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Differential expression of G-protein-coupled estrogen receptor-30 in human myometrial and uterine leiomyoma smooth muscle

      Objective

      To determine differential expression of G-protein-coupled receptor 30 (GPR30) in uterine leiomyoma and its matched myometrium.

      Design

      GPR30 expression examined in both tissues and cultured cells.

      Setting

      Research laboratories.

      Patient(s)

      Women 35 to 50 years old with uterine leiomyomas.

      Intervention(s)

      Hysterectomy.

      Main Outcome Measure(s)

      GPR30 expression profile.

      Result(s)

      Using Western blot and real-time quantitative polymerase chain reaction analyses, we found that GPR30 was highly expressed in uterine leiomyomas compared with their matched myometrium. In only three out of nine patients examined was GPR30 protein detectable by Western blot analysis in myometrial tissues, but at statistically significantly lower levels than in their leiomyomas. Confocal microscopy revealed the nuclear localization of GPR30 in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs). Treatment with 0.1 μM 17β-estradiol increased mRNA expression of GPR30 in leiomyoma SMCs but decreased expression in myometrial SMCs. Treatment with G-1, a GPR30 agonist, stimulated phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in both SMC types. PD98059, the MEK inhibitor, completely inhibited G-1-induced phosphorylation of p44/42 in myometrium SMCs, but not in SMCs from leiomyoma.

      Conclusion(s)

      GPR30 is abundantly expressed in uterine leiomyomas, likely resulting from estrogen stimulation.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/tianr-gpr30-smooth-muscle-cells-uterine-leiomyoma/
      Uterine leiomyoma, characterized by proliferation of smooth muscle cells (SMCs), is the most common benign tumor in women. Because leiomyomas occur in women at reproductive age and regress after menopause, estrogen is believed to play a critical role in the pathogenesis of the tumor. Estrogen receptor-α (ER-α) is abundantly expressed in uterine leiomyomas compared with myometrium (
      • Hermon T.L.
      • Moore A.B.
      • Yu L.
      • Kissling G.E.
      • Castora F.J.
      • Dixon D.
      Estrogen receptor alpha (ERα) phospho-serine-118 is highly expressed in human uterine leiomyomas compared to matched myometrium.
      ), and ER-β is expressed in both myometrium and leiomyoma tissues (
      • Jakimiuk A.J.
      • Bogusiewicz M.
      • Tarkowski R.
      • Dziduch P.
      • Adamiak A.
      • Wrobel A.
      • et al.
      Estrogen receptor alpha and beta expression in uterine leiomyomas from premenopausal women.
      ). However, the expression of the novel ER G-protein-coupled estrogen receptor 1 (GPER1) or G-protein-coupled receptor 30 (GPR30) (
      • Prossnitz E.R.
      • Arterburn J.B.
      • Smith H.O.
      • Oprea T.I.
      • Sklar L.A.
      • Hathaway H.J.
      Estrogen signaling through the transmembrane G protein-coupled receptor GPR30.
      ) has not been investigated in human uterine leiomyomas and their matched myometrial tissues.
      Activation of GPR30 promotes endometrial cell proliferation (
      • Lin B.C.
      • Suzawa M.
      • Blind R.D.
      • Tobias S.C.
      • Bulun S.E.
      • Scanlan T.S.
      • et al.
      Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation.
      ). In endometrial cancer cells, GPR30 mediates the proliferative effects induced by 17β-estradiol (E2) (
      • Vivacqua A.
      • Bonofiglio D.
      • Recchia A.G.
      • Musti A.M.
      • Picard D.
      • Ando S.
      • et al.
      The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17β-estradiol and hydroxytamoxifen in endometrial cancer cells.
      ). GPR30 also mediates E2-induced proliferation of several other cells, such as the mouse spermatogonial GC-1 cell line (
      • Sirianni R.
      • Chimento A.
      • Ruggiero C.
      • De Luca A.
      • Lappano R.
      • Ando S.
      • et al.
      The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17β-estradiol on mouse spermatogonial GC-1 cell line.
      ). The proliferative effect of GPR30 involves the mitogen-activated protein kinase (MAPK) pathway (
      • He Y.Y.
      • Cai B.
      • Yang Y.X.
      • Liu X.L.
      • Wan X.P.
      Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway.
      ). Additionally, GPR30 signaling induces proliferation and migration of breast cancer cells through connective tissue growth factor (
      • Pandey D.P.
      • Lappano R.
      • Albanito L.
      • Madeo A.
      • Maggiolini M.
      • Picard D.
      Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF.
      ).
      In the reproductive tissues, expression of GPR30 was detected in uterus epithelial cells and mediated a cell proliferative response (
      • Dennis M.K.
      • Burai R.
      • Ramesh C.
      • Petrie W.K.
      • Alcon S.N.
      • Nayak T.K.
      • et al.
      In vivo effects of a GPR30 antagonist.
      ). GPR30 mRNA was also detected in mouse uterine tissue (
      • Otto C.
      • Fuchs I.
      • Kauselmann G.
      • Kern H.
      • Zevnik B.
      • Andreasen P.
      • et al.
      GPR30 does not mediate estrogenic responses in reproductive organs in mice.
      ). More recently, the presence of GPR30 mRNA and protein was reported in human myometrium obtained at term cesarean deliveries before or after the onset of labor (
      • Maiti K.
      • Paul J.W.
      • Read M.
      • Chan E.C.
      • Riley S.C.
      • Nahar P.
      • et al.
      G-1-Activated membrane estrogen receptors mediate increased contractility of the human myometrium.
      ). The present study was designed to determine the expression pattern of GPR30 in human myometrium and uterine leiomyoma smooth muscle tissues.

      Materials and methods

      Tissue samples were obtained from patients as detailed in the next section. G-1 was purchased from Cayman Chemical. Antibody for ER-α was purchased from Santa Cruz Biotechnology. GPR30 antibody and peptide were obtained from Novus Biologicals. Antibodies for p44/42 MAPK and phospho-p44/42 MAPK were purchased from Cell Signaling Technology. Alexa Fluor 568-conjugated secondary antibody was obtained from Invitrogen. The α-tubulin antibody and all other reagents were obtained from Sigma-Aldrich.

       Tissue Sample

      Uterine leiomyoma and adjacent normal myometrial tissues were obtained from patients (35 to 50 years old) undergoing hysterectomies at the Tianjin Central Hospital for Obstetrics and Gynecology, Tianjin, People's Republic of China, with approval from the hospital ethics board and patients' consent, as previously described elsewhere (
      • Cui L.
      • Ren Y.
      • Yin H.
      • Wang Y.
      • Li D.
      • Liu M.
      • et al.
      Increased expression of tuberin in human uterine leiomyoma.
      ,
      • Ren Y.
      • Yin H.
      • Tian R.
      • Cui L.
      • Zhu Y.
      • Lin W.
      • et al.
      Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma.
      ). The endometrium of those patients was in the proliferative phase, which was diagnosed through the dilation and curettage (D&C) procedure and by the pathologists in the Pathology Department of Tianjin Central Hospital for Obstetrics and Gynecology. The patients did not receive any hormone therapy or other medications for at least 3 months before surgery. After surgery, tissues were collected and stored in liquid nitrogen for further extraction of protein and RNA for Western blot and real-time quantitative polymerase chain reaction (RT-qPCR) analyses, respectively. Another fraction of fresh tissues were used for primary tissue culture with enzymatic digestion, as previously described elsewhere (
      • Ren Y.
      • Yin H.
      • Tian R.
      • Cui L.
      • Zhu Y.
      • Lin W.
      • et al.
      Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma.
      ).

       Western Blot Analysis

      Cells or homogenized tissues were lysed with ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM β-glycerophosphate, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail (Roche Pharmaceuticals). Protein concentration was determined using the bicinchoninic acid assay (BCA) with protein assay reagent (Pierce) according to the manufacturer's instructions. Equal amounts of protein were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to nitrocellulose membrane (Millipore), followed by blocking with 5% nonfat milk, and incubation with primary antibodies and then horseradish peroxidase-conjugated secondary antibodies. The blot signals were visualized using West Pico Chemiluminescent Substrate Kit (Pierce), imaged by Molecular Image Chemidoc XRS System (Bio-Rad Laboratories) and analyzed using Quantity One software (Bio-Rad Laboratories).
      In the experiment using a GPR30 blocking peptide, primary antibodies for GPR30 were incubated with a fivefold excess of the blocking peptide in a small volume of Tris buffer saline Tween-20 (TBST) for up to 2 hours at room temperature. After blocking with 5% nonfat milk, two polyvinylidene difluoride membranes with transferred proteins were incubated with untreated GPR30 antibodies and blocking peptide-incubated antibodies separately.

       RT-qPCR

      Total RNA from tissues or cultured cells was extracted using Trizol Reagent (Takara Biotechnology). Reverse-transcription was performed using 1 μg of total RNA in a 25 μL reaction volume at 42°C for 60 minutes using a polymerase chain reaction machine (Hangzhou Jingle Scientific Instruments). All real-time PCR experiments were performed on a real-time PCR machine (Bio-Rad Laboratories) with QuantiTect SYBR Green PCR kit and specific primers purchased from Takara Biotechnology and Invitrogen. Quantification of gene expression was assessed with the comparative cycle threshold (Ct) method. The relative amounts of mRNA for the target genes were determined by subtracting the Ct values for these genes from the Ct value for the housekeeping gene β-actin (ΔCt). Data are depicted as 2−ΔCt. qPCR was performed as previously described elsewhere (
      • Cui L.
      • Ren Y.
      • Yin H.
      • Wang Y.
      • Li D.
      • Liu M.
      • et al.
      Increased expression of tuberin in human uterine leiomyoma.
      ).

       Primary Cell Culture

      Human leiomyoma (HL) and matched myometrium (HM) SMCs were isolated from uterine leiomyoma and adjacent normal myometrial tissues, respectively. Leiomyoma and myometrial tissues were cut into small pieces (∼1 mm3), which were then immersed in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS), 0.2% collagenase, and 50 mg/mL of trypsin inhibitor for 2 to 6 hours at 37°C with continuous agitation until cell suspension became evident. Cells were cultured in phenol red-free DMEM with 10% heat-inactivated FBS. Cells from passages three to five were used for the experiments. Extraction of protein and RNA was performed after cells were treated with estrogen for 24 hours in the presence of 10% FBS.

       Immunostaining

      After collection, the tissues were fixed overnight with 4% paraformaldehyde at room temperature and then processed through a graded series of alcohols, followed by embedding in paraffin. Paraffin-embedded tissue sections were cut at 5 μm thickness, dewaxed, and rehydrated in graded alcohol concentrations. Sections were then washed in phosphate-buffered saline (PBS) three times and treated with 0.5% TritonX-100 in PBS (10 minutes). Antigen retrieval was performed in heated 0.01 M citrate buffer (pH 6.4) at 95 to 98°C for 10 minutes, followed by blocking with 10% FBS in PBS for 1 hour at room temperature. Thereafter, the sections were incubated with primary antibodies for GPR30 (1:200 dilution) at 37°C for 1 hour and fluorescein isothiocyanate (FITC) conjugated secondary antibody (1:200 dilution). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Image stacks were acquired by confocal microscopy (Olympus, FV1000).

       Statistical Analysis

      The data were presented as the mean ± standard error of the mean (n = replicate number of experiments). Differences were analyzed using two-tailed paired t-test using Instat 3.0 (Graphpad software), and P<.05 was considered statistically significant.

      Results

       High Expression of GPR30 in Uterine Leiomyoma Tissues

      We analyzed the expression of GPR30 in uterine leiomyomas and their matched myometrial tissues from 13 patients at the Tianjin Central Hospital for Obstetrics and Gynecology. Using Western blot analysis, we detected high expression of GPR30 in leiomyoma tissues compared with matched myometrium. Pretreatment of GPR30 antibody with blocking peptides prevented the appearance of signals at approximately 38 kd in Western blots, confirming the specificity of the GPR30 antibody (Fig. 1A). The position of the GPR30 signal was the same as that observed in MCF-7 breast cancer cells (data not shown). In only three out of nine patients examined was GPR30 expression detectable in myometrial tissues, but the levels were statistically significantly lower than those in the matched leiomyoma tissues. Cumulative results from nine patients showed that GPR30 protein levels in uterine leiomyomas were statistically significantly higher than those in myometrium (see Fig. 1B).
      Figure thumbnail gr1
      Figure 1G-protein-coupled estrogen receptor-30 (GPR30) is overexpressed in uterine leiomyomas. (A) Western blot detection of GPR30 expression using anti-GPR30 antibody without (left) and with (right) blocking peptide treatment. (B) Cumulative Western blot results showing GPR30 protein levels in human myometrium (HM) and leiomyoma (HL) tissues, standardized with α-tubulin. Insert: Representative Western blots (*P<.05, n = 9). (C) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of GPR30 mRNA expression in myometrial and uterine leiomyoma tissues. The y-axis shows the expression level of GPR30 mRNA normalized with β-actin (*P<.05, n = 13).
      Subsequently, we examined GPR30 mRNA levels in 13 patients using RT-qPCR. Consistently, as shown in Figure 1C, the mRNA levels of GPR30 were higher in leiomyoma tissues. In addition, Western blot and RT-qPCR results showed that ER-α was overexpressed in uterine leiomyomas (Supplemental Fig. 1, available online).

       Nuclear Localization of GPR30 in Leiomyoma Tissues

      To visualize the expression of GPR30 in tissue sections of leiomyoma and myometrial tissues, we used immunostaining to detect GPR30 followed by confocal microscopy. Representative micrographs of myometrial and leiomyoma tissue sections from the same patient are shown in Figure 2. The DAPI nuclear staining showed the presence of cells in the tissue sections. Most cells were positively stained for GPR30 in the tissue sections of uterine leiomyomas, but only a few cells were positive in the myometrial tissue sections. Merged images show that GPR30 signals in uterine leiomyoma tissue overlapped with DAPI staining, suggesting their nuclear localization. In myometrium, however, the GPR30 signals were outside the nuclei, indicating a cytosolic localization.
      Figure thumbnail gr2
      Figure 2Immunohistochemical detection of G-protein-coupled estrogen receptor-30 (GPR30) in uterine leiomyoma tissues. Tissue sections were stained with anti-GPR30 antibody and the secondary antibody conjugated with Alexa Fluor 568 (red) with the nuclei counterstained with DAPI (blue). Representative micrographs from tissue sections of uterine leiomyoma (HL) and matched myometrium (HM) were taken under a confocal microscope.

       Estradiol Stimulation of GPR30 mRNA Expression in Cultured SMCs Derived from Uterine Leiomyomas

      Considering that E2 has a potential role in the pathogenesis of uterine leiomyomas, we investigated whether high expression of GPR30 in uterine leiomyoma tissue is related to the presence of E2, or whether E2 can stimulate GPR30 expression. To this end, we performed primary culture of SMCs from uterine leiomyoma and its matched myometrium. The SMCs were characterized by immunostaining and Western blot analyses for SM α-actin and calponin. The SMCs from both myometrium and leiomyoma were stained positive for these smooth muscle marker proteins (Supplemental Fig. 2A, available online), as described in our previous study (
      • Ren Y.
      • Yin H.
      • Tian R.
      • Cui L.
      • Zhu Y.
      • Lin W.
      • et al.
      Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma.
      ). Western blot results showed less SM α-actin and calponin present in leiomyoma SMCs (see Supplemental Fig. 2B). We first evaluated the basal expression levels of GPR30 in cultured HL-SMCs and HM-SMCs using Western blot and RT-qPCR analyses. Our results showed that GPR30 protein and mRNA were much higher in SMCs derived from leiomyoma tissues than myometrium (Fig. 3A and B). The nuclear localization of GPR30 in HL-SMCs was further confirmed by use of confocal microscopy, as shown in Supplemental Figure 3 (available online).
      Figure thumbnail gr3
      Figure 3Estradiol (E2) increases G-protein-coupled estrogen receptor-30 (GPR30) mRNA in cultured smooth muscle cells (SMCs) derived from uterine leiomyoma. Primary cell culture of SMCs from myometrium and uterine leiomyoma was performed using the enzyme digestion approach. Protein and total RNA were extracted from cells from passages 3–5 for Western blot analysis and RT-qPCR, respectively. (A) Cumulative Western blot results showing the expression of GPR30 in cultured SMCs derived from human myometrium (HM) and uterine leiomyoma (HL) (*P<.01, n = 4). Insert: Representative Western blots showing the levels of GPR30 and α-tubulin. (B) Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the mRNA levels of GPR30 in HL-SMCs and HM-SMCs (*P<.01, n = 4). (C) RT-qPCR data showing the mRNA levels of GPR30 in HL-SMCs and HM-SMCs in response to E2 at different concentrations as indicated on the x-axis for 24 hours. The y-axis represents the relative levels of GPR30 normalized by the HM-SMC control (*P<.05, n = 3). (D) RT-qPCR data showing the mRNA levels of GPR30 in HM-SMCs and HL-SMCs in response to treatment with E2 (100 nM) with and without ICI 182,780 (1 μM) as indicated (*P<.05, n = 3).
      Subsequently, we examined the effects of E2 at various concentrations on GPR30 expression in both SMC types. As shown in Figure 3C, treatment with E2 (0–1 μM) for 24 hours stimulated the expression of GPR30 mRNA in SMCs derived from leiomyoma, but decreased the mRNA levels in SMCs from myometrium. However, 24-hour treatment with E2 (0.1 μM) did not statistically significantly regulate GPR30 protein levels in either HL-SMCs or HM-SMCs (data not shown). Pretreatment with ICI 182,780 (1 μM) reversed E2-induced inhibition of GPR30 mRNA expression in HM-SMCs, but further increased the stimulatory effect of E2 on GPR30 expression in HL-SMCs (see Fig. 3D).

       Activation of the MAPK Pathway by G-1

      G-1 has been reported to specifically stimulate GPR30 and activate the MAPK pathway (
      • He Y.Y.
      • Cai B.
      • Yang Y.X.
      • Liu X.L.
      • Wan X.P.
      Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway.
      ). Therefore, we examined the effects of G-1 on phosphorylation of p44/42 MAPK in primary cultured HL-SMCs and HM-SMCs. Representative Western blots showed that 1 μM G-1 treatment for 15 minutes increased the ratio of phospho-p44/42 MAPK and total p44/42 MAPK in both HL-SMCs and HM-SMCs (Fig. 4A). Pretreatment with PD98059 (10 μM), an inhibitor for MEK, abolished phosphorylation of p44/42 induced by G-1 in HM-SMCs. The phosphorylation levels of p44/42 MAPK in the presence of PD98059 were even lower than the control without any treatment. However, the same treatment with PD98059 only partially reduced G-1- induced p44/42 phosphorylation with remaining phosphorylation levels being statistically significantly higher than the control in HL-SMCs (see Fig. 4B).
      Figure thumbnail gr4
      Figure 4Activation of the mitogen-activated protein kinase (MAPK) pathway by G-protein-coupled estrogen receptor-30 (GPR30) agonist G-1. Cultured SMCs (passage 3–5) derived from human leiomyoma smooth muscle cells (HL-SMCs) and myometrium (HM-SMCs) were treated with and without G-1 (1 μM), E2 (100 nM), and PD98059 (10 μM) for 15 minutes as indicated, followed by extraction of protein for Western blot analysis. (A) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without G-1 and PD98059. (B) Cumulative Western blot results showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs with various treatments as indicated. The y-axis represents the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 4, vs. cells without any treatment). (C) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without E2 (100 nM). (D) Cumulative data showing the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 4, vs. control cells). (E) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without G-1 and E2. (F) Cumulative data showing the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 3).
      However, E2 differentially regulated the phosphorylation of p44/42 in HM-SMCs and HL-SMCs. As shown in Figure 4C and D, E2 treatment reduced the basal levels of phospho-p44/42 in HM-SMCs but increased phospho-p44/42 in HL-SMCs. Cotreatment with E2 and G-1 did not increase phospho-p44/42 in HM-SMCs but more potently stimulated p44/42 phosphorylation in HL-SMCs (see Fig. 4E and F).

      Discussion

      For the first time, our study has revealed a high expression level of GPR30 in smooth muscle from uterine leiomyoma compared with matched myometrium using Western blot, RT-qPCR, and immunohistochemical analyses. Stimulation of GPR30 expression by E2 in leiomyoma SMCs suggests a role for E2 in the overexpression of GPR30 in uterine leiomyoma tissues.
      GPR30 expression has been previously reported in vascular SMCs (
      • Haas E.
      • Meyer M.R.
      • Schurr U.
      • Bhattacharya I.
      • Minotti R.
      • Nguyen H.H.
      • et al.
      Differential effects of 17β-estradiol on function and expression of estrogen receptor alpha, estrogen receptor beta, and GPR30 in arteries and veins of patients with atherosclerosis.
      ). It is well known that GPR30 is associated with the cell membrane. However, our confocal microscopy results have demonstrated that GPR30 was localized in the nuclei of leiomyoma cells in tissue sections. GPR30 signals were detected outside the nuclei in a few cells in myometrial tissue. The nuclear localization of GPR30 was further detected in cultured SMCs derived from uterine leiomyoma. The potential significance of its nuclear location in uterine leiomyomas remains to be investigated. Because GPR30 mediates the proliferation of various cancer cells, such as breast cancer (
      • Pandey D.P.
      • Lappano R.
      • Albanito L.
      • Madeo A.
      • Maggiolini M.
      • Picard D.
      Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF.
      ), sperm cell tumor (
      • Sirianni R.
      • Chimento A.
      • Ruggiero C.
      • De Luca A.
      • Lappano R.
      • Ando S.
      • et al.
      The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17β-estradiol on mouse spermatogonial GC-1 cell line.
      ), and endometrial cancer (
      • Vivacqua A.
      • Bonofiglio D.
      • Recchia A.G.
      • Musti A.M.
      • Picard D.
      • Ando S.
      • et al.
      The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17β-estradiol and hydroxytamoxifen in endometrial cancer cells.
      ), our findings also suggest a role for GPR30 in the development of smooth muscle tumors.
      GPR30 was discovered using the technique of differential cDNA library screening from an ER-positive MCF-7 cell cDNA library (
      • Carmeci C.
      • Thompson D.A.
      • Ring H.Z.
      • Francke U.
      • Weigel R.J.
      Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer.
      ). This study revealed that GPR30 is expressed in cancer cells with overexpression of the classic nuclear ER, but not in an ER-negative cell line (MDA-MB-231). However, other studies have shown that GPR30 is also expressed in ER-negative cells, such as SkBr3 breast cancer cells (
      • Thomas P.
      • Pang Y.
      • Filardo E.J.
      • Dong J.
      Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells.
      ). Our results reveal that both GPR30 and ER-α are highly expressed in uterine leiomyoma tissues. Notably, coexpression of GPR30 with ER-β is also associated with disease progression in uterine carcinosarcoma (
      • Huang G.S.
      • Gunter M.J.
      • Arend R.C.
      • Li M.
      • Arias-Pulido H.
      • Prossnitz E.R.
      • et al.
      Co-expression of GPR30 and ERbeta and their association with disease progression in uterine carcinosarcoma.
      ). Our finding is consistent with reports showing high expression of ER-α in leiomyoma tissues (
      • Valladares F.
      • Frías I.
      • Báez D.
      • García C.
      • López F.J.
      • Fraser J.D.
      • et al.
      Characterization of estrogen receptors alpha and beta in uterine leiomyoma cells.
      ).
      GPR30 deficient mice appear to be as normal and fertile as wild-type mice (
      • Otto C.
      • Fuchs I.
      • Kauselmann G.
      • Kern H.
      • Zevnik B.
      • Andreasen P.
      • et al.
      GPR30 does not mediate estrogenic responses in reproductive organs in mice.
      ,
      • Windahl S.H.
      • Andersson N.
      • Chagin A.S.
      • Mårtensson U.E.A.
      • Carlsten H.
      • Olde B.
      • et al.
      The role of the G protein-coupled receptor GPR30 in the effects of estrogen in ovariectomized mice.
      ). In humans, up-regulation of GPR30 has been detected in the pregnant uterus (
      • Maiti K.
      • Paul J.W.
      • Read M.
      • Chan E.C.
      • Riley S.C.
      • Nahar P.
      • et al.
      G-1-Activated membrane estrogen receptors mediate increased contractility of the human myometrium.
      ). It is interesting that E2 differentially regulates GPR30 mRNA expression in HM-SMCs and HL-SMCs. That ICI 182,780, an ER-α and ER-β antagonist, reverses E2-induced inhibition of GPR30 expression suggests an involvement of the classic ER(s) in the E2 effect on HM-SMCs. As ICI 182,780 can also activate GPR30 (
      • Thomas P.
      • Pang Y.
      • Filardo E.J.
      • Dong J.
      Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells.
      ,
      • Boulware M.I.
      • Weick J.P.
      • Becklund B.R.
      • Kuo S.P.
      • Groth R.D.
      • Mermelstein P.G.
      Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein.
      ,
      • Filardo E.J.
      • Quinn J.A.
      • Frackelton Jr., A.R.
      • Bland K.I.
      Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis.
      ), our finding that ICI 182,780 treatment further stimulates E2-induced GPR30 expression suggests a potential involvement of GPR30 activation in its own gene expression. Taken together, our results have revealed a connection between E2 and the high level of GPR30 in leiomyoma tissues.
      Activation of GPR30 was previously reported to stimulate phosphorylation of p44/42 MAPK in various cells, including human endometrial cancer cells (
      • He Y.Y.
      • Cai B.
      • Yang Y.X.
      • Liu X.L.
      • Wan X.P.
      Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway.
      ). Consistently, treatment with G-1, a specific activator of GPR30 (
      • Bologa C.G.
      • Revankar C.M.
      • Young S.M.
      • Edwards B.S.
      • Arterburn J.B.
      • Kiselyov A.S.
      • et al.
      Virtual and biomolecular screening converge on a selective agonist for GPR30.
      ), increased phosphorylation of p44/42 MAPK in SMCs cultured from either uterine leiomyoma or myometrium. It is important to note that PD98059, the MEK inhibitor, not only inhibited G-1-induced phosphorylation of p44/42 but also reduced its phosphorylation below the basal level in HM-SMCs. In HL-SMCs, however, PD98059 only inhibited G-1-induced phosphorylation of p44/42, further suggesting a differential activation of the MAPK pathway in both cell systems as we previously described for epidermal growth factor (EGF) (
      • Ren Y.
      • Yin H.
      • Tian R.
      • Cui L.
      • Zhu Y.
      • Lin W.
      • et al.
      Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma.
      ). Treatment of HL-SMCs with both E2 and G-1 more significantly increased phospho-p44/42 than G-1 alone. Unlike E2 stimulation of p44/42 phosphorylation in HL-SMCs, E2 itself reduces the basal activity of the MAPK in HM-SMCs. In the presence of E2, G-1 did not further increase phospho-p44/42, suggesting a potential interaction between ER-α and GPR30 in HM-SMCs.
      This finding suggests that up-regulation of GPR30 by E2 may alter intracellular signaling in leiomyoma SMCs. However, caution must be taken in interpreting the role of GPR30 in the pathogenesis of uterine leiomyoma, because GPR30 has been also reported to inhibit proliferation of ER-positive breast cancer cells (
      • Ariazi E.A.
      • Brailoiu E.
      • Yerrum S.
      • Shupp H.A.
      • Slifker M.J.
      • Cunliffe H.E.
      • et al.
      The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells.
      ). Nevertheless, our study has uncovered differential expression of GPR30 in SMCs from uterine leiomyoma and myometrium. Although there are challenges, such as the limited cell growth of leiomyoma and myometrium SMCs in primary culture, toward understanding the role of GPR30 in uterine leiomyomas, further investigation of GPR30 in SMCs is warranted.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Estrogen receptor-α (ER-α) is highly expressed in uterine leiomyoma tissues. Human myometrial (HM) and uterine leiomyoma (HL) tissues were sampled from patients, and protein and RNA were extracted for Western blot and RT-qPCR, respectively. (A) Cumulative data showing the protein levels of ER-α normalized by α-tubulin in HM and HL tissues. Insert: Representative Western blots. (*P<.05, n = 9). (B) Real-time quantitative polymerase chain reaction (RT-qPCR) results showing the mRNA levels of ER-α in both tissues (*P<.05, n = 13).
      Figure thumbnail fx2
      Supplemental Figure 2Characterization of primary cultured smooth muscle cells (SMCs) isolated from human leiomyoma (HL) and matched myometrial tissues (HM). SMC marker proteins, such as SM α-actin and calponin, were detected in cultured HL-SMCs and HM-SMCs using (A) immunostaining and (B) Western blot analysis. In immunostaining and fluorescent microcopy, the nuclei were counterstained with propidium iodide (PI).
      Figure thumbnail fx3
      Supplemental Figure 3Immunocytochemical detection of G-protein-coupled estrogen receptor-30 (GPR30) in cultured smooth muscle cells (SMCs). The SMCs were cultured from human leiomyoma and its matched myometrium, fixed with 100% methanol and then stained with anti-GPR30 and the secondary antibody conjugated with Alexa Fluor 568 (red) with the nuclei counterstained with 4′,6-Diamidino-2-phenylindole (DAPI) (blue). Representative micrographs were acquired by confocal microscopy.

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