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-

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

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

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

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

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

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

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

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.
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