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Biology of Reproduction 63, 933-941 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Expression of Cyclooxygenase 2 by Prostaglandin E2 in Human Endometrial Adenocarcinoma Cell Line HEC-1B1

Iqbal Munira,b, Kohji Fukunagaa, Haruhiko Kanasakia,c, Kohji Miyazakic, Takashi Ohbab, Hitoshi Okamurab, and Eishichi Miyamoto2,,a

a Department of Pharmacology and b Department of Obstetrics and Gynecology, Kumamoto University Schoolof Medicine, Kumamoto 860-0811, Japan c Department of Obstetrics and Gynecology, Shimane Medical University, Izumo 693-8501, Japan

ABSTRACT

The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE2 in human endometrial adenocarcinoma cell line HEC-1B. One µM PGE2 could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE2 also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE2-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE2. PGE2 could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE2-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE2, and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE2-induced activation of MAP kinase in HEC-1B cells.

kinases, signal transducers, signal transduction, uterus

INTRODUCTION

Cyclooxygenase (COX) isozymes 1 and 2 are the rate-limiting steps in the biosynthesis of prostaglandins (PGs). Both of the enzymes convert arachidonic acid to PGH2. PGH2 is then converted to various PGs by specific synthases. COX-2 is induced by a variety of stimulants, whereas COX-1 represents its noninducible counterpart. COX-2 is expressed in both of the epithelial [1] and stromal cells of the human endometrium. Human endometrial glandular epithelium contains the highest levels of COX, although whether it is COX-1, COX-2, or both has not been determined [2]. COX-2 plays an obligatory role in blastocyst implantation and in the decidualization of endometrial stromal cells, whereas fertilization proceeds normally without COX-1 [3].

PGs such as PGE2 and PGF2{alpha} are locally produced in endometrium. Numerous PGE2 binding sites are widely present in endometrium. PGF2{alpha} binding sites, although present in endometrium, are few in number [4]. PGs, mainly PGE2, are necessary for increased vascular permeability at the site of implantation and for increased local blood flow [5]. A COX inhibitor, indomethacin, can block implantation in rodents, and this blockade can be overcome by administration of PGs [6]. PGs also initiate endometrial plasminogen activator expression in the rat and thus may be involved in tissue remodeling associated with trophoblast invasion [7]. Transcripts coding PG receptors have been found in mouse luminal endometrial epithelium, the expression of which is coincident with the time of expected implantation [8].

A recent study showed the involvement of PGF2{alpha} in the expression of COX-2 in ovine luteal cells, indicating positive feedback stimulation in PG synthesis [9]. PGs bind with specific receptors and stimulate the intracellular signal transduction system. It is likely that other PGs, including PGE2, may also cause a similar effect, provided that specific receptors and the signaling molecules are present. For the effects of PGF2{alpha}, Ca2+- and protein kinase C (PKC)-mediated signaling pathways were believed to be responsible. But depending on the agonist, other protein kinases were found to be involved in the expression of COX-2, including mitogen-activated protein (MAP) kinase [10, 11] and a recently reported phosphatidylinositol-3-OH kinase, PI3K [12].

MAP kinase or extracellular signal-regulated kinase (ERK) is widely distributed in eukaryocytes as a family of serine/threonine protein kinases. MAP kinase was originally reported to be activated by growth factors and to be involved in proliferation and differentiation of cells through stimulation of gene expression. Subsequently, it was found that agents that bind with G protein-coupled receptors can also increase the activation of MAP kinase. In previous work, we reported that COX-2 expression increased, probably through the MAP kinase pathway, in response to stimulation of hCG receptors in HEC-1B cells [12]. Similarly, in GH3 cells, the secretion of prolactin was stimulated by treatment with thyrotropin releasing hormone (TRH) in correlation with activation of MAP kinase [13]. Furthermore, activation of MAP kinase protected apoptosis of GH3 cells induced with bromocriptine [14].

PGE2 binds with a type of G protein-coupled receptor, and most of the cellular effects mediated by PGE2 were explained by its ability to increase cAMP, and thereby activate protein kinase A (PKA) [15, 16]. cAMP has a variable effect on MAP kinase activity, either increasing [17] or decreasing [18] the activity, depending on cell types.

The HEC-1B cell line originated from endometrial adenocarcinoma and is a substrain of HEC-1A. HEC-1B cells are morphologically epithelial-like, and retain many of the characteristics of endometrial epithelial cells. HEC cells are reported to have both estradiol [19] and progesterone receptors [20] and can express COX-2 upon stimulation. Thus, because the HEC-1B cell line has many properties that are common to normal endometrial epithelial cells, this cell line is a valuable model for the study of regulation of COX-2. Our aim was to examine the effect of PGE2 in the expression of COX-2 and the signaling pathways involved, using HEC-1B cells.

MATERIALS AND METHODS

The following chemicals and reagents were obtained from the indicated sources: fetal bovine serum (FBS), JRH Biosciences (Lenexa, KS); [{gamma}-32P], ATP, and [125I] protein A, DuPont-New England Nuclear (Wilmington, DE); PGE2, 17ß-estradiol, and medroxyprogesterone acetate, Sigma Chemical Company (St. Louis, MO); MEM and Na-pyruvate, GIBCO BRL (Gaithersburg, MD); PD098059, RBI (Natick, MA); wortmannin, Wako Pure Chemical Industries (Osaka, Japan); H-89, Seikagaku Corporation (Tokyo, Japan); ERK-2, COX-2, and COX-1 antibodies, Transduction Laboratories (Lexington, KY); rabbit anti-mouse immunoglobulin G1 (IgG1), Zymed Laboratories (South San Francisco, CA); PKB and phospho-PKB antibodies, New England Biolabs Inc. (Beverly, MA). Myelin basic protein (MBP) was purified from bovine brain [21].

Cell Culture

HEC-1B cell line was obtained from ATCC, Bethesda, MD. Cells were cultured in MEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 60 mg/L kanamycin. Cell cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was replaced every 3–4 days and the cells were split every 4–5 days using trypsin (0.05% v/v) in Mg2+-free and Ca2+-free PBS. Cells were plated in 35-mm dishes for MAP kinase assay and in 100-mm dishes for immunoblot analysis and were cultured for 3–5 days in the same medium, including 10 nM estradiol and 1 µM medroxyprogesterone acetate to reach around 60% confluency.

Immunoblot Analyses of COX-2 and COX-1

In our previous experiments, we found that the expression of COX-2 significantly increased by treatment with PAF and hCG in HEC-1B cells cultured in the presence of both of 10 nM estradiol and 1 µM medroxyprogesterone acetate [12]. A similar result was also obtained when the cells were treated with PGE2 (data not shown). Therefore, we used cells pretreated with 10 nM estradiol and 1 µM medroxyprogesterone acetate for all of the following experiments.

HEC-1B cells were incubated for 12 h in the absence or presence of 1 µM PGE2 after the cells were cultured for 3–5 days in presence of estradiol and medroxyprogesterone acetate. After incubation, the medium was quickly aspirated off and the cells were frozen in liquid N2 and kept at -80°C until assayed.

The frozen cells were scraped off the dishes and solubilized in 0.30 ml of 50 mM HEPES (pH 7.4), 0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM Na4P2O7, 100 mM ß-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 75 µM pepstatin A, 1 mM dithiothreitol, and 1 mM (p-amidinophenyl)-methanesulfonyl fluoride hydrochloride (solubilization solution). The procedures for treatment of cells were carried out at 0–4°C. After sonication (Sonifier 250; Branson, Danbury, CT), the insoluble material was removed by centrifugation at 15 000 x g for 5 min. The extracts were treated with SDS sample buffer [22] and boiled for 3 min. Fifty µg of these protein samples were then subjected to SDS-PAGE in 10% acrylamide and then electrophoretically transferred at 75 V for 2 h to a nitrocellulose membrane. After incubating the membrane in 4.5% skimmed milk-Tris buffered saline (TBS; 10 mM Tris-buffered isotonic saline, pH 7.4) for 2 h to block nonspecific binding sites, the membrane was incubated overnight at 4°C with the monoclonal COX-2 antibody at a 1:125 dilution. After washing, the membrane was incubated with the anti-mouse IgG1 at a 1:200 dilution for 2 h and finally with [125I] protein A at room temperature for 1 h. After the membrane was dried, the amount of radioactivity was quantified by a Bio-imaging analyzer (BA100; Fujifilm, Tokyo, Japan).

For COX-1 immunoblotting, the procedure was essentially similar as described for COX-2, with the exception that COX-1 antibody (1:200 dilution) was used instead of COX-2 antibody and anti-mouse IgG1 was omitted from the procedure.

Assay for MAP Kinase

Cells cultured in the presence of 10 nM estradiol and 1 µM medroxyprogesterone acetate were washed once with Krebs-Ringer HEPES buffer (KRH buffer) containing 128 mM NaCl, 5 mM KCl, 1 mM sodium phosphate, 1.2 mM MgSO4, 10 mM glucose, 2.7 mM CaCl2, and 20 mM HEPES (pH 7.4), and were preincubated at 37°C for 1 h in the same buffer. Then the cells were incubated at 37°C for the specified times with none (control) or specified test agents in KRH buffer. After incubation for the indicated times, the medium was quickly aspirated off, and the cells were frozen in liquid N2 and kept at -80°C until the assay was performed.

The frozen cells were scraped off the dishes and solubilized in 0.15 ml solubilization solution having the same composition as described earlier for COX-2 assay, with the exception that 1 mM Na3VO4 and 100 nM calyculin A were also included. Cells were sonicated and centrifuged as described earlier. Samples containing the same amount of protein (10–15 µg of protein) were subjected to elecrophoresis in 10% polyacrylamide gel containing myelin basic protein (MBP). The procedures following this step is essentially similar to those described by Kurino et al. [23]. Briefly, after electrophoresis, the gel was washed, denatured, and renatured by appropriate buffers. The gel was then preincubated for 1 h with 40 mM HEPES, 2 mM DTT, 10 mM MgCl2, and 0.1 mM Na-orthovanadate at 30°C. Incubation of the gel was performed for 30 min in a buffer described for preincubation, in which 25 µCi [{gamma}32P]ATP, 25 mM cold ATP, and 25 mM EGTA were also added. The gel was washed and dried, and the amount of 32P incorporation into MBP at the position of MAP kinase in the gel was quantified with a Bio-imaging analyzer (BA 100; Fujifilm).

For immunoblot analysis of MAP kinase, cells were cultured in 100-mm dishes and incubated at 37°C with none (control) or PGE2 in KRH buffer for the indicated times as described in the figure legends. The rest of the procedure was similar to that described earlier for COX-2, with the exception that the antibody against the 42-kDa isoform of MAP kinase (also called ERK-2) was used (1:500 dilution) and the anti-mouse IgG1 antibody was omitted.

Immunohistochemical Analysis of MAP Kinase

Cells cultured in 35-mm dishes in the presence of 10 nM estradiol and 1 µM medroxyprogesterone acetate were washed once with KRH buffer and preincubated at 37°C for 1 h in the same buffer. Then the cells were incubated at 37°C for 10 min with nothing (control) or PGE2 in KRH buffer. After incubation the medium was quickly aspirated off, kept over dry ice, and enough volume of methanol was added to cover the cell surface in the dish. After 10 min, methanol was aspirated off and the dish was dried, and kept at -30°C until immunohistochemistry was performed.

The frozen cells were washed with PBS and permeabilized with PBS + 0.01% Triton X-100. After incubating the cells in 0.3% BSA in PBS + 0.01% Triton X-100 for 30 min to block nonspecific binding sites, the cells were incubated overnight at 4°C with phospho-MAP kinase antibody (1:50 dilution) and nonphospho-MAP kinase antibody (1:50 dilution). After washing, the cells were incubated again with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:40 dilution) for 1 h. The cells were then stained with propidium iodide for 2 min before mounting, and then examined under a confocal laser scanning light microscope (CLSM; Olympus, Japan).

Immunoblot Analysis of Protein Kinase B

Cells cultured in 100-mm dishes in the presence of 10 nM estradiol and 1 µM medroxyprogesterone acetate were washed once with KRH buffer and preincubated at 37°C for 1 h in the same buffer. Then the cells were incubated at 37°C for 3 min with nothing (control) or specified test agents in KRH buffer. After incubation for the indicated times, the medium was quickly aspirated off, and the cells were frozen in liquid N2 and kept at -80°C until the assay was performed.

The frozen cells were scraped from the dishes, solubilized in 0.2 ml RIPA solution (50 mM Tris-HCl pH 7.4], 0.15 M NaCl, 0.5% Triton X-100, 10 mM EDTA, 1 mM Na3VO4, 30 mM Na4P2O7, 50 mM NaF, and 4 mM EGTA) containing 0.1 mM leupeptin, 75 µM pepstatin A, 1 mM dithiothreitol, 1 mM (p-amidinophenyl)-methanesulfonyl fluoride hydrochloride, and 100 nM calyculin A. The rest of the procedures were essentially the same as was performed for COX-2 immunoblot with some modifications. One hundred µg of the protein samples were subjected to SDS-PAGE in 10% acrylamide and then transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with phospho-PKB (1:200 dilution) or nondiscriminating PKB antibody (1:1000 dilution). For phospho-PKB, the membrane was incubated again in [125I] protein A for 1 h. For total PKB, after incubation with the nondiscriminating PKB antibody, the membrane was washed in TBS + NP40 and incubated for 1 h with horseradish peroxidase-conjugated anti-sheep IgG. The bands were then visualized using an ECL detection kit.

Other Procedures

Protein concentrations were determined according to the method of Bradford [24] with bovine serum albumin as the standard.

Statistical Analyses

Each experiment was performed in triplicate and repeated at least two to three times. Results were consistent experiment to experiment. Values are expressed as mean ± SEM. Statistical significance of data was analyzed by using either two-way ANOVA or one-way ANOVA followed by Duncan's test for multiple comparisons. P < 0.05 was considered significant.

RESULTS

Expression of COX-2 by PGE2

We examined the expression of COX-2 at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h following stimulation of the cells with 1 µM PGE2 (Fig. 1, A and C). The expression of COX-2 protein significantly increased by 180% of control 8 h after PGE2 stimulation and continuously increased with increasing times. On the other hand, COX-1, the noninducible isoform of COX protein, did not change after PGE2 stimulation (Fig. 1B). The cells that were not treated with PGE2 showed no change in COX-2 expression during the period examined (Fig. 1C).



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  		FIG. 1. Time course of COX-2 and COX-1 expression and concentration-dependence of COX-2 expression with PGE2. HEC-1B cells were grown for 3–5 days in culture medium containing 10 nM estradiol and 1 µM medroxyprogesterone acetate. Cells were incubated for the indicated times without (circles) or with (squares) 1 µM PGE2. A) A representative autoradiogram of the COX-2 immunoblot. B) A representative autoradiogram of the COX-1 immunoblot. C) Graphic representation of PGE2-induced COX-2 expression. Values are expressed as a percentage of control (without PGE2 at zero time). We analyzed the response for two treatments of nonstimulation (dotted line) and stimulation (solid line) without and with PGE2, respective, over time by two-way ANOVA, which was significant (P < 0.0001). D) Cells were incubated without (control) or with various concentrations of PGE2 for 12 h. Data are expressed as a percentage of control, which is taken as 100%. Values are mean ± SEM (bars; n = 3). *P < 0.05, **P < 0.01 versus control (without PGE2 at zero time)

HEC-1B cells were treated for 12 h with PGE2 at concentrations ranging from 0.01 µM to 10 µM to determine whether the effect of PGE2 on COX-2 expression was dose-dependent (Fig. 1D). Maximum COX-2 expression was obtained with 100 nM PGE2. There was no significant difference in the expression of COX-2 between 100 nM and 1 µM concentrations when analyzed statistically. A higher concentration of PGE2 (10 µM) inhibited COX-2 expression.

Time Course of PGE2-Stimulated MAP Kinase Activation

MAP kinase is also called extracellular signal-regulated kinase (ERK) and includes a 44-kDa ERK-1 and a 42-kDa ERK-2. HEC-1B cells contain the 42-kDa isoform of MAP kinase [12]. The time course of PGE2-induced MAP kinase activation is shown in Figure 2, A and C. The cells were incubated with 1 µM PGE2 for up to 30 min. MAP kinase activation was measured from 5 to 30 min after stimulation with PGE2. The maximal activation of MAP kinase was obtained 10 min after stimulation with PGE2 and then decreased gradually. A significant level of MAP kinase activity was still observed even at 30 min. The cells that were not treated with PGE2 showed no change in MAP kinase activation during the period examined (Fig. 1C). The MAP kinase protein level was same along the time course as shown by immunoblot analysis (Fig. 2B).



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  		FIG. 2. Time course of MAP kinase activation with PGE2. A) A representative autoradiogram of the time course of 1 µM PGE2-induced MAP kinase activation. B) Immunoblotting with the MAP kinase antibody. Cells were stimulated with PGE2 for the indicated times and immunoblotting was carried out with MAP kinase antibody. C) Graphic representation of PGE2-induced MAP kinase activation. Values are expressed as a percentage of control (without PGE2 at zero time), which is taken as 100%. Values are mean ± SEM (bars; n = 3). *P < 0.05, **P < 0.01 versus control (without PGE2 at zero time). We analyzed the response for two treatments of nonstimulation (dotted line) and stimulation (solid line) without and with PGE2, respectively, over time by two-way ANOVA, which was significant (P < 0.0001)

Immunohistochemical Analysis of MAP Kinase Activation by PGE2

MAP kinase is a cytosolic enzyme that is translocated in the nucleus upon activation. To supplement the observation of MAP kinase activation by in-gel kinase assay, the cells were immunostained with the phospho-MAP kinase antibody by comparing the staining of the nuclei with propidium iodide (Fig. 3). MAP kinase, a cytosolic protein during its inactivated form, translocates to the nucleus of the cell upon activation. In the event of nuclear translocation of activated MAP kinase, double staining of the nucleus of the cell will occur at the images recorded simultaneously through the FITC and propidium iodide channel. Although the cytoplasms of HEC-1B cells were immunostained with the phospho-MAP kinase antibody (Fig. 3A), the immunoreactivity strongly increased following PGE2 stimulation (Fig. 3E). It is also noteworthy that the nuclei of HEC-1B cells were double-stained with the phospho-MAP kinase antibody and propidium iodide, indicating that activated MAP kinase is localized in the nuclei (Fig. 3G). On the other hand, when MAP kinase antibody was used, which cannot discriminate the phosphorylated and nonphosphorylated forms, there was no difference in immunoreactivity after PGE2 stimulation (Fig. 3, D and H). Thus, it is apparent that the activation of MAP kinase was increased after stimulation with PGE2.



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  		FIG. 3. Confocal laser scanning light microscope images of HEC-1B cells showing activation of MAP kinase. Cells were stimulated for 10 min without (control, upper panel) or with 1 µM PGE2 (lower panel). A and E) Phospho-MAP kinase antibody and FITC-conjugated anti-rabbit antibody. B and F) Propidium iodide. C and G) Pseudocolored confocal image obtained after combining the images recorded through the FITC and propidium iodide channels. Double-level cells are orange. D and H) MAP kinase antibody (nonphosphorylated) and FITC-conjugated anti-rabbit antibody

Effects of Protein Kinase Inhibitors on PGE2-Mediated MAP Kinase Activation

To determine the activation pathway of MAP kinase by stimulation with PGE2, effects of PD098059, H-89, calphostin C, and wortmannin were examined (Fig. 4). The concentrations of these inhibitors used for our experiments were based on previous reports [25–28]. PD098059, a specific inhibitor of MEK [25], could completely inhibit activation of MAP kinase stimulated by PGE2 at 75 µM concentration (Fig. 4, A and C). Activation of MAP kinase was also entirely inhibited by the addition of 10 µM H-89 (Fig. 4, A and C), which selectively inhibits PKA [26]. On the other hand, calphostin C, a selective inhibitor of PKC [27], failed to inhibit MAP kinase activation by PGE2 (Fig. 4, A and C). When used alone, none of the inhibitors showed any effect on MAP kinase activation compared with controls (data not shown). The inhibitors did not affect viability of HEC-1B cells (data not shown). There was no change in MAP kinase protein levels in different treatment conditions (Fig. 4B). Collectively, our data indicate that the inhibitors affected the activity of MAP kinase and that the activation of MAP kinase by PGE2 is dependent on a cAMP pathway rather than PKC.



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  		FIG. 4. Effects of inhibitors on PGE2-induced MAP kinase (MAPK) activation. A) A representative autoradiogram showing the effects of the inhibitors on PGE2-stimulated COX-2 expression. The cells were preincubated for 1 h in KRH buffer and nothing, 75 µM PD098059 (PD), 10 µM H-89, 500 nM calphostin C (Cal. C), or 200 nM wortmannin (Wort) were added for the last 30 min of preincubation. Cells were then incubated for 10 min without (control) or with 1 µM PGE2 in the presence of the indicated inhibitors in KRH buffer. B) Immunoblotting with the MAP kinase antibody. Cells were stimulated as described in the text and the immunoblotting was carried out with MAP kinase antibody. C) Graphic representation of the data obtained from the autoradiogram (A). Values are mean ± SEM (bars; n = 3). Data are expressed as a percentage of the control value, which is taken as 100%. *P < 0.05, **P < 0.01 versus control

We also wanted to know whether PI3K is involved in this MAP kinase activation pathway. Wortmannin is a specific inhibitor of PI3K at nanomolar concentrations [28]. Although wortmannin was found to be involved in some G protein-coupled receptor-mediated MAP kinase activation [29], in our study, it had no effect on PGE2-induced activation of MAP kinase (Fig. 4, A and C).

Activation of PKB by PGE2

PKB is a serine/threonine kinase lying downstream of PI3K. Activation of PKB is indirect evidence of PI3K stimulation. To investigate whether PGE2 can activate PKB, we measured phosphorylated PKB levels by immunoblot analysis. As shown in Figure 5, A and C, phosphorylated PKB levels increased significantly at 3 min after stimulation with PGE2, as recognized by phospho-PKB antibody directed against Ser-473 residue. Wortmannin, a specific inhibitor for PI3K, completely inhibited the effect. However, when the anti-PKB antibody was used, which cannot discriminate phosphorylated and nonphosphorylated forms, there was no change in protein level following PGE2 stimulation (Fig. 5B). This result indicates the activation of PKB by PGE2 in HEC-1B cells. It also suggests that this activation is dependent on PI3K, because wortmannin completely abolished the effect.



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  		FIG. 5. Activation of PKB in response to PGE2. Cells were preincubated for 1 h in KRH buffer and 200 nM wortmannin (Wort) were added for the last 30 min of preincubation. Cells were then incubated for 3 min without (control) or with 1 µM PGE2 in the presence or absence of wortmannin in KRH buffer. A) A representative autoradiogram showing immunoblot analyses of PKB using phospho (Ser-473)-specific (P-PKB) antibody. B) A representative autoradiogram showing immunoblot analyses of PKB using nondiscriminating PKB (PKB) antibody. C) Graphic representation of data obtained from immunoblot analyses of PKB using phospho (Ser-473)-specific (P-PKB) antibody. Data are expressed as a percentage of the control value, which is taken as 100%. Values are mean ± SEM (bars; n = 3). *P < 0.05, **P < 0.01 versus control

Effects of Protein Kinase Inhibitors on PGE2-Induced Expression of COX-2

To examine functional consequences of protein kinases on PGE2-induced expression of COX-2, we used specific pharmacologic inhibitors of signal transduction pathways (Fig. 6). Approximately 50% of COX-2 stimulated by PGE2 was inhibited by PD098059, suggesting involvement of MAP kinase (Fig. 6, A and C). H-89 also partially inhibited COX-2 expression (Fig. 6, A and C). Because H-89 could also inhibit MAP kinase activation stimulated by PGE2, it was likely that inhibition of MAP kinase by H-89 is related to the inhibition of COX-2 expression. Calphostin C failed to inhibit PGE2-induced COX-2 expression (Fig. 6, A and C), suggesting that PKC is not involved in the expression of COX-2 by PGE2. When inhibitors were used alone, none showed any effect on COX-2 expression compared with controls (Fig. 6A).



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  		FIG. 6. Effects of inhibitors on PGE2-induced COX-2 expression. A) A representative autoradiogram of the effects of the inhibitors on PGE2-stimulated COX-2 expression. Cells were preincubated for 30 min in the medium in the presence of nothing, 75 µM PD098059 (PD), 10 µM H-89, 500 nM calphostin C (Cal. C), or 200 nM wortmannin (Wort). Cells were then incubated for 12 h without (control) or with 1 µM PGE2 in the presence of the indicated inhibitors in the medium. B) Immunoblotting with the COX-1 antibody. Cells were stimulated as described in the text and then immunoblotting was carried out. C) Graphic representation of the effects of the inhibitors on PGE2-stimulated COX-2 expression. Values are mean ± SEM (bars; n = 3). Data are expressed as a percentage of the control value, which is taken as 100%. *P < 0.05, **P < 0.01 versus control

In a view to understand any contribution of PI3K pathway to COX-2 expression, we treated the cells with wortmannin and examined the ability of PGE2 in COX-2 expression. It is interesting that wortmannin could inhibit approximately 40% of COX-2 induced by PGE2 (Fig. 6, A and C), suggesting a role for PI3K. In addition, when wortmannin and PD098059 were used together, a total inhibition of COX-2 expression was observed (Fig. 6, A and C). As expected, none of the inhibitors had any effect on COX-1 protein levels (Fig. 6B). Our data show collectively that both MAP kinase and PI3K pathways are involved in the expression of COX-2 by PGE2.

DISCUSSION

In the present study, we have demonstrated that PGE2 can stimulate the expression of COX-2 in HEC-1B cells. If PGE2 stimulates COX-2 expression in vivo, the present finding will suggest that in addition to its role in implantation and decidualization, PGE2 can regulate endometrial functions by inducing COX-2 protein. Tsai et al. [9] demonstrated the ability of PGF2{alpha} in the expression of COX-2 in ovine luteal cells. Our results and results published by Tsai et al. provide evidence of the presence of a positive feedback loop in the production of PGs.

Given that both human endometrial and HEC-1B cells express COX-2, in addition to a range of similarity between these two cells [19, 20, 30], the use of HEC-1B cells in the present study is appropriate and relevant to human endometrial epithelial cell COX-2 physiology. In the present study, we demonstrated that PGE2 in 10 nM to 1 µM concentrations, which are well within the physiological levels, was effective in expressing COX-2 in HEC-1B cells. Similarly, we observed the activation of MAP kinase and PKB with 1 µM PGE2. These protein kinases were found to be involved in the expression of COX-2 in our study. PGE2 is released from both proliferative and secretory endometrial cells in culture [31]. Both of the epithelial and stromal cells of human endometrium contain numerous PGE2 and very few or no detectable PGF2{alpha} binding sites [4]. Therefore, the present findings suggest that locally produced PGE2 may increase the expression of COX-2 in endometrial epithelial cells by involving MAP kinase and PKB pathways.

Cellular effects of PGE2 are known to be mediated by a subfamily of G protein-coupled receptors, designated EP receptors. Four distinct EP receptor subtypes (EP1, EP2, EP3, and EP4) have been cloned and expressed [32]. EP1 and EP3 receptors are coupled to Ca2+ mobilization and inhibition of adenylate cyclase, respectively, whereas EP2 and EP4 receptors are both coupled to stimulation of adenylate cyclase [32]. In rat luminal epithelium, EP2 receptor mRNA expression occurs around the time of implantation [8]. Most of the cellular effects mediated by PGE2 were explained by its ability to increase cAMP, and thereby, activation of PKA [15, 16]. There is only one report of the activation of MAP kinase with PGE2 in COS7 cells transfected with EP3 receptors [33]. In the present study, 1 µM PGE2 could activate MAP kinase in HEC-1B cells in a time-dependent manner. By using the specific inhibitors of the protein kinases we demonstrated that MAP kinase activity was dependent on MEK and PKA. This indicates the ability of cAMP in the MAP kinase activation process. CPT-cAMP, a potent analogue of cAMP, also activates MAP kinase in HEC-1B cells [12]. In neurons, which express B-Raf rather than Raf-1, cAMP-activated MAP kinase was dependent on the activation of Rap1, a small GTP-binding protein [17]. We did not examine the activation of B-Raf by PGE2 in HEC-1B cells, but the ineffectiveness of calphostin C and the effectiveness of H-89 in PGE2-induced activation of MAP kinase indicates a role for cAMP in the MAP kinase activation process, which may be mediated by EP2 or EP4 subtypes of PGE2 receptor. Given the presence of EP2 receptor in endometrial epithelial cells, this study indicates the potential for MAP kinase activation after binding with PGE2.

In addition to MAP kinase, we provided evidence, for the first time, of the activation of PKB by PGE2. PKB, which is also known as Akt, is a serine/threonine kinase. PKB activation is a widespread phenomenon occurring through intrinsic tyrosine kinase receptors for growth factor- and insulin-associated tyrosine kinase receptors for IL-5 and IL-4 receptors, and G protein-coupled serpentine receptors for PAF. PKB is a downstream target for PI3K and has been shown to bind to and be activated by PtdIns(3,4,5)P3 and PtdIns(3,4)P2 signaling molecules generated by PI3K after lipid phosphorylation [34]. In the present study, wortmannin, a specific inhibitor of PI3K in nanomolar concentrations, could completely inhibit the activation of PKB by PGE2. This suggests the involvement of PI3K in PKB activation in HEC-1B cells. The activated PKB influences the metabolism through phosphorylation of glycogen synathase kinase-3 and phosphofructokinase, as well as transmission of a potent antiapoptotic signal [35]. The antiapoptotic signal may be mediated in part by phosphorylation and inactivation of Bad, a proapoptotic BCL-2 family member [35]. In endometrium there is expression of BCL-2 during the proliferative phase in endometrial glandular cells [36]. Expression of BCL-2 in endometrial stromal cells has also been described. There is a rise in stromal BCL-2 expression from the proliferative to the menstrual phase [37]. The in vivo regulation of the expression of this gene is not hormonally driven [36] and it is possible that PGE2, which is present in endometrium regardless of hormonal status, can take part in this process. In nonendometrial cells, PGE2 was found to inhibit apoptosis, in which PKA has been implicated [38].

In our study, MAP kinase was found to be partially responsible for the expression of COX-2. A MAP kinase inhibitor, PD098059, partially inhibited PGE2-mediated COX-2 expression in this study. This is consistent with previous reports in which PD098059 was found to block the stimulation of lysophosphatidic acid-mediated COX-2 expression in rat mesangial cells [39] and lipopolysaccharide-induced COX-2 expression in a murine macrophage cell line [40]. We also found that CPT-cAMP directly stimulates COX-2 expression in HEC-1B cells [12] and that PGE2-induced COX-2 expression is partially inhibited by H-89. Because H-89 also inhibited PGE2-induced MAP kinase activation, it appeared that MAP kinase is the ultimate effector in this kinase cascade.

Although recent studies have paid considerable attention to the role of MAP kinase in the expression of COX-2, evidence is lacking to show the role of the PI3K pathway in the expression of COX-2. Previously, we demonstrated the existence of a wortmannin-sensitive pathway in PAF-mediated COX-2 expression in the HEC-1B cell line [12]. In the present study, we extended our observation and found that wortmannin could partially inhibit the PGE2-induced expression of COX-2. The ability of PGE2 to activate PKB, which is inhibited by wortmannin, provides evidence of a novel PI3K-PKB cascade, which may play a role in the expression of COX-2. The function of PKB still remains elusive. It was previously shown that the rapamycin-sensitive p70s6 kinase can be activated by an oncogenic form of PKB, whereas PI3K inhibitors have been shown to inhibit p70s6 kinase activation [41, 42]. p70s6 Kinase participates in the translational control of mRNA transcripts encoding ribosomal proteins for the protein synthetic machinery. It is interesting that wortmannin was also found to inhibit protein synthesis by affecting translation [43]. This raises a possibility of the involvement of the PI3K-PKB pathway in the translation of COX-2. On the other hand, PKB was translocated into the nucleus [44] and could activate CREB following activation [45]. The COX-2 promoter region has the binding site for CREB and raises the possibility of a transcriptional control.

The present findings may also be important for understanding endometrial hyperplasias and carcinomas. It is a question whether PGE2 receptor is overexpressed in these conditions, which may lead to the continuous stimulation of COX-2 synthesis. COX-2 was found to be overexpressed in some form of cancers [46]. The high PG levels produced from constant COX-2 stimulation can increase cell proliferation, decrease apoptosis, and decrease adhesion to extracellular matrix proteins [46, 47]. PGE2-mediated activation of PKB can be an alternate mechanism for the decrease of apoptosis.

In summary, treatment of the human uterine epithelial cell line, HEC-1B, with PGE2 results in a time- and dose-dependent increase in COX-2 expression. PGE2 also activates MAP kinase and PI3K pathways, and they were found responsible for mediating the PGE2 signal for the expression of COX-2. Further experiments are required to confirm the significance of these findings in a physiological situation in endometrium.

FOOTNOTES

First decision: 12 October 1999.

1 This work was supported in part by Grants-in-Aid for Scientific Research and for Scientific Research on Priority from the Ministry of Education, Science, Sports and Culture, Japan, and by a research grant from the Human Frontier Science Program to E.M. and K.F. Back

2 Correspondence: Eishichi Miyamoto, Department of Pharmacology, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto City 860-0811, Japan. FAX: 81 96 373 5078; emiyamot{at}gpo.kumamoto-u.ac.jp Back

Accepted: May 1, 2000.

Received: July 27, 1999.

REFERENCES

  1. Zhou XL, Lei ZM, Rao ChV. Treatment of human endometrial gland epithelial cells with chorionic gonadotropin/luteinizing hormone increases the expression of the cyclooxygenase gene. J Clin Endocrinol Metab 1999; 84:3364–3377.[Abstract/Free Full Text]
  2. Rao ChV, Chegini N, Lei ZM. Immunocytochemical localization of 5- and 12-lipooxygenases and cyclooxygenase in nonpregnant human uteri. In: Zor U, Naor Z, Danon A (eds.), Leukotrienes and Prostanoids in Health and Disease. New Trends in Lipid Mediators Research. Basel, Switzerland: Karger; 1989: 283–286.
  3. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997; 91:197–208.[CrossRef][Medline]
  4. Chegini N, Rao CV, Wakim N, Sanfilippo J. Prostaglandin binding to different cell types of human uterus: quantitative light microscope autoradiographic study. Prostaglandins Leukot Essent Fatty Acids 1986; 22:129–138.
  5. Kennedy TG. Prostaglandins and the endometrial vascular permeability changes preceding blastocyst implantation and decidualization. Prog Reprod Biol 1980; 7:234–243.
  6. Lau IF, Saksena SK, Chang MC. Pregnancy blockade by indomethacin, an inhibitor of prostaglandin synthesis: its reversal by prostaglandins and progesterone in mice. Prostaglandins 1973; 4:795–803.[CrossRef][Medline]
  7. Zhang X, Shu MA, Ross HE, Kennedy TG. Regulation of plasminogen activator in rat endometrial stromal cells: the role of prostaglandin E2. Biol Reprod 1996; 54:1046–1051.[Abstract]
  8. Lim H, Dey SK. Prostaglandin E2 receptor subtype EP2 gene expression in the mouse uterus coincides with differentiation of the luminal epithelium for implantation. Endocrinology 1997; 138:4599–4606.[Abstract/Free Full Text]
  9. Tsai SJ, Wiltbank MC. Prostaglandin F2alpha induces expression of prostaglandin G/H synthase-2 in the ovine corpus luteum: a potential positive feedback loop during luteolysis. Biol Reprod 1997; 57:1016–1022.[Abstract]
  10. Xie W, Herschman HR. v-src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. J Biol Chem 1995; 270:27622–27628.[Abstract/Free Full Text]
  11. Xie W, Herschman HR. Transcriptional regulation of prostaglandin synthase 2 gene expression by platelet-derived growth factor and serum. J Biol Chem 1996; 271:31742–31748.[Abstract/Free Full Text]
  12. Munir I, Fukunaga K, Miyazaki K, Okamura H, Miyamoto E. Mitogen-activated protein kinase activation and regulation of cyclooxygenase 2 expression by platelet-activating factor and hCG in human endometrial adenocarcinoma cell line HEC-1B. J Reprod Fertil 1999; 117:49–59.[Abstract]
  13. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E. Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells. Biol Reprod 1999; 61:319–325.[Abstract/Free Full Text]
  14. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E. Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells. Biol Reprod 2000; 62:1486–1494.[Abstract/Free Full Text]
  15. Kuver R, Savard C, Oda D, Lee SP. PGE generates intracellular cAMP and accelerates mucin secretion by cultured dog gallbladder epithelial cells. Am J Physiol 1994; 267:G998–G1003.
  16. Wang HS, Keese CR, Giaever I, Smith TJ. Prostaglandin E2 alters human orbital fibroblast shape through a mechanism involving the generation of cyclic adenosine monophosphate. J Clin Endocrinol Metab 1995; 80:3553–3560.[Abstract]
  17. Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ. cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 1997; 89:73–82.[CrossRef][Medline]
  18. Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, Krebs EG. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci U S A 1993; 90:10300–10304.[Abstract/Free Full Text]
  19. Satyaswaroop PG, Fleming H, Bressler RS, Gurpide E. Human endometrial cancer cell cultures for hormonal studies. Cancer Res 1978; 38:4367–4375.[Medline]
  20. Oyama S, Izutsu T. Cell cycle analysis of endometrial cancer cells in vitro treated with growth factor and steroid hormone [in Japanese]. Hum Cell 1992; 5:363–371.[Medline]
  21. Deibler GE, Martenson RE, Kies MW. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem 1972; 2:139–165.[Medline]
  22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  23. Kurino M, Fukunaga K, Ushio Y, Miyamoto E. Activation of mitogen-activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors. J Neurochem 1995; 65:1282–1289.[Medline]
  24. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254.[CrossRef][Medline]
  25. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 1995; 270:27489–27494.[Abstract/Free Full Text]
  26. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 1990; 265:5267–5272.[Abstract/Free Full Text]
  27. Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 1989; 159:548–553.[CrossRef][Medline]
  28. Grammer TC, Blenis J. Evidence for MEK-independent pathways regulating the prolonged activation of the ERK-MAP kinases. Oncogene 1997; 14:1635–1640.[CrossRef][Medline]
  29. Lopez IM, Crespo P, Pellici PG, Gutkind JS, Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI3 kinase gamma. Science 1997; 275:394–397.[Abstract/Free Full Text]
  30. Ahmed A, Sage SO, Plevin R, Shoaibi MA, Sharkey AM, Smith SK. Functional platelet-activating factor receptors linked to inositol lipid hydrolysis, calcium mobilization and tyrosine kinase activity in the human endometrial HEC-1B cell line. J Reprod Fertil 1994; 101:459–466.[Abstract]
  31. Smith SK, Kelly RW. The release of PGF2 alpha and PGE2 from separated cells of human endometrium and decidua. Prostaglandins Leukot Essent Fatty Acids 1988; 33:91–96.[CrossRef][Medline]
  32. Negishi M, Sugimoto Y, Ichikawa A. Molecular mechanisms of diverse actions of prostanoid receptors. Biochim Biophys Acta 1995; 1259:109–119.[Medline]
  33. Burkey TH, Regan JW. Activation of mitogen-activated protein kinase by the human prostaglandin EP3A receptor. Biochem Biophys Res Commun 1995; 211:152–158.[CrossRef][Medline]
  34. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 1995; 81:727–736.[CrossRef][Medline]
  35. Downward J. Lipid-regulated kinases: some common themes at last [comment]. Science 1998; 279:673–674.[Free Full Text]
  36. Harada M, Suganuma N, Furuhashi M. Detection of apoptosis in human endometriotic tissues. Mol Hum Reprod 1996; 2:307–315.[Abstract/Free Full Text]
  37. Koh E, Illingworth P, Duncan W. Immunolocalization of Bcl-2 protein in human endometrium in the menstrual cycle and stimulated early pregnancy. Mol Hum Reprod 1995; 10:1557–1562.
  38. Ottonello L, Gonella R, Dapino P, Sacchetti C, Dallegri F. Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels. Exp Hematol 1998; 26:895–902.[Medline]
  39. Reiser CO, Lanz T, Hofmann F, Hofer G, Rupprecht HD, Goppelt-Struebe M. Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins. Biochem J 1998; 330:1107–1114.
  40. Hwang D, Jang BC, Yu G, Boudreau M. Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappaB signaling pathways in macrophages. Biochem Pharmacol 1997; 54:87–96.[CrossRef][Medline]
  41. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 1995; 376:599–602.[CrossRef][Medline]
  42. Petritsch C, Woscholski R, Edelmann HM, Parker PJ, Ballou LM. Selective inhibition of p70 S6 kinase activation by phosphatidylinositol 3-kinase inhibitors. Eur J Biochem 1995; 230:431–438.[Medline]
  43. Pedersen S, Celis JE, Nielsen J, Christiansen J, Nielsen FC. Distinct repression of translation by wortmannin and rapamycin. Eur J Biochem 1997; 247:449–456.[Medline]
  44. Meier R, Alessi DR, Cron P, Andjelkovic M, Hemmings BA. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase B beta. J Biol Chem 1997; 272:30491–30497.[Abstract/Free Full Text]
  45. Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem 1998; 273:32377–32379.[Abstract/Free Full Text]
  46. Subbaramaiah K, Zakim D, Weksler BB, Dannenberg AJ. Inhibition of cyclooxygenase: a novel approach to cancer prevention. Proc Soc Exp Biol Med 1997; 216:201–210.[Abstract]
  47. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995; 83:493–501.[CrossRef][Medline]



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