The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells

Toxicology and Applied Pharmacology 245 (2010) 83–90

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells Xinyuan Yu a,1, Edward J. Filardo b, Zahir A. Shaikh a,⁎ a Department of Biomedical and Pharmaceutical Sciences, and Center for Molecular Toxicology, 41 Lower College Road, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA b Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, RI 02903, USA

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Article history: Received 24 September 2009 Revised 1 February 2010 Accepted 3 February 2010 Available online 11 February 2010 Keywords: Cadmium Endocrine disruptors Estrogen receptors GPR30 Breast cancer Cell proliferation SKBR3 cells

a b s t r a c t Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrinedisrupting element which mimics estrogen, binds to estrogen receptor alpha (ERα), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells. © 2010 Elsevier Inc. All rights reserved.

Introduction Cd is produced mainly as a byproduct of mining, smelting, and refining of zinc ore. Since World War II, it has been widely used in the manufacture of batteries, pigments, coating and alloys. The large-scale use in industry and disposal of waste containing Cd has resulted in a gradual increase of its concentration in water and soil. For the general population, major sources of exposure to Cd are food products and cigarette smoke. The metal accumulates in the human body with age because of its extremely low excretion rate (Shaikh and Smith, 1980). Prolonged excessive environmental and occupational exposure to Cd can lead to renal dysfunction and osteomalacia in humans (Satoh et al., 2002). Recent studies have shown that Cd is not only a

Abbreviations: Cd, cadmium; E2, 17-β estradiol; ER, estrogen receptor; GPR30, Gprotein coupled receptor 30; cAMP, cyclic adenosine monophosphate; mek, mitogenactivated protein kinase kinase; erk, extracellular signal regulated kinase; rsk, ribosomal S6 kinase; elk, E-26 like protein kinase. ⁎ Corresponding author. E-mail address: [email protected] (Z.A. Shaikh). 1 Present address: Dana-Farber Cancer Institute, Boston, MA, USA. 0041-008X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2010.02.005

carcinogen but also a mutagen (Jin et al., 2003; Waalkes, 2003). In experimental animals, Cd has been reported to cause cancer in a number of tissues (Waalkes, 2000). In epidemiological studies, Cd exposure has been linked to various types of cancers including breast cancer (Cantor et al., 1994; Ursin et al., 1994; Antila et al., 1996; Verougstraete et al., 2003, McElroy et al., 2006; Huff et al., 2007; Akesson et al., 2008). In ovariectomized female Sprague–Dawley rats, Cd demonstrates potent estrogen-like activity and increases uterine weight, promotes growth and development of the mammary glands, and induces hormone-regulated genes (Johnson et al., 2003). The increase in the uterine weight is accompanied by proliferation of the endometrium. In addition, the in utero exposure of female offspring to Cd causes an earlier onset of puberty, and an increase in the epithelial area and the number of terminal end buds in the mammary gland. Cd is a well-known xenoestrogen (Safe, 2003). According to Stoica et al. (2000), it binds to estrogen receptor alpha (ERα) with an equilibrium dissociation constant of 4–5 × 10−10 M and blocks the binding of 17β-estradiol (E2) in a noncompetitive manner (Ki = 2.96 × 10−10 M). The metal appears to interact with the hormone-binding domain of the receptor. Not surprisingly, when

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treated with Cd, the growth of human breast cancer derived MCF-7 cells is stimulated (Garcia-Morales et al., 1994; Brama et al., 2007). A proposed mechanism for Cd-induced activation of ERα is discussed in a review by Byrne et al. (2009). Estrogen is an important steroid hormone that is involved in the regulation of differentiation and proliferation of normal breast epithelial cells (Russo and Russo, 2006). It interacts with the ER, eliciting a cascade of transcriptional regulatory activity (Ikeda and Inoue, 2004). Estrogen is also well known for its ability to directly modulate the expression of cell-cycle regulatory genes (Russo and Russo, 2006). Furthermore, ER mutations that alter receptor expression in breast cancer are associated with cancer progression and hormonal resistance (Herynk and Fuqua, 2004). ER is a nuclear receptor that exists in two forms, ERα and ERβ (Rollerova and Urbancikova, 2000). Upon binding to E2, the receptors form dimers. Since the two forms are co-expressed in many cell types, the receptors may form ERα (αα) or ERβ (ββ) homodimers, or ERαβ (αβ) heterodimers (Matthews and Gustafsson, 2003). The dimers then regulate the transcription of target genes through ER elementdependent and ER element-independent pathways (Wood et al., 1998). In addition to its capacity to promote gene transactivation, estrogen also stimulates rapid nongenomic signaling including the stimulation of second messenger cascades and epidermal growth factor receptor signaling pathways. The G-protein-coupled receptor GPR30 has been linked to specific estrogen binding and rapid signaling (reviewed in Filardo and Thomas, 2005; Prossnitz et al., 2007). GPR30 is a seven-transmembrane-spanning receptor that specifically binds to E2 and causes rapid intracellular signaling, including EGFR transactivation and increased intracellular cyclic AMP (cAMP) (reviewed in Filardo and Thomas, 2005; Prossnitz et al., 2007). The cellular localization of this membrane receptor has been controversial. For example, Revankar et al. (2005) reported that GPR30 is localized in the endoplasmic reticulum where it specifically binds to E2 and its derivatives. In contrast, studies by other groups concluded that this receptor is localized in the plasma membrane (Thomas et al., 2005; Funakoshi et al., 2006; Filardo et al., 2007). An earlier study examined three breast cancer derived cell lines for signal transduction in response to treatment with estrogen. Rapid activation of erk-1/2 was reported in MCF-7 cells (ERα+, ERβ+, GPR30+) and SKBR3 cells (ERα−, ERβ−, GPR30+), but not in MDAMB-231 cells (ERα−, ERβ+, GPR30-). However, forced overexpression of recombinant GPR30 in MDA-MB-231 cells restored estrogenmediated erk-1/2 activation (Filardo et al., 2000). A similar pattern of erk-1/2 activation was observed when these cell types were treated with Cd (Liu et al., 2008). It is well known that the rapid activation of erk-1/2 is a nongenomic event that leads to cell proliferation (Zhang and Liu, 2002; Brama et al., 2007). Whether Cd promotes breast cancer cell proliferation through its interaction with GPR30 is not known. The purpose of the present study was to investigate the possible role of endogenous GPR30 in Cd-induced breast cancer cell proliferation. To address, its role in Cd action, we compared the influence of Cd on paired SKBR3 cell lines that expressed either an interfering mutant of GPR30 (Quinn et al., 2009) or control vector.

Cell culture. Previously described human SKBR3 breast cancer cells expressing dominant negative GPR30 (GPR30-inactive) or vector (GPR30-active) (Quinn et al., 2009) were maintained in phenol redfree DMEM/Ham's F12 medium (1:1) containing 1.2 g/L sodium bicarbonate, 10% FBS and 50 mg/L gentamicin. Stock cultures were maintained in the complete medium in a humidified atmosphere of 95% air–5% CO2 at 37°C and used within 15 serial passages. The cells were harvested using a mixture of 0.25% trypsin and 0.03% EDTA and subcultivated every 6–7 days. Cell conditions for stimulation with estradiol or Cd and preparation of cell extracts. Cells (1 105) were seeded in T-75 flasks in phenol redfree DMEM/F-12 medium containing 10% FBS for 24 h. To deplete intracellular estrogen levels, cells were incubated for 48 h in serum-free medium containing 2% bovine serum albumin (BSA). Subconfluent cultures of serum-starved cells were either exposed to Cd (0.01– 1.0 μM) or (E2) 10 nM), or left untreated. At the end of Cd and E2 treatments, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed with ice-cold RIPA buffer (150 mM sodium chloride, 100 mM Tris, pH 7.5, 1% deoxycholatic sodium, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 100 mM sodium pyrophosphate, 3.5 mM NaVO4, 2 mM phenylmethylsulphonyl fluoride (PMSF), 50 mM NaF, 2 mM EDTA, and protease inhibitor cocktail at a ratio of 1000:1). The cell lysates were centrifuged at 13,000 g for 15 min and the supernatants were stored at −80°C until analyzed. The protein concentration was determined by the micro BCA kit according to the manufacturer's instructions. Cell proliferation assay. In each well of a 96-well plate, 5 × 103 cells were seeded and incubated overnight in phenol red-free DMEM/F-12 medium containing 10% FBS for 24 h. Next, the cells were serumstarved for 48 h using the medium containing 0.2% BSA, followed by treatment with 0–1.0 μM Cd for 24 h. At the end of the treatment, 20 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to each well and the plates were placed on a shaking platform at 150 rpm for 5 min, followed by incubation at 37 °C for 2 h. The medium was discarded, 100 μl DMSO was added to each well to resuspend formazan, and the plates were placed on a shaking platform at 150 rpm for 5 min. Using a 96-well plate reader (Molecular Devices, Sunnyvale, CA) the optical density at 560 nm was measured and the background reading taken at 670 nm was subtracted. Cell proliferation was also measured by counting the cell number. In this case, a 24-well plate was seeded with 1 × 104 cells/well and the cells were incubated overnight in serum-containing medium. The cells were serum-starved for 48 h prior to treatment with 500 nM Cd or 10 nM E2 (positive control). Morphologically, after 1 week of treatment with E2, the confluence of GPR30-active cells was about 80% and that of GPR30-inactive cells about 50%. The actual cell number was recorded using a hemocytometer.

Materials and methods

Measurement of cAMP. The serum-starved cells were treated with 500 nM Cd or 10 nM E2 for 2.5 or 5.0 min. After treatment, the cells were digested with 0.1 M HCl and centrifuged at 1000 g for 10 min. cAMP concentration was measured in the supernatant using an EIA kit following the manufacturer's instructions.

Chemicals and biochemicals. Phenol red-free DMEM/Ham's F12 was obtained from Sigma-Aldrich (St. Louis, MO). Charcoal-treated fetal bovine serum (FBS) was purchased from HyClone (Logan, UT). EIA kit for cAMP was from Cayman Biochemicals (Ann Arbor, MI). Phosphoerk-1/2 Pathway Sampler Kit was purchased from Cell Signaling Technology (Danvers, MA). Blocking buffer was obtained from Li-Cor (Lincoln, NE). Micro BCA reagent kit was purchased from Pierce Biotechnology (Rockford, IL). All other chemicals and reagents were obtained either from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Suwannee, GA).

Western blot analysis. Prior to electrophoresis, the cell extracts in Laemmli buffer containing 5% β-mercaptoethanol were heated at 95– 100°C for 5 min. The amount of protein in the extract used for electrophoresis was kept constant in the same experiment but varied between the experiments (15–30 μg). After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane at 20 V for 20 min by using Trans-Blot SD Semi-dry Transfer Cell (Bio-Rad, Hercules, CA). The membranes were then blocked with the Li-Cor blocking buffer for 1 h at room temperature and incubated with the primary antibodies diluted in PBS–0.1% Tween-20 containing 2.5% BSA

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overnight at 4°C or for 1 h at room temperature. After washing, the membranes were incubated with the secondary antibody labeled with Alexa Fluor 680 (1:15,000 diluted in blocking buffer containing 0.1% Tween-20) for 1 h, followed by extensive washing with PBS-0.1% Tween-20. The washed membranes were scanned with the Odyssey Infrared Imager (Li-Cor, Lincoln, NE) and the optical density of the bands was quantified by using the application software version 2.0.40. Data analysis. Each experiment was repeated at least three times and data were analyzed for statistical significance by one-way ANOVA followed by the Tukey-Kramer post hoc test at p b 0.05. Results Cell proliferation Chronic Cd exposure promotes hyperplastic and dysplastic responses in reproductive tissue (Johnson et al., 2003), suggesting that it functions as an endocrine-disrupting agent. We have previously shown that Cd stimulates proliferative responses and stimulates extracellular kinases, erk-1/2 in a manner that is similar to that reported for estrogen; however these responses did not require ER expression (Liu et al., 2008). Recent evidence has suggested that estrogen may also promote estrogen-dependent proliferation via the G-protein-coupled receptor GPR30 in breast and ovarian cancer cells (Albanito et al., 2007, 2008). To test whether GPR30 promotes Cddependent erk-1/2 activation and proliferation, we measured its relative capacity to stimulate these activities in paired human SKBR3 breast cancer cells (ERα−, ERβ−, GPR30+) in which GPR30 was maintained in an active state or inactivated. The Cd-induced proliferation of breast cancer cells was examined by the MTT assay and by cell number. For the MTT assay, vector-transfected SKBR (GPR30-active) and SKBR3 HA-Δ154 (GPR30-inactive) cells were treated with up to 1.0 μM Cd for 24 h. Even for this relatively short treatment period, the SKBR3 cells expressing functionally active GPR30 clearly exhibited cell proliferation (increase in absorbance) in response to Cd and the effect was concentration dependent (Fig. 1). The proliferative effect was significant at concentrations up to 500 nM, and the maximum effect was approximately 2-fold as compared to SKBR3 cells expressing the dominant interfering mutant of GPR30. Comparison of total cell number as a measure of cell proliferation yielded results that were similar to those obtained with the MTT assay in that only the vector transfectants showed significant increase in cell growth (Fig. 2). After 7 days of treatment with 500 nM Cd or 10 nM E2 there was a 17 and 29% increase, respectively, in cell number over the untreated controls in vector control cells, but no significant change in the HA-GPR30Δ154 cells.

Fig. 1. Concentration dependence of SKBR3 cell proliferation in response to Cd treatment as measured by the MTT assay. Quiescent GPR30-active and GPR30-inactive cells were incubated in the presence of up to 1.0 μM CdCl2 for 24 h and the cell proliferation was determined by the MTT assay. Absorbance values (mean ± SD) from three independent experiments are plotted. ⁎Significantly higher than the GPR30mutant cells at the same Cd concentration (p b 0.05).

Cd or 10 nM E2 for up to 60 min. As shown in Fig. 4A, Cd increased phosphorylated raf-1 (p-raf) level about 3.8-fold in vector control cells, followed by a return to basal level by 60 min. The positive control, E2, also activated raf-1 at 2.5 min to the same extent. In contrast, in cells expressing GPR30 mutant both Cd and E2 caused only about 1.4-fold increase in raf-1 at 2.5 min (Fig. 4B), possibly representing the contribution of residual endogenous GPR30. Since mek-1 is a dedicated substrate of raf-1 the phosphorylation status of mek was evaluated following Cd or E2 stimulation in SKBR3 cells expressing vector or HA-GPR30. Indeed, in control cells, both Cd and E2 activated mek-1 about 4- and 3.8-fold at 2.5 min, respectively (Figs. 5A and B), and phosphorylated mek-1 returned to basal level by 60 min. In the GPR30-inactive cells, however, both Cd and E2 caused only about 1.4-fold increase in mek activation at 2.5 min. The extracellular-regulated kinases, erk-1/2 are members of the mitogen activated protein kinase(MAPK family and are serine/ threonine protein kinases that play important roles in the signaling cascades regulating various cellular processes. As shown in Fig. 6A, the activation of erk-1/2 by Cd was 4.2-fold at 2.5 min in the GPR30-active cells and about 1.4-fold in the GPR30-inactivated SKBR3 cells. Similarly, E2 caused about 3.9- and 1.4-fold increase in p-erk-1 at

Stimulation of cAMP production To determine whether Cd acted by promoting GPR30-mediated rapid cell signaling, cAMP concentration was measured in cells treated with 500 nM Cd for 2.5 or 5 min. E2 (10 nM) was used as a positive control. Treatment of GPR30-active cells with Cd caused 1.5- and 1.7fold increase in cAMP over the untreated controls at 2.5 and 5 min, respectively (Fig. 3A). Similarly, E2 caused a 2.4- and 2.5-fold increase in cAMP at these time points (Fig. 3B). In contrast, there was no significant change in the cAMP levels in HA-Δ154 cells upon either Cd or E2 treatment. Activation of erk signaling pathway Since the cAMP levels were elevated upon Cd and E2 treatments in SKBR3 cells expressing active GPR30, the phosphorylation of downstream erk-1/2 was investigated upon treatment with 500 nM

Fig. 2. Proliferation of SKBR3 cells in response to treatment with Cd or E2 as measured by the total cell number. Quiescent GPR30-active and GPR30-inactive cells were incubated in the presence of 500 nM CdCl2 or 10 nM E2 (positive control) and harvested 7 days later. Cell proliferation was determined by counting the total number of cells. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the respective untreated cells (p b 0.05). †Significantly lower than the GPR30-active cells given the same treatment (p b 0.05).

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Fig. 3. Stimulation of cAMP production in the SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2, or (B) 10 nM E2 (positive control) and harvested 2.5 or 5.0 min later to determine the cAMP levels. Data relative to the untreated controls are plotted as mean ± SD from three independent experiments. ⁎Significantly higher than the respective untreated control group (p b 0.05). †Significantly lower than the GPR30active cells at the same time point (p b 0.05).

2.5 min in the GPR30-active and GPR30-inactive cells, respectively (Fig. 6B). rsk is a downstream kinase that is activated by erk-1/2. Similar to all the other kinases examined in this study, within 2.5 min Cd and E2 activated rsk about 4.1- and 3.7-fold, respectively (Figs. 7A and B) in SKBR3 cells expressing uninhibited GPR30. In comparison, in the SKBR3 cells expressing inactivated GPR30, the elevation in p-rsk level by either treatment was relatively minor (about 1.4-fold). Similar results were also observed for elk-1, a substrate of rsk-1. In the GPR30-active cells, Cd once again activated elk-1 about 4.1-fold at 2.5 min (Fig. 8A). Also, the positive control, E2, caused about 3.9-fold increase in the phosphorylation of elk (Fig. 8B). As with the other kinases, there was only a slight change in p-elk (1.4-fold) in the GPR30-inactive cells. Discussion

Fig. 4. Time-course of raf-1 activation in SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2, or (B) 10 nM E2 (positive control) for up to 60 min and raf-1 was analyzed by Western blotting. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the basal level in the same cells (p b 0.05). †Significantly higher than the GPR30-inactive cells at the same time point (p b 0.05).

It has been suggested that Cd disrupts the endocrine system by mimicking the effects of E2 (Takiguchi and Yoshihara, 2006; Byrne et al., 2009). This view is based on the observation that Cd functionally acts like E2 in breast cancer cells as a result of its ability to bind to the ligand-binding domain of ERα with high affinity (Garcia-Morales et al., 1994; Stoica et al., 2000). Byrne et al. (2009) have proposed a schematic model that shows that the metal interacts with cysteine, histidine, glutamic acid, and aspartic acid residues located in close proximity to the ligand binding site of the receptor. They speculate that such interactions of Cd with the specific amino acids induce structural changes in ERα that are similar to those occurring upon E2 binding and are thus responsible for its activity.

The results of the present study reveal that Cd, like E2, causes cell proliferation in breast cancer cells that contain no ERα or ERβ, but only GPR30. Besides a previous report from our group (Liu et al., 2008), there are no other studies involving Cd and GPR30. It is conceivable that Cd interacts with this estrogen receptor in a manner that is similar to the one proposed for ERα. The amino acid residues that take part in such interaction remain to be identified. It is important to note that the lowest Cd concentration that produced significant cell proliferation in the present study in 24 h was 50 nM. This concentration is comparable to the blood Cd level (140 nM) reported by Fell et al. (1977) in occupationally exposed workers.

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Fig. 5. Time-course of MEK activation in SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2 or (B) 10 nM E2 (positive control) for up to 60 min and MEK was analyzed by Western blotting. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the basal level in the same cells (p b 0.05). †Significantly higher than the GPR30-inactive cells at the same time point (p b 0.05).

Using E2-Alexa dye complexes, Revankar et al. (2005) found GPR30 binding sites both in the plasma membrane and in the intracellular compartment. In comparison, using a classic radioreceptor assay and isotopically labeled [3H]E2, Filardo et al. (2007) found no evidence for the presence of a functional membrane estrogen receptor in the intracellular compartment and concluded that GPR30 was coupled to its G protein in the plasma membrane and not in the endoplasmic reticulum. Furthermore, the binding affinity of GPR30 with E2, like that of other seven-transmembrane-spanning receptors, was dramatically decreased when the receptor was not coupled with its Gα protein (Thomas et al., 2005). Apparently, there is a conformational change in the ligand binding pocket after E2 binds to GPR30 (Karnik et al., 2003; Herrmann et al., 2004; May et al., 2004). We hypothesize that like E2, binding of Cd with GPR30 triggers G

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Fig. 6. Time-course of erk activation in SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2 or (B) 10 nM E2 (positive control) for up to 60 min and erk was analyzed by Western blotting. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the basal level in the same cells (p b 0.05). †Significantly higher than the GPR30-inactive cells at the same time point (p b 0.05).

protein uncoupling and activation of the protein. Furthermore, that the conformation of the receptor changes and it transactivates the EGF receptor, as it does upon E2 binding (Filardo et al., 2007). However, experimental evidence for this possibility awaits further study. The second messenger cAMP reflects the activity of adenyl cyclase. Pang et al. (2008) reported that cAMP was significantly increased after 5-min treatment of croaker oocyte membranes with 100 nM E2 or GPR30-specific ligand G-1. A similar pattern of cAMP production by estrogens was observed in HEK-293 cells transfected with GPR30. Moreover, ER antagonists (either ICI 182,780 or tamoxifen) significantly increased cAMP production. To delineate whether Cd acted at the cell surface to promote GPR30-dependent intracellular signaling, intracellular cAMP concentrations were measured in the present

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Fig. 7. Time-course of rsk activation in SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2 or (B) 10 nM E2 (positive control) for up to 60 min and rsk was analyzed by Western blotting. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the basal level in the same cells (p b 0.05). †Significantly higher than the GPR30-inactive cells at the same time point (p b 0.05).

Fig. 8. Time-course of elk activation in SKBR3 cells in response to Cd or E2 treatment. Quiescent GPR30-active and GPR30-inactive cells were incubated with (A) 500 nM CdCl2 or (B) 10 nM E2 (positive control) for up to 60 min and elk was analyzed by Western blotting. Data from three independent experiments are plotted as mean ± SD. ⁎Significantly higher than the basal level in the same cells (p b 0.05). †Significantly higher than the GPR30-inactive cells at the same time point (p b 0.05).

study. In GPR30-active, but not in GPR30-inactive SKBR3 cells, treatment with 500 nM Cd resulted in a significant increase in the intracellular cAMP in 2.5 min. Similar increase in intracellular cAMP was observed after short-term treatment with 10 nM E2. These results suggest that functional GPR30 is required for rapid signaling in response to E2 and Cd. It is noteworthy that intrauterine administration of cholera toxin in mice causes an increase in uterine mass (Stewart and Webster, 1983) similar to the effect measured in rats subjected to chronic Cd exposure (Johnson et al., 2003). E2 rapidly activates erk-1/2 in MCF-7 cells that contain ERα, ERβ, and GPR30, and in SKBR3 cells that contain only GPR30, but not in MDA-MB-231 cells that lack both ERα and GPR30 (Filardo et al., 2000;

Liu et al., 2008). Similar to E2, Cd also causes rapid activation of erk1/2 and akt in MCF-7 and SKBR3 cells, but not in the MDA-MB-231 cells (Liu et al., 2008). Reconstitution of GPR30 in GPR30-deficient MDA-MB-231 cells restores the non-genomic effects of E2 (Filardo et al., 2000). In a complementary approach, Quinn et al. (2009) recently described a mutant SKBR3 cell line with functionally inactive GPR30. In the present study, we utilized this cell line and have established that functionally active GPR30 is responsible for not only E2-induced cell proliferation, but also cell proliferation in response to Cd treatment. At least 36 genes have been identified as the target genes for GPR30-mediated cell signaling. Of these, the connective tissue growth factor gene is activated more than 16-fold and contributes to the E2-

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induced promotion of breast cancer cell proliferation (Pandey et al., 2009). Furthermore, E2 disrupts the function of pro-oncogenic chemokine, transforming growth factor-beta (TGFβ), via GPR30. Silencing the GPR30 gene in MCF-7 cells suppresses the ability of E2 to inhibit the TGF-β pathway (Kleuser et al., 2008). Conversely, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 suppresses TGFβ signaling only after transfection with GPR30-encoding plasmids. The activation of erk-1/2 is also involved in this process. Rapid activation of erk-1/2 and akt (Liu et al., 2008), and other members of the MAPK pathway in the present study, strongly suggests that GPR30 is involved in the endocrine disruptive effects of Cd. Activation of erk-1/2 has been shown to lead to cell proliferation (Zhang and Liu, 2002; Brama et al., 2007). Lobenhofer et al. (2000) reported inhibition of DNA synthesis in MCF-7 cells by treatment with the mek-1 inhibitor, PD 98059. Similarly, Razandi et al. (2004) observed significant inhibition of E2-induced cell proliferation in the MCF-7 cells treated with the same inhibitor. Since Cd-induced MCF-7 cell proliferation is also blocked by PD 98059, Brama et al. (2007) suggested that E2 and Cd act in a similar manner. Our results show that Cd-induced increase in cAMP levels, activation of erk, and promotion of cell proliferation occurs only in the cells that contain functionally active GPR30. There are several kinases in the erk pathway, such as raf-1, mek-1, and downstream effectors, rsk and elk (McCubrey et al., 2007). Rapid activation of these kinases in response to E2 treatment is reported by several investigators. For example, in MCF-7 cells raf-1 activation occurs within 10 min and rsk activation within 15 min (Gilad et al., 2005). Similarly, in T84 cells elk activation is reported as early as 1 min (Hennessy et al., 2005). The present study demonstrates that, like E2, treatment with Cd also rapidly activates all of the above kinases. Although peak activation times for the various kinases could not be determined precisely due to limited sampling intervals at the early time points, all of the kinases underwent significant activation within 2.5 min. Also, while only phosphorylated and not total protein levels were determined in the present study, we have shown previously that neither E2 nor Cd has any remarkable effect on the total erk or akt expression in the SKBR3 cells (Liu et al., 2008). Based on the results presented in the present study, it is concluded that Cd acts like a xenoestrogen by elevating cAMP levels, activating kinases in the erk pathway, and stimulating breast cancer cell proliferation by interacting with the estrogen receptor, GPR30. Additional studies directed at identifying the Cd binding site on GPR30 and early events in the signaling cascade are in progress to further elucidate the underlying mechanism. Acknowledgment Xinyuan Yu received a partial Graduate Fellowship from the RIINBRE Program supported by grant P20RR016457 from NCRR, NIH. This research was made possible by use of the RI-INBRE Centralized Research Core Facility. References Akesson, A., Julin, B., Wolk, A., 2008. Long-term dietary cadmium intake and postmenopausal endometrial cancer incidence: a population-based prospective cohort study. Cancer Res. 68, 6435–6441. Albanito, L., Madeo, A., Lappano, R., Vivacqua, A., Rago, V., Carpino, A., Oprea, T.I., Prossnitz, E.R., Musti, A.M., Andò, S., Maggiolini, M., 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 Res. 67, 1859–1866. Albanito, L., Sisci, D., Aquila, S., Brunelli, E., Vivacqua, A., Madeo, A., Lappano, R., Pandey, D.P., Picard, D., Mauro, L., Andò, S., Maggiolini, M., 2008. Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells. Endocrinology 149, 3799–3808. Antila, E., Mussalo-Rauhamma, H., Kantola, M., Atroshi, F., Westermarck, T., 1996. Association of cadmium with human breast cancer. Sci. Total Environ. 186, 251–256.

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