Introduction

Estrogen is a pleiotropic hormone with a broad spectrum of physiological functions ranging from regulation of the menstrual cycle and reproduction to modulation of bone density, brain function, and cholesterol mobilization. As a mitogen, estrogen also promotes the development of endometrial cancer and breast cancer, which account for about 40% of cases of cancer in women (Shang 2007). The classical mechanism of estrogen action involves binding to the estrogen receptors (ERs) α and β, which are members of the nuclear hormone receptor superfamily with modular structures and discrete domains for their specific functions as ligand-regulated transcription factors. The sequential activation of genes occurs via multiple mechanisms, either through direct binding at estrogen response elements in the promoter region of estrogen-responsive genes or through protein–protein interactions with sites that are canonical to other well-known transcription factors such as AP1, SP1, and nuclear factor κB (Shang 2006; Pietras et al. 2005; Kumar and Chambon 1988). Following a large number of studies, G protein-coupled receptor (GPR) 30 is now widely recognized as an estrogen receptor that appears to alter gene expression in a rapid, “nongenomic” manner independently of the nuclear ERs and mediates a wide range of responses to estrogen in different cell types (Revankar et al. 2005).

GPR30 is a member of the seven-transmembrane GPR family, which signals through heterotrimeric guanine nucleotide-binding proteins to alter the activity of effector proteins. Cellular activation by GPR30 occurs through a mechanism involving transactivation of epidermal growth factor receptors (EGFRs) via a G protein-dependent pathway and activation of mitogen-activated protein kinase (MAPK), adenylyl cyclase, and phosphoinositide 3-kinase (PI3K) (Thomas et al. 2005; Filardo et al. 2000, 2002). GPR30 has been found to act in physiological processes such as the attenuation of liver injury and pubertal adrenal development (Wang et al. 2007; Baquedano et al. 2007) and has also been shown to mediate the proliferative effects of estrogen in a number of cancer cell lines (Vivacqua et al. 2005, 2006; Albanito et al. 2007). Furthermore, we have found that 17β-estradiol (E2) and G1, a GPR30-specific ligand, stimulate the production and activity of matrix metalloproteinase (MMP), the proteolysis of which is involved in cancer metastasis, via the GPR30-mediated MEK/ERK MAPK pathway, and increased secretion of interleukin-6, the activation of which is associated with tumor growth (He et al. 2009). In the present study, we show that GPR30 mediates invasion induced by E2 in an endometrial cancer cell line and plays a crucial role in endometrial tumorigenesis. Our results provide new evidence for the role of GPR30 in endometrial carcinogenesis.

Materials and methods

Cell culture

The human endometrial cell line RL95-2 was obtained from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (11030; Gibco, Auckland, New Zealand) supplemented with 10% fetal bovine serum (FBS) (16000-44; Gibco, Carlsbad, CA), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2/95% air at 37°C.

Stable transfection

One hundred thousand cells were plated into 24-well dishes with 500 μL regular growth medium per well on the day before transfection. The cells were transfected with short hairpin RNA (shRNA) constructs against GPR30 in pGFP-V-RS (ShGPR30-pGFP-V-RS vector, TG316565; OriGene Technologies, Rockville, MD) (Fig. 1) and the HuSH 29-mer noneffective against enhanced GFP vector (shiv-pGFP-V-RS vector, pRS.TR30003; OriGene Technologies) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. To obtain a stable cell line, selection pressure was maintained with puromycin at a concentration of 0.5–1.0 μg/mL in the growth medium for 6 weeks. Clonal populations of cells were selected by transferring well-isolated single clumps of cells (the clonal ancestor and cells divided from it) into a six-well plate. Growth of these cells was continued in the selection medium for two additional passages.

Fig. 1
figure 1

The pGFP-V-RS plasmid vector pattern and interferential efficiency of ShGPR30-pGFP-V-RS vectors. a The pGFP-V-RS plasmid vector was created with an integrated turboGFP element to readily verify transfection efficiency. It also incorporates both a kanamycin and puromycin resistance elements for greater selection capabilities. The shRNA expression cassette consists of a 29-nt GPR30-specific sequence, a 7-nt loop, and another 29-nt reverse complementary sequence, all under the control of the human U6 promoter. A termination sequence (TTTTTT) is located immediately downstream of the second 29-nt reverse complementary sequence to terminate the transcription by RNA Pol III. b The RNA expression levels of RL95-2 cells transfected with each of four different ShGPR30-pGFP-V-RS vectors were determined using real-time RT-PCR. RNA expression of RL95-2 cells transfected with shiv-pGFP-V-RS (negative control) was set at 1, from which the RNA expression of the other cells was calculated. c RL95-2 cells were visualized by fluorescence microscopy after stable transfection with pGFP-V-RS(b). a Cells transfected with ShGPR30-pGFP-V-RS under white light. b Cells transfected with ShGPR30-pGFP-V-RS under fluorescence. c Cells transfected with shiv-pGFP-V-RS (negative control) under white light. d Cells transfected with shiv-pGFP-V-RS (negative control) under fluorescence. Magnification, (a, b) ×200; (c, d) ×100

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Real-time RT-PCR

Total RNA was extracted from the transfected RL95-2 cells using TRI Reagent (TR118; Molecular Research Center, Cincinnati, OH). cDNA was generated with oligo(dT)18 primers using a Revert Aid First Strand cDNA Synthesis Kit (K1622; Fermentas Life Science, St. Leon-Rot, Germany). A 50-μL PCR amplification of single-strand cDNA was performed with 35 cycles of denaturation (94°C) for 60 s, annealing (55°C) for 30 s, and elongation (72°C) for 30 s using PerfectShot Ex Taq (Loading Dye Mix) (DRR05TA; Takara, Dalian, China). The primer sequences are shown in Table 1.

Table 1 Primer sequences of G protein-coupled receptor (GPR) 30 and β-actin used for real-time RT-PCR
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Proliferation assay

RL95-2 cells were seeded in 96-well plates at 2 × 105 cells/mL and cultured for 24 h. The cells were then switched to phenol red-free and serum-free medium (DMEM/F12, 11039; Gibco, Carlsbad, CA) and cultured for 24 h, followed by the addition of G1, a nonsteroidal GPR30-specific agonist (10−9 ~ 10−5 M) (371705; Calbiochem), or E2 (10−9 ~ 10−5 M) (E2758-250MG; Sigma Aldrich, St. Louis, MO), in the presence or absence of the MEK/ERK MAPK inhibitor U0126 (30 μM) (9903; Cell Signaling Technology) or pertussis toxin (PTX) (200 ng/mL; 516560; Calbiochem). The cells were further cultured for 48 h with phenol red-free DMEM/F12 containing 5% dextran-coated charcoal-treated FBS (S181F; Biowest, Nuaillé, France). Cell proliferation was evaluated using a Cell Counting Kit-8 (CK04-11; Dojindo Molecular Technologies, Gaithersburg, MD). The controls were treated with vehicle (0.1% dimethyl sulfoxide/phenol red-free DMEM/F12).

Transwell® assay

The Matrigel invasion assay was carried out as previously described with slight modification (Sato et al. 2002). The cell culture inserts (8 μm pore size, 6.5 mm diameter; Corning, Corning, NY, USA) coated with 5 μL pure Matrigel (356237, BD Biosciences) were placed in a 24-well plate. Before use, the Matrigel was rehydrated with 100 μL warm DMEM for 2 h. The lower chamber was filled with 800 μL DMEM with 1% FBS. The cells were allowed to invade for 12 h in 5% CO2 at 37°C. RL95-2 cells were plated in the upper chamber. The cells were treated as in “Proliferation assay”. The lower chamber was filled with 800 μL DMEM/F12 containing 5% dextran-coated charcoal-treated FBS. The cells were then incubated at 37°C for 48 h. After swabbing of noninvaded cells in the upper chambers, cells that migrated to the lower chambers were fixed with 3.7% paraformaldehyde in PBS and stained with hematoxylin. For quantification, the cells that had migrated to the lower surface were counted under a light microscope in five predetermined fields at a magnification of ×200. The assay was repeated three times, and the results are expressed as a percentage to the mean number of three controls. Each experiment was carried out in triplicate and repeated three times.

Xenograft tumor formation assays

Intact, female athymic nu/nu mice of 5–6 weeks of age were obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Science. The tumorigenicity of the cells was assessed by subcutaneously injecting 10 × 106 cells bilaterally into five mice for each treatment; a total of 10 mice were thus used. Tumor measurement began one week after injection and was conducted weekly using digital calipers. Tumor volume was calculated as follows: (1/2 × length)(width2). All procedures were approved by the University of Shanghai Jiao Tong University Affiliated First People’s Hospital Animal Care and Use Committee.

Statistics

Statistical analysis was performed using the t test or one way analysis of variance. All tests were performed using Statistical Product and Service Solutions software version 15.0 (SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant.

Results

Expression of GPR30 is stably downregulated by transfection with ShGPR30-pGFP-V-RS

The commercial plasmid pGFP-V-RS (Fig. 1a) contains an inserted sh sequence that can either suppress the expression of GPR30 (ShGPR30-pGFP-V-RS) or is invalid (shiv-pGFP-V-RS). Four designed and sequence-verified shRNA vectors (construct 1–4) are available for GPR30. The vector also contains the pCMV driven tGFP gene, which encodes the tGFP protein constitutively in mammalian cells. This feature makes it possible to monitor the efficiency of transfection.

It took 42 days to obtain stably transfected RL95-2 cells. The interferential efficiency of the different vectors was then determined using real-time RT-PCR. Construct 2 showed the best interference effect (Fig. 1b). The successfully transfected cells were also examined under a microscope, through which green fluorescence from GFP protein expressed by pGFP-V-RS could be seen (Fig. 1c).

GPR30 signaling stimulated proliferation of endometrial cancer cell line RL95-2

To determine whether GPR30 signaling could induce growth of endometrial cancer cells, proliferation assays were conducted. We carried out a proliferation assay to test whether GPR30 signaling could have effects on endometrial cancer cell proliferation. We found that E2 (10−8 M) and G1 (10−8 M) increased the proliferation of endometrial cancer cell line RL95-2 (Fig. 2a). A longer duration of treatment (48 h vs. 24 h) had a greater proliferation-promoting effect (Fig. 2c), suggesting that the ability of G1 and E2 to promote proliferation is time dependent in RL95-2 cells. The stimulation of proliferation by E2 and G1 was disrupted by PTX, an inhibitor of G-protein signaling, and U0126, an inhibitor of MEK/ERK MAPK. When RL95-2 cells were transfected with ShGPR30-pGFP-V-RS, their proliferation declined even in the presence of E2 and G1, versus the negative control group (Fig. 2d). These results suggest that GPR30 has a crucial signaling role in the proliferation of the endometrial cancer cell line RL95-2. Through the GPR30-mediated MEK/ERK MAPK pathway, G1 and E2 could stimulate the proliferation of RL95-2.

Fig. 2
figure 2

G protein-coupled receptor 30 via the MEK/ERK mitogen-activated protein kinase (MAPK) pathway stimulates proliferation of an endometrial cancer cell line. a RL95-2 cells were treated with 17β-estradiol (E2, 10−9 ~ 10−5M) or G1 (10−9 ~ 10−5 M) for 24 h in medium containing 5% charcoal-stripped fetal bovine serum then analyzed on the indicated cultured times. # P < 0.05 versus the other five control groups, * P < 0.05 versus the other five RNAi groups; b RL95-2 cells were treated with E2 (10−8M) and G1(10−8M) for 24 and 48 h. # P < 0.05 versus vehicle 24 h; * P < 0.05 versus vehicle 48 h. c, d: RL95-2 cells were treated with E2 (10−8 M) or G1 (10−8 M) with or without pertussis toxin (PTX) (200 ng/mL) or U0126 (30 μM) in medium containing 5% charcoal-stripped fetal bovine serum. Cells were further cultured for 24 h before evaluation of cell proliferation. Vehicle: 0.1% dimethyl sulfoxide/phenol red-free and serum-free Dulbecco’s modified Eagle’s medium/F12. Negative control: cells transfected with HuSH 29-mer noneffective against enhanced green fluorescent protein (shiv-pGFP-V-RS vector). RNAi: cells transfected with HuSH 29-mer short hairpin RNA against GPR30 in pGFP-V-RS vector (ShGPR30-pGFP-V-RS vector). * P < 0.05; Δ P > 0.05; & P < 0.05 versus the negative control. Medium was refreshed and treatments were renewed every 2 days. Each data point is the mean ± SD of three independent experiments

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GPR30 signaling promoted invasion by endometrial cancer cell line RL95-2

The effect of E2 and G1 on RL95-2 invasion was determined by a Transwell® assay. To determine the role of GPR30 in endometrial cancer cell invasion induced by E2 and G1 in vitro, RL95-2 cells transfected with ShGPR30-pGFP-V-RS or shiv-pGFP-V-RS were treated with E2 (10−8M) or G1 (10−8 M) with or without PTX or U0126. The results revealed that knockdown of GPR30 inhibited RL95-2 cell invasion induced by G1 or E2. Furthermore, incubation with PTX or U0126 markedly decreased cell invasion compared to the control (Fig. 3). These data suggest that GPR30 signaling promoted endometrial cancer cell invasion via the MEK/ERK MAPK pathway.

Fig. 3
figure 3

G protein-coupled receptor 30 stimulates invasion by an endometrial cancer cell line via the MEK/ERK mitogen-activated protein kinase (MAPK) pathway. RL95-2 cells stably transfected with shiv-pGFP-V-RS (negative control; a–g) or ShGPR30-pGFP-V-RS (h–n) were treated as follows. a, h Control group (vehicle 0.1% dimethyl sulfoxide/phenol-free Dulbecco’s modified Eagle’s medium/F12; b, i 17β-estradiol (E2) (10−7 M); c, j E2 (10−7 M) with U0126 (30 μM); d, k E2 (10−7 M) with pertussis toxin (PTX) (200 ng/mL); e, l G1 (10−8 M); f, m G1 (10−8 M) with U0126 (30 μM); g, n G1 (10−8 M) with PTX (200 ng/mL). Cell invasion was then assessed and photomicrographs taken. Quantitation (o, p) and representative photomicrographs (a–n) show inhibition of invasion by RL95-2 cells transfected with ShGPR30-pGFP-V-RS vector (RNAi) compared with the negative control (shiv-pGFP-V-RS). Data are expressed as the mean ± SD. * P < 0.05 compared with the negative control

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GPR30 signaling in endometrial carcinoma cells enhanced tumorigenic capacity

To determine whether GPR30 signaling has a direct effect on tumor cell proliferation in vivo, we implanted RL95-2 cells transfected stably with ShGPR30-pGFP-V-RS or shiv-pGFP-V-RS into female athymic mice using PBS as a vehicle. Significant differences were observed in the mean tumor volumes. Both cell lines formed palpable tumors in less than one week. However, compared with the control group, significant (P < 0.05) decreases in volume were observed from week 4 to week 8 in tumors produced by RL95-2 cells transfected with ShGPR30-pGFP-V-RS, and this difference increased with time (Fig. 4). All animals survived for the duration of the study in apparent good health.

Fig. 4
figure 4

G protein-coupled receptor 30 (GPR30) signaling in endometrial carcinoma cells enhances their tumorigenic capacity. a Volume of tumors produced by RL95-2 cells stably transfected with shiv-pGFP-V-RS (control) or ShGPR30-pGFP-V-RS (GPR30 RNAi) in nude mice over 56 days and b tumors taken from nude mice 56 days after inoculation of RL95-2 cells. Data are expressed as the mean ± SD

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Discussion

The classic or genomic mode of ER action is slow compared with rapid or nongenomic estrogen responses, which can occur within minutes and are mediated by two distinct mechanisms involving membrane ERs. One mechanism involves the localization of nuclear ERs at the plasma membrane (Pedram et al. 2006; Levin 2005; Harrington et al. 2006), whereas the second involves the receptor GPR30.

GPR30 transcripts are reported to be widely distributed in normal human tissues, with high levels found in the heart, lung, liver, intestine ovary, and brain. Accordingly, GPR30 has extensive functions. For example, in the immunological system, studies of rat models have shown GPR30 to be involved in thymocyte apoptosis, and in the circulatory system, the reduction in blood pressure induced by E2 may be mediated by GPR30.

Expression of GPR30 has been observed in several cancer cell lines, and a number of reports have shown estrogen-mediated cell proliferation to be dependent on GPR30 (Vivacqua et al. 2005, 2006; Albanito et al. 2007). In the present study, we found that GPR30 signaling promotes proliferation of the endometrial cancer cell line RL95-2 via the MEK/ERK MAPK pathway. Invasion is a major marker of malignancy and prognosis. In our previous study, we showed that GPR30 signaling via the MEK/ERK MAPK pathway increased MMP production and activity in endometrial cancer cells. In the present study, a Transwell® assay showed that GPR30 signaling promoted endometrial cancer cell invasion though the MEK/ERK MAPK pathway. Together, these findings suggest that GPR30 plays a role in carcinogenesis.

Several studies have examined the expression of GPR30 in vivo. Filardo et al. (2006) performed immunohistochemistry for GPR30 in 361 breast carcinomas and found that overexpression of GPR30 was significantly associated with tumor size (>2 cm), the presence of distant metastases, and HER-2/neu overexpression. Similar studies (Smith et al. 2007; Ignatov et al. 2010) have been conducted on tumors from patients with endometrial adenocarcinoma, and GPR30 overexpression has been found to occur more frequently in endometrial carcinomas exhibiting deep myometrial invasion, high grade, biologically aggressive histological subtype, advanced stage, and significantly worse overall survival rate. Interestingly, there was a significant correlation between G protein-coupled estrogen receptor expression and endometrial pathology in breast cancer patients receiving tamoxifen, an estrogen antagonist with antiproliferative properties in the breast but proliferative properties in the endometrium (Ignatov et al. 2010). In the present study, we have demonstrated for the first time that GPR30 signaling in endometrial carcinoma cells enhances their tumorigenic capacity. All the in vivo studies give strong support to an association between GPR30 and the occurrence and development of endometrial and breast cancer.

In summary, our findings demonstrate that GPR30 signaling promotes the proliferation of endometrial cancer cells and increases their potential for invasion through the MEK/ERK MAPK pathway, providing further evidence for the crucial role of GPR30 in endometrial carcinogenesis. Recently, several groups have provided more details of the mechanism of GPR30 signaling. Lin et al. (2009) found a novel signaling paradigm that is initiated by activation of GPR30 by estrogen, with signal transduction cascades (PI3K and MAPK) that converge on nuclear hormone receptors (steroidogenic factor-1/liver receptor homolog-1) to modulate their transcriptional output. GPR30 expression has been found to be induced by EGF and transforming growth factor through the EGFR/ERK transduction pathway in endometrial and tamoxifen-resistant breast cancer cells (Vivacqua et al. 2009). Considering that GPR30 might activate the EGFR/ERK pathway through the release of heparin-binding EGF, we could assume that a positive feedback loop occurs within the ligand/EGFR/GPR30 signaling network leading to the stimulation of estrogen-sensitive tumors. Like the ER signaling network, the GPR30 network must be complicated and needs further research.