From the
The subcellular locations of prostaglandin endoperoxide
synthase-1 and -2 (PGHS-1 and -2) were determined by quantitative
confocal fluorescence imaging microscopy in murine 3T3 cells and human
and bovine endothelial cells using immunocytofluorescence with
isozyme-specific antibodies. In all of the cell types examined, PGHS-1
immunoreactivity was found equally distributed in the endoplasmic
reticulum (ER) and nuclear envelope (NE). PGHS-2 immunoreactivity was
also present in the ER and NE. However, PGHS-2 staining was twice as
concentrated in the NE as in the ER. A histofluorescence staining
method was developed to localize cyclooxygenase/peroxidase activity. In
quiescent 3T3 cells, which express only PGHS-1, histofluorescent
staining was most concentrated in the perinuclear cytoplasmic region.
In contrast, histochemical staining for PGHS-2 activity was about
equally intense in the nucleus and in the cytoplasm, a pattern of
activity staining distinct from that observed with PGHS-1. Our results
indicate that there are significant differences in the subcellular
locations of PGHS-1 and PGHS-2. It appears that PGHS-1 functions
predominantly in the ER whereas PGHS-2 may function in the ER and the
NE. We speculate that PGHS-1 and PGHS-2 acting in the ER and PGHS-2
functioning in the NE represent independent prostanoid biosynthetic
systems.
PGH
PGHS-1 and PGHS-2 are 60% identical at the amino acid level. The
most pronounced differences occur in the signal peptides and membrane
binding domains near the N termini and in the C-terminal 15-25
amino acids
(1, 2, 4, 19) . The kinetic
properties of the two enzymes are quite similar. Both enzymes catalyze
( a) a cyclooxygenase reaction in which arachidonic acid is
converted to PGG
Treatment of quiescent murine 3T3 cells with serum causes an
increase in PGHS-2 protein levels which peak approximately 3 h
post-serum treatment. During this period, there are no apparent changes
in the levels of PGHS-1 protein
(8, 10, 16) .
However, measurements of cyclooxygenase activities in quiescent and
serum-stimulated (3 h) 3T3 cells have established that platelet-derived
growth factor and serum cause only a 1.5- to 2-fold increase in total
activity
(8, 29, 30, 31) . Similar
measurements comparing the cyclooxygenase activities of control and
LPS-treated macrophages have yielded similar results
(16) .
Thus, in those cells which already express PGHS-1, there is no dramatic
increase in the total PGH
There is recent biochemical evidence that PGHS-1 and
PGHS-2 function as separate biosynthetic systems. Reddy and Herschman
(32) reported that in murine 3T3 cells and macrophages PGHS-1
and PGHS-2 utilize different pools of arachidonate for synthesizing
prostanoids. In murine bone marrow-derived mast cells, PGHS-1 and
PGHS-2 are coupled to different stimulus-initiated pathways
(33) . These results argue that the arachidonate used by PGHS-1
and PGHS-2 for prostanoid biosynthesis is derived from different
endogenous lipid pools.
If PGHS-1 and PGHS-2 do use different
intracellular pools of arachidonate, there must be mechanisms for
compartmentalizing PGHS-1 and PGHS-2 such that each isozyme would only
have access to its own arachidonate substrate pool. One possibility is
that the PGHS isozymes are located in different subcellular
compartments. Previous studies, including those from our laboratory,
have indicated that PGHS-1 and PGHS-2 are both found in the endoplasmic
reticulum (ER) and nuclear envelope (NE) of fibroblasts and macrophages
and that there is no particularly obvious difference in the subcellular
distributions of the isozymes
(34, 35, 36, 37) . We have now
reinvestigated this issue using a variety of cell types, several
antibodies for each isozyme, and a newly developed histofluorescence
assay for PGHS activity. Our results confirm the previous observations
that both PGHS-1 and PGHS-2 are located within the ER and NE. However,
PGHS-2 protein was found to be more concentrated in the NE than PGHS-1.
And when the distributions of PGHS-1 and PGHS-2 activities were
monitored by histofluorescence, only PGHS-2 caused staining within the
cell nucleus.
NIH 3T3 cells expressing PGHS-2 constitutively were prepared as
follows. A 1.8-kilobase SalI restriction fragment containing
the entire murine PGHS-2 coding sequence
(39) was subcloned
into the Lac-inducible plasmid pRSVNOT
(40) . The pRSVNOT
plasmid, part of the LacSwitch gene expression system (Stratagene),
contains a combination Rous sarcoma virus promoter (RSV)-bacterial
Human umbilical vein
endothelial cells (HUVECs) (Cell Systems, passage 3-4) were
cultured in Lab-Tek chambered coverglasses in Cell System CS-C venous
endothelial cell growth medium for 3 days. The cells were then treated
with 2 ng/ml of phorbol myristate acetate (PMA) for 3 h and used for
localization experiments.
Bovine arterial endothelial cells (BAECs)
were isolated from freshly excised carotid artery obtained at a local
abattoir as described previously
(41) . The cells were seeded on
100-mm Corning culture dishes in DMEM supplemented with 20% fetal calf
serum and subcultured in DMEM containing 10% fetal calf serum. The
cells were identified as endothelial cells from their typical
cobblestone appearance at confluency. The endothelial cells (passages
20-22) were cultured in Lab-Tek chambered coverglasses for 2
days, and then cells were treated with 2 ng/ml phorbol 12-myristate
13-acetate (PMA) for 3 h before use in localization experiments.
The studies reported here provide evidence for differences in
the subcellular compartmentation of PGHS-1 and PGHS-2. Our results
provide support for an emerging view that PGHS-1 and PGHS-2 represent
two independently operating prostanoid biosynthetic systems which
produce prostanoids for extracellular housekeeping and intranuclear
differentiative events, respectively. This concept derives from the
following observations. First, PGHS-1 is expressed constitutively by
many cells and is available for acute, on demand prostanoid formation
to modulate processes such as platelet aggregation
(45, 46) and renal water reabsorption
(47, 48) ; in
contrast, PGHS-2 is normally absent from most cells but can be induced
in association with differentiative or replicative events
(2, 4, 7, 10) . Further supporting the
idea that there are two distinct prostanoid systems are findings which
suggest that PGHS-1 and PGHS-2 utilize different arachidonate pools
that are mobilized in response to different cellular stimuli
(32, 33) . Finally, in fibroblasts and macrophages
expressing PGHS-1 constitutively, induction of PGHS-2 leads to a modest
1.5- to 2-fold increase in prostanoid biosynthetic capacities
(8, 16, 29, 30, 31) ; this,
coupled with the fact that the kinetic properties of the isozymes are
very similar, makes it hard to argue that PGHS-2, at least in
fibroblasts and macrophages, exists solely to supplement PGHS-1
activity.
It had been established by immunoelectron microscopy that
PGHS-1 is associated with the ER and NE but not the plasma membrane or
mitochondrion
(34) . When examined by either conventional or
confocal microscopy, PGHS-1 and PGHS-2 exhibit quite similar patterns
of cytoplasmic and perinuclear immunofluorescent staining in both
murine 3T3 cells and macrophages
(35, 36, 37) .
These results have implied that both PGHS-1 and PGHS-2 are associated
with the same intracellular membrane systems, the ER and NE. Our
present results are in qualitative agreement with previous findings.
However, repeated visual observations suggested that there were subtle
differences between the relative levels of cytoplasmic and perinuclear
immunostaining of PGHS-1 and PGHS-2 in 3T3 cells and in bovine and
human endothelial cells. More precise quantitation made possible by
confocal microscopy and digital imaging confirmed these observations.
The intensities of staining for PGHS-1 immunoreactivity,
DiOC
In the
case of PGHS-1, we interpret the results of the immunochemical staining
simply to reaffirm the concept that this isozyme is a resident ER
protein which binds to the lumenal surface of the ER
(36) .
Membrane binding purportedly involves a series of amphipathic helices,
residues 70-116 of the ovine PGHS-1
(49) .
In the case of
PGHS-2, our immunocytochemical localization studies raised the
possibility that this isozyme might form PGH
At this stage, we can only speculate about the implications
of our findings that PGHS-2 is present in ER but is concentrated in the
NE. First, we suggest that PGHS-2 present in the ER of serum-stimulated
3T3 cells and PMA-treated endothelial cells serves to augment
PGHS-1-mediated PGH
If PGHS-2 is uniquely present on the inner membrane of
the nuclear envelope, it may have the ability to produce prostanoids
that function within the nucleus. This is an attractive possibility
based simply on the observations that PGHS-2 is an immediate early gene
(9) whose induction is consistently associated with cell
differentiation or replication, events typically attributable to cell
nuclear activity. The question of the whether PGHS-1 and PGHS-2
proteins have different locations in the NE can best be addressed by
high resolution immunoelectron microscopy using isozyme-specific
antibodies. Equally important would be the ability to visualize the
sites of PGH
Based on
studies with other resident ER proteins, we presume that PGHS-1 is
initially translocated during its synthesis into the ER lumen and then
either retained in the ER
(52) or circulated through the
cis-Golgi ER retention system
(53, 54, 55) . The identity of the ER targeting
signal for PGHS-1 is unknown. In fact, contrary to expectations, the
C-terminal -P/STEL sequence present in all PGHSs appears not to
be involved in ER targeting.
(
)
synthases catalyze the formation of
PGH
from arachidonic acid. Newly formed PGH
is
subsequently converted to what are considered to be the biologically
active prostanoids, PGD
, PGE
,
PGF
, thromboxane A
, or PGI
.
There are two PGH synthase isozymes called PGHS-1 (COX-1) and PGHS-2
(COX-2). Each isozyme is encoded by a different gene
(1, 2) . PGHS-1 is a constitutive enzyme present in many
but not all mammalian cells
(3, 4, 5) . Its
expression appears to be regulated developmentally
(6) . PGHS-2
is undetectable in most mammalian tissues
(4, 5) , but
expression of this isozyme can be induced by cytokines, growth factors,
and tumor promoters
(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
and ( b) a peroxidase reaction in
which PGG
is reduced to PGH
(20, 21) . Moreover, PGHS-1 and PGHS-2 have
similar V
and K
values with arachidonate
(21, 22, 23) ,
both enzymes form a tyrosyl radical in the presence of hydroperoxides
(24) , and each isozyme undergoes suicide inactivation
(22, 23) . Finally, those amino acids required for
catalysis by PGHS-1 are conserved in PGHS-2
(19, 25) .
Despite the overall enzymic similarities between PGHS-1 and PGHS-2, it
should be noted that there are subtle differences between the active
sites of PGHS-1 and PGHS-2 as evidenced by their different affinities
toward some fatty acid substrates
(
)
and
nonsteroidal anti-inflammatory drugs
(21, 22, 23, 26, 27, 28) .
biosynthetic capacity following
the induction of PGHS-2. Coupled with the fact that PGHS-1 and PGHS-2
have very similar kinetic properties, it is difficult to argue that
PGHS-2 is induced simply to augment the biosynthetic capacity of
PGHS-1. Rather, PGHS-2 seems likely to have a function(s) independent
of PGHS-1.
Materials
Dulbecco's modified
Eagle's medium (DMEM) was from Life Technologies, Inc.
Chloroquine, horse myoglobin, saponin, digitonin, fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, FITC-conjugated
concanavalin A (ConA) and DEAE-dextran were from Sigma. Ellman's
reagent, Sulfolink maleimide-activated gel, and maleimide-activated KLH
coupling kits were from Pierce Chemical Co. Fetal calf serum and calf
serum were obtained from Hyclone. Peptides were from the Howard Hughes
Research Center at Harvard Medical School. Arachidonic acid and
linoleic acid were from Cayman Chemical Co. 5-(and
-6)-Carboxyl-2`,7`-dichlorodihydrofluorescein diacetate (CDCF-DA) and
3,3`-dihexyloxacarbocyanine iodide (DiOC(3) ) were
purchased from Molecular Probes. Valerylsalicylate was synthesized as
described previously
(38) . Other reagents were from common
commercial sources.
Cell Culture
NIH 3T3 fibroblasts were grown on
100-mm Corning culture dishes in DMEM supplemented with 8% calf serum
and 2% fetal calf serum. The cells were grown to approximately 80%
confluence, detached from the monolayer with 0.25% trypsin, 0.02% EDTA,
diluted into 50 ml of serum-supplemented DMEM, and used to inoculate
Lab-Tek chambered coverglasses. NIH 3T3 cells cultured in DMEM
supplemented with 0.2% calf serum for 48 h were designated
``quiescent'' cells. To obtain ``serum-activated''
cells, the serum-starved cells were changed to a medium containing 8%
calf serum and 2% fetal calf serum in DMEM and incubated for 3 h.
-galactosidase promoter (RSV/
-gal) to drive transcription of
the donor gene. The pRSVNOT-PGHS-2 plasmid was transfected into a
murine NIH 3T3 cell line expressing a Lac repressor protein, modified
so that it functions as an efficient eukaryotic transcription factor.
In theory, the Lac repressor protein tonically suppresses gene
expression from the RSV/
-gal promoter, and suppression can be
relieved by addition of
isopropyl-1-thio-
-D-galactopyranoside, allowing regulated
expression of the target gene. During characterization of
pRSVNOT-PGHS-2 transformants, however, one cell line was identified
that constitutively expressed PGHS-2. This line was designated as the
3T3-cPGHS-2 cell line. 3T3-cPGHS-2 cells constitutively express PGHS-1
and PGHS-2 under serum-starved culture conditions. 3T3-cPGHS-2 cells
were routinely grown in DMEM supplemented with 10% fetal calf serum and
600 µg/ml geneticin and hygromycin. NIH 3T3-cPGHS-2 cells were
transferred to Lab-Tek chambered coverglasses and cultured for 2 days,
the medium was removed, and the cells were then cultured in DMEM
supplemented with 0.2% calf serum for 24 h. These cells were used for
immunocytochemistry as described below.
Isozyme-specific Anti-peptide
Antibodies
Affinity-purified isozyme-specific antibodies to
PGHS-1 and PGHS-2 were generated as described previously
(36) .
Antibodies reactive against both the murine and human PGHS-1 were
prepared using the following KLH-coupled peptides derived from the
murine protein: ( a)
Leu-Met-Arg-Tyr-Pro-Pro-Gly-Val-Pro-Pro-Glu-Arg-Gln-Met-Ala-Cys
(residues 274-289) or ( b)
Cys-Asn-Thr-Ser-Met-Leu-Val-Asp-Tyr-Gly-Val-Glu-Ala-Leu-Val-Asp-Ala-Phe-Ser
(residues 412-429). An additional anti-peptide antibody reactive
with human, ovine, and bovine PGHS-1, but not murine PGHS-1, was
prepared against the ovine PGHS-1 sequence,
Leu-Met-His-Tyr-Pro-Arg-Gly-Ile-Pro-Pro-Gln (residues 272-283)
(36) . We have previously described an anti-peptide antibody
reactive with human, bovine, and murine PGHS-2
(10, 22, 35, 36, 39) ; this
antibody was prepared against the peptide
Ser-His-Ser-Arg-Leu-Asp-Asp-Ile-Asn-Pro-Thr-Val-Leu-Ile-Lys (residues
584-598), a sequence unique to PGHS-2. Another antibody to murine
PGHS-2 was prepared against the KLH-coupled murine PGHS-2-derived
peptide, Cys-Gln-Asp-Pro-Gln-Pro-Thr-Lys-Thr-Ala-Thr-Ile-Asn (residues
569-580). Peptide-specific antibodies were purified by affinity
chromatography using peptide-coupled Sulfolink matrix as described
previously
(35, 36) and were diluted to approximately
0.5 mg/ml.
Immunocytochemistry of PGHS
Murine NIH 3T3 cells,
murine NIH 3T3-cPGHS-2 cells, HUVECs, and BAECs cultured in Lab-Tek
chambers were washed in phosphate buffered saline (PBS), fixed by
incubation for 10 min in 2% formaldehyde in PBS, and washed in 10% calf
serum in PBS
(35) . Affinity-purified anti-peptide antibodies to
either PGHS-1 or PGHS-2 diluted 1:20 in PBS containing 0.2% saponin and
10% calf serum were added to the chambers, and the samples were
incubated for 30 min. After washing in PBS containing 10% calf serum,
the samples were incubated for 30 min with a 1:40 dilution of
FITC-conjugated goat anti-rabbit IgG in PBS containing 0.2% saponin and
10% calf serum. The samples were washed with PBS containing 10% calf
serum and then rinsed with PBS. For negative control staining, the same
procedure as described above was performed ( a) without primary
antibody or ( b) with primary antibody together with a 10
µM concentration of its cognate peptide.
Cell Labeling with DiOC
In one experiment, after murine 3T3 cells
were subjected to immunocytofluorescence with anti-peptide antibodies,
the cells were then stained using either DiOC(3) or
FITC-labeled ConA
(3) or
FITC-labeled ConA. The cells were treated with 1 µg/ml
DiOC
(3, 42) for 10 s or with 5 µg/ml
FITC-conjugated ConA
(43) for 1 min at room temperature. The
samples were then rinsed with PBS, pH 7.4. These procedures were
performed on the platform of the microscope, and the samples were
visualized immediately.
Histochemical Measurements of PGHS
Activity
Quiescent or serum-activated NIH 3T3 cells were washed
with PBS containing 0.1 mg/ml of both calcium chloride and magnesium
chloride (PBS+), incubated with 2 µM CDCF-DA in
PBS+ for 5 min, and washed with PBS+. The fluorescence
intensity in the cells at 0 time and 20 s after the addition of 10
µM arachidonic acid was quantified by fluorescence
confocal microscopy.
Fluorescence Confocal Microscopy
An Insight
Bilateral scanning confocal microscope (Meridian Instruments, Okemos,
MI) was used with an argon ion laser as the excitation source
(44) . A 60 objective and a laser power setting of 1
milliwatt were used for subcellular localization of enzyme activity by
histofluorescence and subcellular localization of DiOC
(3) and FITC-labeled ConA. A 60
objective lens and a
laser power setting of 30 milliwatts were used for subcellular
localization of PGHSs by immunocytochemistry.
Subcellular Localization of PGHS-1 and PGHS-2 in Murine
3T3 Cells
Shown in Fig. 1are fluorescence
photomicrographs of quiescent and serum-activated murine 3T3 cells
stained by indirect immunofluorescence using primary antibodies
specific for either PGHS-1 or PGHS-2. Staining for PGHS-1 was performed
separately using two different anti-peptide antibodies directed against
residues 274-289 and residues 412-429. In both quiescent
and serum-activated 3T3 cells, PGHS-1 immunoreactivity was observed in
the cytoplasm near the nucleus and also more peripherally. PGHS-1 is
known to be present in the endoplasmic reticulum (ER) of 3T3 cells
(34) , and, thus, fluorescent staining in the cytoplasm is due
to staining of the ER. Anti-peptide antibodies reactive with residues
569-580 or 584-598 of murine PGHS-2 caused fluorescence
staining of both the cytoplasm and the nuclear envelope (NE) of
serum-activated 3T3 cells (Fig. 1); staining of the NE was
clearly more prominent for PGHS-2 than for PGHS-1. No staining for
PGHS-2 was observed in quiescent, serum-starved 3T3 cells which do not
express this second PGHS isozyme
(8, 10, 11, 35) . Moreover,
preincubation of either of the anti-PGHS-1 or anti-PGHS-2 antibodies
with their cognate peptides completely eliminated staining, and no
staining was observed in the absence of the primary antibody.
Figure 1:
Immunofluorescence staining for PGHS-1
and PGHS-2 in quiescent and serum-activated murine NIH 3T3 cells.
Murine NIH 3T3 cells were subjected to indirect immunofluorescent
staining using affinity-purified rabbit anti-peptide antibodies
directed against PGHS-1 or PGHS-2 and then FITC-labeled goat
anti-rabbit IgG as described in the text. The final concentrations of
the anti-peptide antibodies used in each experiment were 25 µg/ml.
Cognate peptides used to block antibodies were used at a concentration
of 10 µM. A, serum-activated 3T3 cells and
antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues
274-288); B, computer enhancement shadowing of
A; C, serum-activated 3T3 cells and antibodies to
murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-288) in the
presence of cognate peptide; D, quiescent 3T3 cells and
antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues
274-288); E, serum-activated 3T3 cells and antibodies to
murine PGHS-1 ((C)-NTSMLVDYGVEALVDAFS; residues 412-429);
F, serum-activated 3T3 cells and antibodies to murine PGHS-2
(SHSRLDDINPTVLIK; residues 584-598); G, computer
enhancement shadowing of F; H, serum-activated 3T3
cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK; residues
584-598) in the presence of cognate peptide; I,
quiescent 3T3 cells and antibodies to murine PGHS-2 (SHSRLDDINPTVLIK;
residues 584-598); and J, serum-activated 3T3 cells and
antibodies to murine PGHS-2 ((C)-GDPQPTKTATIN; residues 569-580).
Magnification, 200.
To
quantify the relative distributions of PGHS-1 and PGHS-2 in the ER and
the NE of 3T3 cells, the cells were stained first with an antibody to a
single PGHS isozyme, and staining intensities in the ER and on the NE
were then quantified using confocal imaging microscopy. The same cells
were then stained with either FITC-labeled ConA or a fluorescent dye
(DiOC(3) ), both of which partition uniformly among
intracellular membranes
(42, 43) , and again the
staining intensities were quantified in the ER and NE.
(
)
As shown in Fig. 2, the relative fluorescence
intensities in the ER and NE were the same for PGHS-1, FITC-labeled
ConA, and DiOC
(3) . In contrast, the intensity of
PGHS-2 immunofluorescence was two times higher in the NE than the ER.
Figure 2:
Subcellular distribution of fluorescence
staining for PGHS-1, PGHS-2, DiOC(3), and concanavalin A in
serum-activated NIH 3T3 cells. Serum-activated murine NIH 3T3 cells
were first subjected to indirect immunocytofluorescence staining using
antibodies to murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues
274-288) or using antibodies to murine PGHS-2 (SHSRLDDINPTVLIK;
residues 584-598) essentially as described in the legend to Fig.
1. The same samples used to analyze PGHS immunoreactivity were
subsequently treated with DiOC
(3) (42) or FITC-conjugated
ConA (43) as described in the text. Under the conditions used for
detecting DiOC
(3) or FITC-conjugated ConA fluorescence, the
fluorescence attributable to PGHS staining was negligible. The
intensities of fluorescence were quantified in 20 different areas (1
µm
) within the cytoplasmic region neighboring the
nucleus ( ER) and in 20 different areas (1 µm
)
over the nuclear envelope ( NE) in each cell.
DiOC
(3), open bars; FITC-labeled ConA, striped
bars; PGHS-1, stippled bars; and PGHS-2, solid dark
bars. The data represent mean ± S.E. of two cells in six
individual culture chambers and are expressed as fluorescence
intensities. For comparison of mean values between two sets of data,
Student's t test was used, employing standard criterion
of p < 0.01 ( double asterisk) to indicate
statistical significance.
In a related experiment, we determined if time-dependent changes
occurred in the distribution of either PGHS-1 or PGHS-2 in the NE
relative to the ER of 3T3 cells after serum activation. As shown in
Fig. 3
, the relative fluorescence intensities in the ER and NE
were 2.0, 2.0, 1.7, and 1.7 for PGHS-2 during a 4-h treatment of
initially quiescent 3T3 cells with fetal calf serum; in contrast, the
relative intensities were 1.0 for PGHS-1 at all time points. When
measurements were conducted at 3, 6, 9, and 12 h post-serum
stimulation, the relative intensities were also the same (± 0.1)
for PGHS-2 at all time points, and the relative intensities remained at
1.0 for PGHS-1. Overall, the intensities of immunostaining for PGHS-2
increased with time after serum activation, but the intensities of
ER/NE staining remained relatively constant.
Figure 3:
Subcellular distribution of PGHS-1 and
PGHS-2 immunoreactivities during stimulation of murine NIH 3T3 cells
with fetal calf serum. Murine 3T3 cells were serum-starved for 48 h and
then treated with fetal calf serum for the indicated times. The cells
were fixed with 2% formaldehyde and subjected to indirect
immunocytofluorescent staining for either PGHS-1 ( upper panel)
or PGHS-2 ( lower panel), and the intensities of fluorescence
were quantified in the cytoplasm adjoining the nucleus (ER) and in the
NE as described in the legend to Fig. 2; open circles, ER;
closed circles, NE. The data represent mean ± S.E. of
two cells in six individual culture chambers and are expressed as the
percentage of the intensities in the NE relative to a cytoplasmic
( i.e. ER) fluorescence of 100%. For comparison of mean values
between two sets of data, Student's t test was used,
employing standard criterion of p < 0.05 ( single
asterisk) and p < 0.01 ( double asterisk) to
indicate statistical significance.
Subcellular Localization of PGHS-1 and PGHS-2 in Murine
3T3-cPGHS-2 Cells, Human Umbilical Vein Endothelial Cells, and Bovine
Aortic Endothelial Cells
The subcellular locations of PGHS-1 and
PGHS-2 in murine 3T3-cPGHS-2 cells, bovine aortic endothelial cells,
and human umbilical vein endothelial cells were also determined by
indirect immunofluorescence. The purpose of these studies was to
determine if the distributions of PGHS-1 and PGHS-2 immunoreactivities
in murine 3T3 cells were representative of other cells which express
both isozymes. NIH 3T3-cPGHS-2 cells express PGHS-2 constitutively.
Staining of serum-starved NIH 3T3-cPGHS-2 cells with antibodies to
PGHS-2 showed intense staining of the NE with some cytoplasmic staining
(Fig. 4). No staining occurred when the anti-PGHS-2 antibodies
were preincubated with their respective cognate peptides (data not
shown). Thus, the pattern of PGHS-2 staining observed with NIH
3T3-cPGHS-2 cells cultured under serum-starved conditions is the same
as that seen under serum-activated conditions in the parent NIH 3T3
cell line. These results suggest that no secondary factor induced by
serum treatment of 3T3 cells is required for PGHS-2 to become
concentrated in the NE.
Figure 4:
Immunofluorescence staining for PGHS-1 and
PGHS-2 in quiescent murine NIH 3T3-cPGHS-2 cells which express PGHS-2
constitutively. Quiescent, serum-starved murine NIH 3T3-cPGHS-2 cells,
which constitutively express both PGHS-1 and PGHS-2, were subjected to
indirect immunocytofluorescence essentially as described in the legend
to Fig. 1. A, quiescent 3T3-cPGHS-2 cells and antibodies to
murine PGHS-1 (LMRYPPGVPPERQMA-(C); residues 274-289);
B, quiescent 3T3-cPGHS-2 cells and antibodies to murine PGHS-2
(SHSRLDDINPTVLIK; residues 584-598). Magnification,
500.
Anti-peptide antibodies to ( a) the
trypsin cleavage sequence of ovine PGHS-1 (residues 272-283)
(36) and ( b) the 18 amino acid cassette unique to
PGHS-2 (residues 584-598 of murine PGHS-2)
(35, 36) were used to stain nonstimulated and PMA-activated human
umbilical vein endothelial cells (HUVECs) and bovine arterial
endothelial cells (BAECs) by indirect immunofluorescence
(Fig. 5). HUVECs and BAECs not treated with PMA only stained with
antibodies to ovine PGHS-1; in both cases, the patterns of PGHS-1
staining were similar to that observed for PGHS-1 in NIH 3T3 cells
(data not shown). Antibodies to PGHS-1 caused staining of the cytoplasm
( i.e. ER) of PMA-activated HUVECs and BAECs. This contrasted
with the results observed for the PGHS-2 antigen where a very distinct
nuclear ring of staining was also apparent. Again, in all cases, the
immunofluorescent staining was blocked when the primary antibody was
used in the presence of its cognate peptide. These data indicate that
the subcellular locations of PGHS-1 and PGHS-2 observed in NIH 3T3
cells are common to endothelial cells from two different sources and
species.
Figure 5:
Immunofluorescence staining for PGHS-1 and
PGHS-2 in PMA-treated human umbilical vein endothelial cells (HUVECs)
and PMA-treated bovine arterial endothelial cells (BAECs). HUVECs and
BAECs were activated by treatment with PMA as described in the text and
then subjected to indirect immunofluorescent staining for PGHS-1 and
PGHS-2 as described in the text and essentially as described for 3T3
cells in the legend to Fig. 1. A, PMA-treated HUVECs stained
with antibodies to PGHS-1 (LMHYPRGIPPQ-(C); residues 272-282 of
ovine PGHS-1); B, PMA-treated HUVECs stained with antibodies
to PGHS-2 (SHSRLDDINPTVLIK; residues 584-598 of murine PGHS-2);
C, PMA-treated BAECs stained using antibodies to PGHS-1
(LMHYPRGIPPQ-(C); residues 272-282 of ovine PGHS-1); and
D, PMA-treated BAECs stained with antibodies to PGHS-2
(SHSRLDDINPTVLIK; residues 584-598 of murine PGHS-2).
Magnification, 400.
Histochemical Staining for PGHS-1 and PGHS-2
Activities
To determine if there is a correspondence between the
locations of PGHS-1 and PGHS-2 immunoreactivities and catalytic
activities, we developed a histochemical procedure for visualizing PGHS
activities. Briefly, NIH 3T3 cells were preloaded with CDCF-DA, and
fluorescence measurements were made following the addition of
arachidonic acid. CDCF is a common co-substrate for peroxidases and
will serve as a reducing substrate for the peroxidase activities of
PGHS-1 and PGHS-2. PGG generated by the cyclooxygenase
activities of PGHSs upon the addition of arachidonate is reduced to
PGH
by the peroxidase activities of PGHSs with resultant
oxidation of CDCF to a fluorescent compound which can be detected by
confocal microscopy. As shown in Fig. 6, addition of 10
µM arachidonate to quiescent, serum-starved NIH 3T3 cells
preloaded with CDCF led to an increase in fluorescence which was
apparent in the cytoplasm of the cell but not in the cell nucleus. No
fluorescence was observed upon addition of arachidonate to cells that
had been pretreated with either 1 mM aspirin or 1 mM
valerylsalicylate for 30 min prior to the addition of arachidonate;
under these conditions, both aspirin and valerylsalicylate inhibit the
cyclooxygenase activity (but not the peroxidase activity) of PGHS-1
(38) . When serum-activated NIH 3T3 cells were subjected to this
same cytochemical assay, fluorescent staining was observed in the
cytoplasm and, additionally, across the surface of the nucleus. Aspirin
prevented arachidonate-induced fluorescence
(
)
in
the serum-activated 3T3 cells. Valerylsalicylate, which is a relatively
specific inhibitor of PGHS-1
(38) , appeared to reduce the
intensity of the cytoplasmic fluorescence in serum-activated cells but
did not block the nuclear staining. Thus, the nuclear staining and part
of the cytoplasmic staining observed with serum-activated 3T3 cells is
a result of the activity of PGHS-2, whereas only cytoplasmic
fluorescence results from the activity of PGHS-1 ( cf.
Fig. 6A versus 6 F). As noted earlier, quiescent
3T3-cPGHS-2 cells express PGHS-2 constitutively. When subjected to
histofluorescence staining, the patterns of staining were similar to
those of serum-activated 3T3 cells. These results, in which the
subcellular locations of PGHS-1 and PGHS-2 activities were determined,
provide support for the concept that PGHS-1 occurs mainly in
association with the ER and the portion of the NE which is contiguous
with the ER, whereas PGHS-2, while present in the ER, is more highly
concentrated than PGHS-1 on the NE.
Figure 6:
Subcellular localization of
histofluorescence staining for PGHS-1 and PGHS-2 activities in
quiescent and serum-activated murine NIH 3T3 cells and quiescent NIH
3T3-cPGHS-2 cells. Cells were loaded with 5-(and
-6)-carboxyl-2`,7`-dichlorodihydrofluorescein (CDCF) by preincubation
with the diacetate derivative (CDCF-DA) as described in the text.
Arachidonate (10 µM) was added, and confocal fluorescence
imaging microscopy was performed 20 s later. A, quiescent 3T3
cells incubated with 10 µM arachidonate; B,
quiescent 3T3 cells pretreated for 30 min with 1 mM aspirin
prior to the addition of arachidonate; C, quiescent 3T3 cells
pretreated for 30 min with 1 mM valerylsalicylate prior to the
addition of arachidonate; D, serum-activated 3T3 cells
incubated with arachidonate; E, serum-activated 3T3 cells
pretreated for 30 min with 1 mM aspirin prior to the addition
of arachidonate; F, serum-activated 3T3 cells pretreated for
30 min with 1 mM valerylsalicylate prior to the addition of
arachidonate; G, quiescent 3T3-cPGHS-2 cells incubated with 10
µM arachidonate; H, quiescent 3T3-cPGHS-2 cells
pretreated for 30 min with 1 mM aspirin prior to the addition
of arachidonate; I, quiescent 3T3-cPGHS-2 cells pretreated for
30 min with 1 mM valerylsalicylate prior to the addition of
arachidonate. Magnification, 250.
(3) , and FITC-labeled ConA were the same in the
ER and NE. In contrast, the intensities of staining for PGHS-2
immunoreactivity were 2-fold higher in the NE than the ER.
(
)
Because the outer membrane of the NE is continuous with the
ER
(50) , PGHS-1 would be expected to be present in the NE and
presumably at a concentration similar to that in the ER. To determine
if there is a correspondence between the subcellular locations of the
PGHS antigens and PGHS activity, we developed a histofluorescence assay
for PGHS activity. This assay represents a modification of a
histochemical method originally described by Janszen and Nugteren
(51) but one which can be used to measure PGHS activity in
intact cells with a fluorescent probe. When applied to the measurement
of PGHS-1 activity, histofluorescent staining was observed throughout
the cytoplasm, but there was little or no staining over the surface of
the nucleus. Thus, there is a correspondence between the location of
PGHS-1 immunoreactivity and enzyme activity, and we conclude again that
PGHS-1 is present and functions primarily in the ER.
at a different
subcellular location than PGHS-1. Indeed, histofluorescence
measurements of enzyme activity showed PGHS-2 staining, but not PGHS-1
staining, of the nucleus. In serum-stimulated NIH 3T3 cells, PGHS-1
continues to be present at constant levels when expression of PGHS-2 is
induced. Valerylsalicylate selectively blocks PGHS-1 activity
(38) , and, similarly, valerylsalicylate blocked PGHS-1
histofluorescence in quiescent 3T3 cells. However, in serum-stimulated
NIH 3T3 cells pretreated with valerylsalicylate, histofluorescent
staining was observed, and staining was present in the nucleus as well
as in the cytoplasm. This pattern is distinct from that seen with
PGHS-1.
synthesis. PGHS-1 and PGHS-2 may
utilize the same pool of arachidonate. Second, we speculate that at
least a portion of the PGHS-2 associated with the NE is in a different
subcellular location than PGHS-1. Because as discussed above, PGHS-1 is
likely on the lumenal surface of the outer membrane of the NE, a subset
of PGHS-2 molecules may be on the lumenal surface of the inner
membrane. Differential compartmentation of PGHS-1 and PGHS-2 in the NE
could easily explain why these isozymes utilize different arachidonate
pools
(32, 33) and raises the possibility that there
are different lipases coupled to the release of arachidonate from these
pools. Furthermore, in some cases there may even be different PGH
metabolizing enzymes ( e.g. thromboxane A and PGI
synthases).
synthesis from endogenous as opposed to
exogenous arachidonate using histofluorescence staining.
(
)
We speculate that
PGHS-2 also contains an ER targeting signal and that the enzyme then
moves from the ER to the inner membrane of the NE directed by a
secondary NE targeting signal. In this regard, it will be of particular
interest to determine if the regions having the greatest sequence
divergence between PGHS-1 and PGHS-2 ( e.g. the signal
peptides, membrane binding domains, and the C-terminal 15-25
amino acids) are involved in either ER or NE targeting.
(3),
3,3`-dihexyloxacarbocyanine iodide; CDCF-DA, 5-(and
-6)-carboxyl-2`,7`-dichlorodihydrofluorescein diacetate; CDCF, 5-(and
-6)-carboxyl-2`,7`-dichlorodihydrofluorescein; DMEM, Dulbecco's
modified Eagle's medium; ConA, concanavalin A; HUVEC, human
umbilical vein endothelial cell; BAEC, bovine arterial endothelial
cell.
(3) were much greater than those for
immunofluorescence staining for PGHS-1 and PGHS-2.
C]arachidonic acid.
I-TID
(3-trifluoromethyl-3-( m-[
I]iodophenyl)diazirine)
labeling of microsomal PGHS-1 indicates that the enzyme is anchored to
the ER membrane via a domain located between Ala
(the N
terminus of the mature enzyme) and Lys
(J. C. Otto and W.
L. Smith, unpublished results).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.