Progestin upregulates G-protein-coupled receptor 30 in breast cancer cells Tytti M. Ahola 1 , Sami Purmonen 1 , Pasi Pennanen 1 , Ya-Hua Zhuang 1 , Pentti Tuohimaa 3 and Timo Ylikomi 1,2 1 Department of Cell Biology, Medical School, 33014 University of Tampere, Finland; 2 Department of Clinical Chemistry, Tampere University Hospital, Finland; 3 Department of Anatomy, University of Tampere, Finland A differential display method was used to study genes the expression of which is altered during growth inhibition induced by medroxyprogesterone acetate (MPA). A tran- script of G-protein-coupled receptor 30 (GPR30) was upregulated by MPA in estrogen-treated MCF-7 breast cancer cells. Northern-blot analysis showed a progestin- specific primary target gene, which was enhanced by prog- esterone and different progestins, but not by dihydrotestos- terone or dexamethasone, and which was abrogated by antiprogestin RU486. The dose-dependent and time- dependent increase in GPR30 mRNA expression correlated with MPA-induced growth inhibition in MCF-7 cells. Additionally, GPR30 upregulation by progestin correlated with growth inhibition when a comparison was made between different breast cancer cell lines. The ERK1/ERK2 pathway is capable of inducing progesterone receptor- dependent and ligand-dependent transcription. Thus we sought to establish whether different MAPK pathway inhibitors affect progestin-induced GPR30 mRNA regula- tion. The regulation of GPR30 was independent of ERK pathway activation, but the p38 pathway inhibitor induced GPR30 expression, which suggested a potential gene regu- lation pathway. These data demonstrate a new progestin target gene, the expression of which correlates with growth inhibition. Keywords: breast cancer; differential display; G-protein- coupled receptor; progestin; proliferation. Progestins are widely used for therapeutic purposes such as contraception and hormonal replacement therapy. In the uterus they oppose the proliferative effect induced by estrogens and are therefore combined with estrogen in hormone replacement therapy. The role of progestins in the normal mammary epithelium in vivo is controversial [1], whereas in in vitro studies with human normal breast [2] and breast cancer cell lines [3–5], progestins inhibit estrogen- stimulated cell proliferation. The mechanism of progestin-induced growth is mostly unknown. Progestin regulates many cell cycle proteins such as cyclinD1, D3 and A [6,7]. It is not known, however, which of these phenomena are direct or indirect effects of progesterone and what is the role of progestin-induced early response genes such as c-myc,c-fos [5], c-jun and jun-B[8]. Progestins can also regulate cell growth through autocrine and paracrine factors. Progestins as well as estrogen enhance the expression of growth factors such as trans- forming growth factor a and epidermal growth factor [1,5]. Interestingly, recent studies show that the mitogen-activated protein kinase (MAPK) pathway regulates progesterone receptor-dependent transcription [9] and may have a role in progestin-induced growth inhibition [10] as it does in growth stimulation [11]. An orphan transmembrane G-protein-coupled receptor 30 (GPR30) is expressed preferentially in estrogen receptor- positive breast cancer cells as well as in endocrine tissues [12–14]. Some G-protein-coupled receptors are known to be involved in growth regulation, but the function of GPR30 is not known. Conservation of GPR30 during evolution and expression in embryogenesis [13] would imply an important role in development. Interestingly, it has also been shown that GPR30 is involved in estrogen-mediated ERK1/ERK2 pathway regulation in estrogen receptor-negative breast cancer cells [15,16]. It was decided to use a differential display technique to detect genes, the expression of which is altered during growth inhibition induced by medroxyprogester- one acetate (MPA). A comparison was made of gene expression patterns in MCF-7 cells cultured in the presence and absence of MPA. An account is given of the isolation of a transcript of the orphan GPR30, which is induced by progestins and progesterone. These results reveal a new progestin primary target gene which may have a potential role in progestin-induced growth inhibition. MATERIALS AND METHODS Hormones 17b-Estradiol, MPA, dexamethasone, cycloheximide and actinomycin D were purchased from Sigma. Dihydrotesto- sterone and progesterone were purchased from Merck (Darmstadt, Germany). Insulin was provided by Gibco/ BRL. Mifepristone (RU486) was gift from Roussel Uclaf. Promegesterone (R5020) was provided by Schering Aktiengesellschaft (Berlin, Germany). PD98059, U0126 Correspondence to T. M. Ahola, Medical School, Department of Cell Biology, 33014 University of Tampere, Finland. Fax: + 358 3 215 6170, Tel.: + 358 3 215 8942, E-mail: ta55935@uta.fi Abbreviations: MAPK, mitogen-activated protein kinase; GPR30, G-protein-coupled receptor 30; MPA, medroxyprogesterone acetate; TBP, TATA binding protein. (Received 11 February 2002, accepted 3 April 2002) Eur. J. Biochem. 269, 2485–2490 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02912.x and SB202190, SAPK2/p38 MAPK inhibitor were from Calbiochem. Cell culture MCF-7 and ZR-75-1 cells were provided by Professor P. Ha ¨ rko ¨ nen, University of Turku. CAMA-1 and BT-474 cells were a gift from Professor J. Isola, University of Tampere. Before the experiment, MCF-7, ZR-75-1, CAMA-1 and BT-474 cells were cultured for two to three passages in phenol red free in Dulbecco’s modified Eagle’s medium/ Ham’s nutrient mixture F12 (1 : 1) supplemented with 5% dextran-coated, charcoal-stripped fetal bovine serum, 100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin and insulin (phenol red free and DCC medium; dextran-coated, charcol-stripped) with 1 n M estrogen. Cell growth assay Cells were seeded in 96-well plates at a density of 3 · 10 3 cells per well in the phenol red free and DCC medium. They were allowed to attach overnight and the medium was replaced. After 24 h, appropriate steroid hormones in 100% ethanol or ethanol were added. The final ethanol concen- tration did not exceed 0.1%. The number of cells was measured using the crystal violet method [17]. Absorbance was measured at a wavelength of 590 nm using a Victor 1420 Multilabel counter (Wallac). Differential display MCF-7 cells (1.0 million) were placed in 150-cm 2 plates and treated as in the cell growth assay. RNA was extracted using TRIzol Reagent (Gibco/BRL) according to the manufac- turer’s protocol, and then digested with DNase I (Promega) for 1 h at 37 °C. The digested RNA was extracted with phenol/chloroform and precipitated with ethanol. The differential display was carried out using the RNAmap KIT (GenHunter, Brookline, MA, USA) according to the manufacturer’s instructions. DNase- treated RNA (1 lg) was reverse-transcribed and subse- quently amplified using an upstream primer (AP1, AP2, AP3, AP4 or AP5), a downstream primer T 12 MN (N ¼ T, A, C or G) and [ 33 P]dATP (1000 CiÆmmol )1 ) (Amersham). PCR products (5 lL) were run on 6% polyacrylamide sequencing gels for 6 h at 1000 V using Poker Face II (Hoefer Scientific Instruments, San Francisco, CA, USA). After autoradiography, differen- tially expressed products were extracted from the gel with boiling water and reamplified. The reamplified PCR product was subcloned in a PCR-TRAP vector (Pharma- cia Biotech). The insert was sequenced using the Dye Terminator cycle sequencing Ready Reaction kit (ABI Prism), and the samples were then processed on an automated 310 Genetic Analyser (ABI Prism). Northern blot Total RNA (30–40 lg) was electrophoresed on a denaturing 1% agarose/2% formaldehyde gel. The RNA was blotted on to a nitrocellulose membrane (MSI, Westborough, MA, USA) using overnight capillary transfer in 10 · NaCl/P i / EDTA. The membrane was hybridized with a randomly primed 32 P-labeled cloned PCR product for 16 h at 42 °Cin the hybridization solution (6 · NaCl/P i /EDTA/5 · Den- hardt’s solution/50% deionized formamide/0.1% SDS/ 100 lgÆmL )1 denatured salmon sperm DNA). The filters were rehybridized with oligonucleotide complementary to 18S rRNA (Ambion) for normalization. The radiolabeled filters were exposed on X-ray film (Kodak BioMax). Signal density was measured using a Wallac densitometer. Proliferation assay Cells were placed on top of glass slides in four-well plates at adensityof10 5 cells per well. Bromodeoxyuridine (BrdU) was added at a final concentration of 20 l M at the indicated time points. After 2 h, the cells were washed three times with NaCl/P i ,fixedwith4%paraformaldehydefor 15 min at room temperature, and washed with NaCl/P i . The fixed cells were permeabilized, and the DNA was denatured as previously described [18]. Thereafter, the cells were immunohistochemically stained using the ABC method [19] with the following modifications. After 20 min blocking with 10% normal horse serum, monoclo- nal anti-BrdU (Sigma) 36 lgÆmL )1 or anti-Ki-67 (Boehrin- ger-Mannheim), 0.2 lgÆmL )1 was added in NaCl/P i containing 1% normal horse serum and 100 UÆmL )1 nuclease S1 (Promega) for 1 h. LightCycler RT-PCR analysis One-step RT-PCR was performed with a LightCycler instrument (Roche) in a total volume of 20 lL containing 100 ng total RNA, 3.25 m M manganese acetate and 0.5 l M each primer. LightCycler RNA Master SYBR Green I kit (Roche) was used. The reverse transcription was performed at 61 °C for 20 min, and the denaturation at 95 °Cfor 2 min. Forty cycles of PCR were carried out. Each cycle of PCR included denaturation at 95 °C, 5 s of primer anneal- ing at 58 °C and 10 s of extension at 72 °C. After amplifi- cation, a melting curve was obtained by heating at 20 °C/s to 95 °C, cooling at 20 °C/s to 65 °C and slowly heating at 0.1 °C/s to 95 °C with fluorescence data collection at 0.1 °C intervals. The following primers (Amersham Pharmacia Biotech) were used for PCR: GPR30-forward, 5¢-AGTCGG ATGTGAGGTTCAG-3¢; GPR30-reverse, 5¢-TCTGTGT GAGGAGTGCAAG-3¢; TBP-forward, 5¢-TTTGGAAG AGCAACAAAGG-3¢; TBP-reverse, 5¢-AAGGGTGCAG TTGTGAGAG-3¢. TBP was used to normalize the RNA samples. These primer pairs result in PCR products of 240 bp (GPR30) and 243 bp (TBP). LightCycler data were quantitatively ana- lysed using LightCycler analysis software. The final results, expressed as N-fold differences in GPR30 gene expression between untreated and MPA-treated samples, were deter- mined as follows: N GPR30 ¼ (GPR30 treated =TBP treated Þ= (GPR30 untreated =TBP untreated Þ The relative concentrations used for expression difference calculations were obtained from the calibration curves. Concentration values for TBP were taken from the calib- ration curve made with TBP, and, in the same way, concentration values for GPR30 were taken from the 2486 T. M. Ahola et al.(Eur. J. Biochem. 269) Ó FEBS 2002 calibration curve made with GPR30. In both cases the serial dilutions were 500 : 100 : 20 : 5 ng of the total RNA. Statistical analysis Data for the correlation analysis were handled using a regression analysis of SSPI and/or a correlation analysis of EXCEL . Data for the growth studies and immunochemistry were analysed by a paired two-tailed t-test. Differences of P < 0.05 were considered significant (*), and P <0.01 (**) and P < 0.001 (***) highly significant. RESULTS Cloning of genes, the expression of which is altered during progestin-induced growth inhibition To study genes associated with progestin-induced growth inhibition, a comparison was made of the gene expression patterns in MCF-7 breast cancer cells cultured inthe presence of MPA and estrogen with estrogen-treated control cells by the differential display PCR technique. Using 20 different primer combinations, cDNA fragments were detected. These were regulated by MPA 24or 48 h after administration ofthe MPA. The amplification products were isolated and ream- plified. The cDNA fragments were subsequently cloned and sequenced. In a BLAST search, two of the transcripts showed 96–100% identity with the GPR30, and there was no significant similarity to any other genes. These were expressed in MPA-treated cells, but not in the control cells at either 24 or 48 h in differential display analysis. Progestins upregulate GPR30 mRNA in MCF-7 breast cancer cells Northern-blot analysis was used to confirm the MPA- dependent regulation and to study the steroid specificity of GPR30 expression in MCF-7 breast cancer cells. Northern- blot analysis revealed a single GPR30 mRNA species of approximate size 3 kb. As shown in Fig. 1A, MPA, R5020 and progesterone had increased GPR30 expression three- fold to fourfold 48 h after administration, whereas dexa- methasone and dihydrotestosterone failed to induce a significant increase in GPR30 expression. A 10-fold excess of antiprogestin RU486 abrogated MPA-induced GPR30 expression (Figs 1B and 2A, lanes 1, 5, 7 and 8), which further indicated progestin specificity of GPR30 expression. When administered alone, RU486 did not induce GPR30 mRNA expression. Treatment with cycloheximide (a protein synthesis inhib- itor) did not prevent progestin-induced upregulation of GPR30 and had no effect on GPR30 mRNA levels at 24 h in MCF-7 cells (Fig. 1C). An RNA synthesis inhibitor did not regulate GPR30 mRNA, but abrogated the induction of GPR30 mRNA. GPR30 expression correlated with progestin-induced growth inhibition in breast cancer cells Figure 2A shows that GPR30 mRNA expression correlated with cell growth as the dose of MPA increased; this further indicated progestin-dependent GPR30 regulation. GPR30 mRNA expression was increased approximately threefold by 0–0.1 n M MPA, and cell growth was decreased according to the dose of MPA. There was a significant correlation (P <0.01,r ¼ )0.91) between growth and the level of expression of GPR30. MCF-7 cells were grown in the presence of 10 n M MPA to determine the time sensitivity of the hormone action, in particular, to determine whether there was a correlation between the onset of the reduced rate of cell proliferation and GPR30 mRNA upregulation. RNA was extracted at 2, 8, 18 and 24 h for the Northern-blot analysis. GPR30 mRNA started to increase between 8 and 18 h after MPA Fig. 1. GPR30 mRNA is a direct target gene of progestin. GPR30 mRNA expression was analysed from the total RNA of MCF-7 cells treated with the indicated factors using Northern blotting and 32 P-labeled GPR30 cDNA fragment as a probe. The intensity of the bands was quantified by densitometry and normalized using ribosomal 18S mRNA bands. (A) MCF-7 cells were incubated with 10 n M MPA, R5020, progesterone, dihydrotestosterone or dexamethasone for 48 h. (B) Cells were incubated with 10 n M MPA with and without 100 n M RU486 for 48 h to study the effect of antiprogestin on MPA-induced GPR30 expression. (C) MPA-treated and untreated cells were incubated for 24 h with 20 lgÆmL )1 cycloheximide and 5 lgÆmL )1 actinomycin D to investigate the effects of protein synthesis inhibitor and RNA synthesis inhibitor on GPR30 expression. Results are the means of two separate experiments. Ó FEBS 2002 GPR30 and progestin (Eur. J. Biochem. 269) 2487 administration and had been induced threefold at 24 h, as revealed by Northern blotting (Fig. 2B). Cell proliferation, measured using BrdU and immunohistochemistry, was decreased to below control levels 12–24 h after MPA administration. BrdU incorporation was decreased 32%, 54% and 44% by MPA at 24, 48 and 72 h, respectively. There was correlation between the onset of growth inhibi- tion and MPA-induced GPR30 mRNA upregulation (r ¼ )0.94). We cultured different breast cancer cell lines with MPA to study how GPR30 is regulated by progestin, and how the expression of GPR30 is related to growth in these cells. Relative cell growth was measured at 120 h and GPR30 mRNA upregulation was detected at 48 h using Light- Cycler analysis in CAMA-1, MCF-7, ZR-75-1 and BT-474 cells. Different cell lines showed different responses to the progestin treatment. The magnitude of GPR30 expression correlated (r ¼ )0.91) with progestin-induced growth inhibition when a comparison was made between the cell lines (Fig. 2C). Progestin-induced GPR30 regulation is not mediated through the ERK pathway To investigate whether the ERK1/ERK2 pathway mediates progestin-induced GPR30 mRNA regulation, MCF-7 cells were treated with the ERK pathway inhibitors PD98059, U0126 and SAPK2/p38 MAPK inhibitor SB202190, with and without MPA. MEK inhibitors neither markedly regulated GPR30 mRNA nor altered MPA-stimulated GPR30 expression analysed by quantitative PCR Light- Cycler (Fig. 3). However, p38 inhibitor enhanced GPR30 mRNA expression threefold, fourfold and 40-fold in the three separate experiments. On average, MPA did not have any additional affect on SB202190-induced GPR30 mRNA upregulation. DISCUSSION A differential display technique was used to isolate cDNA fragments from MPA-treated MCF-7 breast cancer cells. On the basis of preliminary verification of discovered clones, we were able to detect gene regulation by MPA in the case of six genes. Most of the PCR fragments showed homology with mitochondrial enzymes; one was unknown and two of them showed similarity to potentially growth-associated genes. An orphan transmembrane GPR30 was selected for Fig. 2. GPR30 mRNA upregulation correlates with progestin-induced growth inhibition. MCF-7 cells were treated with MPA to study whe- ther GPR30 mRNA upregulation and growth are related. Relative cell numbers were measured at 120 h and RNA extracted for Northern- blot analysis at 48 h unless otherwise indicated. Cells grown without MPA were used as a control. (A) The increase in GPR30 mRNA expression and progestin-induced growth inhibition were dose- dependent. The values in the growth curve (j)arethemeanvaluesof four replicates, and in the Northern analysis (h)themeanvaluesof two replicates. (B) The time sensitivity of GPR30 mRNA expression correlated with the beginning of progestin-induced growth inhibition. MCF-7 cells were treated with MPA for 2–24 h and RNA was extrac- ted for Northern blotting to analyse the mRNA. Cell proliferation was measured using BrdU and immunostaining. (C) GPR30 upregulation (h) in different breast cancer cell lines correlated with growth (j). RNA was analysed by quantitative PCR using a LightCycler. 2488 T. M. Ahola et al.(Eur. J. Biochem. 269) Ó FEBS 2002 further studies because it has been shown to be expressed preferentially in endocrine tissues [12–14], and some G protein-coupled receptors are known to be involved in growth regulation. The regulation of GPR30 expression in MCF-7 cells was progestin specific and was not upregulated by other steroid hormones. Very few progestin-specific genes are reported in breast cancer cells [20,21]. The expression of GPR30 was enhanced late (between 8 and 18 h) after progestin admin- istration, which suggested that it is not the primary target gene for progestins. Surprisingly, however, the protein synthesis inhibitor cycloheximide did not abolish the induction, which suggested that GPR30 is directly regulated by progestins. We were not able to locate any progestin response elements in the promoter and regulatory region of GPR30 gene. Only the AP-1-binding site has been mapped in the promoter. It is possible that this could mediate the effect of progestin on gene transcription [12,22,23]. It has been previously established that the ERK pathway activates progestin-dependent transcription in the presence of ligand [9]. Because GPR30 mRNA was expressed in MCF-7 cells relatively late, although in a progestin- dependent manner, we decided to investigate whether MAPK activation is responsible for GPR30 mRNA regulation by progestin. Progestin-induced GPR30 mRNA regulation was not altered by MEK inhibitors, which suggested that GPR30 regulation is not dependent on the ERK pathway. Unexpectedly however, p38 inhibitor induced GPR30 expression. Progestin did not have any significant additional effect on p38 inhibitor-regulated GPR30 mRNA expression. The fact that progestin [3–5] and p38 pathway inactivation [24,25] have the same kind of effect on proliferation of MCF-7 cells may suggest a common target of the growth factors and steroid hormones. Progestin administration causes an initial increase in cell proliferation lasting about 12–24 h and subsequent inhibition of proliferation [6,26]. The late expression of GPR30 after progestin administration was thus associated with the beginning of growth inhibition by progestins. GPR30 mRNA is regulated according to the dose of progestin, which also correlated with progestin-induced growth inhibition. More importantly, when a comparison was made between different breast cancer cell lines, there was a correlation between GPR30 upregulation and prog- estin-induced growth inhibition. The ligand of GPR30 and its role in growth regulation are not known. In amino acid sequence comparisons, GPR30 shows similarity to a number of proteins; the highest identity is with angiotensin II type 1, interleukin 8 and C-C chem- okine receptors. Chemokines [27,28] and angiotensin II [29] have both shown growth-inhibiting properties with different cancers. Interestingly, an orphan receptor (GPR41 in the rat), which shows a high degree of similarity to GPR30, induces apoptosis during ischemic hypoxia [30]. The results show a progestin target gene, an orphan receptor GPR30, the expression of which correlated with growth inhibition. The potential importance of the G-proteins in progestin-mediated signaling is also highligh- ted by a study showing that half of the progesterone- regulated genes were involved in membrane-initiated events [31]. It is still not known, however, which pathway mediates the progestin effect on the cell cycle regulatory molecules during growth inhibition. Thus our results establish a candidate gene that may be involved in progestin-induced growth inhibition. ACKNOWLEDGEMENTS We thank A. Vienonen for advice. This work was supported by the Medical Research Foundation of Tampere University Hospital, Biomed 2 project PL 963433 and the Cancer Foundation in Pirkanmaa. REFERENCES 1. Clarke, C.L. & Sutherland, R.L. 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(2001) Orphan G protein-coupled receptor, GPR41, induces apoptosis via a p53/Bax pathway during ischemic hypoxia and reoxygenation. J. Biol. Chem. 276, 26453–26460. 31. Richer, J.K., Jacobsen, B.M., Manning, N.G., Abel, M.G., Wolf, D.M. & Horwitz, K.B. (2001) Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J. Biol. Chem. 20, 20. 2490 T. M. Ahola et al.(Eur. J. Biochem. 269) Ó FEBS 2002 . Progestin upregulates G-protein-coupled receptor 30 in breast cancer cells Tytti M. Ahola 1 , Sami Purmonen 1 , Pasi Pennanen 1 , Ya-Hua Zhuang 1 ,. during growth inhibition induced by medroxyprogesterone acetate (MPA). A tran- script of G-protein-coupled receptor 30 (GPR30) was upregulated by MPA in