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Endocrinology Vol. 139, No. 3 1268-1277
Copyright © 1998 by The Endocrine Society

ARTICLES

Prostaglandin Endoperoxide Synthase-2 Contributes to the Endothelin/Sarafotoxin-Induced Prostaglandin E2 Synthesis in Mouse Osteoblastic Cells (MC3T3-E1): Evidence for a Protein Tyrosine Kinase-Signaling Pathway and Involvement of Protein Kinase C1

Hans J. Leis, Doris Zach, Evelyn Huber and Werner Windischhofer

University Children’s Hospital, Department of Biochemical Analysis and Mass Spectrometry, University of Graz, A-8036 Graz, Austria

Address all correspondence and requests for reprints to: Dr. Hans Jörg Leis, University Children’s Hospital, Department of Biochemical Analysis and Mass Spectrometry, Auenbruggerplatz 30, A-8036 Graz, Austria. E-mail: hans.leis{at}kfunigraz.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin (ET) peptides are potent growth factors binding to G protein-coupled receptors. Sarafotoxins (S6) isolated from Atractaspis engaddensis are highly homologous to endothelins. In this study, we have investigated the effects of endothelin/sarafotoxin peptides on the prostaglandin synthesizing system in an osteoblast-like cell line, MC3T3-E1. ET-1, ET-2, ß-ET, and S6b rapidly stimulated prostaglandin E2 production within 5 min, whereas ET-3, S6a, and S6c did not. ET-1, ET-2, ß-ET, S6b, and S6a induced prostaglandin synthesis after 3 h of incubation. Antagonizing these effects with BQ-123, PD 142893, BQ-788, and S6c suggests signaling through an ETA receptor subtype in osteoblasts. Long-term prostaglandin synthesis was blocked by NS-398, and reduced to short-term levels by cycloheximide and actinomycin D, indicating induction of PGHS-2. There was only minor enhancement of cAMP accumulation by the agonists, which had no effect on prostaglandin synthesis. Induction of PGHS-2 was furthermore demonstrated by Northern blot analysis of PGHS-2 messenger RNA. Depletion of protein kinase C with TPA largely blunted the response. Genistein, an inhibitor of protein tyrosine kinases, also blocked long-term prostaglandin E2 formation. We conclude that in osteoblast-like MC3T3-E1 cells, ET-1, ET-2, ß-ET, S6b, and S6a peptides induce PGHS-2 through a protein tyrosine kinase-dependent and protein kinase C-dependent pathway, signaling through ETA receptor occupancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOTHELINS (ET), originally isolated from cultured media of porcine endothelial cells represent a family of 21-amino acid isopeptides (ET-1, ET-2, ET-3) encoded by distinct genes (1, 2, 3). There is a functional and structural relationship to mouse vasoactive intestinal contractor (VIC, or ß-ET) (4) and to sarafotoxin peptides isolated from Atractaspis engaddensis (5, 6). In mammals, sarafotoxins cause intense vasoconstriction frequently associated with cardiac arrest (7). Because of their sequence homology and common bioactivity, ETs and sarafotoxins are believed to constitute a supergene family with common evolutionary origin (8). ETs bind to specific G protein-coupled receptor subtypes. ETA receptors are selective for ET-1 and ET-2 over ET-3, whereas ETB receptors bind all three isopeptides with equal affinity (9, 10). Recently, also a ET-3 selective subtype (ETC) has been cloned and characterized (11). Sarafotoxin 6c (S6c) has been demonstrated to be a highly selective ligand for the ETB receptor subtype (12). ET receptor occupation may result in activation of phospholipase C, phospholipase A2, phospholipase D (13, 14, 15, 16, 17, 18), as well as receptor-operated and voltage-gated calcium channels (15, 19). One of the important cellular responses to this ET-evoked signaling cascade is the production of prostanoids, which can be linked to ET-stimulated contractility and mitogenesis (20, 21, 22). Whereas effects on prostaglandin synthesis are well described for ET-1 in several systems (19, 20, 23, 24, 25), only scarce information exists for the other ET-isopeptides and sarafotoxins. Recently, it has been shown, that ET-1 may stimulate prostaglandin endoperoxide synthase-2 (PGHS-2) messenger RNA (mRNA) expression in cultured rat mesangial cells (22, 26), but no data are available on the effect of ET-isopeptides and sarafotoxins.

It is now recognized that at least ET-1 plays an important role in bone metabolism. It induces bone resorption and stimulates collagen and noncollagen protein synthesis through the phosphoinositide transduction pathway in cultured neonatal mouse calvaria (18). ET-1 reduces cellular alkaline phosphatase activity and stimulates the formation of inositol phosphates and DNA synthesis in rat osteoblastic cells (27). In osteoblast-like MC3T3-E1 cells, ET-1 induces stimulation of phospholipase C-mediated phosphoinositide hydrolysis, mobilization of calcium from extra- and intracellular pools, and stimulation of DNA synthesis (15), activation of phospholipase D (16), and stimulation of tyrosine phosphorylation (28). In osteoblast-like UMR-106 rat osteosarcoma cells, ET-1 modulates calcium signaling by epidermal growth factor, thrombin, and prostaglandin E1 (29) and PTH (30) and activates calcium and inositol phosphate second messenger systems (31). Furthermore, ET-1 inhibits osteoclastic bone resorption by a direct effect on cell motility (32). ET-1 induced bone resorption in cultured neonatal mouse calvaria is prone to inhibition by indomethacin, and thus a role for prostaglandins in the actions of ET-1 on bone was suggested (18). In rat mesangial cells, ET-1-related mitogenic signaling has been attributed to induction of PGHS-2 (22), and the discovery that ET-1 is a growth factor (33, 34) provides a new perspective for considering the biological activity of endothelins and likewise sarafotoxin peptides in bone tissue.

In the present study, we therefore describe the kinetics of prostaglandin formation for the endothelin/sarafotoxin isopeptides in murine osteoblast-like cells and the PGHS enzymes involved. The contribution of cAMP- and tyrosine kinase signaling pathways are evaluated. Furthermore, the receptor signaling system involved is investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ET-1, ET-2, ET-3, ß-ET, S6b, S6a, S6c, PD 142893, BQ 788, 12-decatetranoyl phorbol-13-acetate (TPA), PGE2, bovine PTH, fragment 1–34, 3-isobutyl-1-methylxanthine, cycloheximide, genistein, herbimycin A, actinomycin D, BSA, and HEPES buffer were from Sigma Chemical Co. (Vienna, Austria). BQ-123 was purchased from Bachem (Bubendorf, Switzerland). NS-398 was from ICN (Eschwege, Germany). {alpha}-MEM and FCS were obtained from Sera-lab (Vienna, Austria). L-glutamine was from Serva (Vienna, Austria). Trypsin-EDTA was purchased from Böhringer (Mannheim, Germany). Trizol reagent was from GIBCO BRL. Complementary DNA (cDNA) probes for PGHS-1 and PGHS-2 were from Oxford Biomedical (Oxford, MI), cDNA probe for GAPDH from Clontech (Heidelberg, Germany). 32P-dCTP Prime-a-Gene labeling system was obtained through Promega (Mannheim, Germany). Rp-cAMPs and SQ 22536 were from Biomol (Vienna, Austria). Pentafluorobenzyl bromide (PFBBr), bis-(N, O-trimethylsilyl) trifluoroacetamide (BSTFA), silylation grade pyridine, acetonitrile, and O-methoxyamine hydrochloride (MOX) were from Pierce Chemical Co. (Rockford, IL). Culture dishes were from Falcon via Szabo (Vienna, Austria). MC3T3-E1 cells were kindly donated by Dr. Klaushofer (LBI, Vienna, Austria). Deuterated PGE2 was obtained through MSD Isotopes via IC Chemikalien GmbH (Munich, Germany). Fura-2/AM was from Lambda Probes and Diagnostics (Graz, Austria). cAMP radio receptor assay system, Hybond 228 -N+ nylon membranes, and Hyperfilm 228 MP were obtained through Amersham (Vienna, Austria). N-methyl-D-glucamine was from Fluka (Vienna, Austria). All other chemicals and reagents were from Merck (Darmstadt, Germany).

Cell culture
MC3T3-E1 cells (passage number 10–20) were cultured routinely in {alpha}-MEM containing 5% FCS, gentamycin-sulfate (83.4 mg/liter), 50 µg/ml ascorbate and L-glutamine (0.584 g/liter) in a humidified atmosphere of 5% CO2 in 80 cm2 flasks (initial plating density 2 x 104 cells/cm2) and transferred to 4 cm2 12-well culture dishes before experiments. Experiments were carried out at confluency (day 7 of culture). Twenty-four hours before experiments, FCS in the culture medium was reduced to 0.2%. Experiments were carried out in {alpha}-MEM without gentamycin-sulfate, containing 0.2% FCS and 4 mM Ca2+.

PGE2 analysis
Incubations with test compounds or vehicle were carried out for the indicated time periods. The incubation medium was removed and PGE2 measured by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) (35). Briefly, PGE2 was converted to its PFB ester-TMS ether-O-methyloxime derivative. Quantitation was carried out by use of tetradeuterated PGE2. A Fisons Trio 1000 GC-MS system was used. GC was performed on a 15 m DB-5MS fused silica capillary column (Fisons Instruments). The temperature of the splitless Grob injector was kept at 290 C, initial column temperature was 160 C for 1 min, followed by an increase of 40°/min to 310 C. NICI was carried out in the single ion recording mode with methane as a moderating gas.

cAMP accumulation
Medium (1 ml) was removed and the cell monolayer incubated in 1 ml of HEPES-buffered (10 mM) HBSS, containing 0.5 mM 3-isobutyl-1-methylxanthine. Incubations with PTH (20 nM), PGE2 (10 µM), or vehicle were carried out for 5 min at 37 C. The incubation buffer was removed and the cell layer treated with 1 ml ice-cold 1-propanol (90%). The dishes were left at 4 C for 4 h and placed at -70 C overnight to disrupt cells. The extraction solvent was removed under a stream of nitrogen and the residue reconstituted in assay buffer. cAMP was measured by radio receptor assay.

RNA preparation
Cells were cultured in 35-mm wells containing 4 ml {alpha}-MEM supplemented with 5% FCS. No medium exchange was performed within 60 h before the experiments to prevent induction of PGHS-2 mRNA levels by serum factors as previously described (36). After reaching confluency total RNA was extracted using Trizol reagent following the manufacturers instructions. RNA of six 35-mm wells was pooled. RNA content was determined by measuring the absorbance at 260 nm.

Northern blot Analysis
Northern blot analysis of PGHS-1, PGHS-2, and GAPDH was performed following a protocol previously described by Pilbeam et al. (36). Twenty to 25 µg of total RNA was run on a 1% agarose/2.2 M formaldehyde gel followed by transferring the RNA to Hybond 228 -N+ nylon membranes by capillary blotting overnight. RNA was cross-linked to the membrane by UV irradiation. Prehybridization was performed in a 50% formamide solution at 42 C for 3 h followed by hybridization at 42 C overnight in a similar solution containing cDNA probes for PGHS-1, PGHS-2 and GAPDH (Clontech), which were 32P-labeled by random priming using 32P-dCTP. After hybridization, membranes were washed with 1 x SSC/1% SDS at room temperature for 30 min, once with 0.1 x SSC/0.1% SDS at 65 C for 15 min and three times with 0.1 x SSC/0.1%SDS at room temperature. Autoradiography was performed by exposure of the membranes to Hyperfilm 228 MP at -70 C for 72 h. For reprobing, filters were washed with boiling 0.1 x SSC/0.1% SDS for 15 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rapid PGE2 production (<30 min)
When MC3T3-E1 cells were stimulated with endothelin/sarafotoxin peptides for 30 min, the following PGE2 synthesis profile was obtained: ET-3, S6c, and S6a did not show any PGE2 synthesis up to an agonist concentration of 100 nM. In contrast, ET-1, ET-2, ß-ET, and S6b displayed concentration-dependent and saturable kinetics of prostaglandin formation, as shown in Fig. 1Go. The calculated EC50 values were 9.8 ± 1.1 nM (ET-1), 24.1 ± 2.5 nM (ET-2), 16.7 ± 0.6 nM (ß-ET), and 15.4 ± 0.9 nM (S6b). Because MC3T3-E1 cells show large variations in their prostaglandin response to various agonists depending on passage number, cell number and culture conditions, the maximal PGE2 levels from different experiments (as the kinetics in Fig. 1Go) cannot directly be correlated to each other. We therefore estimated the relative potencies of the agonists at maximal effective doses (50 nM) during one single experiment. ET-1, ET-2, ß-ET were found to yield virtually identical PGE2 values, and S6b producing slightly less, as shown in Table 1Go. In another experiment, however, all four agonists stimulated prostaglandin production to the same extent (results not shown). ET-1, ET-2, ß-ET and S6b-stimulated PGE2 synthesis was maximal after 5 min, as shown in Fig. 2Go.



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  		Figure 1. Stimulation of PGE2 production by endothelin/sarafotoxin peptides in osteoblast-like MC3T3-E1 cells. Cells were cultured as described in Materials and Methods and stimulated with the indicated concentration of peptides for 30 min. PGE2 in the incubation medium was determined as described in Materials and Methods. Values are given as means ± SEM of six determinations.

 

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  		Table 1. Effects of ET-receptor agonists and antagonists endothelin/sarafotoxin-induced "short-term" PGE2-production in MC3T3-E1 cells

 


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  		Figure 2. Time course of endothelin/sarafotoxin-induced PGE2 production in osteoblast-like MC3T3-E1 cells. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of ET-1 ({blacksquare}), ET-2 (•), S6b ({blacktriangleup}), and ß-ET (196) for the indicated periods of time. PGE2 in the incubation medium was determined as described in Materials and Methods. Values are given as means ± SEM of six determinations.

 
Preincubation with the nonselective ET-receptor blocker PD 142893 (37, 38) and the ETA-selective antagonist BQ-123 (39, 38) completely abolished short-term PGE2 production in MC3T3-E1 cells, whereas none of the endothelins was inhibited by the ETB-selective inhibitor BQ-788 (40, 38). Interestingly, PGE2-response to S6b was markedly reduced by 80% with BQ-788. The data are summarized in Table 1Go.

Delayed PGE2 production
When MC3T3-E1 cells were stimulated with S6a and S6c (each 50 nM) for time intervals up to 3 h, PGE2 synthesis occurred as shown in Fig. 3AGo. There was no PGE2 production measurable for S6c, but S6a-stimulated prostaglandin synthesis increased time-dependent reaching maximal levels after 3 h. PGE2 production was blocked by the translation inhibitor cycloheximide (1 µM) and reduced by 83% after pretreatment with actinomycin D (1 µM), as indicated in Fig. 3BGo. There was no PGE2 synthesis measureable after 3 h with ET-3. ET-1, ET-2, ß-ET, and S6b were also able to increase PGE2 formation up to 3 h in addition to their short-term response, as shown in Fig. 4Go. In any case, cycloheximide blocked the stimulatory effect and reduced PGE2 synthesis to the levels obtained after 30 min As already demonstrated for S6a, actinomycin D was not able to prevent prostaglandin production but reduced values as shown in Fig. 5Go. Both agents had no effect on basal prostaglandin production. It is also noteworthy, that relative potencies of stimulation after 3 h are practically identical for ET-1 (8, 905 ± 0, 417 ng/106 cells), ET-2 (9, 530 ± 0, 443 ng/106 cells), and ß-ET (8, 690 ± 0, 624 ng/106 cells), in contrast to S6a (6, 313 ± 0, 207 ng/106 cells) and S6b (14, 638 ± 0, 844 ng/106 cells). The nonspecific ET-receptor blocker PD 142893 clompletely inhibited PGE2 synthesis stimulated with ET-1, ET-2, ß-ET, S6a, and S6b after 3 h, as shown in Table 2Go. Antagonizing the ETA-receptor with BQ-123 (0, 5 µM) largely blunted the stimulatory activity of ß-ET (95% inhibition), S6b (97, 7% inhibition), and S6a (97, 6% inhibition). In contrast, PGE2 synthesis induced by ET-2 was only reduced to 64% of controls, and the stimulatory effect of ET-1 was unaffected with 0,5 µM BQ-123. The concentration-dependent inhibition of PGE2 synthesis by BQ-123 after endothelin/sarafotoxin-stimulation for 3 h is shown in Fig. 6Go.



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  		Figure 3. Long-term stimulation of PGE2 production in osteoblast-like MC3T3-E1 cells. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of the agonist for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. A, Time-dependent formation of PGE2 after stimulation with S6a ({blacksquare}) or S6c ({square}). B, Basal (solid bars) and S6a-induced (open bars) PGE2 production. Cells were pretreated with cycloheximide (CHX, 1 µM), actinomycin D (Ac D, 1 µM), or vehicle (none). Values are given as means ± SEM of six determinations.

 


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  		Figure 4. Long-term stimulation of PGE2 production in osteoblast-like MC3T3-E1 cells. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of (A) ET-2, (B) S6B, (C) ß-ET, and (D) ET-1 for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. Values are given as means ± SEM of six determinations.

 


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  		Figure 5. Basal (solid bars) and agonist-induced (open bars) PGE2 production in osteoblast-like MC3T3-E1 cells after long-term stimulation with (A) S6b, (B) ET-2, (C) ß-ET, and (D) ET-1. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of the agonist for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. Cells were pretreated with cycloheximide (CHX, 1 µM), actinomycin D (Ac D, 1 µM), or vehicle (none). Values are given as means ± SEM of six determinations.

 

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  		Table 2. Effects of ET-receptor agonists and antagonists on endothelin/sarafotoxin-induced delayed PGE2-production in MC3T3-E1 cells

 


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  		Figure 6. Concentration-dependent inhibition of PGE2 production by BQ-123 in osteoblast-like MC3T3-E1 cells after long-term stimulation with ET-1 ({blacksquare}), ET-2 (•), ß-ET ({blacktriangleup}), S6b ({square}), and S6a ({circ}). Cells were cultured as described in Materials and Methods and stimulated with 50 nM of the agonist for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. Cells were pretreated with BQ-123 at the indicated concentrations for 15 min. Values are given as means ± SEM of six determinations.

 
The ETB-receptor antagonist BQ-788 (0, 5 µM) had no effect on PGE2 synthesis induced by ET-1, ET-2, and ß-ET, whereas the effect of S6b was reduced to 51.6%. Interestingly, S6a-induced PGE2 synthesis was markedly inhibited (96.1% inhibition) by BQ-788. The data are summarized in Table 2Go.

When MC3T3-E1 cells were pretreated with the adenylate cyclase inhibitor SQ 22536 (41) and the specific competitive inhibitor of the activation of cAMP-dependent protein kinases Rp-cAMPS (42), both at 50 µM, stimulation with ET-1 (50 nM) for 3 h yielded PGE2 values of 6,18 ± 0,26 ng/106 cells and 6,02 ± 0,23 ng/106 cells, respectively. Untreated controls (ET-1 without inhibitor) produced 5,97 ± 0,11 ng PGE2/106 cells. Preincubation with acetylsalicylic acid (ASA, 100 µM) followed by medium change and stimulation with ET-1 resulted in a reduction of PGE2 production by 32%.

Inhibition of PGHS-2 with NS-398
Pretreatment of the cells with NS-398, a specific inhibitor of PGHS-2 (43), completely abolished rapid and delayed PGE2 synthesis stimulated by the abovementioned agonists, when used at 15 µM. To rule out some possible effects on PGHS-1 as well, concentration kinetics were established with NS-398 after ET-1-stimulation for 30 min and 3 h. The data are shown in Fig. 7AGo and 7BGo, respectively. The 30-min response was dose dependently inhibited with IC50 = 447 ± 38 nM. After 3 h, concentration kinetics showed a biphasic shape with the first IC50 = 0.35 ± 0,29 nM and the second IC50 = 412 ± 24 nM.



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  		Figure 7. NS-398-dependent inhibition of PGE2 production stimulated by ET-1 (50 nM) in osteoblast-like MC3T3-E1 cells after 30 min (A) and 3 h (B). Cells were cultured as described in Materials and Methods and pretreated with the indicated concentration of NS-398 for 30 min PGE2 in the incubation medium was determined after stimulation as described in Materials and Methods. Values are given as means ± SEM of six determinations.

 
Down-regulation of PGHS-2 induction
Down-regulation was accomplished by exhaustive serum induction of PGHS-2 by preculture with 10% FCS for 24 h (36), followed by medium change to 0.2% FCS and stimulation with agonists and inhibitors. The results are shown in Table 2Go. After stimulation for 3 h with ET-1, ET-2, ß-ET, S6a and S6b, PGE2 production was diminished to 21%, 13%, 8%, 5%, and 7% of untreated controls, respectively.

cAMP accumulation
Control incubations with PTH (20 nM) and PGE2 (10 µM) yielded a cAMP response of 207 pmol/106 cells and 63 pmol/106 cells, respectively. ET-3, S6c, and S6a did not show cAMP accumulation above basal levels (2 pmol/106 cells). ET-1, ET-2, ß-ET and S6b (all at 50 nM) produced only minimal cAMP response (12, 11, 3.5 and 4.2 pmol/106 cells), which was readily reduced to basal levels by pretreatment with PD 142893 (5 µM) and BQ-123 (500 nM). In contrast, BQ-788 (500 nM) did not alter cAMP accumulation induced by ET-1 and ET-2.

Protein tyrosine kinase (PTK) inhibition
Preincubation with genistein (25 µM) for 30 min effectively reduced PGE2 synthesis after stimulation with ET-1, ET-2, ß-ET, S6b, and S6a for 3 h, as shown in Fig. 8Go. The chemically and mechanistically dissimilar PTK inhibitor herbimycin A (44) did not show any inhibition in our incubation system (results not shown).



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  		Figure 8. Inhibition of PGE2 production by genistein in osteoblast-like MC3T3-E1 cells after long-term stimulation. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of the agonist for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. Cells were pretreated with genistein (solid bars, 25 µM) or vehicle alone (open bars) for 30 min. Values are given as means ± SEM of six determinations.

 
Depletion of protein kinase C (PKC)
Pretreatment of MC3T3-E1 cells with TPA (100 nM) virtually abolished PGE2 synthesis after stimulation with ET-1, ET-2, ß-ET, S6b, and S6a for 3 h. The results are shown in Fig. 9Go.



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  		Figure 9. Effect of PKC-depletion with TPA on endothelin/sarafotoxin-induced PGE2 production in osteoblast-like MC3T3-E1 cells after long-term stimulation. Cells were cultured as described in Materials and Methods and stimulated with 50 nM of the agonist for 3 h. PGE2 in the incubation medium was determined as described in Materials and Methods. Incubations were carried out with untreated controls (open bars) and cells pretreated with TPA (100 nM) for 24 h (solid bars). Values are given as means ± SEM of six determinations.

 
Northern blot analysis of PGHS-2 mRNA expression
The results of Northern blot analysis of PGHS-2 mRNA expression are given in Fig. 10Go. PGHS-2 mRNA could be induced in MC3T3-E1 cells within 2 h by the addition of ET-1, ET2, ß-ET, S6a, and S6b. There was no PGHS-2 mRNA detectable in unstimulated controls, as well as in cultures treated with S6c and ET-3 (results not shown). Rough comparison of mRNA band intensities clearly shows higher induction by the sarafotoxin peptides on a molecular basis, with S6b being the most potent agonist. In contrast, PGHS-1 mRNA expression was found to be present also in unstimulated controls and was constant for the above agonists, although the bands observed after stimulation were more intense than control bands.



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  		Figure 10. Effect of sarafotoxin/endothelin peptides on the PGHS-2 mRNA expression in MC3T3-E1 cells. RNA extraction and Northern blot analysis were performed as described in Materials and Methods. Northern blots were hybridized with PGHS-2 cDNA and then stripped and probed for PGHS-1 cDNA and GAPDH cDNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Locally acting autocrine and paracrine factors play a crucial role in the bone remodeling cycle. For ET-1, it has been shown to act on bone and bone cells. Thus, in osteoblastic cells, ET-1 stimulates DNA synthesis and decreases alkaline phosphatase (15, 45). It stimulates tyrosine phosphorylation in MC3T3-E1 cells, which possibly contributes to its mitogenic activity (28). In UMR-106 osteoblastic cells, ET-1 modulates calcium signaling by epidermal growth factor, {alpha}-trombin, and prostaglandin E1 (29) and enhances PTH-induced calcium signals by ETB receptor activation (30). Because the effects of ET-1 on bone resorption are prone to inhibition with indomethacin (18), ET-evoked synthesis of the powerful osteogenic PGE2 can be suggested to be one major route to the autocrine and paracrine modulation of bone cell activity. Our first set of experiments was thus aimed to definitely provide evidence of ET-1 induced PGE2 production and define its cellular origin to the osteoblast, with the use of MC3T3-E1 cells. Furthermore, we have aimed on the effects exerted by other endothelin peptides and the highly conserved structural related sarafotoxins. Our results reveal a characteristic pattern of rapid PGE2 synthesis (<30 min). The lack of S6c, a selective ETB receptor agonist (46), and ET-3 to stimulate prostaglandin release and to antagonize the effect of ET-1, ET-2, ß-ET and S6b is consistent with ETA receptor activation by these peptides. This is also confirmed by the antagonism observed with PD142893 (nonselective) and BQ-123 (ETA-selective). In this context, S6a would also have to be considered as ETA-selective in MC3T3-E1 cells. Antagonizing the ETB receptor with BQ-788 expectedly had no effect on prostaglandin formation induced by endothelins. We must thus conclude that short-term PGE2 synthesis evoked by endothelin peptides is exclusively due to ETA receptor activation. For S6b, the effect of BQ-788 surprisingly was inhibitory. This would suggest some yet undefined pharmacological properties of the compound BQ-788 beyond the ETB receptor antagonism. Another explanation would be that S6b besides binding to the ETA receptor, occupies an additional sarafotoxin receptor, or there might exist some further subtypes to the known receptors, as suggested by several other groups as well (47, for review see Ref. 48). Distribution and activation of ET receptor subtypes can be regarded as a tissue-specific task, as both, ETA and ETB receptors mediate prostanoid release in the isolated kidney of the rat (25) and ETB receptor occupancy is responsible for PGE2 synthesis in the inner medullary collecting duct (49).

When MC3T3-E1 cells are incubated for extended periods of time (3 h), the pattern of PGE2 production changes. Prostaglandin synthesis is significantly elevated in any case. While the stimulatory activity of the endothelin isopeptides is about the same, S6b-induced PGE2 formation strikingly exceeds that of all other agonists. Additionally, S6a is now able to contribute considerable to prostaglandin formation in these cells. Antagonizing this effect with PD 142893 was quantitative, whereas the potency of BQ-123 decreased in the order S6a>S6b>ß-ET>>ET-2>ET-1. This is in good agreement with previous published data, where BQ-123 was shown to antagonize S6b by far more effectively than ET-1 (47) and would again account for a ETA-mediated process. Because the distribution pattern of high affinity binding sites for the endothelins and sarafotoxins was found to be very similar in human, porcine, and rat tissues, a common receptor system can be assumed. However, recent reports as reviewed by Bax and Saxena (48), demonstrate that endothelin antagonists, in particular BQ-123, are less effective against ET-1 than against other members of the endothelin/sarafotoxin family and thus may imply the presence of additional receptors or receptor subtypes for the individual peptides (48).

ETA receptor occupation is certainly underlined for the endothelin peptides, as BQ-788 has no effect. On the other hand, the reduction of S6b-induced PGE2 formation and inhibition of S6a-related PGE2 formation again points toward some additional binding sites besides the ETA receptor. If one assumes induction of PGHS-2 after long-term exposure to the agonists, as discussed later, the reduction of S6b-induced PGE2 formation observed with BQ-788 could be attributed to blockage of the constitutive (short term) response, as already shown above. Thus, the remaining PGE2 synthesis should be due to enzyme induction. Inhibition of prostaglandin formation induced by S6a with BQ-788 further supports the concept of pharmacological side effects of the compound and/or receptor heterogeneity for sarafotoxin peptides, a topic to be evaluated in more detail in a separate study.

There is, however, one crucial point to be discussed, regarding the homology of the endothelin/sarafotoxin peptides. The amino acid sequence of S6b and S6a differs only at position 13, where a tyrosine residue (S6b) is substituted by asparagine, and this asparagine is critical for ETB selectivity (38). If we consider short-term PGE2 formation and long-term effects to be differentially regulated, which would suggest different signal transduction pathways, one main prerequisite of rapid PGE2 response is represented by a tyrosine13 in the amino acid sequence of the peptides. Indeed, ET-1, ET-2, and ß-ET have tyrosine13 residues, whereas S6c does not.

To characterize the enzymes involved in endothelin/sarafotoxin-induced PGE2 formation, experiments with NS-398, a specific inhibitor of PGHS-2 (43), were conducted. The fact that prostaglandin formation is completely attenuated with any of the agonists strongly suggests PGHS-2 to be the enzyme involved. On the other hand, short-term PGE2 response was also completely abolished by the compound at 15 µM. In a recent paper, however, NS-398 was described to be a potent inhibitor of PGHS-2 with an IC50 of approximately 3 nM (Py1a cells). In contrast, the IC50 in ROS 17/2.8 cells expressing only PGHS-1 was about 80 nM (50). Our data obtained with MC3T3-E1 cells clearly demonstrate such a differential effect of the drug, and furthermore provide evidence for the involvement of PGHS-1 in rapid PGE2 formation, as well as contribution of PGHS-2 in delayed production.

In another experiment, PGHS-2 was down-regulated by exhaustive serum-induction, as described recently to be the case in MC3T3-E1 cells (36). As expected, induced delayed production of prostaglandins was markedly reduced. It is therefore concluded that PGHS-1 contributes to the rapid PGE2 formation in response to endothelin/sarafotoxin stimulation, whereas PGHS-2 is responsible for the delayed production.

Induction of PGHS-2 after endothelin/sarafotoxin stimulation was actually confirmed with cycloheximide and actinomycin D. While translational inhibition completely abolished PGE2 response to short-term levels, modification at the transcriptional level was only partially effective. This phenomenon has also previously been reported to occur with ET-1 in rat mesangial cells (26) and is possibly due to some PGHS-2 mRNA already synthesized.

Finally, the PGHS-2 mRNA expression induced by ET-1, ß-ET, ET-2, S6b, and S6a was demonstrated by Northern blot analysis. The absence of virtually any PGHS-2 mRNA in untreated controls and S6c- and ET-3 treated cells, as well as the ubiquitous appearance of the constitutive PGHS-1 confirm the results above. The results are also in good agreement with data from prostaglandin measurements, where S6b is by far the most potent stimulator of delayed PGE2 production. The increase of the constitutive PGHS-1 mRNA compared with untreated controls can be attributed to some induction of this enzyme by endothelin/sarafotoxin peptides, as already described to be the case for certain serum factors in MC3T3-E1 cells (50). Nevertheless, the results clearly demonstrate induction of PGHS-2 by ET-1, ET-2, ß-ET, S6a, and S6b.

We have further investigated the signal transduction pathways that are involved in this PGHS-2 induction by endothelin/sarafotoxins. The transcriptional role of cAMP is now well recognized for the gene expression in several enzymes and peptides, like phosphoenolpyruvate carboxykinase (51), vasoactive intestinal polypeptide (52), somatostatin (53), and ß-adrenergic receptor (54). Additionally, induction of PGHS-2 mRNA in mouse osteoblastic cells, MC3T3-E1, by epinephrine was found to be cAMP dependent (55). We have therefore investigated the role of cAMP in endothelin/sarafotoxin-dependent induction of PGHS-2 activity. PTH and PGE2 were used as positive controls. Because there was no measurable accumulation of cAMP with S6a, a contribution of this signaling pathway to PGHS-2-induction can be ruled out. Furthermore, the minimal response observed with ET-1, ET-2, ß-ET, and S6b is much more likely to occur via autocrine feedback regulation by the PGE2 produced by these agonists through PGHS activity, an effect already recognized for ET-1 in other cell systems (56, 57). Reduction to basal levels after pretreatment with the nonselective ET-receptor antagonist PD 142893 and the ETA-selective antagonist BQ-123 is consistent with the expected inhibition of PGE2 synthesis. The lack of inhibition after BQ-788 pretreatment (ETB-selective antagonist) observed for ET-1 and ET-2-dependent cAMP accumulation underlines the sole involvement of ETA-receptors for PGHS-1 stimulation with these compounds. Absent cAMP response to S6b treatment after inhibition with BQ-788 is in correlation with our data on PGE2 production, thus suggesting other pharmacological activities of BQ-788 beyond the selective ETB-receptor antagonism. Involvement of the adenylate cyclase pathway in mediating the effects of these peptides on stimulating delayed PGE2 production can furthermore be ruled out by the findings, that inhibition of adenylate cyclase and cAMP-dependent protein kinases does not have any effect on prostaglandin formation stimulated by ET-1 for 3 h. Irreversible inhibition of PGHS-1 by pretreatment with ASA reduced PGHS-2-related PGE2 formation (induced by ET-1) only by 32%. Thus, the interesting concept of autocrine PGHS-2 induction by initially formed PGE2 does not seem to apply in this case. Although some contribution cannot be ruled out, the reduction in PGE2 formation is more likely to occur due to some residual ASA after medium change. It should be pointed out that repeated washing steps may significantly induce PGHS-2 and hence were kept to a minimum.

The recent finding that ET peptides increase tyrosine phosphorylation of multiple cellular proteins (28, 22, 58, 59) prompted us to ask whether PTK activity was required for PGHS-2-related PGE2 production stimulated by endothelin/sarafotoxin peptides in MC3T3-E1 cells. Tyrosine phosphorylation has previously been implicated in th PGHS-2 mRNA expression induced by interleukin-1 (60) and ET-1 (22). The striking inhibition of prostaglandin production by the PTK inhibitor genistein suggests a crucial role for transcriptional regulation of PGHS-2 activity and hence endothelin/sarafotoxin-dependent PGE2 formation in MC3T3-E1 cells. It was somewhat surprising to observe no such inhibitory effect with another selective PTK inhibitor, herbimycin A. Reevaluating the experimental system however, provides the explanation for this phenomenon: herbimycin A is thought to exert its inhibitory action on PTK via irreversible modification of thiol groups, and hence is itself easily deactivated in the presence of thiol compounds (61). The incubation medium in our experiments, {alpha}-MEM, contains large amounts of cysteine, and thus precludes herbimycin A to be used as an inhibitor of PTK. The concept of seven-transmembrane domain receptors inducing an immediate early gene response via tyrosine kinases is gaining more and more attention, as recent studies have revealed, that vasoconstrictor peptides, including ET-1, can activate nonreceptor-linked tyrosine kinases (22, 28, 58). Hence, osteoblastic prostaglandin production might contribute to the mitogenic signaling of endothelin/sarafotoxin peptides.

The data concerning the role of PKC in mitogenic signaling by endothelins are mainly restricted to ET-1; no data are available for the other endothelins and sarafotoxins. Thus, PKC-depletion led to attenuation of ET-1-stimulated [3H]thymidine uptake in rat 1 fibroblasts (62) and rat mesangial cells (58). On the other hand, PKC activity was found to be neither necessary nor sufficient for ET-1-stimulated PGHS-2 mRNA expression in rat mesangial cells (22), whereas in the same cell system PGHS-2 gene expression induced by serum was found to be regulated via a PKC-dependent mechanism (63). Our results clearly demonstrate a crucial involvement of PKC in endothelin/sarafotoxin-induced PGE2 production in MC3T3-E1 cells, as prostaglandin formation is largely blunted by PKC-depletion with TPA. The contradiction to previous investigations (22) could be due to 1) different regulatory mechanism in different cell systems and 2) that PKC acts on a point downstream of PGHS-2 mRNA expression.

In conclusion, our data provide evidence for PGE2 formation by endothelin/sarafotoxin peptides in osteoblast-like cells. This is, at least for endothelins, attributable to ETA receptor occupancy. A differentially regulated activity exists for a short-term rapid response and long-term prostaglandin formation, most likely depending on differences in receptor binding by sarafotoxins and endothelins. Long-term PGE2 response is solely due to PGHS-2 activity, and enzyme induction is involved. Rapid response is due to the activity of constitutive PGHS-1. Furthermore, this PGHS-2 induction is mediated by PTK and PKC, whereas cAMP is not involved, which might be a crucial event in the mitogenic signaling by the peptides investigated.


   Footnotes
 
1 This work was supported by grants from the Austrian Science Foundation (FWF), Project No. P-12589. Back

Received July 31, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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