G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid Timo Ylikomi 1,2 , Annika Vienonen 1 and Tytti M. Ahola 1 1 Department of Cell Biology, Medical School, 33014 University of Tampere, Finland; 2 Department of Clinical Chemistry, Tampere University Hospital, Tampere, Finland G protein-coupled receptor 30 (GPR30) has previously been described to b e important in s teroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) ce lls using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They e xpressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished th e expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. W hen HME cells were transiently transfected with the glucocorticoid response element MM TV-luc reporter p lasmid, stable expression of GPR30 r esulted in the abolition of ligand- induced transactivation of t he promoter. I n COS cells, transient transfection of GPR30 with glucocorticoid recep- tor a resulted in an abrogation of the MMTV-luc and GRE- luc r eporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling i nterferes with steroid signaling. Keywords: cofactor; glucocorticoid; G PR; proliferation; transcription. Glucocorticoids play a vital role throughout physiology. The physiological response a nd sensitivity t o glucoco rticoids varies among species, individuals an d cell types and duri ng the cell cycle [1,2]. Moreover , several pathological condi- tions are a result of glucocorticoid resistance [3]. The molecular basis of glucocorticoid resistance, however, varies widely and is not completely understood. Glucocorticoids b elong to a member of the steroid hormone family which bind to receptors triggering their conformational change and nuclear translocation. The hormone–receptor complex binds to specific response elements in target genes and interacts with several coacti- vators, including steroid receptor c oactivator 1 (SRC-1) family [4]. SRC-1 coactivators such as transcription intermediary factor 2 (TIF2) are able to enhance transcriptional activa- tion by the receptor via mechanisms that include recruit- ment of the general coac tivator, cAMP-response element binding protein-binding protein (CBP), and histone acety- lation [5]. TIF2 coactivates all steroid, t hyroid, retinoid a nd vitamin D receptors [6,7]. It i s widely e xpressed in different human tissues, implying an important role for TIF2 in cell function [6]. Two reports have addressed its role in growth regulation. Its mouse homologue, glucocorticoid receptor interacting protein-1 (GRIP-1), is critical for skeletal muscle differentiation in the mouse [8]. The reduction of TIF2 expression by antisense oligodeoxynucleotides inhibits lig- and-stimulated ER transcriptional activity and DNA syn- thesis in MCF-7 cells, this constituting evidence of the role of TIF2 in growth stimulation [9]. As coactivators function as transcription a mplifiers, changes i n their expression levels would markedly alter receptor-mediated transcriptional activity. There is some evidence on the r egulation of c ofactors. A potential pathway for TIF2 regulation was suggested by Borud and associates. In transient transfection assays, the function and protein level of over-expressed TIF2 was impaired by activation of protein kinase A (PKA) [10]. Indeed, TIF2-mediated coactivation of the nuclear receptor peroxisome prolifera- tor-activated receptor, liver X nuclear receptor and retinoid X receptor was repressed by PKA catalytic subunit over- expression. Steroid hormones regulate SRC-1 expression in Correspondence to T. M. Ahola, Medical School, 33014 University of Tampere, Finland, Fax: +358 3 215 6170; Tel.: + 358 3 215 8942; E-mail: Tytti.Ahola@uta.fi Abbreviations: BPE, bovine pituitary extract; BrdU, bromodeoxyuri- dine; CBP, cAMP-response element binding protein-binding protein; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; GDI, guanine nucleotide dissociation; GR, glucocorticoid receptor; GPR, G protein-coupled receptor; GRE, glucocorticoid response element; GRIP-1, glucocorticoid receptor interacting protein-1; hrEGF, human recombinant epidermal growth factor; HME, human normal mammary epithelial cells; hTERT, human telomerase reverse transcriptase subunit; IL, interleukin; MAPK, mitogen-activated protein kinase; MMTV, murine mammary tumor virus; PR, progestrone receptor; PI3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A; SUMO-1, small ubiquitin-related modifier-1BrdU, bromodeoxy- uridine; SRC-1, steroid receptor coactivator 1; TBP, TATA binding protein; TIF2, transcription intermediary factor-2. (Received 1 7 March 2004, revised 13 August 2004, accepted 2 September 2004) Eur. J. Biochem. 271, 4159–4168 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04353.x rats. Dexamethasone [4] and estrogen down-regulate, and thyroid hormone up-regulates SRC-1 cofactor expression in rat tissues [11]. The regulation of GR activity by growth factors and cAMP is well established, and in most cases req uires the presence of gluco corticoid. T he role of GPR in gluco- corticoid-mediated signaling w as suggested b y Schmidt and coworkers [12]. b 2 -adrenergic receptors were able to modulate GR transactivation by stimulating phosphati- dylinositol 3-kinase (PI3-K) [12]. In general, GPR signals a large variety of stimuli g enerated by different hor- mones, neurotransmitters, sensory stimuli and odorants [13]. The GPR superfamily numbers close t o 2000 G protein-coupled receptors [14,15], with a major contribu- tion in the growth regulation of different normal and cancer cells. The major e vidence to date concerns the role of GPR in enhancing cell proliferation [16–20]. There are also examples suggesting t he antiproliferative activity of GPRs [21–23]. The role of GPR30 in glucocorticoid-mediated signaling has not hitherto been described . Many groups indep end- ently demonstrated GPR30 in different tissues in the late 1990s, and confirmed its ubiquitous expression pattern. Based on the amino acid sequence of GPR30, it was identified as an orphan transmembrane receptor bearing some degree of similarity to chemokine receptors such as interleukin (IL) 8 and angiotensin II [24]. GPR30 belongs to the rhodopsin-like peptide receptor family, and the chemo- kine receptor-like 2 subfamily, w hich also includes as its members GPR41 (lung; rat) and a receptor similar to GPR30 (uterus, leiomyosarcoma; human). GPR30 is pref- erentially expressed in ER-positive breast cancer cells as well as in endocrine tissues, but also, e.g. in endothelial cells, lung, heart, lymphoid tissues/cells and in the central nervous system [24–28]. The gene is shown t o be up-regulated by fluid shear stress [25], p rogestins and progesterone [29]. GPR30 inhibits proliferation of MCF-7 b reast cancer cells [30], and its close homologue in the rat induces apoptosis through the p53-pathway [31]. It has been suggested t hat GPR30 plays a critical role in progestin- as well as estrogen-mediated signaling [30,32,33]. Here we sought to establish whether GPR30 inhibits growth by affecting steroid receptor activity or through other cell signaling pathways. We thus investigated the effect of GPR30 on the growth an d ste roid receptor activity of immortalized mammary epithelial cells that expressed GR but not estro gen receptors (ER) or progesterone receptors (PR). Unprecedentedly, GPR30 down-regulated the expres- sion o f cofactor TIF2, interfered with the transcriptional activity of GR and inhibited growth. This observation suggests a mechanism by which membrane-initiated signa- ling interferes with steroid signaling. Experimental procedures Hormones and growth factors 17b-Estradiol, medroxyprogesteroneacetate, dexametha- sone, human recombinant epidermal growth factor (hrEGF) and hydrocortisone were obtained from Sigma Chemical Co. Mifepristone (RU486) was a gift from Roussel Uclaf (Paris, France). Insulin, amphotericin-B and bovine pituitary extract (BPE) were purchased from Gibco BRL (Paisley, UK). Cell culture Packaging cell line PT67 and NIH373 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin (Gibco BRL ). hTERT-HME1, a human primary e pithelial cell line stably e xpressing the human telomerase reverse transcriptase subunit (hTERT) was obtained from Clontech and had undergone 134.74 popu- lation doublings at the beginning of the experiment. The cell line was maintained in mammary epithelial basal medium supplemented with 52 lgÆmL )1 BPE, 0.5 lgÆmL )1 hydro- cortisone, 10 ngÆmL )1 hrEGF, 5 lgÆmL insulin, 50 lgÆmL Gentamicin (Gibco BRL) and 5 0 n gÆmL )1 Amphotericin-B. COS cells were maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 supplemented with 5 % fetal bovine s erum and p enicillin/stre ptomycin. In transient transfection assays, fetal bovine serum was replaced by 10% dextran-coated, charcoal-stripped fetal bovine serum. RNA isolation RNA w as isolated from cells using the RNAqueous TM kit (Ambion). Cells were harvested in t rypsin/EDTA a nd washed with NaCl/P i. The c ell pellet w as mixed w ith 900 lL Lysis/Binding solution from th e kit. RNA isolation was carried out according to manufacturer’s instructions. Cell growth assay Cells were seeded in 96-well plates at a density of 1 · 10 3 cells per well in the experimental medium, and were allowed to attach for 1 day. Relative cell numbers were measured using the crystal violet method [34]. The cells were fixated and stained with crystal violet, and d ried cells were diluted with acetic acid. A bsorbance was measured at a 590 n m using a Victor 1420 Multilabel counter (Wallac, Turku, Finland). Cell proliferation assay This assay was carried out as described previously using immunohistochemistry [30]. In brief, cells were plated on glass slides. Bromodeoxyuridine (BrdU) was added to the growth medium, and BrdU was visualized using mono- clonal anti-BrdU Ig (Sigma). Alternatively, the proliferation index was measured using anti-(Ki-67) Ig (Sigma). Establishing retrovirus-producing cell lines GPCR-Br/GPR30 was kindly provided by D. Thompson, Department of Surgery, Standford U niversity, CA, USA [24]. GPR30 was cloned i n the BamHI and NotIsitesofthe pLEGFP-N1 vector (Clontech) replacing enhanced green fluorescent protein (EGFP). In order to create a pL-N1 control vector, restriction sites were filled using Klenow Fragment (MBI Fermentas, Hanover, MD), and the product was blunt-ligated with itself using 300 U T4 ligase (MBI Fermentas). Fusion o f GPR30 to EGFP was effected 4160 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004 in pLEGFP-N1 vector at SalIandBamHI sites. The following primers were used for cloning at the pLEGFP-N1 vector: GPR30-forward 5¢-TAATAAGTCGACGGGTC TCTTCCT-3¢ and GPR30-reverse 5¢-ATTATTGGATC CTACACGGCACTGC-3¢. V iruses capable of i ntroducing pLEGFP-N1, pLEGFP-N1/GPR30, pL-N1 and pL-N1/ GPR30 vectors were established in the PT67 packaging cell line derived from ATCC (American Type Culture Collec- tion, Manassas, VA, USA). PT67 cells (5 · 10 4 )were transfected using 1 lL lipofectamine 2000 ( Gibco BRL) and 1 lg pla smid for 24 h. After 36 h incubation with the growth medium, cells were grown in the presence of 800 lgÆmL )1 Geneticin (Sigma) for 1 week to select those containing pLEGFP-N1, pL-N1 and pL-N1/GPR30 vec- tors. Six colonies were isolated from each plasmid, and a strain showing the highest viral titer was selected for further studies. Viral titer was determined using NIH373 cells as recommended i n the Retroviral Gene Transfer and Expres- sion User Manual (GEU manual, Clontech). PT67 cells, which stably (pLEGFP-N1, pL-N1, pL-N1/GPR30) or transiently (pLEGFP-N1/GPR30) produced viruses, were grown for 4–5 days in the medium, and the viral titer determined as recommended in the GEU manual (Clon- tech) before storage at )80 °C. Cell infection In preliminary studies, optimal infection conditions were determined, and 13 viruses per cell were u sed to infect hTERT-HME1 cells. The cells were infected at two separate times, 12–24 h between infections. Polybrene 8 lgÆmL )1 (Sigma Chemical Co.) was added to reduce charge repul- sion in some infections. After 72 h incubation with the growth med ium, the cells were grown in the presence of 800 lgÆmL )1 Geneticin (Sigma Chemical Co.) to select those containing pLEGFP-N1, pLEGFP-N1/GPR30, pL-N1 and pL-N1/GPR30 vectors. Enhanced green fluorescent protein measurement Cells were plated on 96-well plates as in the cell growth a ssay. At indicated time points the medium was removed an d 20 lL Cell Culture Lysis Reagent (Promega, Madison) was added to the wells. The plates were shaken for 1 5 min at 500 r.p.m. Fluorescence was measured using a Victor 1420 Multilabel counter (Wallac) at wavelengths 485/535 nm. In order to correlate absorbance with protein concentration, a standard curve was made using recombinant EGF protein (Clontech). RT-PCR analysis In order to measure the regulation of mRNA, one-step RT-PCR was p erformed. A LightCycler instrument ( Roche, Mannheim, Germany) a nd LightCycler RNA Master SYBR Green I kit (Roche) were u sed in the a ssay. Specific primers for GPR30 and house-keeping gene TATA binding protein (TBP) were used as described previously [29]. To measure TIF2 exp ression, forward p rimer 5¢-ATCTCCAAGGCAA GATCA-3¢ and reverse prime r 5 ¢-GTGCCATCAGA CAAGGAA-3¢ were used. Primer pair 1 resulted in a PCR product of 216 bp. Additionally, forward primer 5¢-GAGCCCC AAGAAGAAAGA-3¢ and reverse primer 5¢- CATCCAAAATCTCC TCCA-3¢ were used. Primer pair 2 resulted in a PCR product of 230 bp. The forward and reverse primers were designed in different exons. The reverse transcription was performed at 6 1 °C for 20 min and denaturation at 95 °C for 30 s. Forty-two cycles of PCR were carried out. The cycle included d enaturation at 95 °C for 1 s, annealing at 51 °C for 5 s and elongation at 72 °Cfor 10 s. The melting curve was obtained as described elsewhere [29]. The final results, expressed a s N-fold differences in gene expression between the GPR30-transfected and vector- transfected samples, were arrived a t as f ollows: N gene ¼ðTIF2 GPR30-transfected =TBP GPR30-transfected Þ= ðTIF2 vector-transfected =TBP vector-transfected Þ: The relative concentrations used for expression difference calculations were obtained from the calibration curves. Concentration v alues for TI F2 were taken from the calibration curve made from serial dilutions (500 fi 100 fi 20 fi 4 ng) of the total RNA. Immunoblotting Cells were harvested by a cell scraper and calculated in a Burker cell chamber. Nuclear extracts were prepared from 2 to 10 · 10 6 hTERT-HME cells as described previously [35]. Protein concentrations were determined using BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Immunoblot- ting was carried out as previously described [36]. Nuclear extracts or cell pellets were mixed with 2· SDS-sample buffer and boiled for 5 min. Equal amounts of nuclear proteins (196 lg) or cells (3 ·10 5 ) of cell lysates were resolved in 12% polyacrylamide g el and transferred t o a nitrocellulose membrane with an electrophoretic transfer apparatus. After blocking, the membranes were incubated o vernight at 4 °C with GRIP-1 (F-20) antibody at a final concentration of 1 lgÆmL )1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Biotinylated anti-goat IgG (Vector Laboratories I nc., Burlingame, CA) and horseradish peroxidase avidin D (Vector Laboratories Inc.) were used as secondary antibod- ies. Alternatively, membranes were incubated with anti-GR (P20) (Santa Cruz Biotechnology), NCL-L-PR (Novocas- tra), NCL-ER-6F11 (Novocastra) or anti-b-actin (Sigma). Peroxidase-conjugated goat anti-rabbit I gG (Cappel, West Chester, PA, USA) for GR and peroxidase-conjugated goat anti-mouse IgG (Cappel) for PR, ER and b-actin were used as secondary antibodies. After washing, labeled proteins were detected by enhanced chemiluminescene. Transient transfection assays Cells we re transfected using the Lipofectamin 2000 method as recommended ( Gibco B RL). A DNA mixture of pCMVbGal 50 ng, pSG5 or GRa (2.5 lg), pBk-CMV or GPR30/pBk-CMV (0.1, 0 .3, 0.6 lg), G RE-tk-luc ( 2.5 lg) or MMTV-tk-luc (2.5 lg)wasusedtotransfectCOScells;and aDNAmixtureofpCMVbGal ( 50 ng), GRE-tk-luc (2.5 lg) or MMTV-tk-luc (2.5 lg)wasusedtotransfect hTERT-HME cells. Transfection was carried out for 6 h for COS cells and 24 h for hTERT-HME cells. A fter transfec- tion, the cells were incubated for 4 h with the basic medium lacking g lucocorticoid. Luciferase activity in the samples was Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4161 measured 48 h a fter glucocorticoid addition applying the Luciferase Assay System as recommended (Promega) and using a 1450 Microbeta Plus Liquid Scintillation Counter (Wallac). Equal transfection efficiency was confirmed by measuring b-galactosidase activity in heat-treated (10 m in at 50 °C)lysatesasrecommendedintheb-Galactosidase Enzyme Assay system (Promega), with a Victor 1420 Multilabel c ounter at a wavelength of 450 nm (Wallac). Results Mammary epithelial cells expressed GR We characterized the steroid receptor composition of H ME cells. T he cells expressed G R as a ssessed using immuno- blotting analysis (Fig. 1). No PR or ER a was d etected in the cells. MCF-7 cells were used as a positive control. GPR30 inhibited proliferation of mammary epithelial cells GPR30 has a critical role in progestin- and estrogen- mediated signaling [30,32,33]. It also inhibits proliferation of ER- and PR-positive MCF-7 cells [30]. We investigated whether the effect of GPR30 on growth is dependent on steroid receptors and whether GPR30 inhibits the growth of non-neoplastic breast epithelial cells which did not express ER or PR. We stably introduced GPR30 to commercial HME cells immortalized by hTERT, using retroviral- mediated gene delivery. The immortalized HME c ells were cultured without serum, thus avoiding the influence of unknown growth factors. As a control we used cells infected with an empty vector pL-N1. GPR30 mRNA was expressed in HME cells 28-fold compared to control cells as measured by quantitative PCR 96 h after plating (Fig. 2). Interestingly, when relative cell growth was measured, growth was inhibited by 23–34% between 48 and 168 h (Fig. 3A). Corresponding results were obtained from two separate infections. Time point zero taken to be the point when cells were attached to plates, this usually taking 1 day. To further study the mechanism whereby GPR30 regu- lates growth in these cells, we measured the cell proliferation index using a BrdU incorporation assay and KI-67 immu- nostaining. Before the BrdU e xperiment, the cells were allowed to attach, for 1–2 days, the medium was c hanged and 0 h time point measured. BrdU incorporation a nalysis revealed that proliferation was inhibited by 60–87% between 0 and 72 h in H ME cells (Fig. 3B). Cell prolifer- ation was also measured using KI-67 as an indicator of cells in cycle. Prior to the KI-67 assay, HME cells were arrested at the G 0 /G 1 phase by growth factor deprivation (mammary epithelial basal medium present without supplements) for 24 h. Ki-67 staining revealed that GPR30 inhibited prolif- eration by 40% at 2 h and inhibition increased to 53% 12 h after growth factor deprivation (Fig. 3C). A B C D β-actin GR ER PRB MCF-7 HME PRA Fig. 1. HME c ells e xpr ess G R. The receptor composition of immor- talized HME cells was characterized using immunoblotting. Cell lysates o f MCF-7 cells (used as positive control) and HME cells transfected with empty pL-N1 vector w ere run in po lyacrylamide gel. Proteins were transferred to the nitrocellulose membrane and detected with the antibodies against PR A and B (A), ERa (B) an d GR (C). T o confirm equal loading efficiency t he m em branes were incu bated with anti-b-actin (D). 50 * 40 30 20 10 0 C GPR30 GPR30 mRNA fold induction Fig. 2. HME cells express GPR30. Stable expression of GPR30 in HME cells was achieved u sing retroviral-mediated g ene delivery. The relative expression of GPR30 mRNA was stu died u sing q uantitative RT-PCR analysis. Total RNA was extracted from cells grown to 50–70% confluence in thre e independe nt experiments. T he result is presented as mean of three experiments. Statistical significance was calculated using Student’s paired t-test. Differences in expression were considered significant at *P < 0.05. 4162 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004 GPR30 down-regulated cofactor expression in hTERT-HME cells GR affects gene transcription by a mechanism which is enhanced by cofactors. We therefore characterized the effect of GPR30 on the expression of receptors and cofactors. I n our preliminary analysis, GPR30 affected the expression level of TIF2 mRNA, but not of other cofactors (nuclear receptor corepressor, silencing mediator of retinoic acid and thyroid hormone receptor, p300/CBP-associated factor, Ras-related C3 botulinum toxin substrate 3) or receptors (GR, androgen receptor, PR, ER) studied. We thus verified the effect of GPR30 o n the expression level o f TIF2 in HME cells using quantitative RT-PCR analysis a nd two different primer pairs. Expression of TIF2 was down-regulated by 5 C A B C GPR30 * ** *** * * * ** * * * ** * * C GPR30 C GPR30 4 3 2 1 0 0 24 48 72 96 120 144 168 Time (h) 40 30 20 10 0 024487296 Time (h) 20 25 15 5 10 0 0246810 12 14 Time (h) KI-67% BrdU% Relative Cell Number (A 590 nm) Fig. 3. GPR30 inhibits proliferation of HME cells. (A) Cells were plated on 96-well plates and the relative cell number was measured from HME cells transfected either with GPR30 or empty vector using crystal violet staining. (B) Cell proliferation was measured u sing the bromodeoxyuridine (BrdU) incorporation assay, and BrdU-incor- porated cells were visualized by immunostaining with anti-BrdU Ig at indicated time points. (C) Cells were arrested at the G 1 phase by growth factor deprivation. Thereafter, the cells were cultured with growth medium and immunostained with the a ntibody against Ki-67 at indicated time points. The percentage of Ki-67-positive cells was counted. The data presented are the means of three to four replicates. Statistical significance was calculated using the t-test as indicated in Fig. 2. C, control. 100 80 60 40 20 A B C 0 C GPR30 ** * 100 80 60 40 20 0 C GPR30 C GPR30 TIF-2 mRNA % of control TIF-2 TIF-2 mRNA % of control Fig. 4. GPR30 d own-regulates the expression of TIF2 protein. The regulation of cofactor was studied in HME cells stably expressing GPR30. The relative expression of TIF2 mRNA was stud ied using quantitative RT-PCR analysis and two different primer pairs: (A) primer pair 1 and (B) primer pair 2 . W hen c ells were grown to 5 0–70% confluence, total RNA was extracted for analysis. Results are pre- sented as means of three replicates. (C) TIF-2 protein expression was studied the nuclear extracts of HME cells (196 lg) using immuno - blotting. C, control. Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4163 90% a nd 40% by GPR30 a s a nalyzed u sing RT-PCR analysis and two different primer pairs (Fig. 4A,B). To study the c ofactor regulation at p rotein level, we used specific antibodies against cofactor TIF2. In immunoblot- ting analysis GPR30 down-regulated TIF2 expression in HME nuclear extracts (Fig. 4C). GPR30 inhibited transcriptional activity of the glucocorticoid response element in hTERT-HME cells The finding that GPR30 down-regulated the expression of cofactor TIF2 led us to study whether GPR30 affects the transcriptional activation o f the glucocorticoid response element in HME cells which expressed endogenous GR receptor but not PR or ER. We used HME cells infected with GPR30 or pL-N1 control v ector. We transiently transfected the cells with MMTV-luc reporter gene, which contains glucocorticoid response elements. In control cells, MMTV promoter was stimulated 370% by hydrocortisone treatment (Fig. 5A). In GPR30 stable e xpressing cells, the effect of hydrocortisone on MMTV-luc activity w as almost abrogated. Without the presence of hydrocortison, GPR30 had no effect on the activity, indicating that the effect is glucocorticoid-dependent. Equal transfection efficiency was confirmed using a CMV-bgal vector as marker for trans- fection efficiency. GPR30 inhibited the transcriptional activity of the glucocorticoid receptor in COS cells To confirm that the effect of GPR30 on transcriptional activity was mediated through GR, we studied the phe- nomenon in steroid receptor-negative COS cells using the transient transfection approach. COS ce lls were transfected with the expression vectors encoding GRa and GPR30 (or with the corresponding control vectors) together with the reporter construct MMTV-tk-luc. MMTV-tk-luc promoter activity was enhanced 730% by dexamethasone treatment in control cells (Fig. 5B). When the cells were transfected with GPR30 and treated with dexamethasone, the luciferase activity was almost abolished. GPR30 did not affect the target promoter in the absence of dexamethasone, confirm- ing that the effect is glucocorticoid-dependent. To study whether the effect of GPR30 on MMTV activity was c oncentration-dependent, we t ransfected COS cells with increasing amounts of GPR30 plasmid. The 400 AB CD 350 ** ** ** * 300 250 200 150 Luciferase Activity % of control Luciferase Activity % of control Luciferase Activity % of control Luciferase Activity % of control 100 400 350 300 250 200 150 100 400 500 600 700 800 900 1000 300 200 100 400 500 600 700 300 200 100 GPR30 –– –+– + ++ – –– – ++ + ++ +++ COR GPR30 GR DEx – –– – ++ + ++ +++ GPR30 GR DEx – – – + + ++ + + + ++ GPR30 0.1 0.3 0.6 GR DEx Fig. 5. GPR30 i mpairs the ability o f glucocorticoid to a ctivate transcription. (A) H ME ce lls stab ly ex pressing GPR30 (or control vector p LN-1) w ere transfected with a m urine mammary t umor virus-tk-luc reporter construct, and treated with hydrocortisone. (B) COS cells were transfected with MMTV-tk-luc reporter gene, and the expressio n vectors g luc ocorticoid receptor a and G PR30. The cells were treated with dex amethaso ne to ind uce reporter gene transcription. (C) COS cells were transfected w ith MMTV-tk-luc, glucocorticoid receptor a and increasing c onc entrations of GPR30. (D) COS cells were transfected with a glucocorticoid response element-tk-luc reporter construct, and expression vectors containing glucocorticoid receptor a and GPR30. Luciferase assays were p erformed 48 h after addition of dexamethasone. The figure shows the mean v alue of tr iplicate transfections. DEX, dexamethaso ne ; GR, glucocorticoid receptor. 4164 T. Ylikomi et al.(Eur. J. Biochem. 271) Ó FEBS 2004 increase in GPR30 expression was f ound to inhibit t he luciferase activity in a concentration-dependent manner (Fig. 5 C). When COS cells were transfected with the other gluco- corticoid response element, GRE-tk-luc, dexamethasone induced an increase in GRE-tk -luc reporter activity by 360%. Similarly, when the c ells were transfe cted with GPR30 and treated w ith dexamethasone, t he luciferase activity was almost abolished (Fig. 5D). GPR30 by itself had n o effect on transcriptional activation of GR-tk-luc. Equal transfection efficiency was confirmed using a CMV- bgal v ector as marker for transfectio n efficiency. GPR30 fusion protein inhibits growth and cofactor expression We have previously established changes in GPR30 expres- sion level only at the RNA level. In order to confirm t hat the effect of GPR30 on the regulation of cofactor and g rowth was due to GPR30 protein expression, we measured the effects of GPR30 fused with EGFP. As a control, we used HME cells infected with the plasmid-expressing EGFP. Fluorescence was measured in the cell lysates at a wavelength of 485/535 nm. E GFP was expressed in control cells (Fig. 6 A). GPR30-EGFP expression was detected in experimental cells at a high level. We also measured relative cell growth u sing crystal v iolet staining. Cell proliferation was inhibited by GPR30-fusion protein when comparison was made with cells expressing EGFP or parental HME cells (Fig. 6 B). In accord with previous results, TIF2 protein levels were down-regulated by GPR30-fusion protein analyzed by Western b lotting (Fig. 6C). The re sult suggests that the measured effects on growth and the expression of cofactor were due to GPR30 protein expression. Discussion Molecular discrimination of cells differing in their sensitivity to steroids is essential in diseases involving resistance to steroid h ormone treatment. In the present study, we addressed the question of the ability of GPR30 to regulate steroid hormone-induced transcription, particularly as a critical role of GPR30 in steroid-mediated signaling has been described [30,32]. We found that stable expression of transmembrane receptor GPR30 in human immortalized breast cells down-regulated the expression of cofactor TIF2, resulted in abrogation of the transcription activity of GR and inhibited c ell p roliferation. These r esults suggest a new mechanism underlying glucocorticoid resistance. The data also point to cross-talk with G proteins and steroid receptor activity, and provide a mechanism whereby membrane- initiated signaling might diminish cell proliferation. Interestingly, GPR30 inhibited GR-mediated activation of transcription both in non-neoplastic breast epithelial cells and i n COS cells. Other studies have also indicated the role of cytoplasmic s ignaling in s teroid hormone-mediated transcription. b 2 -Adrenergic receptor stimulates GR trans- activation by activating PI3-K [12], and Rho GDP-disso- ciation inhibitor (GDI) modulates ER transcriptional enhancement [36]. Additionally, the activation of PKA is known to lead to s teroid receptor-induced transcription [10]. Inhibition of steroid hormone-induced trans cription by cytoplasmic signaling has not, however, previously been described. We further sought to identify the mechanism by which GPR30 would interfere with GR mediated activation of transcription. We established that GPR30 down-regulated the expression of cofactor TIF2. The crucial r ole of cofactors in the activation of transcription has b een shown in a number of studies. Indeed, over-expression of cofactors HME GPR30 pLEGFP-N1 HME GPR30 pLEGFP-N1 pLEGFP-N1 GRP30 * * * ** 0.01 1.0×10 –04 1.0×10 –06 1.0×10 –08 0244872 Time (h) rEGFP protein mg/ml Relative cell number (A590 nm) 96 120 144 0 0 1 2 3 4 B C A 24 48 72 TIF-2 Time (h) 96 120 14 4 Fig. 6. Fusion protein of GPR30 inhibits proliferation and cofactor expression. GPR30 was fused to EGFP. Retrovirus infection was used to obtain stable expression of this construct in HME cells. Cells stably expressing EGFP were used as control. (A) The amount of GPR30 fusion protein and EGFP in control cells was calculated by measuring fluorescence at wavelengths 485/535. A standard curve was made f rom EGFP recombinant prote in in order to correlate absorbance with protein concentration . R esults ar e p rese nted as means of t hree re pli- cates. (B) Relative c ell g rowth w as measured in GPR30-expressing and control cells using crystal viole t staining. Ab sorbance was m easured at a wavelength o f 590 nm (C) T IF2 protein expression in the nuclear extracts of HME cells (196 lg) was de tected using imm uno blotting . Ó FEBS 2004 GPR30 regulates GR activity (Eur. J. Biochem. 271) 4165 enhances steroid hormone-induced transcriptional activity [5–7], and a r eduction in cofactor TIF2 expression prevents ERa-induced and ligand-induced activation of the promo- ter 3xERE-TATA [9]. Differential expression of cofactors has been proposed in explanation of some of the differential effects of s teroid hormones. The m echanism whereby GPR30 modulates TIF2 expression is not known. We were not able to establish MAPK inactivation or activation by GPR30 in HME cells, although MAPK activation/inacti- vation has been established i n breast cancer cells [32,37]. Other studies have also established t he regulation of cofactor activity by membrane-initiated signaling. Indeed, regulation of GRIP1 and CBP coactivator activity by cytoplasmic Rho GDP-GDI leads to ER activation [36]. Our study therefore suggests that the down-regulation of TIF2 expression by GPR30 i s a mechanism involved in the establishment of steroid hormone resistance by GPR30. GPR30 most probably a lso induces glucocorticoid resistance by mechanisms other than down-regulation of TIF2 expression. Ligand binding is the main inducer of the transcriptional activity, but post-translational modifications have also been shown to play an i mportant role. GR activity is regulated through receptor phosphorylation, e.g. by the PKA and MAPK pathway [38] and through the covalent addition of the small ubiquitin-related modifier-1 (SUMO- 1) peptide [39]. Interestingly, GPR30 has been proposed to activate the PKA p athway in breast cancer cells [33]. Activation of the MAPK pathway has been shown to affect the transcriptional activation o f various receptors by modulating cofactor recruitment [40] and affecting receptor degradation [ 41,42]. I t has also been shown that GPR30 regulates MAPK activity [32,33,37]. In addition to interfering w ith the transcriptio nal activity of GR, GPR30 was seen here to reduce the growth and proliferation of immortalized breast cells. There was a more profound effect on cell proliferation than on cell number, a phenomenon previously described in the case of mutant activated Ga-proteins. Ga inhibits the ability of MCF-7 cells to proliferate and to form tumors in athym ic mice [43]. Additionally, adenovirus-directed expression of ac tivated mutant Ga inhibits the growth of e stablished tumors b y inhibiting the M APK pathway [44]. W e have shown previously that GPR30 is a ble to i nduce G 0 /G 1 phase arrest in MCF-7 breast cancer cells [30]. Thus our result suggests a molecule whose growth effect is independent of the differ- entiation status (normal vs. c ancer) of the epithelial c ells in the mammary gland. We also show here that the abolition of hormone- mediated transactivation of a GRE- responsive element by GPR30 is associated with growth inhibition in HME c ells. Down-regulation of TIF2 expression and the resultant glucocorticoid insensitivity m ight contribute to the growth- inhibitory effects of GPR30 in these cells, because gluco- corticoid has been shown t o inhibit growth in b reast cancer cells [45]. Such a conception is suppo rted by the findings of Cavarretta and associates, who showed that a reduction in TIF2 expression was a ble to p revent estrogen-induced proliferation in MCF-7 cells [9]. We h ave also shown previously that GPR30-regulated growth was independent of the presence of steroid hor- mones [30]. Thus it can be concluded that the ligand for GPR30 is likely to be in the BPE extract or be secreted into the medium by mammary epithelial cells. Computer experiments suggest that the hydrophilic cyclopeptide derived from alpha-fetoprotein interacts with GPR30 and may execute its action by interaction with GPR30 [46]. Interestingly, human alpha-fetoprotein peptides also bind s to the estrogen receptor and estradiol, and suppresses breast cancer [47]. In th e adult, only trace amounts o f alpha- fetoprotein is detected [48], but in a pathological state in adult life, alpha-fetop rotein levels rise (e.g. hepatic tumor). Thus the ligan d of G PR30 might be secreted into the medium by breast cancer and immortalized mammary epithelial cells. In conclusion, our results imply that GPR30 interferes with glucocorticoid-mediated transcriptional activation associated with down-regulation of cofactor TIF2 expres- sion and growth inhibition. 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