seem to be functional as revealed by increased iNOS transcription and nuclear calcium by EP3 agonist, M&B 28,767, which also appears to
involve PTX-sensitive G proteins. The plasma and nuclear membrane
EP3
as well as EP4 receptors appear to be
related because they had similar molecular weights, binding kinetics,
ligand binding properties, and immunoreactivity. Also, the comparable
distribution of ectopically expressed EP3
and
EP4 receptors in HEK 293 cells suggested that the plasma
and nuclear membrane EP receptors may be alike. Radioligand binding
studies have identified the presence of two other classes of G
protein-coupled receptors at the nuclear membrane, the muscarinic
acetylcholine (49) and angiotensin II receptors, AT1 (23);
but the AT1 receptor can be detected in the nucleus only
after stimulation by angiotensin II (50). Other prostanoids, namely
PGD2, its metabolite PGJ2, and PGI2
can activate the peroxisome proliferator-activated receptors (PPARs)
that are members of the nuclear receptor superfamily, but PPARs are not
responsive to PGE2 (51, 52). However, the presence of
EP1 receptors at the nuclear membranes has recently been
suggested (19) albeit its mechanism of action is not clear.
In the newborn brain and cerebral microvasculature, PGE2
receptors and associated functions at the plasma membrane are
down-regulated (2, 20). On the other hand, PGE2 plays a
role in neuroprotection by acting on EP2 and perhaps
EP4 (53). PGE2 also increases the expression of
nitric-oxide synthase via stimulation of EP3 receptors in
the neonate (54). In addition, the perinuclear cyclooxygenase-1 and -2 (7) can produce prostanoids that can act at the nuclear level (11, 12)
and modulate transcription of genes, as had been speculated for iNOS
(41). The present discovery of nuclear EP3 and
EP4 receptors proposes new avenues for the intracellular actions of prostanoids, which may also explain certain effects of
PGE2 especially when plasma membrane EP receptors are
barely detectable. Further studies are needed to clarify the detailed signal transduction mechanisms involved in this action of
prostaglandins via nuclear EP receptors.
 |
ACKNOWLEDGEMENTS |
We thank Hensy Fernandez and Marie Ballak for
technical assistance.
 |
FOOTNOTES |
*
This work was supported by the Medical Research Council of
Canada and the Fonds de la Recherche en Santé du Québec.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.
§
Recipient of a Doctoral Research Award from the Medical Research
Council of Canada.

To whom correspondence should be addressed: Research Center of
Hôpital Ste. Justine, 3175-Côte Sainte-Catherine, Montreal, Quebec H3T 1C5 Canada. Tel.: 514-345-4692; Fax: 514-345-4801; E-mail:
chemtobs{at}ere.umontreal.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
PGE2, prostaglandin E2;
EP, PGE2
receptor;
NEST, nuclear envelope signal transduction;
HEK, human
embryonic kidney cells;
ER, endoplasmic reticulum;
PBS, phosphate-buffered saline;
iNOS, inducible nitric-oxide synthase;
PTX, pertussis toxin;
DiOC6, 3,3'-dihexyloxacarbocyanine iodide;
DAPI, 4'-6-diamino-2-phenylindole.
 |
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