Abstract
Estrogens have significant impact on bone mineral metabolism. Besides the classical estrogen receptors (ERα and ERβ), a trans-membrane G-protein-coupled receptor (GPR30) has been demonstrated to mediate estrogenic effects. We aimed to study whether GPR30 is expressed in bone cells and if so, whether the level of expression is developmentally regulated. Metaphyseal bone biopsies were collected from the tibia in 14 boys and 6 girls, all at different stages of puberty. GPR30 protein expression was studied by immunohistochemistry in paraffin-embedded sections. GPR30-positive osteocytes and osteoblasts were quantified and linear regression analysis was applied. Cytoplasmic GPR30 expression was detected in osteoblasts, osteocytes, and osteoclasts. Osteocytes were more frequently positive for GPR30 than osteoblasts (58±4% vs 46±3% positive cells respectively, P<0.05). Detailed analysis demonstrated that GPR30 positivity declined during pubertal development in osteocytes (R=−0.56, P<0.01) but not in osteoblasts (R=−0.31, P>0.05). No sex difference was observed in the numbers of GPR30-positive osteoblasts or osteocytes. Furthermore, GPR30 expression did not correlate with chronological or bone age. In conclusion, the novel ER GPR30 is expressed in osteoblasts, osteocytes, and osteoclasts suggesting that non-genomic estrogen signaling via GPR30 may exist in bone. However, the functional role of GPR30 in bone tissue remains to be elucidated.
Introduction
Estrogen (17β-estradiol) is an important regulator of bone metabolism, which is illustrated by the low bone mass phenotype in a man carrying a mutation in estrogen receptor-α (ERα; Smith et al. 1994) and in individuals with a deficiency in aromatase activity (Morishima et al. 1995). Furthermore, girls and boys with hypogonadism are known to develop osteopenia, and even though sex steroid replacement in adulthood can increase bone mineral density (BMD) in these individuals, they do not reach adult BMD values (Vanderschueren et al. 2005).
A large proportion of the total bone mass is accumulated during pubertal development in both genders (Bonjour et al. 1994). It has been proposed that bone mass accrual is mediated by the direct action of estrogens on classical ERs. ERα is expressed in osteoblasts and osteocytes in vitro and in vivo (Eriksen et al. 1988, Komm et al. 1988, Braidman et al. 1995, Kusec et al. 1998, Zaman et al. 2006). Mature osteoclasts have been reported to express ERα (Oursler et al. 1991, 1994), while others have found expression in osteoclast precursors but not in mature cells (Kusec et al. 1998, Batra et al. 2003). Another ERβ, has been shown to be expressed in osteoblasts, osteocytes, and osteoclasts (Vidal et al. 1999, Braidman et al. 2001).
Previous in vitro studies in osteoblasts and osteocytes have demonstrated that estrogen can stimulate non-genotropic responses via the Src/Shc/ERK pathway (Kousteni et al. 2001). However, the in vivo role of non-genomic estrogen signaling in bone growth and development is unknown. The description of G-protein-dependent estrogen signaling and membrane localization of estrogen-binding sites has led to the speculation about a trans-membrane ER (Prossnitz et al. 2007). In the late 1990s, a putative receptor was cloned by four different groups (Owman et al. 1996, Carmeci et al. 1997, Takada et al. 1997, O'Dowd et al. 1998). G-protein-coupled receptor 30 (GPR30) mRNA has been demonstrated to be expressed in several tissues, including central nervous system (O'Dowd et al. 1998, Brailoiu et al. 2007), liver (Owman et al. 1996, O'Dowd et al. 1998), and ovary (Wang et al. 2007). Recently, two groups have independently shown that GPR30 acts as a membranous ER and mediates non-genomic estrogen signaling (Revankar et al. 2005, Thomas et al. 2005).
Some functions of estrogen-mediated GPR30 signaling have been suggested previously. These include the non-genomic actions such as MAP kinase activation in breast cancer cells (Filardo et al. 2000), as well as anti-apoptotic (Kanda & Watanabe 2003) and cell growth stimulatory (Kanda & Watanabe 2004) effects on keratinocytes. Recently, GPR30 was demonstrated to mediate the proliferative effects of estrogens in endometrial (Vivacqua et al. 2006a), thyroid (Vivacqua et al. 2006b), and ovarian cancer cells (Albanito et al. 2007), suggesting a role in the development of pathological conditions. Interestingly, GPR30 is differentially regulated by gonadotropins in normal ovarian cells (Wang et al. 2007), indicating that GPR30 may also be important in the regulation of normal estrogen physiology. The cellular and developmental differences in GPR30 expression have been recently demonstrated in human adrenal tissues (Baquedano et al. 2007) and growth plate (Chagin & Savendahl 2007), indicating that GPR30 may mediate local estrogen-dependent effects on cell proliferation and differentiation.
In this study, our aim was to establish whether GPR30 is expressed in human bone cells and whether there is any correlation between the expression level and the pubertal stage.
Materials and Methods
Patients and tissues
Tissue samples were obtained from 14 boys and 6 girls who were subjected to epiphyseal surgery to arrest longitudinal bone growth. The patients suffered from leg length difference or extreme tall stature (Table 1). Biopsies containing metaphyseal bone were taken from the tibial and femoral growth plates employing a bone marrow biopsy needle (8 gauge, Gallini Medical Products and Services, Mirandola, Italy) and immediately fixed in 4% formaldehyde, decalcified, and embedded in paraffin as described previously (Nilsson et al. 2003). The pubertal staging of the patients was performed by a trained pediatric endocrinologist. This study was pre-approved by the local ethics committee at the Karolinska University Hospital, Stockholm, Sweden and informed consent was obtained from each patient and both parents.
Background characteristics of study subjects and levels of G-protein-coupled receptor 30 (GPR30) expression in osteoblasts and osteocytes
| Sex | Age (year: months) | Tanner pubertal stage | GPR30 in osteoblasts (% pos. cells) | GPR30 in osteocytes (% pos. cells) | Diagnosis | |
|---|---|---|---|---|---|---|
| Patient no. | ||||||
| 1 | F | 9:6 | B1 | 52.5 | 79.9 | Leg length difference |
| 2 | F | 12:7 | B1–2 | 65.4 | 78.9 | Leg length difference |
| 3 | F | 12:8 | B2 | 48.4 | 58.3 | Leg length difference |
| 4 | F | 12:6 | B2 | 45.1 | 53.7 | Constitutional tall stature |
| 5 | F | 14:2 | B2–3 | 42.3 | 60.6 | Leg length difference |
| 6 | F | 13:8 | B3 | 46.8 | 46.2 | Leg length difference and CHL |
| 7 | M | 12:3 | G1 | 44.1 | 77.2 | Marfan's syndrome |
| 8 | M | 12:3 | G3 | 51.9 | 60.3 | Leg length difference and FF |
| 9 | M | 14:3 | G3 | 44.6 | 74.8 | Leg length difference |
| 10 | M | 14:10 | G3 | 55.2 | 72.0 | Constitutional tall stature |
| 11 | M | 15:4 | G3–4 | 40.4 | 64.8 | Leg length difference |
| 12 | M | 14:7 | G3–4 | 69.0 | 74.8 | Leg length difference |
| 13 | M | 16:0 | G4 | 25.6 | 18.7 | Leg length difference and FF |
| 14 | M | 13:5 | G4 | 14.6 | 13.1 | Leg length difference |
| 15 | M | 14:7 | G4 | 41.3 | 59.1 | Leg length difference and FTFF |
| 16 | M | 16:4 | G4 | 52.1 | 70.6 | Constitutional tall stature |
| 17 | M | 14:3 | G4–5 | 63.3 | 73.1 | Leg length difference and FF |
| 18 | M | 14:0 | G4–5 | 39.6 | 51.3 | 47, XYY syndrome |
| 19 | M | 14:1 | G4–5 | 64.8 | 44.9 | Leg length difference |
| 20 | M | 12:2 | G5 | 11.4 | 29.5 | Klinefelter's syndrome |
% pos. cells, percentage of positive cells; CHL, congenital hip luxation; FF, femur fracture; FTFF, femur, tibia, and fibula fractures.
Immunohistochemistry
After deparaffinization, immunohistochemical staining of GPR30 in metaphyseal bone sections was performed. Intrinsic peroxidase activity and non-specific binding were blocked by incubation with 3% H2O2 for 10 min, followed by a blocking with 3% horse serum for 1 h. Antigen retrieval was performed in citrate buffer, pH 6.0, for 15 min at +95 °C. The samples were incubated with rabbit anti-serum against human GPR30 (1:1500, a kind gift from Dr E Prossnitz, University of New Mexico, Albuquerque, NM, USA) overnight at +4 °C. After washing, secondary biotin-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch Europe Ltd, Suffolk, UK) was added and incubated for 1 h. Bound antibodies were detected with avidin-conjugated peroxidase (1 h incubation) and 3,3′-diaminobenzidine (3 min; Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with Alcian blue for 2 min. Pre-immune rabbit serum was used as a negative control. Specificity of the anti-GPR30 antibody (Revankar et al. 2005) and its applicability for immunohistochemistry of calcified tissues has been confirmed earlier (Chagin & Savendahl 2007).
In addition, the fluorescent detection of bound antibodies was applied in some sections, where double staining for GPR30 and the osteoclastic marker tartrate-resistant acid phosphatase (TRACP) was performed. Secondary FITC-conjugated anti-rabbit antibody (1:100, Jackson Immunoresearch Europe Ltd) was used for the detection of GPR30-bound antibody and the binding of primary monoclonal anti-human TRACP antibody (1:800, a kind gift from Dr Jussi Halleen, University of Turku, Turku, Finland) was detected with an Alexa633-conjugated mouse antibody (1:300, Molecular Probes Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Some sections were stained for TRACP using a histochemical kit (386-A, Sigma Diagnostics).
Image analysis
Digital images of stained sections were collected by a Nicon Eclipse E800 microscope and an Olympus DP70 digital camera. Three equivalent images of trabecular bone at the primary spongiosa of metaphysis (1.5–2 mm below growth plate) were captured from each sample and used for cell quantification. Osteoblasts were identified as cuboidal cells on trabecular surfaces, while osteocytes were identified as smaller cells within the lacunae inside the bone matrix. The total number of osteocytes (both empty lacunae and lacunae occupied by cells) and the number of GPR30-positive osteocytes were counted manually. For osteoblast quantitation, the total trabecular surface covered with osteoblastic cells and the surface covered by GPR30-positive osteoblasts were measured using the Image-Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA). Cartilaginous areas and areas where bone had detached from the slides were excluded from the analysis. The analysis was performed by a blinded observer. The results are presented as the percentages of cells stained positive for GPR30.
Statistical analysis
Data were expressed as mean±s.e.m. and statistical differences were calculated by Kruskal–Wallis ANOVA on ranks. Regression analysis was used to evaluate developmental trends. Pubertal stage, chronological age, or bone age was used as the independent variable, and the percentage of GPR30-positive cells as the dependent variable. A P value of <0.05 was considered significant.
Results
Immunodetection of GPR30 in human bone
The specificity of the GPR30 antibody used in this study has earlier been well documented, both in human cell lines (Revankar et al. 2005) and tissue sections (Chagin & Savendahl 2007). With an identical immunohistochemical protocol (Chagin & Savendahl 2007), we detected a high level of GPR30 expression in human bone cells. More detailed analysis demonstrated that osteocytes and osteoblasts (Fig. 1A and D) as well as multinucleated osteoclasts (Fig. 1A, B and F) expressed GPR30. GPR30 immunostaining was cytoplasmic in all cells (Fig. 1D and F). Histochemical TRACP staining on comparable sections confirmed the presence of osteoclasts (data not shown). We further confirmed the presence of GPR30 in osteoclasts by performing double staining with TRACP (Fig. 1G) and observed a co-localization of these two proteins (Fig. 1H). No GPR30 staining was observed in negative controls (Fig. 1C and E).
Expression of GPR30 in the human bone. Representative images of bone sections stained for GPR30 (A–E, patient no. 1 in Table 1) and for GPR30 and TRACP (F–H, patient no. 13 in Table 1) as described in Materials and Methods. (A) Positive staining for GPR30 in osteocytes (arrowheads), osteoblasts (block arrows), and an osteoclast (arrow). (B) Positive staining for GPR30 in multiple osteoclasts (arrows). (C) Negative control. (A–C) Scale bar=100 μm. (D) Osteoblasts (arrows) and osteocytes (arrowheads) show cytoplasmic staining of GPR30. (E) Negative control. (D and E) Scale bar=10 μm. (F–H) A multinucleated osteoclast showing cytoplasmic staining of (F) GPR30, (G) TRACP, and (H) a complete co-localization of GPR30 and TRACP (yellow color). (F–H) Scale bar=20 μm. Blue, nuclei; green, GPR30; red, TRACP.
Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-07-0629
Quantitative analysis of GPR30 expression revealed that osteocytes were more often positive than osteoblasts (58±4% vs 46±3% respectively, P<0.05). Furthermore, the newly embedded osteoid–osteocytes appeared to be more frequently positive for GPR30 than osteocytes deeper in the bone matrix (data not shown). When analyzing all samples, we did not observe any gender differences in GPR30 expression. In boys, the percentages of positive osteocytes and osteoblasts were 56±6% and 44±5% respectively, and in girls 63±6% and 50±3% respectively. Even though we observed GPR30 expression in osteoclasts, we could not perform any statistical analysis due to small numbers of these cells.
Expression of GPR30 during pubertal development
When evaluating developmental trends, we observed that the number of GPR30-positive osteocytes varied between different pubertal stages. More cells were positive at earlier than at later pubertal stages. Detailed analysis demonstrated that the number of GPR30-positive osteocytes declined during pubertal development but significantly correlated with the pubertal stage only in girls (R=−0.89, P<0.05, Fig. 2A), although there was a trend toward a decline in boys as well (R=−0.51, P>0.05, Fig. 2A).
Decline in GPR30-positive (A) osteocytes and (B) osteoblasts during pubertal progression. The relationship between the percentages of GPR30-positive (A) osteocytes and (B) osteoblasts in pubertal stage (Tanner) were analyzed by linear regression. The correlation coefficients (R), P values (P), and regression lines for girls (solid) and boys (dashed) are indicated.
Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-07-0629
When separately analyzing patients whose only diagnosis was leg length difference (thus excluding patients with constitutional tall stature and Marfan's, Klinefelter's, and XYY syndromes), a similar decline in the number of GPR30-positive osteocytes was observed during pubertal development (R=−0.61, P<0.05).
In osteoblasts, the level of GPR30 positivity did not correlate with pubertal stage either in boys (R=−0.19, P>0.05 Fig. 2B) or in girls (R=−0.59, P>0.05, Fig. 2B). Similarly, for both boys and girls whose only diagnosis was leg length difference, the number of GPR30-positive osteoblasts did not correlate with pubertal stage (R=−0.19, P>0.05).
Total numbers of bone cells per area were similar in all patients (data not shown), thus confirming that our finding was not dependent on an overall decline in osteocyte or osteoblast density during pubertal development. Furthermore, when the GPR30-positive osteoblasts and osteocytes were plotted against chronological age or bone age, no correlation was observed (data not shown).
Discussion
This study is the first report of GPR30 expression in human bone cells. We observed an intensive GPR30 staining in all three cell types of bone, i.e. osteoblasts, osteocytes, and osteoclasts. The staining was cytoplasmic, which is in accordance with recent reports showing that functional GPR30 has intracellular localization (Filardo et al. 2006, Smith et al. 2007), most likely on the endoplasmic reticulum (Revankar et al. 2007). Since the ligands for GPR30 (i.e. estrogens) are membrane permeable, the intracellular localization is certainly consistent with its function. The fact that osteocytes were more frequently positive for GPR30 than osteoblasts suggests that GPR30 expression is differentially regulated during cell differentiation. Supporting this, the highest level of GPR30 expression was recently observed in terminally differentiated chondrocytes in the human growth plate (Chagin & Savendahl 2007).
Our detailed analysis of the GPR30 staining indicates that the number of GPR30-positive osteocytes negatively correlates with pubertal progression. However, a major limitation of our study is the relatively low number of patients. In our analysis, the lack of girls in late puberty (Tanner stages 4 and 5) and only one male in early puberty (Tanner stage 1) does not allow us to draw any further conclusions on the significance of the observed decline in GPR30 expression during pubertal progression. In addition, since there is a lack of functional data on any effects of GPR30 in bone tissue, it cannot be concluded that such a decline in expression would have any physiological significance.
In line with the previously reported distribution patterns of ERs (van der Eerden et al. 2004), we did not observe any gender difference in the numbers of GPR30-positive bone cells. This suggests that neither classical nor non-genomic estrogen signaling is dependent on gender-specific receptor expression. Moreover, no correlation was observed between the numbers of GPR30 expressing cells and chronological age or bone age, indicating that the level of GPR30 expression might better reflect hormonal status than chronological age or bone age.
In summary, the present study is the first demonstration of GPR30 protein expression in bone cells. Our data clearly show that osteoblasts, osteocytes, and osteoclasts all express GPR30 suggesting that non-genomic estrogen signaling via GPR30 may exist in bone. However, more mechanistic and functional studies are needed to confirm this.
Acknowledgements
This study was supported by Oskar Huttunen Foundation, Jalmari and Rauha Ahokas Foundation, the Swedish Research Council (K2007-54X-15073-04-3), HKH Lovisas Förening för Barnasjukvård, Finnish Cultural Foundation, Research and Science Foundation of Farmos, Sällskapet Barnavård, and Stiftelsen Frimurare Barnhuset i Stockholm. We thank Dr Ola Nilsson and Dr Henrik Wehtje for their tissue collection. The authors declare that there is no conflict of interest.
References
Albanito L, Madeo A, Lappano R, Vivacqua A, Rago V, Carpino A, Oprea TI, Prossnitz ER, Musti AM & Ando S et al. 2007 G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Research 67 1859–1866.
Baquedano MS, Saraco N, Berensztein E, Pepe C, Bianchini M, Levy E, Goni J, Rivarola MA & Belgorosky A 2007 Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues. Journal of Clinical Endocrinology and Metabolism 92 2215–2222.
Batra GS, Hainey L, Freemont AJ, Andrew G, Saunders PT, Hoyland JA & Braidman IP 2003 Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women. Journal of Pathology 200 65–73.
Bonjour JP, Theintz G, Law F, Slosman D & Rizzoli R Peak bone mass Osteoporosis International 4 Suppl. 1 1994 7–13.
Braidman IP, Davenport LK, Carter DH, Selby PL, Mawer EB & Freemont AJ 1995 Preliminary in situ identification of estrogen target cells in bone. Journal of Bone and Mineral Research 10 74–80.
Braidman IP, Hainey L, Batra G, Selby PL, Saunders PT & Hoyland JA 2001 Localization of estrogen receptor beta protein expression in adult human bone. Journal of Bone and Mineral Research 16 214–220.
Brailoiu E, Dun SL, Brailoiu GC, Mizuo K, Sklar LA, Oprea TI, Prossnitz ER & Dun NJ 2007 Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system. Journal of Endocrinology 193 311–321.
Carmeci C, Thompson DA, Ring HZ, Francke U & Weigel RJ 1997 Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 45 607–617.
Chagin AS & Savendahl L 2007 GPR30 estrogen receptor expression in the growth plate declines as puberty progresses. Journal of Clinical Endocrinology and Metabolism 92 4873–4877.
van der Eerden BC, Lowik CW, Wit JM & Karperien M 2004 Expression of estrogen receptors and enzymes involved in sex steroid metabolism in the rat tibia during sexual maturation. Journal of Endocrinology 180 457–467.
Eriksen EF, Colvard DS, Berg NJ, Graham ML, Mann KG, Spelsberg TC & Riggs BL 1988 Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241 84–86.
Filardo EJ, Quinn JA, Bland KI & Frackelton AR Jr 2000 Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Molecular Endocrinology 14 1649–1660.
Filardo EJ, Graeber CT, Quinn JA, Resnick MB, Giri D, DeLellis RA, Steinhoff MM & Sabo E 2006 Distribution of GPR30, a seven membrane-spanning estrogen receptor, in primary breast cancer and its association with clinicopathologic determinants of tumor progression. Clinical Cancer Research 12 6359–6366.
Kanda N & Watanabe S 2003 17beta-estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting Bcl-2 expression. Journal of Investigative Dermatology 121 1500–1509.
Kanda N & Watanabe S 2004 17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression. Journal of Investigative Dermatology 123 319–328.
Komm BS, Terpening CM, Benz DJ, Graeme KA, Gallegos A, Korc M, Greene GL, O'Malley BW & Haussler MR 1988 Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science 241 81–84.
Kousteni S, Bellido T, Plotkin LI, O'Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA & Katzenellenbogen BS et al. 2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104 719–730.
Kusec V, Virdi AS, Prince R & Triffitt JT 1998 Localization of estrogen receptor-alpha in human and rabbit skeletal tissues. Journal of Clinical Endocrinology and Metabolism 83 2421–2428.
Morishima A, Grumbach MM, Simpson ER, Fisher C & Qin K 1995 Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. Journal of Clinical Endocrinology and Metabolism 80 3689–3698.
Nilsson O, Chrysis D, Pajulo O, Boman A, Holst M, Rubinstein J, Martin Ritzen E & Savendahl L 2003 Localization of estrogen receptors-alpha and -beta and androgen receptor in the human growth plate at different pubertal stages. Journal of Endocrinology 177 319–326.
O'Dowd BF, Nguyen T, Marchese A, Cheng R, Lynch KR, Heng HH, Kolakowski LF Jr & George SR 1998 Discovery of three novel G-protein-coupled receptor genes. Genomics 47 310–313.
Oursler MJ, Osdoby P, Pyfferoen J, Riggs BL & Spelsberg TC 1991 Avian osteoclasts as estrogen target cells. PNAS 88 6613–6617.
Oursler MJ, Pederson L, Fitzpatrick L, Riggs BL & Spelsberg T 1994 Human giant cell tumors of the bone (osteoclastomas) are estrogen target cells. PNAS 91 5227–5231.
Owman C, Blay P, Nilsson C & Lolait SJ 1996 Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues. Biochemical and Biophysical Research Communications 228 285–292.
Prossnitz ER, Arterburn JB & Sklar LA 2007 GPR30: a G protein-coupled receptor for estrogen. Molecular and Cellular Endocrinology 265–266 138–142.
Revankar CM, Cimino DF, Sklar LA, Arterburn JB & Prossnitz ER 2005 A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307 1625–1630.
Revankar CM, Mitchell HD, Field AS, Burai R, Corona C, Ramesh C, Sklar LA, Arterburn JB & Prossnitz ER 2007 Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30. ACS Chemical Biology 2 536–544.
Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB & Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. New England Journal of Medicine 331 1056–1061.
Smith HO, Leslie KK, Singh M, Qualls CR, Revankar CM, Joste NE & Prossnitz ER 2007 GPR30: a novel indicator of poor survival for endometrial carcinoma. American Journal of Obstetrics and Gynecology 196 e381–e389.
Takada Y, Kato C, Kondo S, Korenaga R & Ando J 1997 Cloning of cDNAs encoding G protein-coupled receptor expressed in human endothelial cells exposed to fluid shear stress. Biochemical and Biophysical Research Communications 240 737–741.
Thomas P, Pang Y, Filardo EJ & Dong J 2005 Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146 624–632.
Vanderschueren D, Vandenput L & Boonen S 2005 Reversing sex steroid deficiency and optimizing skeletal development in the adolescent with gonadal failure. Endocrine Development 8 150–165.
Vidal O, Kindblom LG & Ohlsson C 1999 Expression and localization of estrogen receptor-beta in murine and human bone. Journal of Bone and Mineral Research 14 923–929.
Vivacqua A, Bonofiglio D, Recchia AG, Musti AM, Picard D, Ando S & Maggiolini M 2006a The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells. Molecular Endocrinology 20 631–646.
Vivacqua A, Bonofiglio D, Albanito L, Madeo A, Rago V, Carpino A, Musti AM, Picard D, Ando S & Maggiolini M 2006b 17beta-Estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the G protein-coupled receptor GPR30. Molecular Pharmacology 70 1414–1423.
Wang C, Prossnitz ER & Roy SK 2007 Expression of G protein-coupled receptor 30 in the hamster ovary: differential regulation by gonadotropins and steroid hormones. Endocrinology 148 4853–4864.
Zaman G, Jessop HL, Muzylak M, De Souza RL, Pitsillides AA, Price JS & Lanyon LL 2006 Osteocytes use estrogen receptor alpha to respond to strain but their ERalpha content is regulated by estrogen. Journal of Bone and Mineral Research 21 1297–1306.
