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GR 63799X, an EP3 receptor agonist, induced S phase arrest and 3T6 fibroblast growth inhibition
European Journal of Pharmacology 529 (2006) 16 – 23 www.elsevier.com/locate/ejphar
GR 63799X, an EP3 receptor agonist, induced S phase arrest and 3T6 fibroblast growth inhibition Teresa Sanchez, Juan J. Moreno ⁎ Department of Physiology, Faculty of Pharmacy, Barcelona University, Avda. Joan XXIII s/n, E-08028 Barcelona, Spain Received 14 July 2005; received in revised form 18 October 2005; accepted 25 October 2005 Available online 28 November 2005
Abstract The importance of arachidonic acid metabolites on the control of cell growth, particularly those derived from cyclooxygenase pathway has long been recognized. Recently, we observed that prostaglandin E2 (PGE2) interaction with EP1 and EP4 receptors is involved in serum-induced 3T6 fibroblast growth due to their effect at various levels of the cell cycle machinery. This study shows that prostanoid EP3 receptor was expressed in 3T6 fibroblast. We studied the role of EP3 receptor agonist GR 63799X in serum-induced 3T6 cell proliferation. This was concentrationdependent inhibit (IC50 ∼10 μM) to a complete inhibition without any cytotoxic or proapoptotic effect. The prostanoid EP3 receptor agonist treatment decreased the G0/G1 and G2/M populations whereas cells were accumulated in S phase. This arrest in S phase was associated with a decrease in cyclin B levels and the enhancement of p21 expression. Our data show that EP3 agonist decreases cAMP levels in our experimental conditions. Interestingly, the S arrest caused by prostanoid EP3 receptor agonist seems to be cAMP dependent, at least in part, because forskolin treatment allowed S-arrested cells to progress through cell cycle and consequently growth. Thus, our results suggest that PGE2 EP3 receptor interaction may be involved in serum-induced 3T6 fibroblast growth due to their effects on cAMP levels and on cell cycle machinery of the S phase. © 2005 Elsevier B.V. All rights reserved. Keywords: Prostaglandin E2; Cell proliferation; Cell cycle; Cyclin; cAMP
1. Introduction Prostaglandins are potent lipid mediators generated from arachidonic acid by the cyclooxygenase isozymes. Prostaglandins are quickly released from cells after synthesis and act as local hormones in the vicinity of their production site to maintain local homeostasis. Thus, these mediators have numerous physiological effects (Smith, 1992), including cell proliferation and differentiation. PGE2 induces DNA synthesis in Swiss 3T3 fibroblasts (Otto et al., 1982). Interestingly, enhanced synthesis of PGEs was observed in a wide variety of tumors (Dubois et al., 1998; Bishop-Bailey et al., 2002). Previous studies have shown that PGE2 is the predominant prostaglandin synthesized and secreted in response to cyclooxygenase-2 up-regulation (Brock et al., 1999). We have suggested elsewhere that PGE2, the major cyclooxygenase pathway metabolite produced by ⁎ Corresponding author. Tel.: +34 93 4024505; fax: +34 93 4035001. E-mail address: [email protected] (J.J. Moreno). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.10.040
fibroblasts, controls 3T6 fibroblast growth (Lloret et al., 1996) and that cyclooxygenase-2 is the main isoform responsible for serum-induced PGE2 synthesis in these cells (Martínez et al., 1997). The ability of each prostaglandin to affect various biological responses is dependent on its binding to specific receptors on the plasma membrane which belong to the rhodopsin family of serpentin receptors. The four subtypes of PGE receptors (EP1– EP4) from various species, previously defined pharmacologically, have been cloned (see Narumiya, 1994, for review). They are encoded by a distinct gene and differ in their amino acid identities, pharmacological characteristics, and signal transduction properties. The prostanoid EP2, EP4 and one isoform of the EP3 receptor can couple to Gs and thus increase intracellular cAMP concentration, whereas other isoforms of EP3 receptor can couple to Gi, causing a decrease in the cAMP levels (Narumiya et al., 1999). Therefore it is likely that physiological consequences of activation of the prostanoid EP3 receptor may vary substantially depending on the cell type examined.
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Several studies have suggested the role of PGE2 receptors in cellular proliferation and tumor development. Experiments on EP1-deficient mice show that this subtype receptor is required for the development of preneoplastic lesions induced in colon epithelium by the carcinogen azoxymethane (Watanabe et al., 1999). The same authors have proposed a chemopreventive effect of EP1 or EP4 selective antagonist on colon cancer development (Mutoh et al., 2002). Moreover, we recently reported that prostanoid EP1 and EP4 receptors are involved in serum-induced 3T6 fibroblast growth, suggesting that PGE2 interaction with both receptors regulates progression through the cell cycle by modulating cyclin D and E and cyclin A levels, respectively (Sánchez and Moreno, 2002). There are contradictory results about prostanoid EP3 receptor presence in fibroblasts. Thus, Watanabe et al. (1996), Noguchi et al. (2002) and Yu et al. (2002) reported that EP1, EP2 and/ or EP4 are expressed in fibroblast but not EP3, whereas Yoshida et al. (2001) observed the expression of EP3 together EP2 and EP4 in fibroblasts. Our results agree with the last authors. We observed that the growth-inhibitory effect of GR 63799X, a selective prostanoid EP3 receptor agonist (Bunce et al., 1991), on serum-stimulated cellular proliferation involve the impairment of cAMP levels, reducing cyclin B levels whereas increasing p21 expression, and induced an S-phase arrest in 3T6 cells. 2. Material and methods 2.1. Reagents
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(100 μg/ml), in a humidified atmosphere of 95% air–5% CO2 at 37 °C. Cells were harvested with trypsin/EDTA and passed to tissue culture 100 or 60 mm dishes (Costar, Cambrigde, MA) for experimental purposes. 2.3. Cell growth The effect of prostanoid EP3 receptor agonist was assessed on 3T6 fibroblasts plated at 104 cells/well in 12-well plates (Costar, Cambrigde, MA) and cultured for 24 h in RPMI supplemented with 10% FCS. Cells were incubated for 2 days in 10% FCS medium in the presence of the different concentration of GR 63799X. At the end of the experiments, cells were washed, trypsinized and counted, using ethidium bromide/acridine orange staining in order to assess viability. To study cell recovery, after a 24 h treatment with prostanoid EP3 agonist, 3T6 fibroblast cultures were washed with phosphate-buffered saline solution (PBS) and fresh 10% FCS medium was added. Growth recovery was assessed after 1, 2 or 3 days, by counting trypsinized cells, as mentioned above. 2.4. [3H]Thymidine incorporation assay DNA synthesis was measured as previously described (Martínez et al., 1997). Briefly, fibroblasts were cultured in 96-well plates at a density of 400 cells/well. 24 h later, cells were incubated with the treatments and [3H]thymidine (1 μCi/ well) for 24 h. [3H]Thymidine-containing media were aspirated, cells were overlaid with 1% Triton X-100, and then cells were scraped off the dishes and the radioactivity present in the cell fraction was measured by liquid scintillation counting.
RPMI 1640, fetal calf serum (FCS), penicilin G, streptomycin and trypsin/EDTA were from Bio Whittaker Europe (Verviers, Belgium). [methyl-3H]thymidine (20 Ci/mmol) was obtained from Du Pont-New England Nuclear (Boston, MA). GR 63799X [1R-[1α (2), 2β (R*), 3α]]-4-(benzoylamino) phenyl 7-[3-hydroxy-2-(2-hydroxy-3-phenoxypropoxy)-5-oxocyclopentyl]-4-heptenoate was kindly provided by Glaxo Welcome (Stevenage Hertfordshire, UK). Aprotinin, leupeptin, diethyldithiocarbamic acid, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride, sodium orthovanadate (Na3VO4), Igepal CA-630, dithiothreitol (DTT), pertussis toxin, neomycin, forskolin, propidium iodide (PI), Hoescht 33258, ethidium bromide, acridine orange and DNase-free ribonuclease A were acquired from Sigma Chemical (St. Louis, MO). Rabbit polyclonal antibodies against cyclins D, E, A, B and p21 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). EP3 and EP4 receptor peptides and rabbit polyclonal antibodies against EP3 and EP4 receptor were from Cayman Chemical Co. (Ann Arbor, MI). Drug was dissolved in dimethylsulfoxide and final concentration of dimethylsulfoxide never exceeded 0.1%.
Cells were seeded and 24 h later, they were washed with PBS and serum starved. After 24 h serum starvation, the percentage of cells in G0/G1 was about 80%. Cells were then cultured in 10% FCS RPMI containing the treatments. After 30 h, cells were trypsinized, fixed with 70% ethanol and stored at 4 °C for at least 24 h. Thapsigargin and forskolin were added to the GR 63799 X-containing culture medium after a 30 h incubation, and 6 h later, cells were harvested and fixed. They were then stained for 1 h at room temperature with a 20 μg/ml propidium iodide solution in PBS containing 0.1% Triton X-100 (Sigma) and 0.2 mg/ml DNase-free RNase A. Cells were analyzed on a Epics XL flow cytometer (Coulter Corporation, Hialeah, Florida). DNA was analyzed (Ploidy analysis) on single fluorescence histograms by Multicycle software (Phoenix Flow Systems, San Diego, CA).
2.2. Cell culture
2.6. Western blot analysis
Murine 3T6 fibroblasts (ATCCC, CL96) were grown as we described elsewhere (Martínez et al., 1997), in RPMI 1640 containing 10% FCS, penicillin (100 U/ml) and streptomycin
3T6 fibroblast cultures were washed twice with ice-cold PBS. Total cellular fraction was obtained by scraping off the cells in lysis buffer containing 200 mM Tris–HCl, 200 mM
2.5. Fluorescent-activated cell sorting (FACS) analysis/flowcytometry cell cycle analysis
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T. Sanchez, J.J. Moreno / European Journal of Pharmacology 529 (2006) 16–23 Table 1 Effect of prostanoid EP3 receptor agonist on [3H]thymidine incorporationa [3H]thymidine incorporation (Dpm × 1000) Control FCS FCS + GR FCS + GR FCS + GR FCS + GR
15 ± 2 183 ± 9 63779X (5 μM) 123 ± 2b 63799X (10 μM) 85 ± 6b 63799X (50 μM) 42 ± 3b 63799X (10 μM) + forskolin (10 μM) 129 ± 8c
a
Fig. 1. The expression of prostanoid EP3 or EP4 receptors in 3T6 fibroblast cultured in 10% FCS was determined by Western blot. EP3 or EP4 receptor peptides (10 μg) were used as standards and total 3T6 fibroblast lysate protein (20 μg) was loaded. Present blot is representative of 3 blots.
NaCl, 2% Igepal CA-630, 400 μM NaF, 200 μM DTT and 400 μM Na3VO4, followed by incubation for 30 min at 4 °C. Total protein was measured by the Bradford method (1976) using the Bio-Rad protein assay, with BSA as standard. Immuno blot
Fig. 2. Effect of prostanoid EP3 receptor agonist on 10% FCS-induced 3T6 fibroblast proliferation. 3T6 fibroblasts (104 cells/well) were plated and cultured in 10% FCS-RPMI. The next day, media were removed and fresh 10% FCSRPMI containing the treatments was added. At the end of experiments, cells were trypsinized and counted. (A) Concentration response effect of the GR 63799X after 48 h incubation. (B) time course of growth cell in presence of GR 63799X 10 μM (triangle), GR 63799X 50 μM (squares) or absence of EP3 receptor agonist (circles). Results are means ± S.E.M. of 3 experiments performed in duplicate. *P b 0.05 vs. control cells.
3T6 fibroblasts were cultured overnight without FCS. The next day, media were removed and fresh 10% FCS-RPMI containing the treatments were added together [3H]thymidine (1 μCi/well). After 24 h, [3H]thymidine incorporation to cells was measured. Results are means ± S.E.M. of three experiments performed in duplicate. b P b 0.05 vs. FCS; cP b 0.05 vs. cells treated with GR 63799X (10 μM).
analysis for cyclins were performed as follows: 20 μg of protein from cell lysates were separated by a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel (Laemmli, 1970) and blotted for 1 h with a constant voltage of 100 V onto a polyvinylidene difluoride membrane (Immun-Blot PVDF membrane, 0.2 m, Bio-Rad) using a MiniProtean II system (Bio-Rad, Hercules, CA). A pre-stained SDS–PAGE protein standard (Bio-Rad) was used to check transfer efficiency and as a molecular weight marker. Membranes were blocked with 5% nonfat milk powder in 0.1%Tween 20 PBS for 1 h.
Fig. 3. Effect of prostanoid EP3 receptor agonist on cell cycle distribution. Synchronized 3T6 fibroblasts were incubated with 10% FCS-RPMI in presence of GR 63799X. Cells were then fixed, stained and analyzed. White bars represent the percentage of cells in G0/G1 phase; vertical striped bars in S phase and horizontal striped bars in G2/M phase. (A) Concentration–response study was performed in presence the GR 63799X for 30 h. (B) Time course of cell cycle distribution in presence of GR 63799X (10 μM). Results are means ± S.E.M. of 3 experiments performed in duplicate. *P b 0.05 vs. Control cells or vs. GR 63799X-treated cells.
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Rabbit polyclonal antibody against cyclins D, E, A, B1 and p21 or EP3 and EP4 receptor were applied in a dilution 1 : 200 for 1 h. The blots were washed several times with PBS-0.1% Tween 20 and were incubated with a goat anti-rabbit antibody in a 1 : 2000 dilution for 1 h. For β-actin immunoblotting, stripped membranes were overlaid with monoclonal anti-actin antibody (1.200) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by rabbit anti-mouse antibody (1 : 1000) (Santa Cruz). All blots were developed using an enhanced chemiluminiscence kit (Supersignal West Dura Extended Duration Substrate), from Pierce (Rockford, IL), using a Bio Max Light-2 film. 2.7. cAMP levels Intracellular cAMP from cell cultures was extracted with ethanol and measured as by suggested the manufacturer of the enzyme immunoassay kit (Amersham Life Science, Buckinghamshire, UK). 2.8. Statistical analysis Results are expressed as mean ± standard error of the mean. Differences between non-treated and treated cells were tested
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by using Student's t-test following by the least significant difference test as appropriate. 3. Results 3.1. Effect of GR 63799X on 3T6 fibroblast proliferation Initially we sought to determine the presence of prostanoid EP3 and EP4 receptor in 3T6 fibroblasts cultured in presence of FCS (Fig. 1). To demonstrate the functionality of prostanoid EP3 receptors, we performed cell proliferation assays in the presence of a selective EP3 receptor agonist, GR 63799X. EP3 receptor agonist caused a significant concentration-dependent decrease in cell growth, with a half inhibition of proliferation at 10 μM approximately and a cell growth blocked at 50 μM (Fig. 2). This action of prostanoid EP3 receptor agonist may be correlated with the inhibition of DNA synthesis measured as [3H] thymidine incorporation (Table 1). GR 63799X did not cause cytotoxicity as high as 50 μM, as was assessed by observing morphological appearance of the cells by light microscopy and ethidium bromide/acridine orange staining. Thus, the effect of EP3 agonist is due to growth inhibition and not to cytotoxic effect. For this reason, one the treatment was removed from the
Fig. 4. Fluorescence-activated cell sorting analysis of the cell cycle. 3T6 fibroblast cultures synchronized by 24 h serum starvation (SS) were incubated with 10% FCS-RPMI (control) or 10% FCS-RPMI containing GR 63799X for 30 h. Forskolin (10 μM) was added 6 h before the end of assay. To determine the reversion of GR 63799X effect, the EP3 agonist was washed 12 h before the end of experiment. Then, cells were trypsinized, fixed, stained and analyzed on a flow cytometer. Representative fluorescence histograms representing cell cycle analyses of three experiments performed in duplicate are shown. *P b 0.05 vs. control cells; **P b 0.05 vs. GR 63799X-treated cells.
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suggesting that the antiproliferative action of prostanoid EP3 receptor agonist did not result from apoptosis. 3.3. Characterization of cell cycle regulator proteins involved in GR 63799X-induced S phase arrest
Fig. 5. Effect of prostanoid EP3 agonist on cyclin D, E, A, B and p21 expression in 3T6 fibroblasts. Cells were synchronized by serum starvation for 24 h. Then, 10% FCS medium (control) or 10% FCS-RPMI containing GR 63799X was added. Forskolin (10 μM) was added 6 h before the end of assay. Cells were harvested 6 (for cyclins D and E), 16 (for cyclin A) or 18 h (for cyclin B and p21) later to carry out Western blot analysis. Total cellular lysate protein (20 μg/ lane) was loaded, separated and immunodetected as described in Material and methods. Western blots representative of 3 blots are shown.
culture medium, 3T6 fibroblast markedly recovered the growth (data not shown). 3.2. Effect of prostanoid EP3 agonist on cell cycle distribution To gain first insight into the mechanisms of prostanoid EP3 receptor agonist-induced growth inhibition, we next analyzed cell-cycle distribution in response to EP3 receptor agonist by flow cytometry. 3T6 fibroblast were synchronized in G0/G1 to allow a more distinct representation of their progression through the individual cell cycle phases. Sinchronization was achieved by overnight serum starvation, which routinely retained approximately 85% to 90% of cells in the G0/G1 phase. 3T6 fibroblasts were then stimulated to re-enter the cell cycle by addition of 10% FCS medium, and cell cycle progression was monitored for up 30 h, a period of time that corresponds to approximately two cycles, in the presence or absence of EP3 receptor agonist. GR 63779X induced a significant and concentration-dependent increase in S population and a decrease on G2/M population and in G1 population (Fig. 3A). The accumulation of cells in S-phase and the impairment of G1 and G2 + M progressed markedly when EP3 agonist treatment was prolonged (Fig. 3B). This S-phase arrest was reverted when GR 63799X was removed from the culture medium (Fig. 4). Moreover, no significant increase of cells with subdiploid DNA-content was noted in EP3 agonist-treated cultures,
Progression of the mammalian cell cycle is governed by cyclins, Cdks and CKIs (Pines, 1995). Cdks form a binary system composed of the inactive catalytic subunit cdk, which is activated by binding to cyclin. We aimed to determine whether the impaired progression through the cycle of EP3 agonist-treated cells was linked to changes in cyclin levels. As shown in Fig. 5, Western blot analyses of total protein lysates showed the impairment of cyclin B content. Interestingly this decrease of cyclin B levels can be related with the enhancement of p21 levels in GR 63799X-treated 3T6 cells (Fig. 5). Previous studies have suggested that prostanoid EP3 receptors could be mediated PGE2-induced changes of cAMP levels (Hatae et al., 2002). To test whether GR 63779X affects to this second messenger, we measured the changes of cAMP in response to EP3 receptor agonist. FCS and PGE2, at micromolar concentrations reached by FCS incubation (Moreno, 1997), increase cAMP levels in 3T6 fibroblat cultures. In contrast, GR 63799X induced a significant impairment of cAMP levels in FCS-cultured fibroblasts (Table 2). Moreover, this impairment of cAMP induced by GR 63799X was reduced by pertussis toxin treatment. Results that suggested that these changes of cAMP could be involved in the cell arrest in S-phase induced by GR 63799X treatment. Some experiments were performed to test this hypothesis. We used forskolin, an adenylate cyclase activator (Seamon and Daly, 1986) to revert the impairment of cAMP levels induced by the prostanoid EP3 receptor agonist. As shown in Table 2, forskolin corrected GR 63799X-treated cell cAMP levels. Interestingly, this effect of forskolin on cAMP levels of 3T6 fibroblast cultures can be correlated with the decrease of cyclin B levels and the enhancement of Table 2 Effect of prostanoid EP3 receptor agonist on cAMP levelsa cAMP (pmol/mg protein) Control FCS FCS + forskolin PGE2 GR 63799X FCS + GR 63799X FCS + GR 63799 X + pertussis toxin FCS + GR 63799X + neomycin FCS + GR 63799X + forskolin a
13 ± 2 55 ± 3b 168 ± 10 52 ± 3b 14 ± 1 21 ± 2c 41 ± 2d 22 ± 3 58 ± 3e
3T6 fibroblast cultures were pre-incubated with pertussis toxin (10 ng/ml) for 3 h or with neomycin (10 μM) for 30 min. Then, cells were stimulated with FCS (10%) or PGE2 (10 μM). In some experiments, cells were co-incubated with GR 63799X (10 μM). GR 63799X and forskolin (10 μM) were incubated together. cAMP levels were measured 1 h after cell cultures were stimulated. Measurements were performed in triplicate and expressed as the mean ± S.E.M. of three experiments. b P b 0.05 vs. control; cP b 0.05 vs. FCS; dP b 0.05 vs. GR 63799X; P b 0.05 vs. FCS plus GR 63799X.
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p21 content (Fig. 5), with a progression through the cell cycle (Fig. 4) and with a recovery of [3H]thymidine incorporation (Table 1). 4. Discussion Calcium-independent cytosolic phospholipase A2 is involved in serum-induced 3T6 fibroblast proliferation (Sánchez and Moreno, 2001), through arachidonic acid release and the subsequent metabolism mainly by the inducible form of cyclooxygenase, cyclooxygenase-2, to synthesize prostaglandins like PGE2 (Martínez et al., 1997). The biological actions of PGE2 have been attributed to results from its interaction with cell surface prostanoid EP receptors (Coleman et al., 1994). PGE2 has versatile and opposing actions due to multiple EP receptor subtypes and the coupling of EP receptor isoforms to a variety of signal transduction pathways. Recently, we provide evidence that PGE2 interaction with EP1 and EP4 receptors are involved in the control of 3T6 fibroblast proliferation which is associated with changes in D, E and A cyclin levels (Sánchez and Moreno, 2002). Thus, the generation and PGE2 interaction with these receptors, in an autocrine or paracrine fashion, may be acted as a necessary comitogenic signal. Here, we demonstrate that prostanoid EP3 receptors are expressed in FCS-cultured 3T6 fibroblasts. To elucidate the functional role of PGE2 EP3 receptors on the control of serum-induced 3T6 fibroblat proliferation and on progression through the cell cycle, we used GR 63799X which has affinity only to the EP3 receptor, indicating its high selectivity for this receptor (Kiriyama et al., 1997). This EP3 receptor agonist reduced FCS-induced 3T6 fibroblast growth in a time- and concentration-dependent manner, and blocked cellular proliferation, without showing cytotoxicity or proapoptotic effects at the concentrations and for the incubation times used. In the present study, we also provide evidence that prostanoid EP3 receptor agonist affect DNA synthesis. Thus, GR 63799X caused the impairment of FCS-induced [3H]thymidine incorporation to cells. In this way, Konger et al. (1998) observed that an EP3 receptor agonist inhibits bromodeoxyuridine uptake in human keratinocytes and Zacharowski and Thiemermann (1999) reported that TEI-3356, an EP3 agonist, inhibits endothelin-induced DNA synthesis in cultured vascular smooth muscle cells. GR 63799X also modified the cell cycle distribution in FCScultured 3T6 fibroblasts. Our results suggest a S phase arrest induced by the interaction of GR 63799X with the PGE2 EP3 receptor in a similar form to the cell cycle distribution changes induced by an PGE2 EP4 receptor antagonist (Sánchez and Moreno, 2002). However, a prostanoid EP4 antagonist induced the impairment of cyclin A levels and an early S phase arrest whereas a prostanoid EP3 agonist induced a delay in the S phase exit into G2/M. The hypothesis that PGE2 EP3 receptor could be regulated the cell cycle progression also was supported by the effects of GR 63799X on cyclin levels. Thus, the S phase arrest induced by GR 63799X could be linked to a significant decrease in cyclin B levels. To the best of our knowledge, this is the first report demonstrating the participa-
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tion of prostanoid EP3 receptor activation in the control of cyclin content. Furthermore, these observations corroborate results from the cell cycle distribution studies indicating a regulatory effect at the S-G2/M phases where cyclin B plays an important regulatory role. Thus, cyclin B is essential for the initiation of mitosis with protein accumulation of cyclin B occurring during S phase to function as a regulatory subunit in cdc2 kinase complexes as cells progress from S into G2/M (Sherr, 1996). The cellular cyclin B content depends on cdk2/ E2F mediated induction (De Gregori et al., 1995), and the ubiquination and proteolysis through the interaction with anaphase-promoting complex (Lukas et al., 1999). The mechanism involved in marked decrease in cyclin B levels in 3T6 cells cultured with prostanoid EP3 agonist need additional study. On the other hand, p21 has been shown inactivates cdc2-cyclinB complex (Chan et al., 2000). Our results also shown the enhancement of p21 levels in GR 63799X-treated cells. We must consider that the increase in p21 levels may inactive the cdccyclin B complex causing growth arrest. In this way, a growth arrest in S phase by the impairment of cyclin B and the enhancement of p21 levels have been recently reported by McGrath-Morrow and Stahl (2001) in A549 cells during hyperoxic stress. In most cells cAMP serves to inhibit cell growth. However, to confound this, in certain cell types, it can stimulate cell growth. Over the years it has proven extremely problematic to try and resolve the role of cAMP in regulating cell growth. Previous studies have suggested that intracellular levels of cAMP are involved in the role of prostanoid EP4 receptor on 3T6 fibroblast growth (Sánchez and Moreno, 2002). Furthermore, Fabre et al. (2001) and Ma et al. (2001) also demonstrated that activation of the murine EP3 receptor for PGE2 inhibits cAMP levels. Our results showed that prostanoid EP3 receptor agonist mediates the decrease in cAMP by a pertussis toxinsensitive and neomycin insensitive pathway which suggested that a G protein of the Gi family (Simon et al., 1991) may be involved in this action of PGE2 EP3 receptor. Interestingly, this impairment of cAMP induced by GR 63799X might be associated with the changes of cyclins and p21 content and with the S phase arrest and the inhibition of [3H]thymidine incorporation by 3T6 fibroblast. Effects that were reverted when forskolin treatment recovered cAMP levels. Thus, prostanoid EP3 receptors may be involved in 3T6 fibroblast growth through the control of cAMP levels. However, we must consider that PGE2 signals through a novel cAMP response element binding protein/CRE pathway, which appears to be independent of cAMP generation (Audoly et al., 1999). Moreover, we must consider that an EP3 receptor is involved in the activation of Ras signal pathway (Yano et al., 2002). Furthermore, the translocation of the NFκB (Meyer-Kirchrath et al., 1998), and a cross talk between extracellular signal-regulated kinase and cAMP signaling appears to be physiologically relevant to drive the proliferation of some cell types (Houslay and Kolch, 2000). Events that could be also involved in the role of PGE2 EP3 receptor on the regulation of cell growth. In summary, we provide evidence that GR 63799X, an PGE2 EP3 receptor agonist, inhibited serum-induced 3T6 fibroblast
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cell cycle progression and 3T6 fibroblast growth. This prostanoid EP3 receptor agonist appears to affect cAMP and cyclin B and p21 levels. These events may be implicated in the cell cycle arrest in S phase induced by GR 63799X in 3T6 fibroblast cultures. Acknowledgments T. Sánchez was a recipient of a pre-doctoral fellowship from the Autonomous Government of Catalonia. FACS analyses were performed at the Serveis Científic-Tècnics of the Barcelona University. GR 63799X was kindly provided by Dr. S.G. Lister from the Compound Supplies Officer, Glaxo Wellcome, Medicines Research Centre, Stevenage, UK. We thank Robin Rycroft for valuable assistance in the preparation of the English manuscript. This study was supported by funding from the Spanish Ministry of Education (PM98-0191), the Spanish Ministry of Science and Technology (BFI2001-3397) and the Autonomous Government of Catalonia (1999SGR00266). References Audoly, L.P., Ma, L., Feoktistov, I., De Foe, S., Breyer, M.D., Breyer, R.M., 1999. Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element mediated gene transcription. J. Pharmacol. Exp. Ther. 289, 140–148. Bishop-Bailey, D., Calatayud, S., Warner, J.D., Hla, T., Mitchell, J.A., 2002. Prostaglandin and the regulation of tumor growth. J. Environ. Pathol. Toxicol. Oncol. 21, 93–101. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brock, T.G., McNish, R.W., Peters-Golden, M., 1999. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J. Biol. Chem. 174, 11660–11666. Bunce, K.T., Clayton, N.M., Coleman, R.A., Collington, E.W., Finch, H., Humphray, J.M., Humphrey, P.P.A., Reeves, J.J., Sheldrick, R.L.G., Stables, R., 1991. GR 63799X a novel prostanoid with selectivety for EP3 receptors. Adv. Prostaglandin Thromboxane Leukotrienes Res. 21A, 379–382. Chan, T.A., Hwang, P.M., Hermeking, H., Kinzler, K.W., Vogelstein, B., 2000. Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev. 14, 1584–1588. Coleman, R.A., Smith, W.L., Narumiya, S., International Union of Pharmacology, 1994. Classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol. Rev. 46, 205–221. Dubois, R.N., Abramson, S.B., Crofford, L., Gupta, R.A., Simon, L.S., Van De Putte, L.B., Lipsky, P.E., 1998. Cycloxygenase in biology and disease. FASEB J. 12, 1063–1073. De Gregori, J., Kowalik, J., Nevins, J.R., 1995. Cellular targets for activation by the E2F1 transcription factor induce DNA synthesis- and G1/S-regulatory genes. Mol. Cell. Biol. 15, 4215–4224. Fabre, J.E., Nguyen, M., Athirakul, K., Coggins, K., McNeish, J.D., Austin, S., Parise, L.K., Fitzgerald, G.A., Coffman, T.M., Koller, B.H., 2001. Activation of the murine EP3 receptor for PGF2 inhibits cAMP production and promotes platelet aggregation. J. Clin. Invest. 107, 603–610. Hatae, N., Sugimoto, Y., Ichikawa, A., 2002. Prostaglandin receptors: advances in the study of EP3 receptor signaling. J. Biochem. 131, 781–784. Houslay, M.D., Kolch, W., 2000. Cell-type specific integration of cross-talk between extracellular signal-regulated kinase and cAMP signaling. Mol. Pharmacol. 58, 659–668.
Konger, R.L., Malaviya, R., Pentland, A.P., 1998. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim. Biophys. Acta 140, 221–234. Kiriyama, M., Ushikubi, F., Kobayashi, T., Hirata, M., Sugimoto, Narumiya, S., 1997. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptor expressed in Chinese hamster ovary cells. Br. J. Pharmacol. 122, 217–224. Laemmli, K.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lloret, S., Torrent, M., Moreno, J.J., 1996. Proliferation-dependent changes in arachidonic acid mobilization from phospholipids of 3T6 fibroblasts. Pflügers Arch. Eur. J. Physiol. 432, 655–662. Lukas, C., Sorensen, C.S., Kramer, E., Santoni-Rugiu, E., Lindeneg, C., Peters, J.M., Bartek, J., Lukas, J., 1999. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401, 815–818. Ma, H., Hara, A., Xiao, C.Y., Okada, Y., Takahata, O., Nakaya, K., Sugimoto, Y., Ichikawa, A., Narumiya, S., Ushikushi, F., 2001. Increased bleeding tendency and decreased susceptibility to thromboenbolism in mice lacking to prostaglandin E receptor subtype EP(3). Circulation 104, 1176–1180. Martínez, J., Sánchez, T., Moreno, J.J., 1997. Role of prostaglandin H synthase2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth. Am. J. Physiol., Cell Physiol. 273, C1466–C1471. McGrath-Morrow, S.A., Stahl, J., 2001. Growth arrest in A549 cells during hyperoxic stress is associated with decreased cyclin B1 and increased p21waf1/Cip1/Sdi1 levels. Biochim. Biophys. Acta 1538, 90–97. Meyer-Kirchrath, J., Kuger, P., Hohlfeld, T., Schror, K., 1998. Analysis of a porcine EP3-receptor: cloning, expression and signal transduction. NaunynSchmiedeberg's Arch. Pharmacol. 358, 160–167. Moreno, J.J., 1997. Regulation of arachidonic acid release and prostaglandin formation by cell–cell adhesive interactions in wound repair. Pflügers Arch. Eur. J. Physiol. 433, 351–356. Mutoh, M., Watanabe, K., Kitamura, T., Shogi, Y., Takahashi, M., Kawamori, T., Tani, K., Kobayashi, M., Maruyama, T., Kobayashi, K., Ohuchida, S., Sugimoto, Y., Narumiya, S., Sugimura, T., Wakabayashi, K., 2002. Involvement of prostaglandin E receptor subtype EP4 in colon carcinogenesis. Cancer Res. 62, 28–32. Narumiya, S., 1994. Prostanoid receptors. Structure, function, and distribution. Ann. N.Y. Acad. Sci. 744, 126–138. Narumiya, S., Sugimoto, Y., Ushikubi, F., 1999. Prostanoid receptors: structures, properties and functions. Physiol. Rev. 79, 1193–1226. Noguchi, K., Shitashige, M., Endo, H., Kondo, H., Ishikawa, I., 2002. Binary regulation of interleukin (IL-)-6 production by EP1 and EP2/EP4 subtypes of PGE2 receptors in IL-1 beta-stimulated human gingival fibroblasts. J. Periodontal Res. 37, 29–36. Otto, A.M., Nilsen-Hamilton, M., Boss, B.D., Ulrich, M.O., Jimenez de Asua, L., 1982. Prostaglandins E1 and E2 interact with prostaglandin F2 alpha to regulate interaction of DNA replication and cell division in Swiss 3T3 cells. Proc. Natl. Acad. Sci. U. S. A. 79, 4992–4996. Pines, J., 1995. Cyclins and cyclin-dependent kinases: a biochemical view. Biochem. J. 308, 697–711. Sánchez, T., Moreno, J.J., 2001. The effect of high molecular phospholipase A2 inhibitors on 3T6 fibroblast proliferation. Biochem. Pharmacol. 61, 811–816. Sánchez, T., Moreno, J.J., 2002. Role of EP1 and EP4 prostaglandin E2 subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation. Am. J. Physiol., Cell Physiol. 282, C280–C288. Seamon, K.B., Daly, J.W., 1986. Forskolin: its biological and chemical properties. Adv. Cycl. Nucleotide Protein Phosphoryl. Res. 20, 1–150. Sherr, C.J., 1996. Cancer cell cycles. Science 274, 1672–1677. Simon, M.I., Strathmann, M.P., Gautam, N., 1991. Diversity of G proteins in signal transduction. Science 152, 802–808. Smith, W.L., 1992. Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol., Renal Fluid Electrolyte Physiol. 263, F181–F191. Watanabe, T., Satoh, H., Togoh, M., Taniguchi, S., Hashimoto, Y., Kurokawa, K., 1996. Positive and negative regulation of cell proliferation through prostaglandin receptors in NIH-3T3 cells. J. Cell. Physiol. 169, 401–409.
T. Sanchez, J.J. Moreno / European Journal of Pharmacology 529 (2006) 16–23 Watanabe, K., Kawamori, T., Nakatsugi, S., Ohta, T., Ohuchida, S., Yamamoto, H., Maruyama, T., Kondo, K., Ushikubi, F., Narumiya, S., Sugimura, T., Wakabayashi, K., 1999. Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res. 59, 5093–5096. Yano, T., Zissel, G., Muller-Qernheim, J., Jae Shin, S., Satoh, H., Ichikawa, T., 2002. Prostaglandin E2 reinforces the activation of ras signal pathway in lung adenocarcinoma cells via EP3. FEBS Lett. 518, 154–158. Yoshida, T., Sakamoto, H., Horiuchi, T., Yamamoto, S., Suematsu, A., Oda, H., Koshihara, Y., 2001. Involvement of prostaglandin E2 in interleukin-1α-
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induced parathyroid hormone-related peptide production in synovial fibroblasts of patients with rheumatoid arthritis. J. Clin. Endocrinol. Metab. 86, 3272–3278. Yu, J., Prado, G.N., Schreiber, B., Polgar, P., Polgar, P., Taylor, L., 2002. Role of prostaglandin E2 EP receptor and cAMP in the expression of connective tissue growth factor. Arch. Biochem. Biophys. 404, 302–308. Zacharowski, K., Thiemermann, C., 1999. Pharmacology of the prostaglandin EP3 receptor agonist TEI-3356. Cardiovasc. Drug Rev. 17, 329–340.
GR 63799X, an EP3 receptor agonist, induced S phase arrest and 3T6 fibroblast growth inhibition Teresa Sanchez, Juan J. Moreno ⁎ Department of Physiology, Faculty of Pharmacy, Barcelona University, Avda. Joan XXIII s/n, E-08028 Barcelona, Spain Received 14 July 2005; received in revised form 18 October 2005; accepted 25 October 2005 Available online 28 November 2005
Abstract The importance of arachidonic acid metabolites on the control of cell growth, particularly those derived from cyclooxygenase pathway has long been recognized. Recently, we observed that prostaglandin E2 (PGE2) interaction with EP1 and EP4 receptors is involved in serum-induced 3T6 fibroblast growth due to their effect at various levels of the cell cycle machinery. This study shows that prostanoid EP3 receptor was expressed in 3T6 fibroblast. We studied the role of EP3 receptor agonist GR 63799X in serum-induced 3T6 cell proliferation. This was concentrationdependent inhibit (IC50 ∼10 μM) to a complete inhibition without any cytotoxic or proapoptotic effect. The prostanoid EP3 receptor agonist treatment decreased the G0/G1 and G2/M populations whereas cells were accumulated in S phase. This arrest in S phase was associated with a decrease in cyclin B levels and the enhancement of p21 expression. Our data show that EP3 agonist decreases cAMP levels in our experimental conditions. Interestingly, the S arrest caused by prostanoid EP3 receptor agonist seems to be cAMP dependent, at least in part, because forskolin treatment allowed S-arrested cells to progress through cell cycle and consequently growth. Thus, our results suggest that PGE2 EP3 receptor interaction may be involved in serum-induced 3T6 fibroblast growth due to their effects on cAMP levels and on cell cycle machinery of the S phase. © 2005 Elsevier B.V. All rights reserved. Keywords: Prostaglandin E2; Cell proliferation; Cell cycle; Cyclin; cAMP
1. Introduction Prostaglandins are potent lipid mediators generated from arachidonic acid by the cyclooxygenase isozymes. Prostaglandins are quickly released from cells after synthesis and act as local hormones in the vicinity of their production site to maintain local homeostasis. Thus, these mediators have numerous physiological effects (Smith, 1992), including cell proliferation and differentiation. PGE2 induces DNA synthesis in Swiss 3T3 fibroblasts (Otto et al., 1982). Interestingly, enhanced synthesis of PGEs was observed in a wide variety of tumors (Dubois et al., 1998; Bishop-Bailey et al., 2002). Previous studies have shown that PGE2 is the predominant prostaglandin synthesized and secreted in response to cyclooxygenase-2 up-regulation (Brock et al., 1999). We have suggested elsewhere that PGE2, the major cyclooxygenase pathway metabolite produced by ⁎ Corresponding author. Tel.: +34 93 4024505; fax: +34 93 4035001. E-mail address: [email protected] (J.J. Moreno). 0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2005.10.040
fibroblasts, controls 3T6 fibroblast growth (Lloret et al., 1996) and that cyclooxygenase-2 is the main isoform responsible for serum-induced PGE2 synthesis in these cells (Martínez et al., 1997). The ability of each prostaglandin to affect various biological responses is dependent on its binding to specific receptors on the plasma membrane which belong to the rhodopsin family of serpentin receptors. The four subtypes of PGE receptors (EP1– EP4) from various species, previously defined pharmacologically, have been cloned (see Narumiya, 1994, for review). They are encoded by a distinct gene and differ in their amino acid identities, pharmacological characteristics, and signal transduction properties. The prostanoid EP2, EP4 and one isoform of the EP3 receptor can couple to Gs and thus increase intracellular cAMP concentration, whereas other isoforms of EP3 receptor can couple to Gi, causing a decrease in the cAMP levels (Narumiya et al., 1999). Therefore it is likely that physiological consequences of activation of the prostanoid EP3 receptor may vary substantially depending on the cell type examined.
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Several studies have suggested the role of PGE2 receptors in cellular proliferation and tumor development. Experiments on EP1-deficient mice show that this subtype receptor is required for the development of preneoplastic lesions induced in colon epithelium by the carcinogen azoxymethane (Watanabe et al., 1999). The same authors have proposed a chemopreventive effect of EP1 or EP4 selective antagonist on colon cancer development (Mutoh et al., 2002). Moreover, we recently reported that prostanoid EP1 and EP4 receptors are involved in serum-induced 3T6 fibroblast growth, suggesting that PGE2 interaction with both receptors regulates progression through the cell cycle by modulating cyclin D and E and cyclin A levels, respectively (Sánchez and Moreno, 2002). There are contradictory results about prostanoid EP3 receptor presence in fibroblasts. Thus, Watanabe et al. (1996), Noguchi et al. (2002) and Yu et al. (2002) reported that EP1, EP2 and/ or EP4 are expressed in fibroblast but not EP3, whereas Yoshida et al. (2001) observed the expression of EP3 together EP2 and EP4 in fibroblasts. Our results agree with the last authors. We observed that the growth-inhibitory effect of GR 63799X, a selective prostanoid EP3 receptor agonist (Bunce et al., 1991), on serum-stimulated cellular proliferation involve the impairment of cAMP levels, reducing cyclin B levels whereas increasing p21 expression, and induced an S-phase arrest in 3T6 cells. 2. Material and methods 2.1. Reagents
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(100 μg/ml), in a humidified atmosphere of 95% air–5% CO2 at 37 °C. Cells were harvested with trypsin/EDTA and passed to tissue culture 100 or 60 mm dishes (Costar, Cambrigde, MA) for experimental purposes. 2.3. Cell growth The effect of prostanoid EP3 receptor agonist was assessed on 3T6 fibroblasts plated at 104 cells/well in 12-well plates (Costar, Cambrigde, MA) and cultured for 24 h in RPMI supplemented with 10% FCS. Cells were incubated for 2 days in 10% FCS medium in the presence of the different concentration of GR 63799X. At the end of the experiments, cells were washed, trypsinized and counted, using ethidium bromide/acridine orange staining in order to assess viability. To study cell recovery, after a 24 h treatment with prostanoid EP3 agonist, 3T6 fibroblast cultures were washed with phosphate-buffered saline solution (PBS) and fresh 10% FCS medium was added. Growth recovery was assessed after 1, 2 or 3 days, by counting trypsinized cells, as mentioned above. 2.4. [3H]Thymidine incorporation assay DNA synthesis was measured as previously described (Martínez et al., 1997). Briefly, fibroblasts were cultured in 96-well plates at a density of 400 cells/well. 24 h later, cells were incubated with the treatments and [3H]thymidine (1 μCi/ well) for 24 h. [3H]Thymidine-containing media were aspirated, cells were overlaid with 1% Triton X-100, and then cells were scraped off the dishes and the radioactivity present in the cell fraction was measured by liquid scintillation counting.
RPMI 1640, fetal calf serum (FCS), penicilin G, streptomycin and trypsin/EDTA were from Bio Whittaker Europe (Verviers, Belgium). [methyl-3H]thymidine (20 Ci/mmol) was obtained from Du Pont-New England Nuclear (Boston, MA). GR 63799X [1R-[1α (2), 2β (R*), 3α]]-4-(benzoylamino) phenyl 7-[3-hydroxy-2-(2-hydroxy-3-phenoxypropoxy)-5-oxocyclopentyl]-4-heptenoate was kindly provided by Glaxo Welcome (Stevenage Hertfordshire, UK). Aprotinin, leupeptin, diethyldithiocarbamic acid, phenylmethylsulfonyl fluoride (PMSF), sodium fluoride, sodium orthovanadate (Na3VO4), Igepal CA-630, dithiothreitol (DTT), pertussis toxin, neomycin, forskolin, propidium iodide (PI), Hoescht 33258, ethidium bromide, acridine orange and DNase-free ribonuclease A were acquired from Sigma Chemical (St. Louis, MO). Rabbit polyclonal antibodies against cyclins D, E, A, B and p21 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). EP3 and EP4 receptor peptides and rabbit polyclonal antibodies against EP3 and EP4 receptor were from Cayman Chemical Co. (Ann Arbor, MI). Drug was dissolved in dimethylsulfoxide and final concentration of dimethylsulfoxide never exceeded 0.1%.
Cells were seeded and 24 h later, they were washed with PBS and serum starved. After 24 h serum starvation, the percentage of cells in G0/G1 was about 80%. Cells were then cultured in 10% FCS RPMI containing the treatments. After 30 h, cells were trypsinized, fixed with 70% ethanol and stored at 4 °C for at least 24 h. Thapsigargin and forskolin were added to the GR 63799 X-containing culture medium after a 30 h incubation, and 6 h later, cells were harvested and fixed. They were then stained for 1 h at room temperature with a 20 μg/ml propidium iodide solution in PBS containing 0.1% Triton X-100 (Sigma) and 0.2 mg/ml DNase-free RNase A. Cells were analyzed on a Epics XL flow cytometer (Coulter Corporation, Hialeah, Florida). DNA was analyzed (Ploidy analysis) on single fluorescence histograms by Multicycle software (Phoenix Flow Systems, San Diego, CA).
2.2. Cell culture
2.6. Western blot analysis
Murine 3T6 fibroblasts (ATCCC, CL96) were grown as we described elsewhere (Martínez et al., 1997), in RPMI 1640 containing 10% FCS, penicillin (100 U/ml) and streptomycin
3T6 fibroblast cultures were washed twice with ice-cold PBS. Total cellular fraction was obtained by scraping off the cells in lysis buffer containing 200 mM Tris–HCl, 200 mM
2.5. Fluorescent-activated cell sorting (FACS) analysis/flowcytometry cell cycle analysis
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T. Sanchez, J.J. Moreno / European Journal of Pharmacology 529 (2006) 16–23 Table 1 Effect of prostanoid EP3 receptor agonist on [3H]thymidine incorporationa [3H]thymidine incorporation (Dpm × 1000) Control FCS FCS + GR FCS + GR FCS + GR FCS + GR
15 ± 2 183 ± 9 63779X (5 μM) 123 ± 2b 63799X (10 μM) 85 ± 6b 63799X (50 μM) 42 ± 3b 63799X (10 μM) + forskolin (10 μM) 129 ± 8c
a
Fig. 1. The expression of prostanoid EP3 or EP4 receptors in 3T6 fibroblast cultured in 10% FCS was determined by Western blot. EP3 or EP4 receptor peptides (10 μg) were used as standards and total 3T6 fibroblast lysate protein (20 μg) was loaded. Present blot is representative of 3 blots.
NaCl, 2% Igepal CA-630, 400 μM NaF, 200 μM DTT and 400 μM Na3VO4, followed by incubation for 30 min at 4 °C. Total protein was measured by the Bradford method (1976) using the Bio-Rad protein assay, with BSA as standard. Immuno blot
Fig. 2. Effect of prostanoid EP3 receptor agonist on 10% FCS-induced 3T6 fibroblast proliferation. 3T6 fibroblasts (104 cells/well) were plated and cultured in 10% FCS-RPMI. The next day, media were removed and fresh 10% FCSRPMI containing the treatments was added. At the end of experiments, cells were trypsinized and counted. (A) Concentration response effect of the GR 63799X after 48 h incubation. (B) time course of growth cell in presence of GR 63799X 10 μM (triangle), GR 63799X 50 μM (squares) or absence of EP3 receptor agonist (circles). Results are means ± S.E.M. of 3 experiments performed in duplicate. *P b 0.05 vs. control cells.
3T6 fibroblasts were cultured overnight without FCS. The next day, media were removed and fresh 10% FCS-RPMI containing the treatments were added together [3H]thymidine (1 μCi/well). After 24 h, [3H]thymidine incorporation to cells was measured. Results are means ± S.E.M. of three experiments performed in duplicate. b P b 0.05 vs. FCS; cP b 0.05 vs. cells treated with GR 63799X (10 μM).
analysis for cyclins were performed as follows: 20 μg of protein from cell lysates were separated by a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gel (Laemmli, 1970) and blotted for 1 h with a constant voltage of 100 V onto a polyvinylidene difluoride membrane (Immun-Blot PVDF membrane, 0.2 m, Bio-Rad) using a MiniProtean II system (Bio-Rad, Hercules, CA). A pre-stained SDS–PAGE protein standard (Bio-Rad) was used to check transfer efficiency and as a molecular weight marker. Membranes were blocked with 5% nonfat milk powder in 0.1%Tween 20 PBS for 1 h.
Fig. 3. Effect of prostanoid EP3 receptor agonist on cell cycle distribution. Synchronized 3T6 fibroblasts were incubated with 10% FCS-RPMI in presence of GR 63799X. Cells were then fixed, stained and analyzed. White bars represent the percentage of cells in G0/G1 phase; vertical striped bars in S phase and horizontal striped bars in G2/M phase. (A) Concentration–response study was performed in presence the GR 63799X for 30 h. (B) Time course of cell cycle distribution in presence of GR 63799X (10 μM). Results are means ± S.E.M. of 3 experiments performed in duplicate. *P b 0.05 vs. Control cells or vs. GR 63799X-treated cells.
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Rabbit polyclonal antibody against cyclins D, E, A, B1 and p21 or EP3 and EP4 receptor were applied in a dilution 1 : 200 for 1 h. The blots were washed several times with PBS-0.1% Tween 20 and were incubated with a goat anti-rabbit antibody in a 1 : 2000 dilution for 1 h. For β-actin immunoblotting, stripped membranes were overlaid with monoclonal anti-actin antibody (1.200) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by rabbit anti-mouse antibody (1 : 1000) (Santa Cruz). All blots were developed using an enhanced chemiluminiscence kit (Supersignal West Dura Extended Duration Substrate), from Pierce (Rockford, IL), using a Bio Max Light-2 film. 2.7. cAMP levels Intracellular cAMP from cell cultures was extracted with ethanol and measured as by suggested the manufacturer of the enzyme immunoassay kit (Amersham Life Science, Buckinghamshire, UK). 2.8. Statistical analysis Results are expressed as mean ± standard error of the mean. Differences between non-treated and treated cells were tested
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by using Student's t-test following by the least significant difference test as appropriate. 3. Results 3.1. Effect of GR 63799X on 3T6 fibroblast proliferation Initially we sought to determine the presence of prostanoid EP3 and EP4 receptor in 3T6 fibroblasts cultured in presence of FCS (Fig. 1). To demonstrate the functionality of prostanoid EP3 receptors, we performed cell proliferation assays in the presence of a selective EP3 receptor agonist, GR 63799X. EP3 receptor agonist caused a significant concentration-dependent decrease in cell growth, with a half inhibition of proliferation at 10 μM approximately and a cell growth blocked at 50 μM (Fig. 2). This action of prostanoid EP3 receptor agonist may be correlated with the inhibition of DNA synthesis measured as [3H] thymidine incorporation (Table 1). GR 63799X did not cause cytotoxicity as high as 50 μM, as was assessed by observing morphological appearance of the cells by light microscopy and ethidium bromide/acridine orange staining. Thus, the effect of EP3 agonist is due to growth inhibition and not to cytotoxic effect. For this reason, one the treatment was removed from the
Fig. 4. Fluorescence-activated cell sorting analysis of the cell cycle. 3T6 fibroblast cultures synchronized by 24 h serum starvation (SS) were incubated with 10% FCS-RPMI (control) or 10% FCS-RPMI containing GR 63799X for 30 h. Forskolin (10 μM) was added 6 h before the end of assay. To determine the reversion of GR 63799X effect, the EP3 agonist was washed 12 h before the end of experiment. Then, cells were trypsinized, fixed, stained and analyzed on a flow cytometer. Representative fluorescence histograms representing cell cycle analyses of three experiments performed in duplicate are shown. *P b 0.05 vs. control cells; **P b 0.05 vs. GR 63799X-treated cells.
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suggesting that the antiproliferative action of prostanoid EP3 receptor agonist did not result from apoptosis. 3.3. Characterization of cell cycle regulator proteins involved in GR 63799X-induced S phase arrest
Fig. 5. Effect of prostanoid EP3 agonist on cyclin D, E, A, B and p21 expression in 3T6 fibroblasts. Cells were synchronized by serum starvation for 24 h. Then, 10% FCS medium (control) or 10% FCS-RPMI containing GR 63799X was added. Forskolin (10 μM) was added 6 h before the end of assay. Cells were harvested 6 (for cyclins D and E), 16 (for cyclin A) or 18 h (for cyclin B and p21) later to carry out Western blot analysis. Total cellular lysate protein (20 μg/ lane) was loaded, separated and immunodetected as described in Material and methods. Western blots representative of 3 blots are shown.
culture medium, 3T6 fibroblast markedly recovered the growth (data not shown). 3.2. Effect of prostanoid EP3 agonist on cell cycle distribution To gain first insight into the mechanisms of prostanoid EP3 receptor agonist-induced growth inhibition, we next analyzed cell-cycle distribution in response to EP3 receptor agonist by flow cytometry. 3T6 fibroblast were synchronized in G0/G1 to allow a more distinct representation of their progression through the individual cell cycle phases. Sinchronization was achieved by overnight serum starvation, which routinely retained approximately 85% to 90% of cells in the G0/G1 phase. 3T6 fibroblasts were then stimulated to re-enter the cell cycle by addition of 10% FCS medium, and cell cycle progression was monitored for up 30 h, a period of time that corresponds to approximately two cycles, in the presence or absence of EP3 receptor agonist. GR 63779X induced a significant and concentration-dependent increase in S population and a decrease on G2/M population and in G1 population (Fig. 3A). The accumulation of cells in S-phase and the impairment of G1 and G2 + M progressed markedly when EP3 agonist treatment was prolonged (Fig. 3B). This S-phase arrest was reverted when GR 63799X was removed from the culture medium (Fig. 4). Moreover, no significant increase of cells with subdiploid DNA-content was noted in EP3 agonist-treated cultures,
Progression of the mammalian cell cycle is governed by cyclins, Cdks and CKIs (Pines, 1995). Cdks form a binary system composed of the inactive catalytic subunit cdk, which is activated by binding to cyclin. We aimed to determine whether the impaired progression through the cycle of EP3 agonist-treated cells was linked to changes in cyclin levels. As shown in Fig. 5, Western blot analyses of total protein lysates showed the impairment of cyclin B content. Interestingly this decrease of cyclin B levels can be related with the enhancement of p21 levels in GR 63799X-treated 3T6 cells (Fig. 5). Previous studies have suggested that prostanoid EP3 receptors could be mediated PGE2-induced changes of cAMP levels (Hatae et al., 2002). To test whether GR 63779X affects to this second messenger, we measured the changes of cAMP in response to EP3 receptor agonist. FCS and PGE2, at micromolar concentrations reached by FCS incubation (Moreno, 1997), increase cAMP levels in 3T6 fibroblat cultures. In contrast, GR 63799X induced a significant impairment of cAMP levels in FCS-cultured fibroblasts (Table 2). Moreover, this impairment of cAMP induced by GR 63799X was reduced by pertussis toxin treatment. Results that suggested that these changes of cAMP could be involved in the cell arrest in S-phase induced by GR 63799X treatment. Some experiments were performed to test this hypothesis. We used forskolin, an adenylate cyclase activator (Seamon and Daly, 1986) to revert the impairment of cAMP levels induced by the prostanoid EP3 receptor agonist. As shown in Table 2, forskolin corrected GR 63799X-treated cell cAMP levels. Interestingly, this effect of forskolin on cAMP levels of 3T6 fibroblast cultures can be correlated with the decrease of cyclin B levels and the enhancement of Table 2 Effect of prostanoid EP3 receptor agonist on cAMP levelsa cAMP (pmol/mg protein) Control FCS FCS + forskolin PGE2 GR 63799X FCS + GR 63799X FCS + GR 63799 X + pertussis toxin FCS + GR 63799X + neomycin FCS + GR 63799X + forskolin a
13 ± 2 55 ± 3b 168 ± 10 52 ± 3b 14 ± 1 21 ± 2c 41 ± 2d 22 ± 3 58 ± 3e
3T6 fibroblast cultures were pre-incubated with pertussis toxin (10 ng/ml) for 3 h or with neomycin (10 μM) for 30 min. Then, cells were stimulated with FCS (10%) or PGE2 (10 μM). In some experiments, cells were co-incubated with GR 63799X (10 μM). GR 63799X and forskolin (10 μM) were incubated together. cAMP levels were measured 1 h after cell cultures were stimulated. Measurements were performed in triplicate and expressed as the mean ± S.E.M. of three experiments. b P b 0.05 vs. control; cP b 0.05 vs. FCS; dP b 0.05 vs. GR 63799X; P b 0.05 vs. FCS plus GR 63799X.
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p21 content (Fig. 5), with a progression through the cell cycle (Fig. 4) and with a recovery of [3H]thymidine incorporation (Table 1). 4. Discussion Calcium-independent cytosolic phospholipase A2 is involved in serum-induced 3T6 fibroblast proliferation (Sánchez and Moreno, 2001), through arachidonic acid release and the subsequent metabolism mainly by the inducible form of cyclooxygenase, cyclooxygenase-2, to synthesize prostaglandins like PGE2 (Martínez et al., 1997). The biological actions of PGE2 have been attributed to results from its interaction with cell surface prostanoid EP receptors (Coleman et al., 1994). PGE2 has versatile and opposing actions due to multiple EP receptor subtypes and the coupling of EP receptor isoforms to a variety of signal transduction pathways. Recently, we provide evidence that PGE2 interaction with EP1 and EP4 receptors are involved in the control of 3T6 fibroblast proliferation which is associated with changes in D, E and A cyclin levels (Sánchez and Moreno, 2002). Thus, the generation and PGE2 interaction with these receptors, in an autocrine or paracrine fashion, may be acted as a necessary comitogenic signal. Here, we demonstrate that prostanoid EP3 receptors are expressed in FCS-cultured 3T6 fibroblasts. To elucidate the functional role of PGE2 EP3 receptors on the control of serum-induced 3T6 fibroblat proliferation and on progression through the cell cycle, we used GR 63799X which has affinity only to the EP3 receptor, indicating its high selectivity for this receptor (Kiriyama et al., 1997). This EP3 receptor agonist reduced FCS-induced 3T6 fibroblast growth in a time- and concentration-dependent manner, and blocked cellular proliferation, without showing cytotoxicity or proapoptotic effects at the concentrations and for the incubation times used. In the present study, we also provide evidence that prostanoid EP3 receptor agonist affect DNA synthesis. Thus, GR 63799X caused the impairment of FCS-induced [3H]thymidine incorporation to cells. In this way, Konger et al. (1998) observed that an EP3 receptor agonist inhibits bromodeoxyuridine uptake in human keratinocytes and Zacharowski and Thiemermann (1999) reported that TEI-3356, an EP3 agonist, inhibits endothelin-induced DNA synthesis in cultured vascular smooth muscle cells. GR 63799X also modified the cell cycle distribution in FCScultured 3T6 fibroblasts. Our results suggest a S phase arrest induced by the interaction of GR 63799X with the PGE2 EP3 receptor in a similar form to the cell cycle distribution changes induced by an PGE2 EP4 receptor antagonist (Sánchez and Moreno, 2002). However, a prostanoid EP4 antagonist induced the impairment of cyclin A levels and an early S phase arrest whereas a prostanoid EP3 agonist induced a delay in the S phase exit into G2/M. The hypothesis that PGE2 EP3 receptor could be regulated the cell cycle progression also was supported by the effects of GR 63799X on cyclin levels. Thus, the S phase arrest induced by GR 63799X could be linked to a significant decrease in cyclin B levels. To the best of our knowledge, this is the first report demonstrating the participa-
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tion of prostanoid EP3 receptor activation in the control of cyclin content. Furthermore, these observations corroborate results from the cell cycle distribution studies indicating a regulatory effect at the S-G2/M phases where cyclin B plays an important regulatory role. Thus, cyclin B is essential for the initiation of mitosis with protein accumulation of cyclin B occurring during S phase to function as a regulatory subunit in cdc2 kinase complexes as cells progress from S into G2/M (Sherr, 1996). The cellular cyclin B content depends on cdk2/ E2F mediated induction (De Gregori et al., 1995), and the ubiquination and proteolysis through the interaction with anaphase-promoting complex (Lukas et al., 1999). The mechanism involved in marked decrease in cyclin B levels in 3T6 cells cultured with prostanoid EP3 agonist need additional study. On the other hand, p21 has been shown inactivates cdc2-cyclinB complex (Chan et al., 2000). Our results also shown the enhancement of p21 levels in GR 63799X-treated cells. We must consider that the increase in p21 levels may inactive the cdccyclin B complex causing growth arrest. In this way, a growth arrest in S phase by the impairment of cyclin B and the enhancement of p21 levels have been recently reported by McGrath-Morrow and Stahl (2001) in A549 cells during hyperoxic stress. In most cells cAMP serves to inhibit cell growth. However, to confound this, in certain cell types, it can stimulate cell growth. Over the years it has proven extremely problematic to try and resolve the role of cAMP in regulating cell growth. Previous studies have suggested that intracellular levels of cAMP are involved in the role of prostanoid EP4 receptor on 3T6 fibroblast growth (Sánchez and Moreno, 2002). Furthermore, Fabre et al. (2001) and Ma et al. (2001) also demonstrated that activation of the murine EP3 receptor for PGE2 inhibits cAMP levels. Our results showed that prostanoid EP3 receptor agonist mediates the decrease in cAMP by a pertussis toxinsensitive and neomycin insensitive pathway which suggested that a G protein of the Gi family (Simon et al., 1991) may be involved in this action of PGE2 EP3 receptor. Interestingly, this impairment of cAMP induced by GR 63799X might be associated with the changes of cyclins and p21 content and with the S phase arrest and the inhibition of [3H]thymidine incorporation by 3T6 fibroblast. Effects that were reverted when forskolin treatment recovered cAMP levels. Thus, prostanoid EP3 receptors may be involved in 3T6 fibroblast growth through the control of cAMP levels. However, we must consider that PGE2 signals through a novel cAMP response element binding protein/CRE pathway, which appears to be independent of cAMP generation (Audoly et al., 1999). Moreover, we must consider that an EP3 receptor is involved in the activation of Ras signal pathway (Yano et al., 2002). Furthermore, the translocation of the NFκB (Meyer-Kirchrath et al., 1998), and a cross talk between extracellular signal-regulated kinase and cAMP signaling appears to be physiologically relevant to drive the proliferation of some cell types (Houslay and Kolch, 2000). Events that could be also involved in the role of PGE2 EP3 receptor on the regulation of cell growth. In summary, we provide evidence that GR 63799X, an PGE2 EP3 receptor agonist, inhibited serum-induced 3T6 fibroblast
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cell cycle progression and 3T6 fibroblast growth. This prostanoid EP3 receptor agonist appears to affect cAMP and cyclin B and p21 levels. These events may be implicated in the cell cycle arrest in S phase induced by GR 63799X in 3T6 fibroblast cultures. Acknowledgments T. Sánchez was a recipient of a pre-doctoral fellowship from the Autonomous Government of Catalonia. FACS analyses were performed at the Serveis Científic-Tècnics of the Barcelona University. GR 63799X was kindly provided by Dr. S.G. Lister from the Compound Supplies Officer, Glaxo Wellcome, Medicines Research Centre, Stevenage, UK. We thank Robin Rycroft for valuable assistance in the preparation of the English manuscript. This study was supported by funding from the Spanish Ministry of Education (PM98-0191), the Spanish Ministry of Science and Technology (BFI2001-3397) and the Autonomous Government of Catalonia (1999SGR00266). References Audoly, L.P., Ma, L., Feoktistov, I., De Foe, S., Breyer, M.D., Breyer, R.M., 1999. Prostaglandin E-prostanoid-3 receptor activation of cyclic AMP response element mediated gene transcription. J. Pharmacol. Exp. Ther. 289, 140–148. Bishop-Bailey, D., Calatayud, S., Warner, J.D., Hla, T., Mitchell, J.A., 2002. Prostaglandin and the regulation of tumor growth. J. Environ. Pathol. Toxicol. Oncol. 21, 93–101. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brock, T.G., McNish, R.W., Peters-Golden, M., 1999. Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2. J. Biol. Chem. 174, 11660–11666. Bunce, K.T., Clayton, N.M., Coleman, R.A., Collington, E.W., Finch, H., Humphray, J.M., Humphrey, P.P.A., Reeves, J.J., Sheldrick, R.L.G., Stables, R., 1991. GR 63799X a novel prostanoid with selectivety for EP3 receptors. Adv. Prostaglandin Thromboxane Leukotrienes Res. 21A, 379–382. Chan, T.A., Hwang, P.M., Hermeking, H., Kinzler, K.W., Vogelstein, B., 2000. Cooperative effects of genes controlling the G(2)/M checkpoint. Genes Dev. 14, 1584–1588. Coleman, R.A., Smith, W.L., Narumiya, S., International Union of Pharmacology, 1994. Classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol. Rev. 46, 205–221. Dubois, R.N., Abramson, S.B., Crofford, L., Gupta, R.A., Simon, L.S., Van De Putte, L.B., Lipsky, P.E., 1998. Cycloxygenase in biology and disease. FASEB J. 12, 1063–1073. De Gregori, J., Kowalik, J., Nevins, J.R., 1995. Cellular targets for activation by the E2F1 transcription factor induce DNA synthesis- and G1/S-regulatory genes. Mol. Cell. Biol. 15, 4215–4224. Fabre, J.E., Nguyen, M., Athirakul, K., Coggins, K., McNeish, J.D., Austin, S., Parise, L.K., Fitzgerald, G.A., Coffman, T.M., Koller, B.H., 2001. Activation of the murine EP3 receptor for PGF2 inhibits cAMP production and promotes platelet aggregation. J. Clin. Invest. 107, 603–610. Hatae, N., Sugimoto, Y., Ichikawa, A., 2002. Prostaglandin receptors: advances in the study of EP3 receptor signaling. J. Biochem. 131, 781–784. Houslay, M.D., Kolch, W., 2000. Cell-type specific integration of cross-talk between extracellular signal-regulated kinase and cAMP signaling. Mol. Pharmacol. 58, 659–668.
Konger, R.L., Malaviya, R., Pentland, A.P., 1998. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim. Biophys. Acta 140, 221–234. Kiriyama, M., Ushikubi, F., Kobayashi, T., Hirata, M., Sugimoto, Narumiya, S., 1997. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptor expressed in Chinese hamster ovary cells. Br. J. Pharmacol. 122, 217–224. Laemmli, K.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lloret, S., Torrent, M., Moreno, J.J., 1996. Proliferation-dependent changes in arachidonic acid mobilization from phospholipids of 3T6 fibroblasts. Pflügers Arch. Eur. J. Physiol. 432, 655–662. Lukas, C., Sorensen, C.S., Kramer, E., Santoni-Rugiu, E., Lindeneg, C., Peters, J.M., Bartek, J., Lukas, J., 1999. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401, 815–818. Ma, H., Hara, A., Xiao, C.Y., Okada, Y., Takahata, O., Nakaya, K., Sugimoto, Y., Ichikawa, A., Narumiya, S., Ushikushi, F., 2001. Increased bleeding tendency and decreased susceptibility to thromboenbolism in mice lacking to prostaglandin E receptor subtype EP(3). Circulation 104, 1176–1180. Martínez, J., Sánchez, T., Moreno, J.J., 1997. Role of prostaglandin H synthase2-mediated conversion of arachidonic acid in controlling 3T6 fibroblast growth. Am. J. Physiol., Cell Physiol. 273, C1466–C1471. McGrath-Morrow, S.A., Stahl, J., 2001. Growth arrest in A549 cells during hyperoxic stress is associated with decreased cyclin B1 and increased p21waf1/Cip1/Sdi1 levels. Biochim. Biophys. Acta 1538, 90–97. Meyer-Kirchrath, J., Kuger, P., Hohlfeld, T., Schror, K., 1998. Analysis of a porcine EP3-receptor: cloning, expression and signal transduction. NaunynSchmiedeberg's Arch. Pharmacol. 358, 160–167. Moreno, J.J., 1997. Regulation of arachidonic acid release and prostaglandin formation by cell–cell adhesive interactions in wound repair. Pflügers Arch. Eur. J. Physiol. 433, 351–356. Mutoh, M., Watanabe, K., Kitamura, T., Shogi, Y., Takahashi, M., Kawamori, T., Tani, K., Kobayashi, M., Maruyama, T., Kobayashi, K., Ohuchida, S., Sugimoto, Y., Narumiya, S., Sugimura, T., Wakabayashi, K., 2002. Involvement of prostaglandin E receptor subtype EP4 in colon carcinogenesis. Cancer Res. 62, 28–32. Narumiya, S., 1994. Prostanoid receptors. Structure, function, and distribution. Ann. N.Y. Acad. Sci. 744, 126–138. Narumiya, S., Sugimoto, Y., Ushikubi, F., 1999. Prostanoid receptors: structures, properties and functions. Physiol. Rev. 79, 1193–1226. Noguchi, K., Shitashige, M., Endo, H., Kondo, H., Ishikawa, I., 2002. Binary regulation of interleukin (IL-)-6 production by EP1 and EP2/EP4 subtypes of PGE2 receptors in IL-1 beta-stimulated human gingival fibroblasts. J. Periodontal Res. 37, 29–36. Otto, A.M., Nilsen-Hamilton, M., Boss, B.D., Ulrich, M.O., Jimenez de Asua, L., 1982. Prostaglandins E1 and E2 interact with prostaglandin F2 alpha to regulate interaction of DNA replication and cell division in Swiss 3T3 cells. Proc. Natl. Acad. Sci. U. S. A. 79, 4992–4996. Pines, J., 1995. Cyclins and cyclin-dependent kinases: a biochemical view. Biochem. J. 308, 697–711. Sánchez, T., Moreno, J.J., 2001. The effect of high molecular phospholipase A2 inhibitors on 3T6 fibroblast proliferation. Biochem. Pharmacol. 61, 811–816. Sánchez, T., Moreno, J.J., 2002. Role of EP1 and EP4 prostaglandin E2 subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation. Am. J. Physiol., Cell Physiol. 282, C280–C288. Seamon, K.B., Daly, J.W., 1986. Forskolin: its biological and chemical properties. Adv. Cycl. Nucleotide Protein Phosphoryl. Res. 20, 1–150. Sherr, C.J., 1996. Cancer cell cycles. Science 274, 1672–1677. Simon, M.I., Strathmann, M.P., Gautam, N., 1991. Diversity of G proteins in signal transduction. Science 152, 802–808. Smith, W.L., 1992. Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol., Renal Fluid Electrolyte Physiol. 263, F181–F191. Watanabe, T., Satoh, H., Togoh, M., Taniguchi, S., Hashimoto, Y., Kurokawa, K., 1996. Positive and negative regulation of cell proliferation through prostaglandin receptors in NIH-3T3 cells. J. Cell. Physiol. 169, 401–409.
T. Sanchez, J.J. Moreno / European Journal of Pharmacology 529 (2006) 16–23 Watanabe, K., Kawamori, T., Nakatsugi, S., Ohta, T., Ohuchida, S., Yamamoto, H., Maruyama, T., Kondo, K., Ushikubi, F., Narumiya, S., Sugimura, T., Wakabayashi, K., 1999. Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res. 59, 5093–5096. Yano, T., Zissel, G., Muller-Qernheim, J., Jae Shin, S., Satoh, H., Ichikawa, T., 2002. Prostaglandin E2 reinforces the activation of ras signal pathway in lung adenocarcinoma cells via EP3. FEBS Lett. 518, 154–158. Yoshida, T., Sakamoto, H., Horiuchi, T., Yamamoto, S., Suematsu, A., Oda, H., Koshihara, Y., 2001. Involvement of prostaglandin E2 in interleukin-1α-
23
induced parathyroid hormone-related peptide production in synovial fibroblasts of patients with rheumatoid arthritis. J. Clin. Endocrinol. Metab. 86, 3272–3278. Yu, J., Prado, G.N., Schreiber, B., Polgar, P., Polgar, P., Taylor, L., 2002. Role of prostaglandin E2 EP receptor and cAMP in the expression of connective tissue growth factor. Arch. Biochem. Biophys. 404, 302–308. Zacharowski, K., Thiemermann, C., 1999. Pharmacology of the prostaglandin EP3 receptor agonist TEI-3356. Cardiovasc. Drug Rev. 17, 329–340.