Prostaglandin H synthase: protein synthesis-independent
regulation in bovine aortic endothelial cells
Moti
Rosenstock,
Abraham
Danon, and
Gilad
Rimon
Department of Clinical Pharmacology, The Corob Center for Health
Sciences, Ben-Gurion University and Soroka Medical Center,
Beer-Sheva 84105, Israel
 |
ABSTRACT |
The objective of
the present study was to examine whether prostaglandin H synthase
(PGHS) can be regulated by pathways independent of de novo synthesis of
PGHS. Incubation of bovine aortic endothelial cells (BAEC) for as short
as 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity.
PGHS activity induced by NaF was unaffected by either 10 µM
cycloheximide or 1 µM actinomycin D. Aspirin (25 µM) completely
inhibited resting PGHS activity, and NaF did not induce further
stimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, was
ineffective. Basic fibroblast growth factor (bFGF) induced a
significant increase in PGHS activity within 30 min and was insensitive
to cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measured
by Western blots, were not affected by NaF or bFGF. The tyrosine kinase
inhibitor genistein attenuated PGHS activity that was induced by NaF
and bFGF, whereas the tyrosine phosphatase inhibitor, sodium
orthovanadate, augmented these responses. The G protein activators
5'-guanylyl imidodiphosphate and guanosine
5'-O-(3-thiotriphosphate) inhibited both resting and
NaF-induced PGHS activities. These results suggest that, in BAEC,
PGHS-1 activity can be regulated by tyrosine kinase and/or G
proteins, independently of de novo protein synthesis.
sodium fluoride; guanyl nucleotides; basic fibroblast growth
factor; tyrosine kinase; phospholipase
A2; cycloheximide
 |
INTRODUCTION |
THE ENDOTHELIUM THAT LINES the vascular system acts not
only as a mechanical barrier but also as a source of several substances that control the tone of the underlying smooth muscle cells (23). One
of the mechanisms involved in maintaining vascular tone by endothelial
cells has been ascribed to the capacity of these cells to synthesize
prostacyclin (PGI2), a potent
inhibitor of platelet aggregation, a powerful dilator of blood vessels,
and an inflammatory mediator. PGI2
synthesis is regulated by two key enzymes: phospholipase A2
(PLA2), which liberates
arachidonic acid (AA) from phospholipid stores, and prostaglandin H
synthase (PGHS), which oxidizes AA to
PGH2.
Endothelial cells, like many other cells, contain two distinct PGHS
isoforms, PGHS-1 and PGHS-2 (6, 12). Although PGHS-1 and PGHS-2 share
60% identity and 75% similarity, the expression of these enzymes is
regulated differently. PGHS-1 has been observed in a variety of
prostanoid-producing cells to be constitutively expressed, whereas
PGHS-2 is inducible in response to agents such as growth factors and
cytokines. This difference suggests that PGHS-1 is involved mainly in
housekeeping processes, whereas PGHS-2 is involved in differentiative
and inflammatory responses. De novo expression of PGHS-2 is generally
assumed to be the sole mode controlling its activity. Although PGHS-2
mRNA and protein concentrations are low in resting cells, stimulation
with growth factors, phorbol esters, inflammatory cytokines, and
lipopolysaccharide dramatically increases PGHS-2 expression (for review
see Ref. 17). Selective inhibitors of transcription and of translation have been used to confirm that transcriptional and posttranscriptional events are responsible for the induction of PGHS activity by
interleukin-1 (IL-1) (18).
Recently, we demonstrated that NaF (40-60 mM) stimulated PGHS
activity as well as AA release from bovine aortic endothelial cells
(BAEC) within 30 min. Higher concentrations of NaF inhibited PLA2 but not PGHS activity (19).
The present study was designed to investigate whether the rapid action
of NaF on PGHS could be mediated by mechanisms other than de novo
synthesis of the PGHS protein, e.g., by signal transduction pathways.
Here we demonstrate that, in BAEC, NaF and basic fibroblast growth
factor (bFGF) increase PGHS activity, whereas GTP analogs acutely
decrease PGHS activity, and these actions are insensitive to
translation and transcription inhibitors.
 |
MATERIALS AND METHODS |
Materials. Cycloheximide, genistein,
AlCl3, sodium orthovanadate,
indomethacin, AA, digitonin, guanosine
5'-O-(2-thiodiphosphate) (GDP
S), and bFGF (from bovine pituitary glands) were purchased from
Sigma Chemical (St. Louis, MO). Guanosine
5'-O-(3-thiotriphosphate) (GTP
S) and
5'-guanylyl imidodiphosphate [Gpp(NH)p] were obtained from Boehringer Mannheim. Actinomycin D was from Merck Sharp & Dohme
International (Rahway, NJ),
[5,6,8,9,11,12,14,15-3H(N)]AA
(200 Ci/mmol) was from DuPont New England Nuclear (Boston, MA), NaF and
aspirin were from BDH (Poole, England, UK), and NS-398 was from
Calbiochem (La Jolla, CA). Arachidonyl trifluoromethyl ketone
(AACOCF3) was from
Biomol Research Laboratories (Plymouth Meeting, PA), and CGP-43187 was
kindly provided by Dr. Breitenstein (Ciba-Geigy, Basel, Switzerland).
Cell cultures. Cultures of BAEC,
obtained through the courtesy of Dr. I. Vlodavski (Hebrew University,
Jerusalem, Israel) (8), were used between passages
8 and 16. BAEC were
seeded in 24-well tissue culture plates (Costar, Cambridge, MA) and
incubated at 37°C in a humidified 8%
CO2-92% air
atmosphere. Each 16-mm-diameter well contained 1 ml of Dulbecco's
modified Eagle's medium (DMEM; Biological Industries, Beit Haemek,
Israel) supplemented with 10% calf serum (Sigma), 2 mM
L-glutamine, and
penicillin-streptomycin (Biological Industries) at final concentrations
of 100 U/ml and 100 µg/ml, respectively. During the subconfluent
period bFGF was added (2 ng/ml) every 2 days until the cultures became
confluent. Cultures were used at least 14 days after reaching
confluency. Experiments were initiated by twice washing with serum-free
DMEM (SFM), and then the cells were preincubated in SFM for 24 h. The cultures were then incubated in 1 ml SFM containing the test agents for
the duration indicated in each experiment. At the end of each experiment the samples were transferred into Eppendorf-type tubes containing 10 µM indomethacin and were frozen pending analysis of
6-ketoprostaglandin F1
(6-keto-PGF1
) and
prostaglandin E2
(PGE2).
Determination of
6-keto-PGF1
and
PGE2.
The 6-keto-PGF1
and
PGE2 that accumulated in the media
were measured in unextracted samples of DMEM by single antibody radioimmunoassays (RIA) with dextran-coated charcoal precipitation. DMEM did not interfere with the assays. The RIA was performed in
duplicate for each sample. Rabbit antisera to
6-keto-PGF1
and
PGE2 were purchased from Sigma.
3H-labeled
6-keto-PGF1
(175 Ci/mmol) and
PGE2 (160 Ci/mmol) were
obtained from The Radiochemical Center (Amersham, UK). The sensitivities of both assays were 0.15 ng/ml.
PGE2 and
6-keto-PGF1
for standard curves
were from Sigma. The
6-keto-PGF1
antiserum cross-reacted with other prostaglandins (PGs; at 50% displacement) as
follows: PGE1, 22%;
PGE2, 10%;
PGF1
, 16%;
PGF2
, 10%; other PGs, <1%.
The PGE2 antiserum cross-reacted
with the following PGs (at 50% displacement):
PGA1, 3%;
PGA2, 1.2%;
PGF1
, 7.7%; PGF2
, 6.8%; other PGs, <1%.
Assay for PGHS activity. PGHS activity
in BAEC was quantified as previously described (13). During the last 30 min of each experiment, 50 µM unlabeled AA was added. This
concentration of exogenous AA overrides the release of endogenous
substrate by PLA2. Thus the
observed changes in release of the major PGHS product, PGI2 (as
6-keto-PGF1
), under these
conditions are ascribed to changes in PGHS activity. The validity of
the assay was confirmed by complete inhibition of PGHS activity by
pretreatment with 25 µM aspirin (see Fig. 3). To exclude a possible
effect on an isomerase or PGI2
synthase activity, PGE2 was
quantified as well in several experiments. In the presence of exogenous
AA, PGE2 production showed
parallel changes as 6-keto-PGF1
after treatment with the different agents (data not shown).
Immunoblot (Western blot) analysis.
BAEC were cultured in six-well plates. After 24 h of incubation with
fresh SFM, cells were treated with test agents for the duration
indicated in each experiment. Cells were then extracted with continuous
shaking for 1 h at 4°C with 200 µl of extraction buffer
containing 20 mM tris(hydroxymethyl)aminomethane
(Tris) · HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 1%
(vol/vol) Triton X-100, 0.5% (wt/vol) sodium deoxycholate, 200 µM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 13.5 µg/ml soybean trypsin inhibitor, 2.5 µg/ml
antipain, and 50 µg/ml bacitracin. Cell lysates were transferred to
Eppendorf-type tubes and centrifuged at 4°C for 15 min at 10,000 g. Protein content was determined
according to Lowry et al. (15), with bovine serum albumin (BSA) as
standard. Cell lysates were mixed (1:1) with Laemmli reagent
[62.5 mM Tris · HCl (pH 6.8), 2% (wt/vol)
sodium dodecyl sulfate (SDS), 10% (vol/vol) glycerol, 5% (vol/vol)
2-mercaptoethanol, 0.05% (wt/vol) bromophenol blue] and were
boiled for 5 min. SDS-polyacrylamide gel electrophoresis was performed
using 10% and 3% N-acrylamide for
the separation and stacking gels, respectively. Samples (20 µg
protein per lane) were subjected to electrophoresis (150 V), and the
separated proteins were transferred to nitrocellulose (100 min at 200 mA; Bio-Rad, Munich, Germany). The blots were incubated for 15 min in
Tris-buffered saline (TBS) containing 3% Tween-20 and then incubated
in blocking solution [TBS containing 0.1% Tween-20 (TTBS) and
5% BSA] for 1 h. After the blots were washed (2 × 20 min)
with TTBS, they were primed with rabbit antibody raised against
synthetic murine PGHS-2 or mouse antibody raised against purified ovine
PGHS-1 (Cayman Chemical, Ann Arbor, MI) diluted 1:100 for 16-18 h.
The blots were further incubated for 1 h with anti-rabbit
immunoglobulin G (IgG) or anti-mouse IgG antibodies conjugated with
horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) diluted
1:10,000. Excess secondary antibody was eliminated by washing with TTBS
(2 × 10 min) followed by a 10-min wash with TBS.
Chemiluminescent substrates were used to reveal positive bands
according to the manufacturer's instructions, and bands were
visualized by exposure to Hyperfilm enhanced chemiluminescence (Fuji
Photo Film, Tokyo, Japan).
AA release. AA release was used to
assess PLA2 activity. BAEC
monolayers in 24-well plates were preincubated for 10-12 h in SFM
containing 0.75 µCi/ml
[3H]AA (200 Ci/mmol)
to label cellular phospholipids (21). Unincorporated [3H]AA was removed by
washing the cells four times with SFM. Test agents were then added to
SFM containing 1% BSA. At the conclusion of each experiment, the
radioactivity released into the media was determined by liquid
scintillation counting (1214 RackBeta LKB; Wallac, Turku, Finland).
Ether extracts of the samples were evaporated under
N2, redissolved in ethanol,
chromatographed on DC-Alufolien silica gel 60 F254 (Merck, Darmstadt,
Germany), and developed with chloroform-methanol-acetic acid-water
(90:8:1:0.7) as previously described (7). Authentic labeled AA,
6-keto-PGF1
, and
PGE2 were used to characterize the
system. About 90% of the radioactivity released into the media
migrated with AA.
Protein determination. After removal
of the medium, the attached cells in each well were dissolved in 1 ml
of 10 mM NaOH and were transferred into test tubes. Protein
concentrations were determined using the Bio-Rad protein assay (acidic
solution of Coomassie brilliant blue G-250 that shifts from 465 to 595 nm when bound to protein; Bio-Rad). BSA was used as a standard.
Cell viability. To exclude the
possibility of general cytotoxic effects of NaF, trypan blue exclusion
was used. In both control and NaF-treated cells, nonviable cells did
not exceed 2%.
Data analysis. Results are means ± SE for each experiment. Statistical analysis of the results was
performed using two-tailed Student's
t-test, and
P
0.05 was considered significant.
 |
RESULTS |
The BAEC cultures as used incorporated 70-90% of exogenous
[3H]AA and synthesized
considerable amounts of PGI2,
measured as the stable hydrolyzed product,
6-keto-PGF1
. PGHS activity was
assessed after addition of exogenous unlabeled AA, as detailed in
MATERIALS AND METHODS.
Effect of NaF on PGHS activity. NaF, a
nonselective activator of both stimulatory and inhibitory
heterotrimeric membrane-bound G proteins (11), was used to evaluate
the possible involvement of G proteins in PGHS activation. After 24 h
of preincubation in SFM, the cells were exposed to different
concentrations of NaF for 30 min or alternatively to 40 mM NaF for
different lengths of time. As shown in Fig.
1A, NaF
induced significant increases in PGHS activity in a concentration- and
time-dependent manner. Incubation with NaF for as short as 5 min
increased PGHS activity by 80% (Fig. 1,
inset).

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Fig. 1.
Effect of NaF on prostaglandin H synthase (PGHS) activity. Bovine
aortic endothelial cell (BAEC) monolayers were preincubated in 24-well
plates with serum-free DMEM (SFM) for 24 h.
A: cells were then treated with
increasing concentrations of NaF + 50 µM AlCl3 or 100 mM NaCl as control,
in the presence of 50 µM arachidonic acid (AA), for 30 min.
Inset: after 24 h in SFM, cells were
treated with 40 mM NaF + 50 µM
AlCl3 for different lengths of
time. In last 5 min, 50 µM AA was added.
B: cells were pretreated with 50 µM
arachidonyl trifluoromethyl ketone
(AACOCF3), CGP-43187, or vehicle
for 15 min. Thereafter, cells were exposed to 40 mM NaF + 50 µM
AlCl3 or 40 mM NaCl as control, in
the presence of 50 µM AA, for 30 min. Media were then collected for
assessing PGHS [as 6-ketoprostaglandin
F1
(6-keto-PGF1 or 6-K)]. Results are means ± SE of 8 determinations. Similar results were obtained in 2 additional experiments.
* P < 0.05 vs. control. Prot.,
protein.
|
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In a previous paper (19), we showed that NaF had a biphasic effect on
PLA2 activity, expressed as
stimulation at low concentrations (40-60 mM) and inhibition at
higher concentrations (80-100 mM). To further ascertain that the
6-keto-PGF1
production that was
measured in the presence of high exogenous AA levels was independent of
cellular PLA2 activity, we tested
the effects of cytosolic and secretory
PLA2 inhibitors,
AACOCF3 and CGP-43187,
respectively. Figure 1B shows that
neither of these inhibitors of
PLA2 had an effect on resting or
NaF-induced PGHS activity, confirming the adequacy of our system as a
sole indication of PGHS activity.
As demonstrated in Fig. 2, preincubation of
the cells with cycloheximide (10 µM), an inhibitor of protein
translation, or with actinomycin D (1 µM), a DNA transcription
inhibitor, did not affect the stimulation of PGHS activity by NaF. To
further exclude de novo protein synthesis, cells were pretreated with aspirin (25 µM) for 30 min and then treated with NaF for 30 min (Fig.
3). As expected, aspirin irreversibly
abolished preexisting PGHS activity, and NaF failed to augment this
activity, confirming that new protein was not elaborated.

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Fig. 2.
Effect of cycloheximide (CHX) and actinomycin D (ACT) on NaF-induced
PGHS activity. BAEC were preincubated with SFM in 24-well plates for 24 h and then were pretreated with 10 µM CHX or 1 µM ACT for 45 min.
Thereafter, cells were exposed to 40 mM NaF + 50 µM
AlCl3 or 40 mM NaCl as control for
30 min in the presence of 50 µM AA. Media were then collected for
assessing PGHS (as 6-keto-PGF1 ). Results are
means ± SE of 7 determinations. * P < 0.05 vs. control.
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Fig. 3.
Effects of aspirin and NS-398 on resting and NaF-induced PGHS activity.
Cell monolayers were preincubated with SFM in 24-well plates for 24 h
and then treated with NS-398 (500 nM) or vehicle for 20 min. After
removal of the supernatant, cells were washed 3 times to remove any
residual NS-398 and to allow specific inhibition of PGHS-2. Medium was
replaced, and cells were treated with aspirin (25 µM) or vehicle for
30 min, and then, for last 30 min, cells were treated with 40 mM NaF + 50 µM AlCl3 or 40 mM NaCl as
control, in the presence of 50 µM AA.
Inset: after 24 h with SFM, cells were
pretreated with increasing concentrations of aspirin for 30 min.
Thereafter, cells were treated with 40 mM NaF + 50 µM AlCl3 in the presence of 50 µM
AA. Media were then collected for assessing PGHS (as
6-keto-PGF1 ). Results are
means ± SE of 6 determinations. Similar results were obtained in 2 additional experiments. * P < 0.05 vs. control, # P < 0.05 vs. NaF.
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Effect of aspirin and NS-398 on resting and
NaF-induced PGHS activity. To get an insight into the
identity of the PGHS isoform that was activated by NaF, we used
aspirin, which at low concentrations inhibits PGHS-1, and NS-398, a
specific PGHS-2 inhibitor. According to Mitchell et al. (16), aspirin
inhibited PGHS-1 and PGHS-2 in BAEC with 50% inhibitory concentrations
(IC50) of 2 and 250 µM,
respectively. We observed that aspirin inhibited resting (not shown)
and NaF-induced PGHS activity with an
IC50 of ~5 µM (Fig. 3,
inset), confirming that NaF-induced
PGHS activation could be ascribed to PGHS-1 rather than PGHS-2.
Copeland et al. (5) reported that NS-398 binds to the active sites of
both PGHS-1 and PGHS-2; however, only the latter was irreversibly
inhibited by NS-398. Therefore, cells were carefully washed after
pretreatment with NS-398 and then were exposed to NaF or NaCl as
control. In contrast to aspirin, which inhibited PGHS activity by 95%,
NS-398 inhibited resting and NaF-induced PGHS activity by only ~12%. The combination of NS-398 and aspirin did not result in further inhibition of PGHS activity (Fig. 3).
Effects of GTP
S, G(pp)NHp, NaF, and
GDP
S on PGHS activity.
To test the possible involvement of G proteins in PGHS regulation, the
cells were permeabilized with digitonin (9) and treated with 100 µM
of the nonhydrolyzable GTP analogs, GTP
S and Gpp(NH)p, for 30 min.
Both GTP
S and Gpp(NH)p significantly reduced resting PGHS activity
by 30-35% (Fig.
4A) and
abrogated the increase in PGHS activity that was induced by NaF (Fig.
4B). On the other hand, GDP
S, an
inhibitor of G proteins, significantly attenuated the inhibitory
effects of GTP
S and Gpp(NH)p on both resting (not shown) and
NaF-induced PGHS activities (Fig.
4B). Pretreatment of the cells with
cycloheximide (10 µM) did not affect Gpp(NH)p and GTP
S inhibition
of PGHS activity (not shown).

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Fig. 4.
Effects of guanosine 5'-O-(3-thiotriphosphate)
(GTP S), 5'-guanylyl imidodiphosphate [Gpp(NH)p],
NaF, and guanosine
5'-O-(2-thiodiphosphate) (GDP S) on PGHS activity. BAEC monolayers were preincubated in 24-well plates with SFM for 24 h. A:
cells were permeabilized with 2 µg/ml digitonin for 30 min and then
were exposed to 100 µM of GTP S or Gpp(NH)p for additional 30 min
in the presence of 50 µM AA. B:
cells were permeabilized with 2 µg/ml digitonin for 30 min with or
without 0.5 mM GDP S and then exposed to 100 µM GTP S or Gpp(NH)p
for additional 30 min. During last 30 min cells were treated with 40 mM
NaF + 50 µM AlCl3 or 40 mM NaCl as control in the presence of 50 µM AA. Media were then collected for
assessing PGHS (as
6-keto-PGF1 ). To account for
possible effects of digitonin, control cells were also permeabilized
with digitonin. Results are means ± SE of 4 determinations. Similar results were obtained in 2 additional experiments.
* P < 0.05 vs. control.
$ P < 0.05 vs. vehicle.
# P < 0.05 vs. NaF (40 mM) alone.
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Involvement of the tyrosine kinase
pathway. In an attempt to further characterize the
transduction pathways involved in PGHS activation by NaF, we utilized
several agents that interfere with the tyrosine kinase signal
transduction pathway. Genistein, a protein tyrosine kinase inhibitor,
abolished NaF-induced PGHS activity (Fig.
5) in a concentration-dependent manner with
an IC50 of 7.5 µM (not shown).
By contrast, sodium orthovanadate, an inhibitor of tyrosine
phosphatase, while not altering resting activity, significantly
augmented the PGHS activity that was induced by NaF (Fig. 5). As shown
in Fig. 5, inset, genistein was
without effect on NaF-induced AA release, excluding
PLA2 as a possible site of
regulation by genistein.

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Fig. 5.
Effects of NaF, genistein (Gen.), and sodium (sod.) orthovanadate on
PGHS activity and
[3H]AA release. BAEC
monolayers were preincubated in 24-well plates with SFM for 24 h and
were pretreated with 50 µM genistein or 1 mM sodium orthovanadate for
15 min. Cells were then treated for 30 min with 40 mM NaF + 50 µM
AlCl3 or 40 mM NaCl as control (Con.), in the presence 50 µM AA, and media were then collected for
assessing PGHS (as
6-keto-PGF1 ).
Inset: for measurement of AA release,
cells were preincubated with 0.75 µCi/ml
[3H]AA for 20 h. To
remove free [3H]AA,
cells were washed 3 times with SFM and then pretreated with 50 µM
genistein for 15 min. For the next 30 min, cells were treated with 40 mM NaF + 50 µM AlCl3 or 40 mM
NaCl as control in the presence of 1% bovine serum albumin (BSA).
Results are means ± SE of 4 determinations. Similar results were
obtained in an additional experiment.
* P < 0.05 vs. control.
# P < 0.05 vs. NaF
(40 mM) alone.
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bFGF-mediated regulation of PGHS
activity. The growth factor bFGF was employed as a
physiological stimulus to PGHS. Incubation of BAEC with bFGF (1.25 × 10
10 M) for 30 min
significantly stimulated PGHS activity (Fig.
6). Cycloheximide (5 µM) did not alter
this response to bFGF (data not shown). Involvement of tyrosine kinase
in the effect of bFGF was probed by testing the effect of genistein.
Figure 6A shows that genistein
inhibited bFGF-induced PGHS activity in a concentration-dependent manner. The higher concentration of genistein (50 µM) actually reduced PGHS activity to below resting levels. AA release was not
altered by the entire range of genistein concentrations, both in the
presence or absence of bFGF. Increasing concentrations of sodium
orthovanadate resulted in augmentation of PGHS activity that was
induced by bFGF (Fig. 6B).

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Fig. 6.
Effect of basic fibroblast growth factor (bFGF), genistein, and sodium
orthovanadate (SOV) on PGHS activity and
[3H]AA release. BAEC
monolayers were preincubated in 24-well plates with SFM for 24 h.
A: AA release was assessed as
described in Fig. 5. Cells were pretreated with increasing
concentrations of genistein for 15 min, and, for the next 30 min, bFGF
(2.5 ng/ml) was added in the presence of either 50 µM AA (for PGHS
activity) or 1% BSA (for AA release). Media were then collected for
assessing PGHS (as
6-keto-PGF1 ) or
[3H]AA release.
B: cells were pretreated with
increasing concentrations of sodium orthovanadate for 15 min, and, for
the next 30 min, bFGF (2.5 ng/ml) was added in the presence of 50 µM
AA. Media were then collected for assessing PGHS (as
6-keto-PGF1 ). Results are
means ± SE of 8 determinations.
* P < 0.05 vs. control. # P < 0.05 vs. bFGF alone.
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Effect of NaF and bFGF on PGHS-1 and PGHS-2
levels. Measurement of PGHS-1 and PGHS-2 izoenzymes by
Western blots failed to reveal any increase in protein levels after
short treatment with NaF or bFGF (Fig. 7).
As a positive control for the induction of PGHS-2 in BAEC, we used
lipopolysaccharide. PGHS-1 was found to be the predominant species both
in resting and NaF- or bFGF-stimulated cells, which is in agreement
with data depicted in Fig. 3.

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Fig. 7.
Effects of NaF, bFGF, CHX, and lipopolysaccharide (LPS) on PGHS-1 and
PGHS-2 protein levels. BAEC were cultured in 6-well plates. Cells were
serum deprived for 24 h and then were treated with vehicle (Veh.) for
75 min, with 40 mM NaF for 30 min, with 2.5 ng/ml bFGF for 30 min, with
10 µM CHX for 45 min, with 10 µM CHX for 45 min + 40 mM NaF for 30 min, with 10 µM CHX for 45 min + 2.5 ng/ml bFGF for 30 min, with
vehicle for 2 h, and 1 µg/ml LPS for 2 h. Immunoblots were performed
with antibody against purified ovine PGHS-1 or with antibody against
synthetic murine PGHS-2, as described in MATERIALS AND
METHODS. Results are representative of 4 independent
experiments performed on different BAEC cultures.
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 |
DISCUSSION |
The present study shows that NaF and bFGF regulate PGHS activity in a
time- and concentration-dependent manner without affecting PGHS-1 or
PGHS-2 expression. Four lines of evidence in this study suggest that
the increased PGHS activity that was affected by NaF and bFGF did not
involve de novo PGHS synthesis: 1)
the time course of increased PGHS activity, occurring as early as 5 min after exposure to NaF, is probably too short to allow protein synthesis; 2) neither cycloheximide,
an inhibitor of mRNA translation, nor actinomycin D, which inhibits DNA
transcription, inhibited the increase in PGHS activity that was induced
by NaF or bFGF; 3) NaF failed to
increase PGHS activity after inhibition of preexisting PGHS by aspirin
pretreatment; and 4) immunoblot
analysis failed to detect any increase in PGHS-1 or PGHS-2 after
stimulation with NaF or bFGF.
The identity of the PGHS species that was activated was inferred from
the observation that aspirin, but not NS-398, at concentrations assumed
to inhibit specifically PGHS-1, inhibited NaF-induced PGHS activity by
~95%. It is therefore concluded that NaF activates primarily PGHS-1
and not PGHS-2.
The fact that both cytosolic and secretory
PLA2 inhibitors,
AACOCF3 and CGP-43187,
respectively, did not affect resting and NaF-induced PGHS activities
confirms that NaF regulates PGHS activity independently of its effect
on PLA2.
NaF has been reported to interfere with cellular transduction pathways
by various mechanisms, namely, by nonselective activation of G
proteins, by inhibition of tyrosine phosphatases, and by activation of
tyrosine kinases (3). The present observations, showing inhibition of
the NaF-induced PGHS activity by genistein, a tyrosine kinase
inhibitor, and enhancement by orthovanadate, a tyrosine phosphatase
inhibitor, suggest that tyrosine phosphorylation may indeed be involved
in the short-term regulation of PGHS by NaF. In addition, the lack of
effect of genistein on AA release in the presence of either NaF or bFGF
suggests that, in BAEC, tyrosine kinases are specifically involved in
the regulation of PGHS, rather than of
PLA2. The involvement of G binding
proteins in the short-term regulation of PGHS is implied from the
observations that nonhydrolyzable analogs of GTP, GTP
S and Gpp(NH)p,
inhibited both resting and NaF-induced PGHS activity and that GDP
S,
a G protein inhibitor, abrogated the effects of GTP
S and Gpp(NH)p on
PGHS. This is in agreement with data by Vandenburgh et al. (22) who
showed that pretreatment of skeletal muscle with pertussis toxin
prevented stretch-induced PGHS activation, indicating involvement of G
protein in PGHS activity. Thus the present results suggest that the
short-term protein synthesis-independent regulation of PGHS activity
comprises at least two distinct pathways: activation of G proteins
inhibits PGHS activity, whereas tyrosine kinase increases PGHS
activity. Involvement of tyrosine kinase in de novo expression of PGHS
protein has been shown in several cell types, including endothelial
cells. Thus induction of PGHS expression in BEAC by IL-1
, tumor
necrosis factor-
, and epidermal growth factor (1) was
inhibited by erbstatin, a tyrosine kinase inhibitor. Likewise, in human
umbilical vein endothelial cells, three inhibitors of protein tyrosine
kinases, genistein, herbimycin, and AG-213, reduced tyrosine
phosphorylation of cell substrates, in parallel with selective
reduction in PGHS-2 that was induced by IL-1
and phorbol ester (2).
Because no consensus sequence for tyrosine phosphorylation is
recognized on PGHS, one may assume the presence of an intermediary
regulatory protein that may be involved in this process.
Alteration of PG synthesis through modulation of
PLA2 activity by a variety of
molecular mechanisms has been extensively studied in numerous
laboratories. Some of these mechanisms involve elevation of
intracellular calcium concentration (20), activation of protein kinase
C (10, 20), activation of membrane-bound G proteins (4, 19), and
activation of p42 mitogen-activated protein kinase (14). In the present
study we demonstrate that the cellular signal transduction systems in
BAEC are also involved in rapid regulation of PGHS-1 activity
independent of PLA2 activity or de
novo synthesis of PGHS.
Taken together, our observations suggest that rapid regulation of
PGHS-1 activity in BAEC by growth factors and other agents may result
from posttranslational activation or inhibition of the PGHS enzyme.
Further studies are required to elucidate the precise mechanism(s) of
this short-term regulation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Breitenstein, Ciba-Geigy Corp., for the generous gift
of CGP-43187 and Mazal Rubin for skillful technical assistance.
 |
FOOTNOTES |
This study was supported in part by grants to A. Danon and G. Rimon
from the Chief Scientist's Office, Ministry of Health, Israel.
Address for reprint requests: G. Rimon, Dept. of Clinical Pharmacology,
Ben-Gurion Univ., PO Box 653, Beer-Sheva 84105, Israel.
Received 21 April 1997; accepted in final form 22 July 1997.
 |
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