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Endocrinology Vol. 143, No. 12 4620-4626
Copyright © 2002 by The Endocrine Society

ARTICLE

Progestin and G Protein-Coupled Receptor 30 Inhibit Mitogen-Activated Protein Kinase Activity in MCF-7 Breast Cancer Cells

Tytti M. Ahola, Niina Alkio, Tommi Manninen and Timo Ylikomi

Department of Cell Biology (T.M.A., N.A., T.M., T.Y.), Medical School, University of Tampere, 33014 Tampere, Finland; and Department of Clinical Chemistry (T.Y.), Tampere University Hospital, FIN-33521 Tampere, Finland

Address all correspondence and requests for reprints to: Tytti M. Ahola, Medical School, University of Tampere, P.O. Box 607, 33014 Tampere, Finland. E-mail: ta55935{at}uta.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that the G protein-coupled receptor (GPR)30 is critical for progestin-induced growth inhibition. In this study, we addressed signal transduction pathways involved in progestin-mediated signaling. Progestin could not provide any additional growth inhibitory effect to MCF-7 cells treated with specific MAPK kinase inhibitors, PD98059 and U0126. Medroxyprogesteroneacetate (MPA) induced a late (22–23 h) decrease in ERK-1 and -2 activities verified by immunoblotting and kinase assay. The inactivation was abrogated by antiprogestin. Transient expression of GPR30 decreased ERK-1 and -2 activity; and in the cells in which GPR30 expression was decreased by the antisense, ERK activities were increased. The antisense-expressing cells were able to significantly resist the growth-inhibitory effect of the MAPK kinase inhibitors PD98059 and U0126 but not that of other factors tested. Interestingly, the decrease of ERK activity induced by MPA was abrogated by GPR30 antisense. Collectively, these results show that MAPK activity is inhibited by progestin and GPR30 and suggest that progestin-induced ERK inactivation is mediated through GPR30. Coupled with our previous findings, the data imply that up-regulation of GPR30 by progestin leads to ERK-1 and -2 inactivation associated with MPA-induced growth inhibition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE HAS MANY important functions, including the regulation of the menstrual cycle in the uterus and maintaining pregnancy during the first trimester. Therefore, its synthetic forms, progestins, are used in contraception and the treatment of dysfunctional uterine bleeding (1). Progestins are also used to minimize the risk of uterine cancer associated with estrogen replacement therapy and are widely employed to treat metastatic endometrial and breast cancer.

Progestins decrease estrogen-induced proliferation in the uterus, but its role in the mammary gland is uncertain. Recent data suggest that progestins increase the risk of breast cancer in postmenopausal women (2, 3, 4, 5). In premenopausal women the measurement of serum progesterone concentrations suggests that higher circulating concentrations may be associated with a lower risk of breast cancer and better prognosis if it does occur (6, 7), although progesterone levels in serum correlate with the increase of proliferation of epithelial cells (8, 9, 10). In xenocraft models medroxyprogesteroneacetate (MPA) treatment reduces tumor incidence, mean weight of tumors, and 3H-thymidine labeling index (11, 12). In breast cancer cells, the effect of progestin on growth is in most cases inhibitory (13, 14, 15, 16, 17). Progestin first enhances the cell cycle and then arrests cells at the G1 phase (13, 14, 18). The growth-inhibitory effect of progestin is associated with a change of activity of cyclin-dependent kinases (Cdk) (19, 20). A concerted model of progestin action is suggested, whereby Cdk inhibitors p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4 (21).

In breast cancer cells progestin as well as estrogen have been shown to rapidly activate the Src/p21ras/ERK pathway (22, 23). This activation is thought to be responsible for a progestin and estrogen growth-stimulatory effect, although in the case of estrogen, this was argued by Caristi et al. (24). Interestingly, it was reported that direct binding of progesterone receptor (PR) with tyrosine kinase receptors, and possible MAPK activation, is critical for progestin-induced growth inhibition (25). However, there is no direct evidence that the MAPK pathway mediates the growth-inhibitory effect of progestin. Growth factors, tyrosine protein kinases, and protooncogene products stimulate the MAPK pathway. Activation of MAPK induces cyclin expression, activates Cdk (26) and usually leads to cell proliferation (27). Signal transduction involves Raf-1, which activates MAPK capable of further phosphorylating two forms of MAPK (ERK-1 and ERK-2) (28). There are fewer reports concerning the mechanism of MAPK inactivation, which is shown to lead to growth inhibition (29, 30, 31).

The family of G protein-coupled receptors (GPRs) are transmembrane receptors that confer a variety of extracellular stimuli such as neuropeptides and chemokines. GPRs make a major contribution to the growth regulation of differing normal and cancer cells. Their most important pathway, by which they regulate cell growth, is the MAPK pathway. GPRs are mainly shown to enhance cell proliferation. For instance, the GPR-2 initiates cell proliferation of human colon cancer cells (32), and Kaposi’s sarcoma-associated herpes virus GPR promotes endothelial cell survival and induces proliferation (33). The growth inhibitory effects of GPRs have also been established. The activation of GPR-1 leads to an inhibition of ovarian cancer cell proliferation through activation of MAPK (34). Angiotensin receptor type 2 is growth inhibitory and cross-talks with the signaling of other GPRs and growth factor receptors (35). Somatostatin, on the other hand, negatively regulates the growth of various normal and tumor cells. Its effects are mediated directly by a family of GPRs (sst1–5), which modulate the MAPK pathway and induce G1 cell cycle arrest or alternatively promote apoptosis by p53-dependent and -independent mechanisms (36). Additionally, a metastasis suppressor gene KiSS-1 was recently shown to encode a peptide ligand of a GPR (37).

GPR30 is widely expressed by, among others, prostate, ovarian, lung, brain, lymphoid, and breast cancer tissues (38, 39, 40, 41). We have previously shown that the expression of GPR30 is up-regulated by progestin (42) and is critical for progestin-induced growth inhibition in breast cancer cells (43). The ligand of GPR30 is not known, but the receptor shows some identity to angiotensin II type 1, IL-8, and other C-C-chemokine receptors. GPR30 is described as regulating rapid membrane effects on the MAPK pathway by estrogen through two different signal transduction pathways. It was shown that estrogen-induced ERK-1 and -2 activation requires the expression of the GPR30 and occurs through the epithelial growth factor (EGF) receptor (44). GPR30 also restores the ability to stimulate adenylyl cyclase and attenuate EGF-induced activation of ERK-1 and -2 by estrogen (45).

In this study we addressed various signal transduction pathways involved in progestin- and GPR30-mediated signaling. We show the inhibition of ERK-1 and -2 activity by progestin and GPR30. The results suggest that progestin regulates MAPK activity through GPR30.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones, inhibitors, and stimulators
PD98059, U0126 [MAPK kinase (MEK) inhibitors], and SB202190 (SAPK2/p38 MAPK inhibitor) were purchased from Calbiochem (La Jolla, CA). 17ß-Estradiol and MPA were provided by Sigma (St. Louis, MO) and insulin by Gibco BRL (Paisley, Scotland, UK). Mifepristone (RU486) was kindly provided by Roussel Uclaf.

Cell culture and cell growth assay
MCF-7 cells were cultured as previously described (42). The culture medium was supplemented with 5% dextran-coated, charcoal-stripped fetal bovine serum, 10 ng/ml insulin, and 1 nM estrogen. In transfected cells, tetracycline 2 µg/ml was used to suppress GPR30 antisense expression. In the cell growth assay, 3 x 103 cells were seeded per well in 96-well plates. After 48 h of plating the cells, appropriate steroid hormones and inhibitors were added. Relative cell numbers were measured using the crystal violet nuclei staining method (46).

Creation of antisense GPR30-expressing MCF-7 cells
MCF-7 cells expressing GPR30 antisense under tetracycline regulation were previously established (43). MCF-7 cells were transfected with plasmids coding tetracycline-regulated transactivator and pCEP4-TET episomal vector containing full-length cDNA of GPCR-Br/GPR30 in the antisense orientation.

Transient transfection
MCF-7 cells (106) were transfected for 24 h with (20 µg) pBk-CMV with and without a GPR30 insert and (10 µg) pCMVßGal using 30 µl Lipofectamine 2000 (Life Technologies, Inc.) according to the manufacturer’s recommendations.

Immunoblotting
Immunoblotting was carried out as previously described (47). Cells were harvested by a cell scraper and calculated in Burker’s cell chamber. Cell samples were mixed with sodium dodecyl sulfate-sample buffer. The viscosity of the samples was reduced by drawing the samples through a 23G needle and thereafter boiling them for 5 min. Equal amounts of cells (3 x 105) were resolved in 12% polyacrylamide gel and transferred to a nitrocellulose membrane with an electrophoretic transfer apparatus. After blocking, the membranes were incubated overnight at 4 C with phospho p44/p42 MAPK antibody or p44/p42 MAPK antibody (New England Biolabs Inc., Beverly, MA) or ß-galactosidase antibody and washed. Peroxidase-conjugated goat antirabbit IgG (Capperl, West Chester, PA) was used as a secondary antibody. After washing, labeled proteins were detected by enhanced chemiluminescence.

Kinase assay
ERK-1 and -2 activities were measured by a nonradioactive method using the p44/p42 MAPK assay kit according to the manufacturer’s instructions (Cell Signaling Technology, Beverly, MA). The kinases were immunoprecipitated from 8 x 105 cells with a phospho p44/p42 MAPK antibody, and because a kinase substrate was used, 2 µg Elk-1 fusion protein. Elk-1 phosphorylation was detected by enhanced chemiluminescence using the kinase assay kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth of MCF-7 breast cancer cells treated with MEK inhibitor, PD98059
We studied different cell signaling pathways with reference to progestin (MPA)-induced growth inhibition. In our preliminary studies, we cultured MCF-7 cells with various small molecule inhibitors and stimulators combined with 100 nM MPA. We tried to find molecules that have a synergistic growth effect with MPA or, alternatively, interfere with MPA-induced growth inhibition. Treatment of MCF-7 cells with a small molecule inhibitor of the MAPK pathway, PD98059 (MEK inhibitor), resulted in growth inhibition (Fig. 1Go). MPA did not induce any additional effects when the cells were treated with the MAPK pathway inhibitor PD98059 in MCF-7 cells.



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  		Figure 1. Progestin and the MEK inhibitor PD98059 inhibit growth of MCF-7 cells. MCF-7 cells were continuously cultured with estrogen (E), seeded in 96-well plates and allowed to attach for 2 d. The cells were treated with 100 nM MPA with and without 50 µM PD98059 (PD). Relative cell numbers were calculated using the crystal violet nuclei staining method at 120 h. The data are means of six replicates; the experiment was repeated three times. Statistical significance was calculated using paired t test and values were compared with estrogen-treated control cells. Differences of P > 0.05 were considered nonsignificant (NS); P < 0.05 was considered significant (*), and P < 0.01 (**) and P < 0.001 (***) highly significant.

 
GPR30 antisense effects on the growth-inhibitory outcome of the MEK inhibitors
We studied whether the effect of the factors affecting MAPK pathways were compromised in GPR30 antisense cells in a similar manner as previously established when the cells were treated with progestin (43). We cultured MCF-7 cells expressing GPR30 antisense under tetracycline regulation with the small molecule inhibitors of MEK and SAPK2/p38 kinase. Antisense GPR30 expression stimulated growth 41.2% when growth was compared with tetracycline-treated control cells. In control cells MPA, PD98059, and U0126 diminished growth by 41%, 41%, and 71%, respectively (Fig. 2Go). Interestingly, in GPR30 antisense cells, MPA and MEK inhibitors (PD98059, U0126) inhibited growth by only 5%, 9%, and 47%, respectively. The p38 pathway inhibitor, SB202190, inhibited growth an equal amount in GPR30 antisense cells and control cells (by 24–25%). Thus, the growth inhibitory effects of MPA as well as MEK inhibitors were decreased by GPR30 antisense. Furthermore, in tetracycline-treated control cells, MPA did not provide any additional growth-inhibitory effect when the cells were treated with MEK inhibitors but further reduced growth when the cells were treated with SB202190 (P < 0.01).



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  		Figure 2. The growth-inhibitory effects of MEK inhibitors were compromised in GPR30 antisense cells. MCF-7 cells expressing GPR30 antisense under tetracycline regulation were cultured with 2 µg/ml tetracycline (tet) to suppress GPR30 antisense expression. When cells were grown without tetracycline, the antisense expression was high. Cells were seeded in 96-well plates and after 2 d of attachment were treated with 100 nM MPA (M), 50 µM PD98059 (PD), 10 µM U0126 (U), and 10 µM SB202190 (S). Relative cell numbers were calculated using the crystal violet nuclei staining method at 120 h. Both GPR30 antisense cells treated with (tet, E) and without tetracycline (E) were used as a reference cells (100%) in statistical analysis. Growth percents were calculated from corresponding reference cells. The data are means of four replicates; the experiment was repeated three times. Statistical significance was calculated using the t test as in Fig. 1Go.

 
The difference in growth-inhibitory response between the two MEK inhibitors (PD98059 and U0126) might be due to the fact that PD98059 more selectively inhibits MEK1 activity (48, 49), but U0126 inhibits MEK1 and MEK2 activities to a similar extent (50). The growth-inhibitory effects of factors that affect other signal transduction pathways (protein kinase C, Ca2+, and phosphatidylinositol 3-kinase), were not compromised in GPR30 antisense-expressing MCF-7 cells (results not shown). The results suggest that GPR30, progestin, and the ERK pathways are converged, leading to growth inhibition.

MPA decreased ERK activity in MCF-7 cells
To study whether MAPK activity was changed by progestin, we treated MCF-7 cells with 100 nM MPA. We extracted proteins for immunoblotting at a time that the beginning of growth stimulation and inhibition could be expected. The cell extract was immunoblotted with antibody against phosphorylated and nonphosphorylated p44/p42 MAPK. ERK-1 and ERK-2 activities were increased at 30 min to 12 h; and diminished at 22 and 23 h by MPA (Fig. 3AGo). The total amount of p44/p42 was not changed by hormone treatments. Progestin resulted one to two MAPK inactivation periods between 22 and 23 h in separate three (of five) experiments. In two experiments the basic MAPK activity was changed but was quite stable in three other experiments.



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  		Figure 3. Progestin decreased late ERK-1 and -2 activity in MCF-7 breast cancer cells. Serum-deprived MCF-7 cells were treated with the experimental medium containing serum, 1 nM estrogen (C, control cells) and the indicated factors. A, To study the time sensitivity of progestin action, the amount of phosphorylated MAPK was measured from MCF-7 cells treated with 100 nM MPA, using immunoblotting and the phospho-MAPK (p42/p44) antibody. As a control, total ERK-1 and -2 was measured using the MAPK (42/44) antibody. B, To study the PR dependency of progestin-induced ERK inactivation, cells were treated with 100 nM MPA, 1 µM RU486, or MPA and RU486. ERK-1/-2 phosphorylation was measured using p42/p44 and 42/44 antibodies. Representative results were established two times. C, To study the activation of ERK-1 and -2 by progestin, the kinases were immunoprecipitated from the positive control, 20 ng ERK2; estrogen-treated control cells; and cells treated with 100 nM MPA at 20, 21, 22, 23, and 24 h and incubated with an Elk-1 substrate. The amount of phosphorylated Elk-1 was measured by Western blotting. Representative results were established two times.

 
To study whether the phosphorylation of ERK-1 and -2 was PR dependent, we cultured cells with MPA and antiprogestin RU486. MPA-induced ERK-1 and -2 inactivation was abrogated by anti-progestin, and RU486 itself had no marked effect on the phosphorylation (Fig. 3BGo).

To establish whether the activity of ERK-1 and -2 is changed according to kinase phosphorylation, we used the kinase assay and measured the amount of Elk-1 fusion protein that was phosphorylated by immunoprecipitated ERK-1 and -2 extracted from MCF-7 cells, treated with and without progestin. The MAPK activity was shown to diminish by progestin at 22 and 23 h (Fig. 3CGo).

ERK activity in MCF-7 cells expressing GPR30 antisense
Because GPR30 antisense cells were able to resist the growth inhibitory effect of MEK inhibitors, suggesting that the MAPK pathway is activated in the cells, we studied the phenomenon by using immunoblotting and antibodies against phosphorylated and nonphosphorylated ERK-1 and -2. Additionally, we examined whether MPA-induced ERK inactivation was abrogated by the antisense in an equal manner of MPA-induced growth inhibition (43). In the control tetracycline-treated cells, ERK-1 and -2 activities were down-regulated in a similar manner by 1 nM, 10 nM, and 100 nM MPA, respectively, whereas in antisense GPR30-expressing cells, down-regulation of ERK-1 and -2 activity was abrogated and further stimulated at 23 h (Fig. 4Go). The total amount of ERKs was not changed by hormone treatment or the antisense expression.



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  		Figure 4. GPR30 antisense stimulates ERK-1 and -2 activity and abrogates progestin-induced ERK-1 and -2 inactivation. MCF-7 cells expressing GPR30 antisense under tetracycline regulation were serum deprived overnight. The cells were treated with the experimental medium containing 1 nM estrogen with and without tetracycline (tet), which suppresses GPR30 antisense expression. Additionally, cells were treated with 1 nM MPA (lane 2), 10 nM MPA (lane 3), or 100 nM MPA (lanes 4 and 6). Cells were lysed at 23 h. The amount of phosphorylated MAPK was measured by immunoblotting using phospho-MAPK (p42/p44) and MAPK (42/44) antibodies. Representative results were established twice.

 
Transient expression of GPR30 inhibits ERK activity
To further study the effect of GPR30 on the ERK pathway, we cotransfected MCF-7 cells with GPR30/pBk-CMV (or with pBk-CMV plasmid) and with ß-galactosidase expression plasmid. ERKs were immunoprecipitated from cell lysates and incubated with the substrate. The amount of phosphorylated Elk-1 was measured by Western blotting. There was a slight transient increase of ERK activity at 1 h; interestingly, GPR30 decreased ERK activity at 2.5 h (Fig. 5Go) and occasionally at 3 h. Transfection efficiency was approximately 50–60% measured by using ß-galactosidase staining. The expression of the transfected plasmids was confirmed by Western blotting and measuring the expression of ß-galactosidase protein.



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  		Figure 5. Overexpression of GPR30 inhibits ERK-1 and -2 activity. MCF-7 cells were cotransfected with GPR30/pBk-CMV (G) or pBk-CMV plasmid (C), and pCMVßGal using Lipofectamine for 24 h. After transfection, the medium was replaced, and, at the indicated time points, the cells were lysed. ERK-1 and -2 proteins were immunoprecipitated from MCF-7 lysates or the positive control (K = 20 ng ERK2), incubated with an Elk-1 substrate, and the amount of phosphorylated Elk-1 measured by Western blotting. To control transfection efficiency, the amount of ß-galactosidase (GAL) protein was measured using immunoblotting. Representative results were established three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MAPK cascade plays a critical role in the progesterone-induced oocyte maturation process in Xenopus oocytes (51, 52, 53, 54). The role of the MAPK pathway in progestin-mediated growth regulation has been studied less. In breast cancer cells, progestin has been shown to induce rapid Src/ERK activity through a nongenomic pathway (22, 25, 55), which is suggested to be responsible for progestin’s growth-stimulatory effect. We also show an early activation of MAPK, correlating with progestin-induced growth stimulation (43). Interestingly, we demonstrate here a transient decrease of ERK-1 and -2 activity between 22 and 23 h after progestin treatment. This late inactivation associated with growth inhibition induced by MPA (established at 24 h) (42). The synergy between the MAPK and the progestin-mediated pathway was also suggested by the result that MPA could not cause any additional growth inhibitory effect in the MCF-7 cells treated with the MEK inhibitors, PD98059 and U0126. Additionally, the decrease of MAPK activity was abrogated by antiprogestin, suggesting PR dependency in ERK inactivation. There is one study in which the relationship of progestin, MAPK cascade, and growth inhibition has been demonstrated. In that study, the binding of the PR with tyrosine kinase receptors was shown to play a critical role in progestin-induced growth inhibition (25). This binding was also shown to mediate rapid MAPK activation, but there is no evidence that the rapid activation of ERK-1 and -2 is responsible for a progestin-induced growth inhibition. Thus, this is the first report to establish an association between progestin-induced growth inhibition and a decrease in ERK activity.

The transient expression of GPR30 inhibited ERK-1 and -2 activity, and GPR30 antisense activated ERK-1 and -2. The change of these activities correlated with cell growth: Expression of GPR30 inhibited growth and GPR30 antisense-stimulated growth (43). Interestingly, GPR30 antisense cells were able to resist the growth-inhibitory effect of MEK inhibitors but not that of the p38 inhibitor. This further suggested that the inactivation of the MAPK pathway might be critical for GPR30-induced growth inhibition. During our study it was established that GPR30 is required for an estrogen-induced MAPK activation. Indeed, the activity of ERK-1 and -2 was regulated through two distinct pathways: by releasing heparan-bound-EGF (44) and stimulating adenylyl cyclase (45). However, the independent role of GPR30 in isolation was not studied in these experiments.

Studies show that MAPK inactivation is involved in growth regulation. Somatostatin and its analog suppress cell growth and MAPK activity of the mouse insulinoma-derived cell line, human neuroblastoma cell line, and human small cell lung carcinoma cell lines (29, 30). The expression of G protein {alpha} inhibits both the ability of MCF-7 cells to form tumors (56) and the growth of established human tumors of breast cancer cells in athymic mice by inhibiting the MAPK pathway (31).

Progestins inhibit the growth of breast cancer cells and change the activity of Cdk. However, it is not known whether this is a primary or secondary effect of progestin. Previously, we have demonstrated that GPR30 expression correlates with a progestin-induced growth inhibition in different breast cancer cells (42) and that GPR30 is critical for a progestin-induced growth inhibition (43). As discussed above, progestin-mediated ERK inactivation was dependent on a PR. The result was in line with the hypothesis that GPR30 up-regulation through PR is essential for an MAPK cascade inactivation by progestin. Additionally, MPA-induced ERK inactivation correlated with the beginning of GPR30 up-regulation induced by progestin (42). MCF-7 cells expressing GPR30 antisense were able to resist the growth-inhibitory effect of progestin, progesterone (43), and MEK inhibitors, suggesting a convergence of progestin, GPR30, and MAPK pathways in regulating the growth of breast cancer cells. Interestingly, when GPR30 expression was diminished by the antisense, progestin was not able to inhibit ERK activity. This further suggested that GPR30 is required for a progestin-induced ERK inhibition.

It is possible that ERK inactivation leads to a change of activity in steroid hormone receptors. The MAPK pathway is seen to phosphorylate the estrogen receptor and catalyze its transcriptional efficiency (57, 58). MAPK is also able to phosphorylate the PR at sites that signal its degradation through ubiquitinization (59). Alternatively, MAPK can induce cyclin expression, activate Cdk (26) and lead to changes in cell proliferation. As a conclusion, the results presented here, as well as our previous results, suggest that up-regulation of GPR30 by progestin leads growth inhibition associated with ERK-1 and -2 inactivation. Whether progestin- or GPR30-induced MAPK inactivation is the main cause of the growth inhibition remains to be shown. Our results provide interesting new insights into progestin-mediated signaling and GPR30 action alone and point out the usefulness of MAPK pathway inhibitors in breast cancer treatment.


   Acknowledgments
 
We thank Professor Kalle Saksela, Ph.D.; Heimo Syvälä; and Laborants Hilkka Mäkinen and Janette Hinkka for their help.


   Footnotes
 
This work was supported by the Medical Research Foundation of Tampere University Hospital, EU Biomed 2 Project PL 963433, and the Cancer Foundation in Pirkanmaa.

Abbreviations: Cdk, Cyclin-dependent kinase; EGF, epithelial growth factor; GPR, G protein-coupled receptor; MEK, MAPK kinase; MPA, medroxyprogesteroneacetate; PR, progesterone receptor.

Received May 7, 2002.

Accepted for publication August 28, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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