A Positive Feedback Loop that Regulates Cyclooxygenase-2 Expression and Prostaglandin F2 Synthesis via the F-Series-Prostanoid Receptor and
http://www.100md.com
《内分泌学杂志》
Medical Research Council Human Reproductive Sciences Unit (H.N.J., K.J.S., S.C.B.), Reproductive and Developmental Sciences (R.A.A.), and Department of Pathology (A.R.W.W.), The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh EH16 4TJ, Scotland, United Kingdom
Abstract
Cyclooxygenase (COX) enzymes catalyze the biosynthesis of eicosanoids, including prostaglandin (PG) F2. PGF2 exerts its autocrine/paracrine function by coupling to its G protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of COX-2 and FP receptor colocalized together within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF2-FP receptor interaction in modulating COX-2 expression and PGF2 biosynthesis using an endometrial adenocarcinoma cell line stably transfected with the FP receptor cDNA (FPS cells). PGF2-FP receptor activation rapidly induced COX-2 promoter, mRNA, and protein expression in FPS cells. These effects of PGF2 on the expression of COX-2 could be abolished by treatment of FPS cells with an FP receptor antagonist (AL8810) and chemical inhibitor of ERK1/2 kinase (PD98059), or by inactivation of ERK1/2 signaling with dominant-negative mutant isoforms of Ras or ERK1/2 kinase. We further confirmed that elevated COX-2 protein in FPS cells could biosynthesize PGF2 de novo to promote a positive feedback loop to facilitate endometrial tumorigenesis. Finally, we have shown that PGF2 could potentiate tumorigenesis in endometrial adenocarcinoma explants by inducing the expression of COX-2 mRNA.
Introduction
IN THE REPRODUCTIVE tract, there are five main endogenous prostanoids produced, namely prostaglandin (PG)D2, PGE2, PGF2, prostacyclin (PGI2), and thromboxane A2 (1). In the uterus, the E- and F-series of PGs are the most abundantly biosynthesized prostanoids, and PGF2 is a major metabolite of cyclooxygenase (COX) enzymes in human endometrium (2, 3). Two predominant isoforms of the COX enzymes have been identified (COX-1 and COX-2). COX-1 is constitutively expressed in many cell types and has been shown recently to be inducible in certain cancers (4, 5, 6, 7). COX-2 is the readily inducible form of the enzyme and is commonly associated with several pathological conditions including tumorigenesis (8, 9). Arachidonic acid, once released from the membrane phospholipids, is converted to the prostanoid intermediate PGH2 by the COX enzymes. PGH2 acts as a substrate for synthases specific to each prostanoid such as PGF synthase for PGF2 (10). After biosynthesis, PGF2 is transported out of the cell by means of a prostaglandin transporter (11) by which it exerts an autocrine/paracrine function through G protein-coupled receptor (GPCR)-mediated interaction. The GPCR for the human PGF2 [F-series-prostanoid (FP)] has been cloned, and its activation leads to coupling to the G protein Gq and release of inositol 1,4,5-trisphosphate (IP) and diacylglycerol (12).
A role for COX enzymes and various prostaglandins and their receptors, including PGF2 and the FP receptor, has been implicated in numerous endometrial pathologies including excessive menstrual bleeding (menorrhagia), endometriosis, painful periods (13, 14, 15, 16, 17, 18), and cancer (7, 19, 20, 21, 22, 23, 24, 25, 26, 27). In all of these pathologies, an aberrant angiogenic and vascular function has been described. Numerous studies have demonstrated that overexpression of prostanoid receptors are associated with enhanced production of angiogenic factors (28, 29). These factors act in a paracrine manner to promote endothelial cell migration and microvascular tube formation (29).
Recently we reported elevated expression and signaling of the human FP receptor in human endometrial adenocarcinomas and have ascertained a role for PGF2-FP receptor interaction in enhancing the proliferation of endometrial epithelial cells (25) and promoting the expression of proangiogenic factors such as vascular endothelial growth factor (30). This study was designed to assess the potential effect of PGF2-FP receptor signaling on expression of COX enzymes in human endometrial adenocarcinoma cells. This was investigated using Ishikawa endometrial epithelial cells stably transfected with the human isoform of the FP receptor. We found that PGF2-FP interaction promoted the transcription and translation of COX-2 and the subsequent de novo biosynthesis of PGF2 into the culture medium via activation of the ERK1/2 signaling pathway. Finally, the pathological significance of such a mechanism was confirmed by using endometrial adenocarcinoma biopsy tissue.
Materials and Methods
Reagents
Culture medium was purchased from Life Technologies (Paisley, UK). Penicillin-streptomycin and fetal calf serum were purchased from PAA Laboratories (Middlesex, UK). COX-1 (sc-1752), COX-2 (sc-1745), -actin (sc-1616), and antibodies were purchased from Santa Cruz Biotechnology (Autogen-Bioclear, Wiltshire, UK). Alkaline phosphatase secondary antibodies, indomethacin, PBS, BSA, AL8810 (10 mM stock in ethanol, used at final concentration of 50 μM), and PGF2 were purchased from Sigma Chemical Co. (Dorset, UK). The enhanced chemifluorescence (ECF) system was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). PD98059 [50 mM stock in dimethylsulfoxide used at a final concentration of 50 μM] and NS398 (10 mM stock in dimethylsulfoxide, used at final concentration of 10 μM) were purchased from Calbiochem (Nottingham, UK) and stored at –20 C.
Patients and tissue collection
Endometrial adenocarcinoma tissue (n = 25) was collected from women undergoing hysterectomy who had been prediagnosed to have adenocarcinoma of the uterus. All women were postmenopausal and had received no treatment before surgery. The ages of the patients ranged from 50 to 71 yr of age with a median age of 60.5 yr. Hysterectomy specimens for adenocarcinoma were collected from the operating room and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist. Small samples (5 mm to 3 cm) of polypoidal adenocarcinoma tissue were collected from the uterine cavity and transferred into neutral buffered formalin and wax embedded for immunofluorescence studies, snap frozen in dry ice, and stored at –70 C (for RNA extraction), and placed in RPMI 1640 culture medium containing 2 mM L-glutamine, 100 U penicillin, and 100 μg/ml streptomycin and 3 μg/ml indomethacin (to inhibit endogenous COX activity) for in vitro culture. The diagnosis of adenocarcinoma was confirmed histologically in all cases, and the percentage of tumor cells to stroma was estimated to be approximately 75:25%. Normal endometrial tissue (n = 10) at different stages of the menstrual cycle was collected from women undergoing surgery for minor gynecological procedures with no underlying endometrial pathology with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) from women with regular menstrual cycles (25–35 d) and processed exactly as described above. The ages of the control women ranged from 21 to 39 yr of age with a median age of 30.5 yr. None of the control women had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes et al. (31). Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.
Cell culture
Ishikawa endometrial adenocarcinoma cells were obtained from the European Collection of Cell Culture (Wiltshire, UK). Stable FP transfectant cells were constructed, characterized, and maintained as described (30), with the addition of a maintenance dose of 200 μg/ml G418.
Immunohistochemistry and confocal laser microscopy
FP receptor and COX-2 protein expression were colocalized in endometrial adenocarcinomas (n = 12) by dual immunofluorescence immunohistochemistry. Tissue sections were prepared as described previously (25) and blocked using 5% normal horse serum diluted in PBS. Subsequently sections were incubated with goat anti-COX-2 antibody at a dilution of 1:50 for 18 h at 4 C. Control sections were incubated with normal goat IgG. Thereafter sections were washed with PBS and incubated with biotinylated horse antigoat (Dako Corp., High Wycombe, UK) followed by incubation with the fluorochrome streptavidin 488 Alexafluor (Molecular Probes Inc., Eugene, OR) diluted 1:200 in PBS. Sections were reblocked with 5% normal goat serum diluted in PBS and incubated with rabbit anti-FP receptor antibody at a dilution of 1:100 at 4 C for 18 h. Control sections were incubated with rabbit IgG. Thereafter the sections were washed in PBS and incubated with the fluorochrome streptavidin 546 Alexafluor (Molecular Probes) diluted 1:200 in PBS at 25 C for 20 min. Sections were mounted and coverslipped, and fluorescent images were visualized and photographed using a laser scanning microscope (LM 510; Carl Zeiss, Jena, Germany).
Protein extraction
For COX-1 and COX-2 protein expression, 1 x 106 cells were seeded in 5-cm dishes and allowed to attach and grow for at least 16 h. Next, cells were incubated in serum-free culture medium, and 8.4 μM indomethacin (a dual COX-enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis) for at least 16 h. Thereafter medium was removed and replaced with fresh medium containing indomethacin, and cells were stimulated with either vehicle or 100 nM PGF2 in the absence or presence of chemical inhibitors for the time indicated in the figure legend. After stimulation with PGF2, proteins were extracted and quantified as described previously (25). The protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad Laboratories, Hemel Hempstead, UK). Data are presented as mean ± SEM from four independent experiments.
Western blot analysis
Approximately 20 μg protein was solubilized in Laemmli buffer [125 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue] and then boiled for 5 min. Proteins were resolved and immunoblotted as described previously (25) and incubated with specific primary and alkaline phosphatase-conjugated secondary antibodies. Immunoreactive proteins were visualized by the enhanced chemifluorescence (ECF) system according to the manufacturer’s instructions. Proteins were revealed and quantified by PhosphorImager analysis using the Typhoon 9400 system (Molecular Dynamics, Amersham Biosciences). Relative density of immunoblots was calculated by dividing the value obtained from the COX-2 blots by the value obtained from -actin blots and expressed as fold above vehicle controls. All data are presented as mean ± SEM from four independent experiments.
Taqman quantitative RT-PCR
FP receptor, COX-1, and COX-2 expression in endometrial adenocarcinoma (n = 25) and normal endometrium (n = 10) was measured by quantitative RT-PCR analysis as described previously (24). Moreover, the effect of PGF2 on COX enzyme expression was assessed in FPS cells and endometrial adenocarcinoma tissue. FPS cells were synchronized by serum withdrawal for at least 12 h in serum-free medium containing 8.4 μM indomethacin. Endometrial adenocarcinoma explants were finely chopped using a sterile scalpel blade and incubated in serum-free medium for at least 12 h. Thereafter medium was removed and replaced with fresh medium containing indomethacin with vehicle, 100 nM PGF2, or 100 nM PGF2 and chemical inhibitor for the time indicated in the figure legends. RNA was extracted using Tri-reagent (Sigma) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed and subjected to RT-PCR analysis using an ABI Prism 7700 (PE Applied Biosystems, Warrington, UK) as described previously (25). COX-1/COX-2 and FP primers and probe for quantitative PCR were designed using the PRIMER express program (PE Applied Biosystems) as described previously (7, 25). Data were analyzed and processed using Sequence Detector (version 1.6.3; PE Applied Biosystems). Expression of COX-1/COX-2/FP was normalized to RNA loading for each sample using the 18S rRNA as an internal standard. Results are expressed as fold increase above vehicle treated from four independent experiments and represented as mean ± SEM.
COX-2 luciferase reporter assays
The COX-2 promoter reporter plasmid consisting of a 966-bp fragment of the COX-2 promoter (C2.1; –917 to +49) ligated to a firefly luciferase construct as described by Bradbury et al. (32) was kindly supplied by Dr. Robert Newton (BioMedical Research Institute, Department of Biological Sciences, The University of Warwick, Warwick, UK). The COX-2 promoter firefly luciferase reporter was cotransfected into Ishikawa cells in triplicate with an internal control pRL-TK (containing the renilla luciferase coding sequence; Promega, Southampton, UK) and either control vector (pcDNA3.0) or vector encoding a dominant-negative (DN) isoform of Ras or mitogen-associated kinase kinase (MEK). The DN mutants cDNAs were kindly supplied by Professor Zvi Naor (Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel) and have been previously characterized and described (33, 34). Empty luciferase vector cDNA (pGL3 basic; Promega) was transfected in parallel as a control. Cells were transfected using Superfect (QIAGEN, Crawley, UK) for 6 h. The following day the cells were serum starved for at leased 16 h with indomethacin before stimulation for 4 h with vehicle, 100 nM PGF2, 100 nM PGF2 and AL8810, or 100 nM PGF2 and PD98059. The activity of both firefly and renilla luciferase was determined using the dual luciferase assay kit (Promega), and total luciferase activity was determined by dividing the relative light units generated by the firefly luciferase by the relative light units generated by the renilla luciferase in the same reaction. Fold increase in luciferase activity was calculated by dividing the total luciferase activity in cells treated with PGF2 by the total luciferase activity in cells treated with vehicle. Data are presented as mean ± SEM from four independent experiments.
PGF2 ELISA
PGF2 secretion in the culture media was assayed using an ELISA as described by Denison et al. (35, 36). Briefly, cells (250 x 105) were seeded in 6-well dishes and allowed to attach and grow. Thereafter cells were starved in serum-free medium for a minimum of 16 h before being preincubated with culture medium in the absence/presence of the COX-2 inhibitor NS398, MEK inhibitor PD98059, or FP receptor antagonist AL8810 for 1 h. After preincubation, cells were stimulated with 100 nM PGF2 in the absence/presence of NS398, AL8810, or PD98059 for 4 h. After stimulation, cells were washed and incubated with serum-free medium containing the same inhibitors for 24 h. Thereafter medium was removed and assayed for PGF2. Data are presented as mean ± SEM from four independent experiments.
Statistics
Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Berkeley, CA).
Results
FP receptor and COX enzyme expression in endometrial adenocarcinoma and normal endometrium
The expression of FP receptor, COX-1, and COX-2 mRNA in human endometrial adenocarcinoma and normal endometrium was determined by Taqman quantitative RT-PCR analysis (Fig. 1A). The expression of FP receptor and COX-2 mRNA was significantly up-regulated in all cases of endometrial adenocarcinoma investigated, compared with normal endometrium (P < 0.05). No correlation was observed between levels of expression of FP receptor and COX-2 mRNA and grade or stage of carcinoma, and no difference was observed in the levels of COX-1 mRNA expression between carcinoma and normal endometrium. The relative expression of FP receptor and COX-2 mRNA in endometrial adenocarcinomas was determined to be 67.9 ± 24.8 and 15.4 ± 4.6, respectively, compared with expression of 0.4 ± 0.1 and 0.4 ± 0.3 in normal endometrium (for FP receptor and COX-2, respectively).
Colocalization of FP receptor with COX-2 in endometrial adenocarcinomas
The site of expression of FP receptor and COX-2 was colocalized in endometrial adenocarcinomas by dual-immunofluorescence immunohistochemistry and confocal laser microscopy (Fig. 1B). FP receptor expression (FP; red) and COX-2 expression (COX-2; green) were localized together (FP/COX-2; yellow) in the glandular epithelial compartment in all poorly, moderately, and well-differentiated endometrial adenocarcinomas. Incubating sections with nonimmune IgG from the host species (inset) abolished the immunoreactivity.
PGF2-FP receptor activation induces COX-2 expression in Ishikawa FPS cells
The role of PGF2-FP receptor signaling on the expression of COX-1 and COX-2 was investigated by quantitative RT-PCR (Fig. 2, A and B) and Western blot (Fig. 2C) analysis. WT and FPS cells were stimulated with vehicle or 100 nM PGF2 for 2, 4, 8, and 24 h. No significant alteration in COX-1 mRNA or protein expression was observed in WT or FPS cells at any of the time points investigated (Fig. 2, A and C). However, PGF2 stimulation resulted in a significant fold increase in the expression of COX-2 mRNA (Fig. 2B) and protein (Fig. 2C) in Ishikawa FPS cells at 2, 4, 8, and 24 h, with the greatest elevation in expression observed at 4 h (P < 0.01). No significant alteration in COX-2 mRNA (Fig. 2B) or protein (data not shown) expression was observed in WT cells at any of the time points investigated.
COX-2 expression is mediated via activation of the Ras-ERK pathway
In a previous study, we characterized the molecular signal transduction pathways activated afterPGF2-FP ligand receptor interaction (30). We found that PGF2 stimulation of FPS cells activated phospholipase C, rapidly mobilizing IP, culminating in the activation of the Ras-ERK1/2 pathway. These effects of PGF2 on ERK activation via the FP receptor could be inhibited with a specific FP receptor antagonist AL8810 or by using chemical inhibitors of phospholipase C and MEK or targeted disruption of kinase signaling with DN mutant isoforms of Ras and MEK. In the present study, we set out to determine the signaling pathways mediating the role of PGF2-FP ligand receptor interaction on COX-2 expression in FPS cells. Using a reporter cDNA construct containing the full-length COX-2 promoter cDNA fused upstream of the firefly luciferase reporter (Fig. 3A), we found that COX-2 promoter activity in FPS cells was dependent on activation of ERK1/2 (Fig. 3A) because cotransfection of FPS cells with the COX-2 luciferase reporter and DN Ras or DN-MEK or targeted chemical inhibition of empty vector-transfected cells with the MEK inhibitor PD98059 (50 μM) or specific FP receptor antagonist AL8810 (50 μM) significantly inhibited COX-2 promoter activity (P < 0.05). Cotransfection of FPS cells with the empty vector luciferase construct (PGL3-basic) showed no significant alteration in luciferase activity in response to any treatments (Fig. 3A, open bars). Similarly, treatment of FPS cells with PGF2 resulted in up-regulation of expression of COX-2 RNA and protein, whereas cotreatment of the FPS cells with PGF2 and AL8810 or PD98059 significantly reduced the PGF2-induced expression of COX-2 mRNA (Fig. 3B, P < 0.05) and protein (Fig. 3C, P < 0.05), confirming that the effect of PGF2 on COX-2 is exerted via the FP receptor and ERK1/2 pathways.
Up-regulated COX-2 expression in FPS cells produces PGF2
We next investigated whether the up-regulated COX-2 protein, brought about by PGF2-FP ligand receptor interaction, could promote the de novo production of PGF2 into the surrounding culture medium, thereby setting up a positive feedback loop to sustain tumorigenesis. FPS cells were stimulated with vehicle or 100 nM PGF2 in the absence/presence of the specific COX-2 inhibitor NS-398 (10 μM), chemical inhibitor of MEK (PD98059, 50 μM), or FP receptor antagonist (AL8810; 50 μM) for 4 h to regulate COX-2. Subsequently the culture medium was removed and replaced with serum-free medium in the absence or presence of vehicle or the various inhibitors for a further 24 h and prostanoid production measured by ELISA. We found that up-regulated COX-2 in FPS cells could biosynthesize PGF2 (Fig. 4) de novo. Treatment of FPS cells with the specific COX-2 inhibitor NS-398, inhibitor of MEK (PD98059), or FP receptor antagonist (AL8810) significantly reduced the de novo biosynthesis of PGF2 (P < 0.05).
PGF2-FP receptor activation induces the expression of COX-2 mRNA in endometrial adenocarcinomas
To correlate our findings obtained using the Ishikawa FP receptor model system with PGF2 signaling to COX-2 in endometrial adenocarcinomas in vivo, we also used endometrial adenocarcinoma tissue explants. We incubated endometrial adenocarcinoma explants with either vehicle or PGF2 in the absence or presence of AL8810 or PD98059 for 24 h and assessed COX-2 mRNA expression by quantitative RT-PCR analysis (Fig. 5). PGF2 significantly elevated the expression of COX-2 in endometrial adenocarcinoma explants, compared with vehicle-treated tissue (P < 0.01). This elevation in expression of COX-2 was significantly inhibited by cotreatment of tissue explants with AL8810 or PD98059 (P < 0.05).
Discussion
Recent studies have demonstrated up-regulated and inducible expression of COX enzymes in different biological models (for review see Refs.37 and 38). COX-2 expression is up-regulated in numerous pathologies including those of the reproductive tract such as ovarian carcinoma, cervical carcinoma, and endometrial adenocarcinoma (7, 22, 26, 39, 40). Similarly, a correlation between tumor progression and COX-1 expression has recently been ascertained and elevated expression of COX-1 has been reported in human breast cancer (5), human prostate cancer (6), murine models of lung tumorigenesis (4), and human cervical carcinomas (7). More recent work has implicated specific prostanoids and their receptors and signaling pathways in reproductive tract carcinomas (27) and animal models of tumorigenesis (41, 42, 43). In these studies enhanced tumorigenic and angiogenic effects have been observed via PGE2-E-series-prostanoid receptor interaction (41, 42, 43). However, the role of the F-series prostanoids and FP receptor in modulating tumorigenesis or angiogenesis is poorly understood.
In addition to PGE2, PGF2 is a major prostanoid in the reproductive tract and a correlation between PGF2 biosynthesis and reproductive tract dysfunction has been ascertained (14, 15, 16, 44). Previously we reported on the expression and localization of COX-2 (26) and FP receptor (25) in human endometrial adenocarcinomas. Moreover, elevated FP receptor expression in endometrial adenocarcinomas can promote tumorigenesis by elevating cell proliferation and angiogenesis (25, 30). The data presented herein further confirm the concomitant up-regulation of COX-2 and FP receptor expression in adenocarcinoma of the human endometrium, as demonstrated by real-time quantitative RT-PCR analysis. Moreover, COX-2 was observed to colocalize with FP receptor in the neoplastic epithelial cells of the endometrial glands in poorly, moderately, and well-differentiated endometrial adenocarcinomas by dual-immunofluorescence immunohistochemistry and confocal laser microscopy. These data suggest an autocrine/paracrine control of neoplastic epithelial cell function by COX-2-derived prostanoids acting via the FP receptor.
In prostate and colorectal cancer cells, PGE2 has been shown to up-regulate the expression of COX-2 and endogenous biosynthesis of PGE2 (45, 46). Furthermore, Fujino and Regan et al. (47) have shown that the ovine FP receptor, which lacks the last 46 amino acids from the ovine FP receptor carboxy terminus tail, can activate a COX-2 promoter construct in human embryonic kidney cells overexpressing the FP receptor. Interestingly in the human, only one seven-transmembrane FP receptor has been cloned, although a six-transmembrane variant with no known biological role has been reported recently (48). The potential regulation of COX-2 expression and its contribution to human endometrial dysfunction via the PGF2-FP receptor signaling system has thus not been investigated. To elucidate the molecular mechanisms whereby PGF2, via the human FP receptor, could modulate the expression of COX-2, and potentially promote tumorigenesis, we overexpressed FP receptor in Ishikawa cells by introducing the FP receptor cDNA in the sense orientation (FPS cells) (30). The levels of FP receptor in the stably transfected FPS Ishikawa cell line are comparable with those observed in endometrial adenocarcinomas in vivo (30). In the present study, we demonstrated that PGF2-FP receptor coupling in the FPS cells induced the mRNA and protein expression of COX-2, but not COX-1, in a time-dependent manner.
The integrated response to GPCR coupling results in activation of numerous effector signaling pathways, including the MAPK pathway (49). The MAPK pathway is a key signaling mechanism that regulates many cellular functions such as growth, differentiation, and transformation (49, 50). The upstream component of the ERK-MAPK pathway is the GTPase Ras, which activates the serine/threonine kinase Raf, which in turn phosphorylates and activates ERK1/2 (51, 52). In a recent study, we found that PGF2 induced a rapid increase in ERK (but not p38 or JNK) phosphorylation. This PGF2-induced effect was significantly elevated in FPS cells, compared with WT cells and was mediated via the FP receptor in a Ras-dependent manner (30). Here we show that the PGF2-FP mediated activation of the COX-2 promoter and mRNA and protein expression of COX-2 via the activation of ERK1/2 in a Ras-dependent manner. Treatment of FPS cells with the FP receptor antagonist or chemical inhibitor of MEK or transfection of cells with a DN isoform of MEK or Ras significantly reduces expression of COX-2.
To determine whether the up-regulated COX-2 expression, induced after PGF2-FP ligand-receptor interaction, could biosynthesize prostanoids de novo, we treated FPS cells for 4 h with 100 nM PGF2 to induce COX-2 protein in the absence or presence of the selective inhibitors of COX-2 and MEK or FP receptor antagonist and then measured the levels of PGF2 secreted into the culture medium over 24 h. We found that PGF2 was biosynthesized and released into the culture medium and that this effect was abolished by treatment of FPS cells with the FP receptor antagonist or inhibitors of MEK or COX-2. The inhibition of prostaglandin biosynthesis by the specific COX-2 inhibitor NS398 and the MEK inhibitor PD98059 confirms that the elevated synthesis of PGF2 is a direct consequence of the up-regulation in expression of the COX-2 gene, which is dependent on phosphorylation of the ERK1/2 pathway. Although we have not demonstrated a direct alteration in phenotype or cell growth in this study, COX-2 has been shown to promote tumorigenesis in many other model systems (8, 28, 29, 43). It is likely that the up-regulated COX-2 in endometrial adenocarcinomas could similarly modulate endometrial tumorigenesis. Hence, endometrial tumorigenesis could be promoted through a self-amplifying loop, triggered by PGF2-FP receptor coupling and activation of the COX-2 gene via a Ras-ERK signaling cascade.
The data presented herein also demonstrate that PGF2-FP receptor activation could potentiate tumorigenesis in vivo. The data obtained from the endometrial adenocarcinoma explants are in agreement with the Ishikawa FPS cell model system and confirm that PGF2-FP receptor interaction can potentiate tumorigenesis in endometrial adenocarcinoma cells by up-regulating the expression of COX-2 and the biosynthesis of prostanoids. Overall, this study outlines the potential application of specific FP receptor antagonist and/or chemical inhibitors targeted toward the ERK1/2 pathway in future therapeutic strategies for treatment of women with endometrial adenocarcinoma (Fig. 6).
Acknowledgments
The authors thank Joan Creiger for tissue collection and Tammy List and Vivien Grant for technical assistance.
Footnotes
Abbreviations: COX, Cyclooxygenase; DN, dominant negative; FP, F-series-prostanoid; GPCR, G protein-coupled receptor; IP, inositol 1,4,5-trisphosphate; MEK, mitogen-associated kinase kinase; PG, prostaglandin.
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Abstract
Cyclooxygenase (COX) enzymes catalyze the biosynthesis of eicosanoids, including prostaglandin (PG) F2. PGF2 exerts its autocrine/paracrine function by coupling to its G protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of COX-2 and FP receptor colocalized together within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF2-FP receptor interaction in modulating COX-2 expression and PGF2 biosynthesis using an endometrial adenocarcinoma cell line stably transfected with the FP receptor cDNA (FPS cells). PGF2-FP receptor activation rapidly induced COX-2 promoter, mRNA, and protein expression in FPS cells. These effects of PGF2 on the expression of COX-2 could be abolished by treatment of FPS cells with an FP receptor antagonist (AL8810) and chemical inhibitor of ERK1/2 kinase (PD98059), or by inactivation of ERK1/2 signaling with dominant-negative mutant isoforms of Ras or ERK1/2 kinase. We further confirmed that elevated COX-2 protein in FPS cells could biosynthesize PGF2 de novo to promote a positive feedback loop to facilitate endometrial tumorigenesis. Finally, we have shown that PGF2 could potentiate tumorigenesis in endometrial adenocarcinoma explants by inducing the expression of COX-2 mRNA.
Introduction
IN THE REPRODUCTIVE tract, there are five main endogenous prostanoids produced, namely prostaglandin (PG)D2, PGE2, PGF2, prostacyclin (PGI2), and thromboxane A2 (1). In the uterus, the E- and F-series of PGs are the most abundantly biosynthesized prostanoids, and PGF2 is a major metabolite of cyclooxygenase (COX) enzymes in human endometrium (2, 3). Two predominant isoforms of the COX enzymes have been identified (COX-1 and COX-2). COX-1 is constitutively expressed in many cell types and has been shown recently to be inducible in certain cancers (4, 5, 6, 7). COX-2 is the readily inducible form of the enzyme and is commonly associated with several pathological conditions including tumorigenesis (8, 9). Arachidonic acid, once released from the membrane phospholipids, is converted to the prostanoid intermediate PGH2 by the COX enzymes. PGH2 acts as a substrate for synthases specific to each prostanoid such as PGF synthase for PGF2 (10). After biosynthesis, PGF2 is transported out of the cell by means of a prostaglandin transporter (11) by which it exerts an autocrine/paracrine function through G protein-coupled receptor (GPCR)-mediated interaction. The GPCR for the human PGF2 [F-series-prostanoid (FP)] has been cloned, and its activation leads to coupling to the G protein Gq and release of inositol 1,4,5-trisphosphate (IP) and diacylglycerol (12).
A role for COX enzymes and various prostaglandins and their receptors, including PGF2 and the FP receptor, has been implicated in numerous endometrial pathologies including excessive menstrual bleeding (menorrhagia), endometriosis, painful periods (13, 14, 15, 16, 17, 18), and cancer (7, 19, 20, 21, 22, 23, 24, 25, 26, 27). In all of these pathologies, an aberrant angiogenic and vascular function has been described. Numerous studies have demonstrated that overexpression of prostanoid receptors are associated with enhanced production of angiogenic factors (28, 29). These factors act in a paracrine manner to promote endothelial cell migration and microvascular tube formation (29).
Recently we reported elevated expression and signaling of the human FP receptor in human endometrial adenocarcinomas and have ascertained a role for PGF2-FP receptor interaction in enhancing the proliferation of endometrial epithelial cells (25) and promoting the expression of proangiogenic factors such as vascular endothelial growth factor (30). This study was designed to assess the potential effect of PGF2-FP receptor signaling on expression of COX enzymes in human endometrial adenocarcinoma cells. This was investigated using Ishikawa endometrial epithelial cells stably transfected with the human isoform of the FP receptor. We found that PGF2-FP interaction promoted the transcription and translation of COX-2 and the subsequent de novo biosynthesis of PGF2 into the culture medium via activation of the ERK1/2 signaling pathway. Finally, the pathological significance of such a mechanism was confirmed by using endometrial adenocarcinoma biopsy tissue.
Materials and Methods
Reagents
Culture medium was purchased from Life Technologies (Paisley, UK). Penicillin-streptomycin and fetal calf serum were purchased from PAA Laboratories (Middlesex, UK). COX-1 (sc-1752), COX-2 (sc-1745), -actin (sc-1616), and antibodies were purchased from Santa Cruz Biotechnology (Autogen-Bioclear, Wiltshire, UK). Alkaline phosphatase secondary antibodies, indomethacin, PBS, BSA, AL8810 (10 mM stock in ethanol, used at final concentration of 50 μM), and PGF2 were purchased from Sigma Chemical Co. (Dorset, UK). The enhanced chemifluorescence (ECF) system was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). PD98059 [50 mM stock in dimethylsulfoxide used at a final concentration of 50 μM] and NS398 (10 mM stock in dimethylsulfoxide, used at final concentration of 10 μM) were purchased from Calbiochem (Nottingham, UK) and stored at –20 C.
Patients and tissue collection
Endometrial adenocarcinoma tissue (n = 25) was collected from women undergoing hysterectomy who had been prediagnosed to have adenocarcinoma of the uterus. All women were postmenopausal and had received no treatment before surgery. The ages of the patients ranged from 50 to 71 yr of age with a median age of 60.5 yr. Hysterectomy specimens for adenocarcinoma were collected from the operating room and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist. Small samples (5 mm to 3 cm) of polypoidal adenocarcinoma tissue were collected from the uterine cavity and transferred into neutral buffered formalin and wax embedded for immunofluorescence studies, snap frozen in dry ice, and stored at –70 C (for RNA extraction), and placed in RPMI 1640 culture medium containing 2 mM L-glutamine, 100 U penicillin, and 100 μg/ml streptomycin and 3 μg/ml indomethacin (to inhibit endogenous COX activity) for in vitro culture. The diagnosis of adenocarcinoma was confirmed histologically in all cases, and the percentage of tumor cells to stroma was estimated to be approximately 75:25%. Normal endometrial tissue (n = 10) at different stages of the menstrual cycle was collected from women undergoing surgery for minor gynecological procedures with no underlying endometrial pathology with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) from women with regular menstrual cycles (25–35 d) and processed exactly as described above. The ages of the control women ranged from 21 to 39 yr of age with a median age of 30.5 yr. None of the control women had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes et al. (31). Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.
Cell culture
Ishikawa endometrial adenocarcinoma cells were obtained from the European Collection of Cell Culture (Wiltshire, UK). Stable FP transfectant cells were constructed, characterized, and maintained as described (30), with the addition of a maintenance dose of 200 μg/ml G418.
Immunohistochemistry and confocal laser microscopy
FP receptor and COX-2 protein expression were colocalized in endometrial adenocarcinomas (n = 12) by dual immunofluorescence immunohistochemistry. Tissue sections were prepared as described previously (25) and blocked using 5% normal horse serum diluted in PBS. Subsequently sections were incubated with goat anti-COX-2 antibody at a dilution of 1:50 for 18 h at 4 C. Control sections were incubated with normal goat IgG. Thereafter sections were washed with PBS and incubated with biotinylated horse antigoat (Dako Corp., High Wycombe, UK) followed by incubation with the fluorochrome streptavidin 488 Alexafluor (Molecular Probes Inc., Eugene, OR) diluted 1:200 in PBS. Sections were reblocked with 5% normal goat serum diluted in PBS and incubated with rabbit anti-FP receptor antibody at a dilution of 1:100 at 4 C for 18 h. Control sections were incubated with rabbit IgG. Thereafter the sections were washed in PBS and incubated with the fluorochrome streptavidin 546 Alexafluor (Molecular Probes) diluted 1:200 in PBS at 25 C for 20 min. Sections were mounted and coverslipped, and fluorescent images were visualized and photographed using a laser scanning microscope (LM 510; Carl Zeiss, Jena, Germany).
Protein extraction
For COX-1 and COX-2 protein expression, 1 x 106 cells were seeded in 5-cm dishes and allowed to attach and grow for at least 16 h. Next, cells were incubated in serum-free culture medium, and 8.4 μM indomethacin (a dual COX-enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis) for at least 16 h. Thereafter medium was removed and replaced with fresh medium containing indomethacin, and cells were stimulated with either vehicle or 100 nM PGF2 in the absence or presence of chemical inhibitors for the time indicated in the figure legend. After stimulation with PGF2, proteins were extracted and quantified as described previously (25). The protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad Laboratories, Hemel Hempstead, UK). Data are presented as mean ± SEM from four independent experiments.
Western blot analysis
Approximately 20 μg protein was solubilized in Laemmli buffer [125 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue] and then boiled for 5 min. Proteins were resolved and immunoblotted as described previously (25) and incubated with specific primary and alkaline phosphatase-conjugated secondary antibodies. Immunoreactive proteins were visualized by the enhanced chemifluorescence (ECF) system according to the manufacturer’s instructions. Proteins were revealed and quantified by PhosphorImager analysis using the Typhoon 9400 system (Molecular Dynamics, Amersham Biosciences). Relative density of immunoblots was calculated by dividing the value obtained from the COX-2 blots by the value obtained from -actin blots and expressed as fold above vehicle controls. All data are presented as mean ± SEM from four independent experiments.
Taqman quantitative RT-PCR
FP receptor, COX-1, and COX-2 expression in endometrial adenocarcinoma (n = 25) and normal endometrium (n = 10) was measured by quantitative RT-PCR analysis as described previously (24). Moreover, the effect of PGF2 on COX enzyme expression was assessed in FPS cells and endometrial adenocarcinoma tissue. FPS cells were synchronized by serum withdrawal for at least 12 h in serum-free medium containing 8.4 μM indomethacin. Endometrial adenocarcinoma explants were finely chopped using a sterile scalpel blade and incubated in serum-free medium for at least 12 h. Thereafter medium was removed and replaced with fresh medium containing indomethacin with vehicle, 100 nM PGF2, or 100 nM PGF2 and chemical inhibitor for the time indicated in the figure legends. RNA was extracted using Tri-reagent (Sigma) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed and subjected to RT-PCR analysis using an ABI Prism 7700 (PE Applied Biosystems, Warrington, UK) as described previously (25). COX-1/COX-2 and FP primers and probe for quantitative PCR were designed using the PRIMER express program (PE Applied Biosystems) as described previously (7, 25). Data were analyzed and processed using Sequence Detector (version 1.6.3; PE Applied Biosystems). Expression of COX-1/COX-2/FP was normalized to RNA loading for each sample using the 18S rRNA as an internal standard. Results are expressed as fold increase above vehicle treated from four independent experiments and represented as mean ± SEM.
COX-2 luciferase reporter assays
The COX-2 promoter reporter plasmid consisting of a 966-bp fragment of the COX-2 promoter (C2.1; –917 to +49) ligated to a firefly luciferase construct as described by Bradbury et al. (32) was kindly supplied by Dr. Robert Newton (BioMedical Research Institute, Department of Biological Sciences, The University of Warwick, Warwick, UK). The COX-2 promoter firefly luciferase reporter was cotransfected into Ishikawa cells in triplicate with an internal control pRL-TK (containing the renilla luciferase coding sequence; Promega, Southampton, UK) and either control vector (pcDNA3.0) or vector encoding a dominant-negative (DN) isoform of Ras or mitogen-associated kinase kinase (MEK). The DN mutants cDNAs were kindly supplied by Professor Zvi Naor (Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel) and have been previously characterized and described (33, 34). Empty luciferase vector cDNA (pGL3 basic; Promega) was transfected in parallel as a control. Cells were transfected using Superfect (QIAGEN, Crawley, UK) for 6 h. The following day the cells were serum starved for at leased 16 h with indomethacin before stimulation for 4 h with vehicle, 100 nM PGF2, 100 nM PGF2 and AL8810, or 100 nM PGF2 and PD98059. The activity of both firefly and renilla luciferase was determined using the dual luciferase assay kit (Promega), and total luciferase activity was determined by dividing the relative light units generated by the firefly luciferase by the relative light units generated by the renilla luciferase in the same reaction. Fold increase in luciferase activity was calculated by dividing the total luciferase activity in cells treated with PGF2 by the total luciferase activity in cells treated with vehicle. Data are presented as mean ± SEM from four independent experiments.
PGF2 ELISA
PGF2 secretion in the culture media was assayed using an ELISA as described by Denison et al. (35, 36). Briefly, cells (250 x 105) were seeded in 6-well dishes and allowed to attach and grow. Thereafter cells were starved in serum-free medium for a minimum of 16 h before being preincubated with culture medium in the absence/presence of the COX-2 inhibitor NS398, MEK inhibitor PD98059, or FP receptor antagonist AL8810 for 1 h. After preincubation, cells were stimulated with 100 nM PGF2 in the absence/presence of NS398, AL8810, or PD98059 for 4 h. After stimulation, cells were washed and incubated with serum-free medium containing the same inhibitors for 24 h. Thereafter medium was removed and assayed for PGF2. Data are presented as mean ± SEM from four independent experiments.
Statistics
Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Berkeley, CA).
Results
FP receptor and COX enzyme expression in endometrial adenocarcinoma and normal endometrium
The expression of FP receptor, COX-1, and COX-2 mRNA in human endometrial adenocarcinoma and normal endometrium was determined by Taqman quantitative RT-PCR analysis (Fig. 1A). The expression of FP receptor and COX-2 mRNA was significantly up-regulated in all cases of endometrial adenocarcinoma investigated, compared with normal endometrium (P < 0.05). No correlation was observed between levels of expression of FP receptor and COX-2 mRNA and grade or stage of carcinoma, and no difference was observed in the levels of COX-1 mRNA expression between carcinoma and normal endometrium. The relative expression of FP receptor and COX-2 mRNA in endometrial adenocarcinomas was determined to be 67.9 ± 24.8 and 15.4 ± 4.6, respectively, compared with expression of 0.4 ± 0.1 and 0.4 ± 0.3 in normal endometrium (for FP receptor and COX-2, respectively).
Colocalization of FP receptor with COX-2 in endometrial adenocarcinomas
The site of expression of FP receptor and COX-2 was colocalized in endometrial adenocarcinomas by dual-immunofluorescence immunohistochemistry and confocal laser microscopy (Fig. 1B). FP receptor expression (FP; red) and COX-2 expression (COX-2; green) were localized together (FP/COX-2; yellow) in the glandular epithelial compartment in all poorly, moderately, and well-differentiated endometrial adenocarcinomas. Incubating sections with nonimmune IgG from the host species (inset) abolished the immunoreactivity.
PGF2-FP receptor activation induces COX-2 expression in Ishikawa FPS cells
The role of PGF2-FP receptor signaling on the expression of COX-1 and COX-2 was investigated by quantitative RT-PCR (Fig. 2, A and B) and Western blot (Fig. 2C) analysis. WT and FPS cells were stimulated with vehicle or 100 nM PGF2 for 2, 4, 8, and 24 h. No significant alteration in COX-1 mRNA or protein expression was observed in WT or FPS cells at any of the time points investigated (Fig. 2, A and C). However, PGF2 stimulation resulted in a significant fold increase in the expression of COX-2 mRNA (Fig. 2B) and protein (Fig. 2C) in Ishikawa FPS cells at 2, 4, 8, and 24 h, with the greatest elevation in expression observed at 4 h (P < 0.01). No significant alteration in COX-2 mRNA (Fig. 2B) or protein (data not shown) expression was observed in WT cells at any of the time points investigated.
COX-2 expression is mediated via activation of the Ras-ERK pathway
In a previous study, we characterized the molecular signal transduction pathways activated afterPGF2-FP ligand receptor interaction (30). We found that PGF2 stimulation of FPS cells activated phospholipase C, rapidly mobilizing IP, culminating in the activation of the Ras-ERK1/2 pathway. These effects of PGF2 on ERK activation via the FP receptor could be inhibited with a specific FP receptor antagonist AL8810 or by using chemical inhibitors of phospholipase C and MEK or targeted disruption of kinase signaling with DN mutant isoforms of Ras and MEK. In the present study, we set out to determine the signaling pathways mediating the role of PGF2-FP ligand receptor interaction on COX-2 expression in FPS cells. Using a reporter cDNA construct containing the full-length COX-2 promoter cDNA fused upstream of the firefly luciferase reporter (Fig. 3A), we found that COX-2 promoter activity in FPS cells was dependent on activation of ERK1/2 (Fig. 3A) because cotransfection of FPS cells with the COX-2 luciferase reporter and DN Ras or DN-MEK or targeted chemical inhibition of empty vector-transfected cells with the MEK inhibitor PD98059 (50 μM) or specific FP receptor antagonist AL8810 (50 μM) significantly inhibited COX-2 promoter activity (P < 0.05). Cotransfection of FPS cells with the empty vector luciferase construct (PGL3-basic) showed no significant alteration in luciferase activity in response to any treatments (Fig. 3A, open bars). Similarly, treatment of FPS cells with PGF2 resulted in up-regulation of expression of COX-2 RNA and protein, whereas cotreatment of the FPS cells with PGF2 and AL8810 or PD98059 significantly reduced the PGF2-induced expression of COX-2 mRNA (Fig. 3B, P < 0.05) and protein (Fig. 3C, P < 0.05), confirming that the effect of PGF2 on COX-2 is exerted via the FP receptor and ERK1/2 pathways.
Up-regulated COX-2 expression in FPS cells produces PGF2
We next investigated whether the up-regulated COX-2 protein, brought about by PGF2-FP ligand receptor interaction, could promote the de novo production of PGF2 into the surrounding culture medium, thereby setting up a positive feedback loop to sustain tumorigenesis. FPS cells were stimulated with vehicle or 100 nM PGF2 in the absence/presence of the specific COX-2 inhibitor NS-398 (10 μM), chemical inhibitor of MEK (PD98059, 50 μM), or FP receptor antagonist (AL8810; 50 μM) for 4 h to regulate COX-2. Subsequently the culture medium was removed and replaced with serum-free medium in the absence or presence of vehicle or the various inhibitors for a further 24 h and prostanoid production measured by ELISA. We found that up-regulated COX-2 in FPS cells could biosynthesize PGF2 (Fig. 4) de novo. Treatment of FPS cells with the specific COX-2 inhibitor NS-398, inhibitor of MEK (PD98059), or FP receptor antagonist (AL8810) significantly reduced the de novo biosynthesis of PGF2 (P < 0.05).
PGF2-FP receptor activation induces the expression of COX-2 mRNA in endometrial adenocarcinomas
To correlate our findings obtained using the Ishikawa FP receptor model system with PGF2 signaling to COX-2 in endometrial adenocarcinomas in vivo, we also used endometrial adenocarcinoma tissue explants. We incubated endometrial adenocarcinoma explants with either vehicle or PGF2 in the absence or presence of AL8810 or PD98059 for 24 h and assessed COX-2 mRNA expression by quantitative RT-PCR analysis (Fig. 5). PGF2 significantly elevated the expression of COX-2 in endometrial adenocarcinoma explants, compared with vehicle-treated tissue (P < 0.01). This elevation in expression of COX-2 was significantly inhibited by cotreatment of tissue explants with AL8810 or PD98059 (P < 0.05).
Discussion
Recent studies have demonstrated up-regulated and inducible expression of COX enzymes in different biological models (for review see Refs.37 and 38). COX-2 expression is up-regulated in numerous pathologies including those of the reproductive tract such as ovarian carcinoma, cervical carcinoma, and endometrial adenocarcinoma (7, 22, 26, 39, 40). Similarly, a correlation between tumor progression and COX-1 expression has recently been ascertained and elevated expression of COX-1 has been reported in human breast cancer (5), human prostate cancer (6), murine models of lung tumorigenesis (4), and human cervical carcinomas (7). More recent work has implicated specific prostanoids and their receptors and signaling pathways in reproductive tract carcinomas (27) and animal models of tumorigenesis (41, 42, 43). In these studies enhanced tumorigenic and angiogenic effects have been observed via PGE2-E-series-prostanoid receptor interaction (41, 42, 43). However, the role of the F-series prostanoids and FP receptor in modulating tumorigenesis or angiogenesis is poorly understood.
In addition to PGE2, PGF2 is a major prostanoid in the reproductive tract and a correlation between PGF2 biosynthesis and reproductive tract dysfunction has been ascertained (14, 15, 16, 44). Previously we reported on the expression and localization of COX-2 (26) and FP receptor (25) in human endometrial adenocarcinomas. Moreover, elevated FP receptor expression in endometrial adenocarcinomas can promote tumorigenesis by elevating cell proliferation and angiogenesis (25, 30). The data presented herein further confirm the concomitant up-regulation of COX-2 and FP receptor expression in adenocarcinoma of the human endometrium, as demonstrated by real-time quantitative RT-PCR analysis. Moreover, COX-2 was observed to colocalize with FP receptor in the neoplastic epithelial cells of the endometrial glands in poorly, moderately, and well-differentiated endometrial adenocarcinomas by dual-immunofluorescence immunohistochemistry and confocal laser microscopy. These data suggest an autocrine/paracrine control of neoplastic epithelial cell function by COX-2-derived prostanoids acting via the FP receptor.
In prostate and colorectal cancer cells, PGE2 has been shown to up-regulate the expression of COX-2 and endogenous biosynthesis of PGE2 (45, 46). Furthermore, Fujino and Regan et al. (47) have shown that the ovine FP receptor, which lacks the last 46 amino acids from the ovine FP receptor carboxy terminus tail, can activate a COX-2 promoter construct in human embryonic kidney cells overexpressing the FP receptor. Interestingly in the human, only one seven-transmembrane FP receptor has been cloned, although a six-transmembrane variant with no known biological role has been reported recently (48). The potential regulation of COX-2 expression and its contribution to human endometrial dysfunction via the PGF2-FP receptor signaling system has thus not been investigated. To elucidate the molecular mechanisms whereby PGF2, via the human FP receptor, could modulate the expression of COX-2, and potentially promote tumorigenesis, we overexpressed FP receptor in Ishikawa cells by introducing the FP receptor cDNA in the sense orientation (FPS cells) (30). The levels of FP receptor in the stably transfected FPS Ishikawa cell line are comparable with those observed in endometrial adenocarcinomas in vivo (30). In the present study, we demonstrated that PGF2-FP receptor coupling in the FPS cells induced the mRNA and protein expression of COX-2, but not COX-1, in a time-dependent manner.
The integrated response to GPCR coupling results in activation of numerous effector signaling pathways, including the MAPK pathway (49). The MAPK pathway is a key signaling mechanism that regulates many cellular functions such as growth, differentiation, and transformation (49, 50). The upstream component of the ERK-MAPK pathway is the GTPase Ras, which activates the serine/threonine kinase Raf, which in turn phosphorylates and activates ERK1/2 (51, 52). In a recent study, we found that PGF2 induced a rapid increase in ERK (but not p38 or JNK) phosphorylation. This PGF2-induced effect was significantly elevated in FPS cells, compared with WT cells and was mediated via the FP receptor in a Ras-dependent manner (30). Here we show that the PGF2-FP mediated activation of the COX-2 promoter and mRNA and protein expression of COX-2 via the activation of ERK1/2 in a Ras-dependent manner. Treatment of FPS cells with the FP receptor antagonist or chemical inhibitor of MEK or transfection of cells with a DN isoform of MEK or Ras significantly reduces expression of COX-2.
To determine whether the up-regulated COX-2 expression, induced after PGF2-FP ligand-receptor interaction, could biosynthesize prostanoids de novo, we treated FPS cells for 4 h with 100 nM PGF2 to induce COX-2 protein in the absence or presence of the selective inhibitors of COX-2 and MEK or FP receptor antagonist and then measured the levels of PGF2 secreted into the culture medium over 24 h. We found that PGF2 was biosynthesized and released into the culture medium and that this effect was abolished by treatment of FPS cells with the FP receptor antagonist or inhibitors of MEK or COX-2. The inhibition of prostaglandin biosynthesis by the specific COX-2 inhibitor NS398 and the MEK inhibitor PD98059 confirms that the elevated synthesis of PGF2 is a direct consequence of the up-regulation in expression of the COX-2 gene, which is dependent on phosphorylation of the ERK1/2 pathway. Although we have not demonstrated a direct alteration in phenotype or cell growth in this study, COX-2 has been shown to promote tumorigenesis in many other model systems (8, 28, 29, 43). It is likely that the up-regulated COX-2 in endometrial adenocarcinomas could similarly modulate endometrial tumorigenesis. Hence, endometrial tumorigenesis could be promoted through a self-amplifying loop, triggered by PGF2-FP receptor coupling and activation of the COX-2 gene via a Ras-ERK signaling cascade.
The data presented herein also demonstrate that PGF2-FP receptor activation could potentiate tumorigenesis in vivo. The data obtained from the endometrial adenocarcinoma explants are in agreement with the Ishikawa FPS cell model system and confirm that PGF2-FP receptor interaction can potentiate tumorigenesis in endometrial adenocarcinoma cells by up-regulating the expression of COX-2 and the biosynthesis of prostanoids. Overall, this study outlines the potential application of specific FP receptor antagonist and/or chemical inhibitors targeted toward the ERK1/2 pathway in future therapeutic strategies for treatment of women with endometrial adenocarcinoma (Fig. 6).
Acknowledgments
The authors thank Joan Creiger for tissue collection and Tammy List and Vivien Grant for technical assistance.
Footnotes
Abbreviations: COX, Cyclooxygenase; DN, dominant negative; FP, F-series-prostanoid; GPCR, G protein-coupled receptor; IP, inositol 1,4,5-trisphosphate; MEK, mitogen-associated kinase kinase; PG, prostaglandin.
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