Instituto de Biología y Medicina Experimental (G.V., J.L.B., P.S.), Consejo Nacional de Investigaciones Científicas y Técnicas and Departamento de Química Biológica (J.L.B., P.S.), Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires, 1428 Buenos Aires, Argentina; and Centre de Regulació Genòmica (G.V., C.B., M.B.), Universitat Pompeu Fabra, 08003 Barcelona, Spain
Address all correspondence and requests for reprints to: Patricia Saragüeta, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Obligado 2490, (1428) Buenos Aires, Argentina. E-mail: sarag{at}dna.uba.ar.
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ABSTRACT |
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INTRODUCTION |
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The pattern of PR expression during the estrous cycle in rat endometrial epithelial cells is markedly different from that in the stromal cells. In immunocytochemical studies, the nuclei of the epithelial cells are intensively PR positive at diestrus, whereas those of the stromal cells stained deeply at proestrus; at metestrus, however, when the estrogen levels are low, the stromal cells invariably show low-intensity PR staining of the nucleus (6). Decidualization is normal in estrogen receptor- (ER
)-deficient (ER
/) mice, indicating that ER
is not involved in this process (7). ERß (ERß) is present in the uterus, especially in the stromal cells (8). Moreover, ERß-deficient mice have a poor reproductive capacity (9). Therefore, it is likely that estrogen effects on stromal decidualization are mediated by ERß.
The molecular mechanisms underlying physiological action of progesterone on uterine function are unclear, although it has been assumed that they involve transcriptional activation of progesterone target genes mediated by binding of its nuclear PR to hormone-responsive elements (10). However, the proof for participation of this mechanism in the proliferative response of uterine stromal cells is missing.
In addition to their transcriptional effects, steroid hormones can act by nongenomic mechanisms involving cross talk with growth factor receptors and other cytoplasmic signaling pathways (11, 12, 13). In human breast cancer cells, rapid and transient activation of Src/p21ras/Erk and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways has been shown to be important in estrogen and progesterone induction of cell proliferation (14, 15). This effect of progesterone is blocked not only by antiprogestins but also by antiestrogens, indicating a cross talk between the receptors for both ovarian hormones in T47D cells (14). This cross talk involves two domains on the N-terminal half of PR isoform B (PRB), designated ER-interacting domains I and II (ERID-I and ERID-II), which interact with the ligand-binding domain of ER and are required for progesterone activation of the c-Src/Ras/Erk pathway in breast cancer cells (16). The N-terminal region of the PR also contains a cluster of proline residues located between ERID-I and ERID-II, which interacts with the SH3 domain of c-Src and can activate its kinase domain (17), but this interaction is not required for progestin activation of the Src/Ras/Erk pathway (16). Instead binding of progestins to the cytoplasmic PRB, which is part of a preformed PRB-ER
complex, activates ER
in the absence of estrogens and leads to an interaction of the ligand-binding domain of ER
with the SH2 domain of c-Src and to activation of the cascade (16).
Here we explore the participation of activation of the Erk and the Akt pathways in the proliferative response of normal uterine stromal cells to progesterone, using as a model the established UIII cell line (18). UIII cells were derived from rat uterus by sequential enzymatic digestion and density fractionation on Percoll gradient and subcultured by trypsinization. They retained estrogen, progesterone, and prolactin receptors, and their proliferation is regulated by progesterone (18). We show that UIII cells cultured in serum-free medium respond to short exposure to picomolar concentrations of progestin with enhanced proliferation, and that this effect is preceded by transient activation of Erk and Akt signaling pathways. Moreover, the proliferative response required both active PR and ER and can be blocked with inhibitors of Erk or Akt activation. In the absence of hormone, UIII cells have low levels of PR located mainly in the cytoplasm and unable to transactivate a progesterone reporter gene. In response to low concentrations of progestins, PR translocates to the cell nucleus along with the activated Erk. UIII cells are negative for ER and positive for ERß, which is cytoplasmic in the absence of hormones and colocalizes with PR. The presence of a PR-ERß complex was also evidenced by coimmunoprecipitation assays. Thus, our results demonstrate for the first time that a proliferative effect of picomolar progestin concentrations, mediated by Erk and Akt activation, needs the cross talk of PR with ERß in a system in which nongenomic pathways are independent of transcriptional effects of steroids.
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RESULTS |
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Using this protocol of short-time exposure to R5020, we analyzed the dose-response curve. R5020 increased cell proliferation at a range of 1012 to 108 M concentration. The statistical differences between the controls in the absence of hormone and in the presence of hormone are significant for 1011, 1010, 109, and 108 M. However, in various dose-response assays the effect on cell number was optimal at 1011 to 1010 M R5020, and the curves had a reproducible tendency to decrease at 109 and 108 M. In the experiment shown in Fig. 1C, the differences between both 1011 M and 107 M (P < 0.01) and between 1010 M and 107 M (P < 0.001) were statistically significant.
This unexpected finding is in contrast with the dose-response curves of transcriptional effects of R5020, which peak at 108 M concentrations (see below, Fig. 5).
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Rapid and Transient Progestin Activation of Erk12 Is Required for Cell Proliferation
In addition to their transcriptional effects, progestins are known to activate the Src/Ras/Erk pathway in breast cancer cell lines, and this activation is important for their effect on cell proliferation (14). We therefore analyzed Erk12 activation after progestin treatment in UIII cells. We found a stimulatory effect of 108 M R5020 on Erk12 activation that was maximum after 5 min treatment, decreased after 10 min (Fig. 2A), and returned to control values after 2030 min (data not shown). These results are similar to those reported in breast cancer cell lines (14) and demonstrate a rapid and transient activation of the Erk12 cascade by progestins in immortalized uterine stromal cells. High activation (
5-fold) was already evident with 1012 M R5020 but the 5 min Erk12 activation was maximal at 1010 M (
10-fold) (Fig. 2
, B and C). This dose-response curve is similar to that found for the progestin effect on cell proliferation (see Fig. 1C
and dotted line in Fig. 2C
), suggesting that Erk12 activation may be related to UIII cell proliferation.
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The Effects of Progestins on Cell Proliferation and Erk12 Activation Are Blocked by an ER Antagonist
The rapid effects of progesterone on Erk activity can be mediated by either a direct interaction of PR with c-Src (17) or by an interaction of PR with the ER, which in turn interacts with c-Src (16). To test for an ER involvement in the progestin signaling in UIII cells, we analyzed the effect of the ER antagonist ICI182.780 on the response to progestins. Erk12 induction by R5020 was inhibited by ICI182.780 to the same extent as by the PR antagonist RU486 (Fig. 3A). The proliferative effect of R5020 was also blocked by ICI182.780, which alone had no effect on cell number (Fig. 3B
). Therefore, we conclude that unliganded ER is required for progestin activation of Erk12 and cell proliferation in uterine stromal cells.
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UIII Cells Contain Levels of PR Insufficient for Transactivation of a Reporter Gene
To estimate the relative level of PR in UIII cells, we used Western blotting to compare extracts from UIII cells with extracts from the breast cancer cell line T47D, which is known to contain high levels of PR (19). The level of PR in UIII cells was considerably lower than in T47D cells (Fig. 5A). However, we cannot exclude differences in the affinity of the antibody for the rat vs. the human PR.
To have an independent evaluation of the relative levels of PR in UIII and T47D cells, we quantitated PR-mRNA by real-time PCR using as primers oligonucleotides that do not distinguish between PRA and PRB. We found that the level of PR-mRNA in UIII cells was 31-fold lower than in T47D cells (Fig. 5B). We also showed by RT-PCR that the levels of PR-mRNA in UIII cells were considerably lower than in rat uterus, the tissue from which they were derived (Fig. 5B
, inset). This result is in accordance with the lower signal detected by Western blotting and suggests that the PR levels in UIII cells are very low.
To test the transcriptional competence of the endogenous PR levels of UIII cells, we performed transient transfection studies with a mouse mammary tumor virus (MMTV)-luc reporter gene. The reporter gene was not activated by treatment of the transfected cells for 36 h with concentrations of R5020 going from 1011 M to 108 M (Fig. 5C, white bars), under conditions that activate the same reporter gene in PR-positive breast cancer cell lines (data not shown). The lack of response was not due to limiting factors in UIII cells other than PR, because the MMTV-luc reporter was induced by R5020 when an expression vector for PRB was cotransfected (Fig. 5C
, gray bars). Optimal induction was detected at 108 M, and no response was observed at 1011 M R5020 (Fig. 5C
), a concentration sufficient for optimal activation of kinase cascades. The induction by 108 M R5020 was blocked by the PR antagonist RU 486 (Fig. 5C
). The lack of transcriptional activation by the endogenous PR was not promoter specific because it was observed in similar experiments with three other transfected reporters of PR; PRE2tk-CAT, consisting of two progesterone response elements (PREs) cloned upstream of the thymidine kinase (tk) promoter-CAT (chloramphenicol acetyl transferase) hybrid gene (20) (Fig. 5D
); P4Bcl-X-Luc consisting of nucleotides from 3288 to 2652 from Bcl-X promoter cloned upstream of LUC (21) (Table 1
); and hydroxysteroid dehydrogenase (HSD)11ß2-Luc, consisting of nucleotides from 1778 to +117 of human 11ß-HSD type II promoter cloned upstream of Luc (22) (Table 1
). R5020 108 M did not transactivate these reporter genes unless expression vectors for PRB were cotransfected (Fig. 5D
and Table 1
). We conclude that the level of endogenous PR in UIII cells is insufficient for transcriptional activation of a transfected reporter.
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Upon activation by phosphorylation, Erk12 is known to translocate to the cell nucleus, where it acts on nuclear targets. Only a small percentage of hormone-treated cells exhibited nuclei stained with anti-p-ERK antibody (Fig. 6B,
p-Erk). These Erk-positive cells also exhibited nuclear staining with antibodies against PR (Fig. 6C
,
PR). An overlay of double staining showed colocalization of p-Erk and PR (Fig. 6C
, merge).
UIII Cells Lack ER and Their ERß Does Not Transactivate a Reporter Gene
Next we used Western blot and RT-PCR to analyze the identity of ER in UIII cells. Western blots showed that UIII cells lack of ER protein (Fig. 7A
, top panel; see COS7 cells as negative control) and have low levels of ERß protein (Fig. 7A
, bottom panel), as compared with MDA-MB-231 cells. In agreement with these results, RT-PCR showed that UIII cells have low levels of ERß-mRNA, as compared with rat ovary, but are negative for ER
-mRNA (Fig. 7A
).
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A Fraction of PR and ERß Form a Complex in Absence of Hormones
To test whether PR and ERß form a complex in UIII cells, as found for PR and ER in breast cancer cells (14), we performed coimmunoprecipitation experiments. Cell lysates were immunoprecipitated with a polyclonal anti-PR antibody and the precipitates were analyzed by Western blots with anti-PR or anti-ERß antibodies. We found that a small fraction of the ERß was precipitated with anti-PR antibody (Fig. 8A
). Quantitation of the bands showed that 6% of the total ERß is associated with PR, a figure comparable to that found in breast cancer cells (16).
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DISCUSSION |
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Transient exposure to progestins for 1 h was sufficient for a substantial proliferative response measured 5 d after hormone addition, even when an inhibitor of PR was added after the first hour. This suggests that all events needed for an irreversible commitment to proliferate are triggered by progestins within 60 min. Rapid and transient activation of Erk12 and Akt kinases is an essential part of the signaling pathway used by progestins, because inhibition of any of these kinases blocked subsequent cell proliferation. Thus, kinase activation could be involved in the generation of a strong positive feedback loop to produce the irreversibility of progesterone-dependent proliferation. A similar bistable memory module has been described to govern Xenopus oocyte maturation (24).
Cross Talk of PR and ERß in Uterine Stromal Cells
Activation of the kinase cascades and enhanced cell proliferation in response to progestins can be inhibited by the PR antagonist RU486, demonstrating the need for the classical nuclear PR. Moreover, the progestin effect on cell proliferation and on kinase activation can be blocked by the ER antagonist ICI 182.780, thus excluding a participation of the newly described transmembrane ER GPR30 (25). From that we conclude that the classical ER mediates progestin activation of Erk12, Akt, and cell proliferation, A similar situation has been described in breast cancer cells, in which progestin activation of Erk12 is mediated by a preformed complex of PRB and ER (13). The lack of ER
and the presence of ERß in UIII cells are consistent with the predominance of this isoform in uterine stromal cells.
There are three potential translational start sites at the 5'-end of rat ERß mRNA, which, if used, would produce proteins 485, 530, and 549 amino acids (aa) in length, differing at their N termini (26). On Western blots with extracts of rat mammary glands using an antibody against the ligand-binding domain of ERß, three bands were observed with the main bands in the 62-kDa range (27). The extracts from UIII cells yielded a similar main band using the same antibody for the Western blots.
Although ERß can inhibit ER-mediated gene transcription, in the absence of ER
, it can partially replace it (28). It has also been reported that ERß can mediate nongenomic response to estradiol of endothelial cells, and that estradiol signaling via endothelial nitric oxide synthase can occur independent of ER
(29). Thus it is possible that in UIII cells, which lack ER
, the nongenomic response to hormones could be mediated by ERß. Coimmunoprecipitation experiments showed that a fraction of PR and ERß form a complex in UIII cells before hormone administration. The potential of ERß to form a complex with another steroid receptor, the androgen receptor, has been reported using glutathione-S-transferase fusion proteins (30). Therefore, it is possible that the cross talk between progestins and estrogens in UIII cells is mediated by a hitherto unreported interaction between PR and ERß.
Estradiol, on its own, has no effect on cell proliferation under serum-free conditions (data not shown), as previously reported in the presence of serum (18). As compared with progesterone alone, a combination of progestins and estrogens does not enhance UIII cell proliferation, but rather induces decidual differentiation (Vallejo, G., D. Maschi, C. Ballaré, M. Beato, and P. Saragueta, manuscript in preparation). This situation is different from that reported in LNCaP prostate cells, where androgens and estrogens induce cell proliferation (30).
Subcellular Localization of PR and ERß
In breast cancer cells with high levels of PR, the majority of PR is nuclear before hormonal treatment. In contrast, the immunocytochemical assays showed that the subcellular localization of the small amount of PR in UIII cells was mainly cytoplasmic in the absence of hormone. A large fraction of PR translocated to the nucleus after R5020 treatment, indicating that the fluorescence signal corresponds to an active PR. However, a fraction of PR remained close to the cell membrane even after hormone addition, suggesting a vicinity to the kinase pathways leading to activation of Erk12 and Akt (14).
ERß has been reported to be localized in the cell nucleus before hormone administration in rat uterus (31) In contrast, in UIII cells the majority of ERß is found in the cytoplasm before hormone addition. We do not know whether this is due to the low levels of ERß in these cells or whether ERß is retained in the cytoplasm by its interaction with PR.
Transcriptional Incompetence of Endogenous PR and ERß
UIII cells with low PR content are reminiscent of uterine stromal cells during metestrus or of endometrial stromal cells from rats depleted of ovarian hormones (6). The levels of PR in UIII cells are indeed so low that they are not sufficient for progestin activation of a transfected PR responsive gene. However, if the levels of receptor are increased by transient transfection of a PR expression vector, we observed transcriptional activation of a progesterone reporter gene in response to nanomolar progestin concentrations. Similarly, in fibroblasts transfected with low concentrations of AR, no transcriptional activation of a transfected androgen-responsive promoter was observed in response to hormone (32). We conclude that low receptor concentration is sufficient for activation of the kinase pathways by picomolar hormone concentrations, but they are not sufficient for mediating a transcriptional response.
Similar to PR, endogenous ERß in UIII cells is unable to transactivate a transfected ERE reporter gene. This is not due to lack of coactivators, because cotransfection of an expression vector for ERß or ER leads to activation of the reporter in response to estrogens. The inability of endogenous ERß to mediate transactivation may be due to its low cellular levels or to its interaction with PR in a complex that is transcriptionally incompetent. The nuclear translocation of PR after progestin treatment shown in Fig. 6
does not invalidate this argument because it is a prerequisite but not sufficient for gene activation. For instance, in several cell types RU486 induces nuclear translocation of PR and GR without leading to transcriptional activation of target genes (33, 34).
The mechanism that maintains low levels of the ERß-PR complex in the cells remains to be established, but it is clear that a fraction of the transfected ER escapes this type of control.
Optimal Response at Picomolar Concentrations of R5020
The proliferative response of UIII cells reaches a maximum at picomolar progestin concentrations (1010 to 101M), which is also the optimal concentration for activation of Erk12 and Akt kinase activity. This finding is in apparent contradiction with the measured dissociation constant (Kd) of PR for R5020, which lies in the nanomolar range. Similarly, fibroblasts transfected with low amounts of an androgen receptor expression vector exhibit hypersensitivity to low doses of androgen in terms of androgen-dependent Erk12 activation (32).
A recent study in mice uterus has revealed the presence of high-affinity, low-capacity, progesterone binding sites (Kd for R5020, 43 ± 9 pM) in addition to the classical PR (Kd 9.2 ± 4.2 nM) (35). The high-affinity sites represent about 3% of the total progesterone binding capacity. As we have shown that less than 5% of the total PR in breast cancer cells exists in a complex with ER (16), it is possible that the PR in this complex exhibits a higher affinity for progestins. If this were the case, low concentrations of progesterone could activate the Erk pathway acting through this high-affinity receptor complex.
Suboptimal Effect at Nanomolar Concentrations of R5020
An unexpected finding is that nanomolar concentrations of R5020 have a less pronounced effect on cell proliferation and kinase activation than picomolar concentrations. One possible explanation would be that nanomolar concentrations of progestins favor PR dimerization and thus reduce the amount of PR in the monomeric conformation required for interaction with ER and kinase activation. Kinetic analysis of estradiol receptor homodimerization showed that formation of the receptor homodimer in vitro is markedly dependent upon the receptor concentration (36) and is promoted by hormone binding (37). Moreover, receptor homodimerization is required for nuclear translocation and ligand-dependent transcriptional activation in vivo (38). Therefore, differential tissue responses in different cells may be obtained depending on their steroid receptor content. T47D cells, the intracellular PR concentration of which is in the nanomolar range (19), may form a substantial amount of the activated dimer receptor in the absence of ligand, and respond to nanomolar concentrations of progestins with optimal nuclear translocation and transcriptional activation. In contrast, in UIII cells, with subnanomolar concentrations of PR, the majority of the receptor will be in the monomeric form in the absence of ligand and will localized to the cytoplasm, poised for cross talk with kinase cascades. We will test these hypotheses by constructing cell lines with different amounts of epitope-tagged PR and ER, allowing detection and biochemical analysis of minute amounts of the different forms of the receptor and their associated proteins.
Our results suggest that the proliferative response of uterine stromal cells to picomolar concentrations of progestins does not require direct transcriptional effects. A progestin response via integration of rapid intracellular signaling and PR that functions as a ligand-activated transcription factor (39) has been recently reported for gene regulation and proliferation in breast cancer cells. The UIII cell line is a useful model to study the effects mediated by the rapid signaling component.
The relevance of the cross talk between steroid hormones and signaling cascades for activation of cell proliferation in cells with very low receptor concentrations force us to reconsider our definition of progesterone target cells. By many classical criteria, including Western blot, steroid binding assays, and conventional immunocytochemistry, UIII cells would have been considered PR negative because their levels of PR are 1 order of magnitude lower than in classical breast cancer model cell lines. Nevertheless they exhibit a clear dependence on progesterone for growth and differentiation (Vallejo, G., D. Maschi, C. Ballaré, M. Beato, and P. Saragueta, manuscript in preparation) and are sensitive to very low concentrations of hormone. Thus, more accurate and sensitive methods are needed to estimate the hormone receptor content of cells, and the spectrum of target cells for various steroid hormones will have to be expanded. This is especially important given the eminent physiological relevance of the nongenomic effects of steroid hormones, which are measurable in a whole variety of cell types not belonging to the traditional target tissues of these hormones (12, 13).
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MATERIALS AND METHODS |
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Plasmids
The pAGEMMTVLu (MMTV-Luc) plasmid, carrying the MMTV promoter linked to the firefly luciferase (luc) gene, PRE2tk-CAT carrying two synthetic PRE oligonucleotides cloned upstream of the thymidine kinase promoter-CAT hybrid gene, and P4Bcl-X-Luc encompassing nucleotides 3288 to 2652 from Bcl-X promoter in pGAW.Luc were described previously (20, 21, 40). pSG5:PR encoding the human PRB, and pSG5:ER (HEG0) encoding the human ER
were a gift from Pierre Chambon (41). pCXN2-ERß encoding the human ERß was a gift from Geoffrey Greene. HSD11ß2-Luc encompassing nucleotides 1778 to +117 of human 11ß-hydroxy steroid dehydrogenase type II promoter was cloned upstream of Luc gene in pGL3-Basic (22).
Cell Culture and Cell Proliferation Experiments
UIII rat normal uterine stromal cells were maintained in M199 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C in humidified 95% air with 5% CO2 (18). Culture media were changed every 2 d.
For hormone treatment experiments in the presence of serum, cells were first cultured in M199 supplemented with 10% dextran-coated charcoal-treated FBS (DCC/FBS). At the beginning of the treatment, media were replaced by fresh DCC/FBS medium with vehicle (ethanol) as control or with hormones. Viable cells were counted after 5 d of culture using the 0.1% trypan blue exclusion method.
For hormone treatment experiments in the absence of serum, cells were cultured in DCC/FBS, and 48 h later, media were replaced by fresh M199 without serum. After 2 d in serum-free conditions, media were replaced by fresh media with either vehicle or hormones. The effects of antagonists or inhibitors were tested in cells pretreated with antagonists, inhibitors, or vehicle during 30 min before hormone treatment. The cell numbers were determined after 5 d of culture in medium without serum.
For studying short-term (1-h) progesterone effect, media containing the hormone were removed after 1-h progesterone treatment, and cells were carefully washed six times (10 min each) with hormone-free medium. After the washings, media were changed every 2 d. The number of cells was determined after 120 h of treatment.
Flow Cytometry
To analyze cell cycle response to progestins, cells at 7080% of confluence were incubated for 48 h in serum-free media and then treated for 24 h with 108 M R5020. After treatments, cells were trypsinized, washed, transferred to 15-ml tubes containing 0.9 ml PBS (1 x 106 cells per tube), and collected by centrifugation. The cell pellets were resuspended and incubated for 30 min at 37 C in 1 ml of freshly prepared analysis solution, containing 5 µg/ml Hoechst 33342. One million cells per time point and treatment were analyzed on a LSR detector flow cytometer (Becton Dickinson and Co., San Jose, CA). Fluorescent data were collected and DNA histograms were analyzed with the ModFit 3.0 software from Verity Software House, Inc. (Topham, ME).
Western Blots
SDS-PAGE and immunoblot analyses were used to identify the Erk12 and Akt proteins. Cell lysates were prepared in 1% sodium dodecyl sulfate, 25 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM EGTA plus protease and phosphatase inhibitors, and analyzed by Western blots with the antibodies described above. The band intensities were measured with ImageQuant 3.3 program (Amersham Pharmacia Biotech, Arlington Heights, IL).
Transient Transfections and Luciferase Activity
UIII cells were transiently transfected with the Lipofectamine Plus reagent (Invitrogen, San Diego, CA) according to the manufacturers instructions.
For studying the transcriptional activity of PR, the MMTV-Luc plasmid (40) carrying the MMTV promoter linked to the luciferase reporter gene was cotransfected together with the PRB expression vector pSG5:PR or the empty plasmid pSG5. RSV:ßGal plasmid was also included in each transfection to normalize transfection efficiency. After transfection, cells were treated for 36 h with R5020 (from 108 to 1011 M), or 108 M R5020 plus 106 M RU486, or 106 MRU486 or ethanol. Additional transfection experiments were also performed with PRE2tk-CAT, P4Bcl-X-Luc, and HSD11ß2-Luc (20, 21, 22) cotransfected together with the PRB expression vector pSG5:PR or the empty plasmid pSG5. After transfection, cells were treated for 36 h with R5020 (108 M) or ethanol.
For studying the transcriptional activity of ER and ß, the ERE-Luc plasmid (42) containing the luciferase reporter gene downstream from a minimum ß-globin promoter and two estrogen-responsive elements was cotransfected together with the ER
or ERß expression vector or the empty plasmid. RSV:ßGal plasmid was also included in each transfection to normalize transfection efficiency. After transfection, cells were treated for 36 h with estradiol (108 M), alone or in combination with ICI182780 (106 M), or with ethanol.
Cell lysates were prepared and luciferase and ß-galactosidase activities were determined with assay kits from Promega Corp. (Madison, WI), according to manufacturers instructions. To correct for differences in transfection efficiencies, luciferase units were normalized for ß-galactosidase activities. For detecting the transcriptional activity of PRE2tk-CAT, total RNA was isolated and the level of CAT mRNA was measured by semiquantitative RT-PCR using as forward primer: 5'-GCC TTT ATT CAC ATT CTT GC-3' and as reverse primer: 5'-CAG GTT TTC ACC GTA ACA CGC-3'. PCR amplification reaction was carried out within the exponential range (28 cycles of 1 min at 52 C for CAT). Relative expression of CAT mRNA were normalized by ß-actin mRNA content as described below.
RNA Extraction and RT-PCR
Total RNA isolation was performed according to the guanidinium thiocyanate-phenol-chloroform extraction single-step method (43). To determine hormone receptors mRNA expression, cDNA was synthesized from equal amounts of total RNA (2.5 µg) with Superscript Reverse Transcriptase (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen), and amplified with Taq DNA polymerase (Invitrogen). The following primers were used: PR forward primer: 5'-CCC ACA GGA GTT TGT CAA GC-3'; PR reverse primer: 5'-TAA CTT CAG ACA TCA TTT C-3'; ER forward primer: 5'-AAT TCT GAC AAT CGA CGC CAG-3'; reverse primer: 5'-GTG CTT CAA CAT TCT CCC TCC TC-3'; ERß forward primer: 5'-GTC CTG CTG TGA TGA ACT AC, reverse primer: 5'-CCC TCT TTG CGT TTG GAC TA-3'; ß-actin forward primer: 5'-GAA GGC CCC TCT GAA CCC CAA-3'; ß-actin reverse primer: 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'. PCR amplification reaction was carried out within the exponential range (35 cycles of 1 min at 50 C for PR, 35 cycles of 1 min at 56 C for ER
and ERß, and 24 cycles of 1 min at 62 C for ß-actin). All primers used are specific for rat steroid receptors. In all cases the receptor mRNAs were compared with mRNAs from rat tissues.
All amplification products were routinely checked by gel electrophoresis on a 1.3% agarose gel and then visualized under UV light after staining with 0.05% ethidium bromide to confirm the size of the DNA fragment and that only one product was formed.
Real-Time PCR
Amplification mixtures were loaded into LightCycler glass capillary tubes containing 1 µl of cDNA, 2 µl of 10x LightCycler FastStart SYBR Green l (Roche Molecular Biochemicals, Mannheim, Germany), 0.3 mM MgCl2 solution, 0.2 µM of PR oligonucleotide forward primer, 0.2 µM of PR oligonucleotide reverse primer, and water to a final volume of 20 µl. PCR program was performed on the LightCycler (Roche Molecular Biochemicals) with an initial cycle of denaturation at 95 C for 300 sec, followed by a 55-cycle program consisting of heating at 20 C/sec to 95 C with a 2-sec hold, cooling at 20 C/sec to 50 C with a 10-sec hold, heating at 20 C/sec to 72 C with a 13-sec hold, and heating at 20 C/sec to 79 C with a 2-sec hold (single acquisition mode). The authenticity of the PCR products was verified by melting curve analyses and agarose gel electrophoresis. Fluorescent values of each capillary were measured at 530 nm. Crossing points were calculated with the fit point method (LightCycler software 3.5.3; Roche Molecular Biochemicals). Assuming that during the log-linear phase the efficiency is constant and that the fluorescence is proportional to the amount of template, these intensities were used to calculate the initial amount of target.
Coimmunoprecipitation of ERß and PR
For the coimmunoprecipitation assay, cells were plated in M199 medium supplemented with 10% DCC/FBS and 48 h later medium was replaced by fresh M199 without serum. After 1 d in serum-free conditions, cells were lysed in 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 5 mM MgCl2; 1% Triton X-100; 0.1% sodium deoxycholate plus protease and phosphatase inhibitors. Immunoprecipitation was conducted as previously described with some modifications (16). Briefly, cell extracts (2 mg/ml for each immunoprecipitation) were precleared 2 h with 30 µl antirabbit IgG beads (eBioscience, San Diego, CA). After centrifugation, aliquots of the supernatants were removed for later analysis (inputs), and the remaining supernatants were incubated overnight at 4 C with 6 µg anti-PR rabbit polyclonal antibody (H190, Santa Cruz), or with 6 µg of an unspecific control antibody (anti-Oct1 rabbit polyclonal antibody) or without antibody, followed by incubation with 60 µl antirabbit IgG beads for 2 h. Samples were centrifuged and the pellets were washed four times with washing buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 5 mM MgCl2; 1% Triton X-100 plus protease and phosphatase inhibitors) and once with PBS plus protease and phosphatase inhibitors. The immunoprecipitated proteins were eluted from the beads by boiling in sodium dodecyl sulfate sample buffer. Inputs and immunoprecipitated proteins were analyzed for PR and ERß by Western blot using anti-PR rabbit polyclonal antibody (H190, Santa Cruz) and polyclonal anti-ERß DNA-binding domain, respectively.
Immunofluorescences
Cells were seeded onto coverslips in six-well multidishes as described above for hormone treatment experiments in the absence of serum. After 30 min of treatment with ethanol or 1011 M R5020, cells were washed, fixed by incubation in 3% paraformaldehyde in 0.1% Tween-PBS for 5 min in ice, and permeabilized by incubation in 0.1% Triton X-100 for 1015 min at room temperature. After rinsing three times for 5 min in 0.1% Tween-PBS, the coverslips were incubated for 2 h with 10% BSA in 0.1% Tween-PBS to reduce nonspecific staining. To detect PR, cells were incubated with polyclonal antibody (C20) from Santa Cruz Biotechnology, diluted 1:100 in 10% BSA 0.1%Tween-PBS at 4 C overnight. After several washes in Tween-PBS, coverslips were exposed to secondary antibody ALEXA 488 goat antirabbit (Molecular Probes), diluted 1:1000 in 0.1% Tween-PBS for 1 h at room temperature. Coverslips were mounted on slides with VectaShield mounting medium (Vector Laboratories, Burlingame, CA) and subjected to Leica DM IRBE inverted research microscope and Leica TCS SP2 spectral confocal module with a HCX PL APO 63x /1.32 Oil Ph3 CS objective (Leica Microsystems, Nussloch, Germany). Samples were excited at 488 nm and analyzed at an emission of 515 nm. Serial z-axis with a 40-nm resolution sections were collected as digitized fluorescent images.
For colocalization of phospho-Erk and PR immunofluorescence, the primary antibodies were diluted 1:400 for monoclonal antiphospho-Erk12 and 1:100 for C20 polyclonal anti-hPR in 10% BSA 0.1% Tween-PBS, and incubation was at 4 C overnight followed by a mixture of secondary antibodies from Molecular Probes: ALEXA 488 goat antimouse and ALEXA 633 goat antirabbit, both diluted 1:1000 in 0.1% Tween-PBS for 1 h at room temperature.
For colocalization of PR and ERß immunofluorescence the primary antibodies, anti-PR rabbit polyclonal antibody (H190, Santa Cruz) and anti-ERß 503 chicken polyclonal antibody, respectively, were diluted 1:100 in 10% BSA 0.1% Tween-PBS, and incubation was at 4 C overnight followed by a mixture of secondary antibodies, fluorescein isothiocyanate rabbit antichicken (Sigma) diluted 1:32 and ALEXA 633 goat antirabbit (Molecular Probes), both diluted 1:1000 in 0.1% Tween-PBS for 1 h at room temperature.
Nuclei were counterstained with To-pro-3 iodine in all cases (Molecular Probes). Samples were excited at 488, 632, and 645 nm and analyzed at an emission of 515, 647, and 661 nm, respectively. Serial z-axis with a 40-nm resolution sections were collected as digitized fluorescent images. Images were analyzed using Leica Confocal Software TCS SP: 3D, Physiology and Multicolor software (Leica Microsystems). Bright-field images (nonconfocal images) were recorded with a transmitted light detector (Leica Microsystems). Images for figures were resized with Adobe Photoshop 7.0 software (Adobe Systems, Inc., San Jose, CA).
Statistical Analysis
Results from progesterones effect on the UIII cell proliferation experiment were analyzed with Kruskal-Wallis nonparametric test and Dunns t multiple-comparisons test. Individual comparisons within a given group of treatments from all other experiments were made, using an ANOVA and Dunnett multiple-comparisons test. Differences were considered significant if P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online July 14, 2005
1 G.V. and C.B. contributed equally to this work.
Abbreviations: CAT, Chloramphenicol acetyl transferase; DCC/FBS, dextran-coated, charcoal treated fetal bovine serum; ER, estrogen receptor; ERID, ER-interacting domain; HSD, hydroxysteroid dehydrogenase; MMTV, mouse mammary tumor virus; PI3-K, phosphatidylinositol 3-kinase; PR, progesterone receptor; PRB, PR-isoform B; PRE, progesterone response element; tk, thymidine kinase.
Received for publication January 10, 2005. Accepted for publication June 28, 2005.
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