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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Cycloheximide, genistein, AlCl3, sodium orthovanadate, indomethacin, AA, digitonin, guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), and bFGF (from bovine pituitary glands) were purchased from Sigma Chemical (St. Louis, MO). Guanosine 5'-O-(3-thiotriphosphate) (GTPgamma 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 F1alpha (6-keto-PGF1alpha ) and prostaglandin E2 (PGE2).

Determination of 6-keto-PGF1alpha and PGE2. The 6-keto-PGF1alpha 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-PGF1alpha and PGE2 were purchased from Sigma. 3H-labeled 6-keto-PGF1alpha (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-PGF1alpha for standard curves were from Sigma. The 6-keto-PGF1alpha antiserum cross-reacted with other prostaglandins (PGs; at 50% displacement) as follows: PGE1, 22%; PGE2, 10%; PGF1alpha , 16%; PGF2alpha , 10%; other PGs, <1%. The PGE2 antiserum cross-reacted with the following PGs (at 50% displacement): PGA1, 3%; PGA2, 1.2%; PGF1alpha , 7.7%; PGF2alpha , 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-PGF1alpha ), 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-PGF1alpha 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-PGF1alpha , 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-PGF1alpha . 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 F1alpha (6-keto-PGF1alpha 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.

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-PGF1alpha 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-PGF1alpha ). 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-PGF1alpha ). 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.

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 GTPgamma S, G(pp)NHp, NaF, and GDPbeta 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, GTPgamma S and Gpp(NH)p, for 30 min. Both GTPgamma 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, GDPbeta S, an inhibitor of G proteins, significantly attenuated the inhibitory effects of GTPgamma 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 GTPgamma S inhibition of PGHS activity (not shown).


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Fig. 4.   Effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), 5'-guanylyl imidodiphosphate [Gpp(NH)p], NaF, and guanosine 5'-O-(2-thiodiphosphate) (GDPbeta 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 GTPgamma 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 GDPbeta S and then exposed to 100 µM GTPgamma 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-PGF1alpha ). 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.

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-PGF1alpha ). 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.

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-PGF1alpha ) 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-PGF1alpha ). Results are means ± SE of 8 determinations. * P < 0.05 vs. control. P < 0.05 vs. bFGF alone.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, GTPgamma S and Gpp(NH)p, inhibited both resting and NaF-induced PGHS activity and that GDPbeta S, a G protein inhibitor, abrogated the effects of GTPgamma 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-1beta , tumor necrosis factor-alpha , 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-1alpha 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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1749-C1755
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