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Medical Research Council Human Reproductive Science Unit (O.P.M.S., S.B., K.J.S., H.N.J.) and Department of Reproductive and Developmental Sciences (O.P.M.S., H.O.D.C.), University of Edinburgh, Center for Reproductive Biology, Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Science Unit, Centre for Reproductive Biology, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.
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Introduction |
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The IP receptor is known to mediate a cAMP rise and has been termed a relaxant receptor (1), with routine activation of the IP receptor activating adenylate cyclase via the Gs subunit in a dose-dependent manner. Knockout studies disrupting the IP gene in mice have demonstrated thrombotic tendencies and decreased inflammatory responses (6, 7). The cyclical regeneration and repair undergone by the human endometrium during the menstrual cycle necessarily involves physiological processes involving clotting and inflammation. Uterine PGI production may be involved in myometrial smooth muscle relaxation, vasodilation, and prevention of clot formation. These physiological actions are all involved in the process of menstruation, and a role for PGI in menstruation and menstrual disturbances is likely. Indeed, endometrium collected from women with excessive menstrual blood loss has a greater capability of enhancing myometrial PGI production than endometrium collected from women with normal menstrual blood loss (8). Previous studies have also demonstrated an increase in the expression of PGIS and IP receptor mRNA during the menstrual stage compared with the proliferative and secretory stages of the cycle (4).
The cyclical remodeling of the human endometrium requires tight control of angiogenic growth factors to coordinate the growth of new vessel formation. Previous studies have demonstrated that COX enzymes, in particular COX-2 (9), and PGs such as PGE2 (10) and PGF2
(11) can modulate the expression of target angiogenic genes within the human endometrium.
This study was designed to investigate the expression of the IP receptor and its role within the human endometrium. Using an endometrial epithelial cell line (Ishikawa) and human endometrial tissue, we investigated the intracellular signaling transduction pathways activated after PGI-IP ligand-receptor interaction. We found that the IP receptor is spatio-temporally regulated within the glandular epithelial compartment of human endometrium and is colocalized with the epidermal growth factor receptor (EGFR) in human endometrial glandular epithelial cells. Investigation of IP receptor signal transduction pathways using Ishikawa cells and human endometrial tissue showed rapid activation of the ERK1/2 signaling pathway in an EGFR-dependent manner. Moreover, activation of the IP receptor was shown to promote changes in the expression of proangiogenic genes, basic fibroblast growth factor (bFGF), angiopoietin-1 (Ang-1), and Ang-2.
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Materials and Methods |
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Tissue culture
Tissue samples were finely minced using sterile forceps and scissors before overnight incubation in serum-free RPMI medium (as described above) and 3 µg/ml indomethacin (a dual COX enzyme inhibitor to inhibit endogenous prostanoid production). The next day, tissue was pretreated with a specific chemical inhibitor of EGFR kinase (AG1478; 100 nM) for 1 h before stimulation with 100 nM iloprost for the time period stated in the figure legends. After stimulation, tissue was either snap-frozen in dry ice and stored at –20 C for subsequent protein extraction or stored at –70 C for RNA extraction. Protein was harvested by homogenization of tissue in protein lysis buffer. Protein content was determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK).
Cell culture
Ishikawa human endometrial epithelial cells (European Collection of Cell Culture, Wiltshire, UK) were maintained as previously described (10, 13). The cells were grown on monolayer in 6-cm dishes to 60–80% confluence, after which the culture medium was replaced with serum-free DMEM containing 3 µg/ml indomethacin for overnight incubation. The next day, cells were pretreated with specific inhibitors of EGFR kinase (AG1478; 100 nM) or MAPK kinase (MEK; PD98059; 50 µM) for 1 h before stimulation with 100 nM iloprost or 100% (vol/vol) ethanol as a vehicle control for the time period specified in the figure legends. After stimulation with iloprost, proteins were harvested and extracted as described previously (10), and the protein content in the supernatant fraction was determined using a protein assay kit (Bio-Rad Laboratories, Inc.).
TaqMan quantitative RT-PCR
The expression of IP receptor across the menstrual cycle and the effects of iloprost on proangiogenic gene expression in Ishikawa cells or endometrial tissue were investigated by TaqMan quantitative RT-PCR analysis. Total RNA was extracted from endometrial biopsies using an RNeasy Midi Kit (Qiagen, Sussex, UK) according to the manufacturer’s instructions. Samples were treated for DNA contamination by DNA digestion during RNA purification. RNA was extracted from Ishikawa cells as described previously (10, 13). Once extracted and quantified, RNA samples were reverse transcribed and subjected to real-time quantitative PCR using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Warrington, UK) as previously described (10, 13). All primers and probes were designed using the PRIMER express program (Applied Biosystems; Table 1
); IP primers and probes have been previously described (4).
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Transient transfections and immunoprecipitation
To confirm the role of EGFR in iloprost-mediated ERK1/2 phosphorylation, we used a dominant negative (DN) mutant EGFR. Ishikawa cells were seeded to a density of 5 x 105/well in 6-cm dishes, then transfected with a c-Myc-tagged ERK1/2 cDNA construct together with either empty vector cDNA (pcDNA3; Invitrogen Life Technologies, Inc., de Schelp, The Netherlands) or DN-EGFR cDNA using Superfect (Qiagen, Crawley, UK) according to the manufacturer’s protocol (DN-EGFR and c-Myc tagged ERK constructs were gifts from Dr. Zvi Naor, Tel-Aviv University, Tel-Aviv, Israel). Optimal concentrations of cDNA for transfection were determined by titration, and the transfection efficiency of the Ishikawa cell line was determined by transfection with a pcDNA6/V5/His/lacZ cDNA construct (Invitrogen Life Technologies, Inc.) and a ß-galactosidase assay. Transfection efficiency, as reported previously for this cell line using standard procedures according to the manufacturer protocol, is 45 ± 5% (11). The following day, cells were starved by overnight incubation in serum-free medium containing 3 µg/ml indomethacin, then treated with 100 nM iloprost or vehicle for 10 min. Cells were lysed, and protein was quantified as described above. The tagged ERK1/2 was immunoprecipitated from whole-cell lysate. For immunoprecipitation, equal amounts of protein were incubated with specific c-Myc antibody preconjugated to protein A Sepharose overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer, and immune complexes were eluted and 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], boiled for 5 min, and subjected to Western blot analysis.
Western blot analysis
Western blot analysis was conducted to investigate ERK1/2 expression in Ishikawa cells and human endometrial tissue. A total of 50 µg protein from whole-cell lysate was resuspended in 20 µl Laemmli buffer. Proteins were resolved on 4–20% Tris-glycine gels (NOVEX, Invitrogen Life Technologies, Inc.), transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Watford, UK), and subjected to immunoblot analysis as previously described (14). Blots were incubated with anti-phospho-p42/p44 ERK (9101, Cell Signaling Technologies/New England Biolabs, Hertfordshire, UK) and alkaline-phosphatase conjugated secondary antibodies (Sigma-Aldrich Corp., Ayrshire, UK). Immunoblots were stripped and reprobed with antibody recognizing total ERK (sc-93, Santa Cruz Biotechnology/Autogen-Bioclear, Wiltshire, UK). Immunoreactive proteins were visualized by the enhanced chemiluminescence system according to the manufacturer’s instructions (Amersham Biosciences, Little Chalfont, UK). Proteins were revealed and quantified by PhosphorImager analysis using a Typhoon 9400 PhosphorImager (Molecular Dynamics, part of Amersham Biosciences). Relative density in immunoblots was calculated by dividing the value obtained from the phosphorylated immunoblots by the value obtained from the total immunoblots in the same experiment and was expressed as the fold increase above the vehicle control value.
Immunohistochemistry
To investigate the expression of the IP receptor in human endometrium, endometrial sections (5 µm) from across the menstrual cycle were dewaxed in xylene and rehydrated using decreasing grades of ethanol, followed by water. All washes were carried out in 0.01 M PBS (Sigma-Aldrich Corp.). Antigen retrieval was performed by pressure-cooking in 0.01 M sodium citrate (pH 6.0) for 5 min. Thereafter, slides were sequentially incubated with 3% hydrogen peroxide (VWR, Inc., Poole, UK) in distilled water for 10 min (to quench endogenous peroxidase activity), followed by a 15-min incubation with avidin and biotin solutions (Vector Laboratories, Inc., Peterborough, UK) to block endogenous streptavidin activity. Nonspecific binding was further reduced by 20-min incubation with nonimmune horse serum (Vector Laboratories, Inc.) in a humidified chamber at room temperature before overnight incubation with the primary antibody at 4 C. For localization of IP receptor, slides were incubated with a goat polyclonal antibody raised against a peptide mapping near the carboxyl terminus of IP receptor of human origin (sc-20436, Santa Cruz Biotechnology, Inc.) at a 1:30 dilution in normal horse serum. Preabsorption of the antibody with a specific blocking peptide (Santa Cruz Biotechnology, Inc.) was used as the negative control in addition to a control goat IgG antibody at a matched protein concentration to the IP antibody. After a wash in PBS with 0.01% Tween 20, the slides were incubated in biotinylated horse antigoat secondary antibody (Vector Laboratories, Inc.) in normal horse serum at a 1:200 dilution for 60 min at room temperature. Tertiary detection was carried out using an avidin-biotin-peroxidase complex (Vectastain Elite, Vector Laboratories, Inc.) for 60 min at room temperature, and visualization was carried out with the substrate and chromagen 3,3'-diaminobenzidine (DakoCytomation, Carpinteria, CA). Sections were counterstained with hematoxylin, dehydrated in xylene, and mounted.
Scoring and analysis of immunoreactivity
The immunostaining intensity of the IP receptor epitope in all tissue sections was assessed in a semiquantitative manner on a 4-point scale: 0, no immunostaining; 1, mild immunostaining; 2, moderate immunostaining; and 3, intense immunostaining. All tissue sections were scored blind by two observers. This scoring system has been previously validated in a subset of tissue sections in which immunoreactivity was measured with a computerized image analysis system; a strong correlation between quantitative data derived from the image analysis and subjective scores determined by a trained observer was obtained (15).
Immunofluorescent confocal laser microscopy
Colocalization of the site of expression of the IP receptor with EGFR or the endothelial cell marker CD31 was performed in human endometrium by dual-immunofluorescence immunohistochemistry and confocal laser microscopy. Human endometrial sections (5 µm) were dewaxed, rehydrated, and washed as described above. The immunohistochemical methodology was repeated for antigen retrieval by pressure cooking, quenching of hydrogen peroxidase activity, and blocking of endogenous streptavidin activity. Nonspecific binding was further reduced by 20-min incubation with 5% nonimmune rabbit serum diluted in PBS before overnight incubation at 4 C with the polyclonal mouse anti-EGFR primary antibody (NCL-EGFR-384; Nova-Castra, Newcastle-upon-Tyne, UK) at a dilution of 1:25. For colocalization of the IP receptor with CD31, a monoclonal mouse anti-CD31 antibody (DakoCytomation) was used at a dilution of 1:20. Control sections were incubated with polyclonal goat anti-IP primary antibody at a dilution of 1:100 to demonstrate the specificity of the secondary antibody for the mouse primary antibodies. The following day, sections were washed with PBS Tween 20 and incubated with a 1:500 dilution of biotinylated rabbit antimouse IgG for 1 h. An additional 1-h incubation with the fluorochrome streptavidin AlexiFluor 488 (Molecular Probes, Inc., Cambridge Bioscience, Cambridge, UK) diluted at 1:200 in PBS was performed. Next, sections were incubated for 20 min in a PBS solution containing biotin to enhance fluorescent signal before reblocking with 5% nonimmune rabbit serum. Incubation with the goat anti-IP antibody (Santa Cruz Biotechnology, Inc.) at a 1:100 dilution at 4 C overnight was then performed. A second control slide was incubated with mouse anti-EGFR or mouse anti-CD31 primary antibody. Incubation with a 1:200 dilution of rabbit antigoat peroxidase (Vector Laboratories, Inc.) secondary antibody was performed for 30 min. Tertiary detection was performed with an 8-min incubation with tyramide Cy3 solution (PerkinElmer Life Sciences, Boston, MA) at a 1:50 dilution according to the manufacturer’s instructions. Slides were counterstained with To Pro (Molecular Probes, Inc.) at a 1:2000 dilution for 2 min, then mounted in Permafluor (Immunotech-Coulter, Buckinghamshire, UK).
Statistics
Unless otherwise stated and where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (StatView 4.0; Abacus Concepts, Inc., Piscataway, NJ), and statistical significance was accepted at P < 0.05. Semiquantitative scoring results for immunohistochemical staining were analyzed by a nonparametric method, the Kruskal-Wallis test, followed by Dunn’s post hoc multiple comparison test.
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Results |
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). IP receptor mRNA was significantly up-regulated during the menstrual stage of the cycle compared with all other stages in the cycle (P < 0.05).
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). Subjective scoring of IP receptor immunoreactivity showed a significant variation of temporal-spatial expression across the menstrual cycle within the glandular epithelium of the functional layer of human endometrium (Fig. 3; P < 0.05). IP receptor immunostaining was greatest during the proliferative (Fig. 2Ai) and early secretory (Fig. 2Aii) phases within the glandular compartment of the functional layer and was observed to decrease during the late secretory stages (Fig. 2Aiii). High magnification views (Fig. 2B) of a representative endometrial section show the plasma membrane localization of the IP receptor, with some cytoplasmic immunoreactivity also present. Minimal basal layer stromal immunostaining was observed in all tissue sections across the menstrual cycle compared with the functional stroma.
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). IP receptor (red) was observed to colocalize (yellow) with CD31 (green) in the blood vessels of all tissue sections investigated, indicating that IP receptor was present in the vascular compartment.
Iloprost activation of the IP receptor in Ishikawa cells
Treatment of Ishikawa cells with 100 nM iloprost elicited a significant time-dependent increase in phosphorylation of the ERK1/2 pathway, with maximal phosphorylation detected at 5 min (Fig. 4A; P < 0.05). Previous studies in our laboratory have demonstrated that prostanoid (including PGE2 and PGF2) signaling to downstream MAPK pathways involves transactivation of the EGFR (10, 11, 13). To investigate the potential involvement of the EGFR in transducing the PGI-IP receptor signal to ERK1/2, we used the selective EGFR tyrosine kinase inhibitor, AG1478. Preincubation of cells for 1 h with the EGFR kinase inhibitor (AG1478; 100 nM) or inhibitor of MEK (PD98059; 50 µM) abolished the phosphorylation of ERK1/2 in response to a 5-min 100-nM iloprost treatment (Fig. 4B). No significant alteration in basal levels of ERK phosphorylation was observed in cells treated with chemical inhibitor alone (data not shown).
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, lane 2). This elevation in ERK1/2 phosphorylation by iloprost was abolished by cotransfection of cells with the DN-EGFR cDNA (Fig. 4C, lane 3).
PGI-IP receptor signaling in Ishikawa cells promotes the expression of proangiogenic genes
Iloprost stimulation of Ishikawa cells caused a significant fold increase in mRNA expression of the proangiogenic genes, bFGF, Ang-1, and Ang-2, at 24 h compared with earlier time points (Fig. 5A). Cotreatment of the cells with the EGFR kinase inhibitor (AG1478; 100 nM) significantly reduced IP receptor-induced mRNA expression of all target genes (Fig. 5B; P < 0.05).
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) confirmed colocalization (merged; yellow) of the IP receptor (Fig. 6, A and D, red) with the EGFR (Fig. 6, B and E, green). Colocalization was most evident within the glandular epithelial compartments in both basal (Fig. 6, A–C) and functional (Fig. 6, D–F) layers of the endometrium, with minimal stromal cell colocalization.
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) caused a significant phosphorylation of ERK1/2 after 10 min. Preincubation of the tissue with 100 nM AG1478 for 1 h abolished the iloprost-induced phosphorylation of ERK1/2.
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). A similar trend in expression of the two angiopoietin genes, Ang-1 and Ang-2, was observed in response to treatment with 100 nM iloprost. Preincubation of endometrial tissue with 100 nM AG1478 reduced the iloprost-mediated elevation of Ang-1 and Ang-2 expression; however, the reduction was not statistically significant (Fig. 7B). |
Discussion |
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The data presented in this manuscript demonstrate the expression and localization of the IP receptor in epithelial, endothelial, and stromal cells of the human endometrium across the menstrual cycle. IP receptor mRNA is dramatically elevated in human endometrium during the menstrual phase of the cycle and precedes the glandular expression of IP receptor protein, which is highest in the proliferative phase of the menstrual cycle. The IP receptor is a Gs-coupled heptahelical transmembrane receptor that has been shown to activate the protein kinase A pathway, resulting in the production of cAMP (4). Activation of prostanoid receptors, such as E- and F-series prostanoid receptors, results in initiation of numerous effector signaling pathways, including the MAPK pathway (10, 13, 21). The MAPK pathway is a key signaling mechanism that regulates many cellular functions, such as growth, differentiation, and transformation (22, 23). The data presented in this report also demonstrate that activation of the IP receptor induces a rapid increase in p42 ERK phosphorylation in Ishikawa cells and of p42/p44 ERK phosphorylation in endometrial tissue. Interestingly, the differential phosphorylation of ERK between Ishikawa cells and endometrial biopsy tissue may be due to the presence of other cells types (stromal and endothelial) within the biopsy tissue compared with the homogeneity of the Ishikawa cell line. Alternatively, Ishikawa cells originate from an endometrial carcinoma and may respond differently from normal endometrial epithelial cells. This phosphorylation of ERK in Ishikawa cells and endometrial tissues is inhibited with the specific chemical inhibitor of EGFR kinase or by transfection of Ishikawa cells with a DN mutant isoform of the EGFR. Moreover the expression of EGFR colocalized with IP receptor in the glandular epithelial compartment of the human endometrium. Thus, as observed with prostanoid receptor signaling to downstream ERK1/2 in other model systems (10, 11, 24, 25, 26), in the present study, EGFR is in close proximity with the IP receptor, and EGFR transactivation is required for the PGI-IP-induced activation of the ERK1/2 signaling pathway within human endometrium. Whether the EGFR is held in a complex of protein-protein interactions with the IP receptor in the glandular epithelial compartment, or transactivation of the EGFR is mediated by intermediary scaffold adapter molecules to facilitate ERK1/2 signaling remains to be determined.
Our data also demonstrate that IP receptor activation by PGI leads to an increase in mRNA expression of several proangiogenic genes, including bFGF, Ang-1, and Ang-2, in both Ishikawa cells and normal human endometrium. bFGF is potent growth factor, which is known to promote the growth and proliferation of numerous cell types by activation of membrane FGF receptor tyrosine kinases. Moreover, bFGF is known to have potent proangiogenic effects in several model systems (27, 28) and has been implicated in promoting tumor angiogenesis (29, 30) and angiogenesis in proliferative lesions of endometriosis (31). The angiopoietins are a family of growth factors that act as ligands for the largely endothelial-restricted Tie-2 receptor tyrosine kinase, which is essential for vascular development. Ang-1 is a Tie-2 receptor agonist that is required for recruitment of perivascular cells, leading to the formation and stabilization of capillaries, vessel maturation, and endothelial cell survival. Ang-1 and other angiogenic factors, such as vascular endothelial growth factor (VEGF) and bFGF, may act synergistically to increase vascular sprouting and branching. In addition, the Ang-1/Tie-2 interaction enhances the mitogenic effect of angiogenic factors, such as VEGF, on endothelial cell growth (reviewed in Ref.32). By contrast, Ang-2 is a natural Tie-2 receptor antagonist, destabilizing cell contacts and thus allowing access to angiogenic factors, such as VEGF. The process of angiogenesis is thus a fine balance among the expressions of numerous proangiogenic factors, all of which may be present concurrently in the cell to regulate vascularization in response to PGI-IP receptor interaction.
Furthermore, in the present study we have demonstrated that the actions of PGI, via the IP receptor, on target angiogenic gene expression are dependent upon the presence of the EGFR. It is also possible that this up-regulation of expression is mediated by ERK1/2 phosphorylation. This mechanism of target gene regulation in reproductive cells and tissues via prostanoid-receptor interaction is in agreement with previous studies. Transactivation of EGFR and ERK1/2 phosphorylation, leading to increased expression of angiogenic genes, including VEGF, have previously been shown for PGE2-E-series prostanoid receptor 2 (10) and PGF2-F-series prostanoid receptor interaction (11), suggesting that EGFR transactivation is a central theme for the promotion of vascular function in the human endometrium by prostanoids. In the present study we have focused on the role of PGI-IP receptor signaling to angiogenic genes using the Ishikawa endometrial epithelial cell line as a model system; however, it is possible that PGI-IP receptor signaling in stromal and endothelial cells may act in a synergistic manner with epithelial cells in the endometrium to favor angiogenesis, because our parallel studies of whole tissue endometrial biopsy explants are in agreement with our data derived from the Ishikawa cell line.
The precise role of PGI in human endometrium remains to be fully explored; however, PGI has been implicated in menstruation (33) and menstrual disturbances, where levels are elevated in endometrial pathologies such as menorrhagia (excessive menstrual blood loss) (8). It is thus possible that vascular disturbances in the endometrium of women with menstrual pathologies such as menorrhagia may be exacerbated by the elevation of proangiogenic genes, such as bFGF, Ang-1, and Ang-2, brought about by enhanced PGI-IP receptor signaling. In other studies, an aberration of expression levels of angiogenic growth factors has been demonstrated in endometrium from women with menorrhagia, such that a decrease in the expression of Ang-1 mRNA (34) and an increase in Ang-2 protein (35) were reported in endometrium collected from women with heavy menstrual blood loss compared with control endometrium. These alterations in Ang expression are coincident with an increase in the expression of bFGF receptor in the endometrium of women with excessive menstrual blood loss compared with control endometrium (36).
Taken together, we have demonstrated a potential role for PGI-IP receptor signaling in the Ishikawa endometrial epithelial cell line and whole human endometrial biopsy explants in regulating the mRNA expression of several proangiogenic genes. These genes can influence angiogenesis by acting on adjacent endothelial cells in an autocrine/paracrine manner. Moreover, these studies have demonstrated a role for PGI-EGFR cross talk in promoting angiogenic gene expression in the endometrium. Blockade of EGFR signaling with an orally active EGFR tyrosine kinase inhibitor has been used successfully in inhibiting angiogenesis in nude mice (37). Additionally, in a mouse model of colorectal cancer, studies have demonstrated that a combinatorial approach using a nonselective COX enzyme inhibitor in combination with an inhibitor of EGFR kinase is of greater therapeutic benefit than either compound alone (38). These observations of EGFR inhibition and ours reported in the present study suggest that targeted inhibition of EGFR function with small molecule chemical inhibitors alone or in combination with a COX enzyme inhibitor may modulate angiogenic activity in the endometrium, with possible benefits for menstrual pathologies that are associated with aberrant expression and signaling of prostanoids and altered angiogenesis or vascular function (39, 40, 41, 42).
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Footnotes |
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O.P.M.S., S.B., K.J.S., and H.N.J. have nothing to declare. H.O.D.C. has received support for invited presentations at scientific meetings on the management of menstrual bleeding problems from Schering, but not on the context or topic of the scientific content of this manuscript.
First Published Online December 22, 2005
Abbreviations: AA, Arachidonic acid; Ang-1, angiopoetin-1; bFGF, basic fibroblast growth factor; COX, cyclooxygenase; DN, dominant negative; EGFR, epidermal growth factor receptor; IP receptor, G protein-coupled PGI receptor; MEK, MAPK kinase; PG, prostaglandin; PGI, prostacyclin; PGIS, PGI synthase; VEGF, vascular endothelial growth factor.
Received August 23, 2005.
Accepted for publication December 13, 2005.
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References |
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-FP receptor interaction in human endometrial adenocarcinomas. Cancer Res 65:7707–7716 receptor in human endometrial adenocarcinoma: regulation of proliferation by activation of the epidermal growth factor receptor and mitogen-activated protein kinase signaling pathways. J Clin Endocrinol Metab 89:986–993 receptor expression and signaling in human endometrium: role of PGF2 in epithelial cell proliferation. J Clin Endocrinol Metab 88:1825–1832This article has been cited by other articles:
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  O.P.M. Smith, H.N. Jabbour, and H.O.D. Critchley Cyclooxygenase enzyme expression and E series prostaglandin receptor signalling are enhanced in heavy menstruation Hum. Reprod., May 1, 2007; 22(5): 1450 - 1456. [Abstract] [Full Text] [PDF]
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S. Battersby, K.J. Sales, A.R. Williams, R.A. Anderson, S. Gardner, and H.N. Jabbour Seminal plasma and prostaglandin E2 up-regulate fibroblast growth factor 2 expression in endometrial adenocarcinoma cells via E-series prostanoid-2 receptor-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase pathway Hum. Reprod., January 1, 2007; 22(1): 36 - 44. [Abstract] [Full Text] [PDF]
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