(Received for publication, August 7, 1995; and in revised form, October 19, 1995)
From the
Human progesterone receptor (PR) expression is controlled by two promoter regions giving rise to transcripts encoding PR A and B proteins. It is unknown whether estrogen and progesterone, the major physiological modulators of PR expression, exert their effects equally on the PR promoters. The aim of this study was to analyze estrogen and progestin effects on PR promoters, PR-encoding transcripts, and PR A and B proteins in T-47D human breast cancer cells. The progestin ORG 2058 caused a prolonged decrease in transcription of the PR gene and also abrogated estrogen stimulation of PR transcription. Estradiol (E2) treatment increased the activity of the B but not the A promoter transfected into T-47D cells. ORG 2058 had no effect on the basal or E2-stimulated activity of either promoter. E2 caused a preferential increase in transcripts derived from promoter B, whereas progestins decreased the levels of all PR transcripts. E2 preferentially increased the concentration of the PR B protein and caused a decrease in the PR A/B ratio. This demonstration that estrogen and progestin independently control the synthesis of transcripts arising from the PR promoters and that estrogen alters the cellular PR A/B ratio provides possible mechanisms underlying the cell and tissue specificity of PR regulation.
Progesterone plays a major role in mammalian reproductive
biology, including development of the normal mammary gland and
expression of its differentiated function during pregnancy, and
promotion of uterine differentiation and preparation for implantation
in pregnancy(1) . Progesterone effects are mediated via the
nuclear progesterone receptor (PR) ()and control of
progesterone action is achieved largely although not exclusively by
control of the concentration of PR. The major physiological modulators
of PR concentration are the ovarian hormone 17
-estradiol (E2), and
progesterone itself, which binds to PR in order to exert progestational
effects but also participates in regulation of its own receptor.
Understanding of PR regulation derives largely from detailed and elegant studies in the mammalian uterus and in breast cancer cells, which led to the generally accepted view that estrogen increases and progestins decrease PR expression(2, 3) . However, the advent of monoclonal antibodies to the steroid hormone receptors and the examination of PR expression at an individual cell level have shown that PR regulation may be more complex than previously suspected. Regulation of PR in the uterus is a cell-specific event, and PR regulation in the normal breast in vivo may be different from its regulation in the uterus and in breast cancer cells. In the endometrium, immunohistochemical evidence supports the view that the cyclical effects of estrogen and progesterone are mediated by estrogen stimulation of PR and progesterone down-regulation of both PR and estrogen receptor (ER)(4, 5, 6, 7) . However, progesterone down-regulation of PR is not a uniform effect in the uterus, as myometrial and stromal PR levels are not decreased by progesterone and persist during the luteal phase of the menstrual cycle(4, 5, 6, 7) . Furthermore, circulating progestins cause a decrease in ER concentration in the normal breast during the menstrual cycle, as observed in the endometrium, but no decrease of PR(8, 9, 10, 11) . PR is expressed in the breast at similar concentrations throughout the menstrual cycle in normal women, and there is no evidence that its synthesis is under estrogen control in that tissue(9, 10, 11) . It is possible that differences in the regulation of PR by estrogen and progestins in the breast and endometrium reflect the differential requirement for persistence of progesterone action in those tissues (1) and may be a consequence of expression of distinct PR isoforms.
The complexity of PR regulation in vivo is paralleled by evidence of complexity in the molecular mechanisms underlying PR expression. The expression of human PR is controlled by two promoters that direct the synthesis of mRNA transcripts originating from two clusters of transcription start sites and coding for the A and B PR proteins(12) . The functional activities of PR A and B differ in a cell type-, promoter-, or ligand-specific manner(13, 14, 15) . In some cases, the N-terminally truncated PR A can act as a repressor of the activity of PR B (16, 17) and more generally of the activity of other members of the nuclear receptor family(18, 19) . PR A and B also differ in their ability to inhibit the activity of ER, in a way that is both promoter- and cell type-specific(19, 20, 21, 22) . The functional implications of N-terminal isoforms are not restricted to PR; androgen receptor mRNA isoforms differing within the region coding for the N-terminal portion of the protein have been described which are either developmentally regulated or are expressed in differentiated tissues in Xenopus laevis(23) . In Drosophila melanogaster, three isoforms of the ecdysone receptor with different N-terminal regions have been documented, which are expressed in different combinations during metamorphosis and may be required to elicit different metamorphic responses(24) .
Regulation of PR concentration by estrogen and progestins is accompanied by increases and decreases, respectively, in PR mRNA levels, which are reflected in alterations in cellular PR levels (25, 26, 27, 28, 29, 30, 31, 32, 33) , but the promoter specificity of these effects on human PR is not known. In the rabbit, estrogenic stimulation and progestin inhibition of PR gene expression take place via the same region in the 5`-untranslated region of the rabbit gene(34) . An estrogen-responsive element has been defined in this region, which binds to ER in gel shifts and mediates E2 induction in transfections. However, PR does not bind to this region even though it suppresses E2 induction in the presence of the progestin R5020(35) .
Given the complexity of human PR regulation in target tissues in vivo and the complexity of regulation of PR gene expression via its two promoters, as well as the emerging evidence of marked functional differences in PR A and B activity, it is essential to gain a deeper insight into the mechanisms that control the regulation of PR by its major physiological modulators, estrogens and progestins. It is not known whether these steroids exert equivalent effects on the two PR promoters and lead to equivalent alterations in the levels of PR A and B proteins in progestin target cells, or whether progestin abrogation of estrogen stimulation of PR expression is mediated via the same mechanism. This question is critical to understanding the in vivo complexity of PR expression and regulation in the breast and endometrium. This study addresses the PR isoform specificity of the actions of estrogen and progestin in human breast cancer cells, which are physiological targets of estrogen and progestin action.
T-47Dsd cells were prepared by maintaining T-47D cells for several weeks in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal calf serum (SFCS) and supplemented as described (42, 43) , except that 1 µg/ml insulin was used. T-47Dsd cells proliferated with doubling times that were significantly slower than the parent line and demonstrated increased sensitivity to estradiol as measured by the lower estradiol concentrations required to increase gene expression of estrogen-sensitive genes(44) .
Nuclear RNA for S1 nuclease protection assays was prepared from nuclei that were isolated from freshly harvested T-47Dsd cells using Nonidet P-40 lysis buffer as described for nuclear run-on analysis (45) and solubilized in guanidinium isothiocyanate. RNA was isolated by the guanidinium isothiocyanate-cesium chloride method as described for total RNA.
Figure 5:
Regulation of PR promoter-specific
transcripts by estrogen, progestin and their antagonists. Panel
A, relative positions on the PR gene of PR B promoter-specific
probe PR(+464,+742), and PR(+814,+1194), which
detects all PR mRNA species, are shown. The position of the first of
the downstream cluster of transcription start sites at +751 with
respect to the upstream transcription start is also indicated. ORF, open reading frame. Panel B, T-47Dsd cells were
cultured in RPMI + 5% SFCS, changed to RPMI + 1% SFCS
(+1 µg/ml insulin) and treated 1 day later with 100 nM ICI 164384 (lanes 3 and 4), 100 nM Tamoxifen (lanes 5 and 6), or vehicle (lanes
1 and 2) in the presence (lanes 2, 3,
and 5) or absence (lanes 1, 4, and 6) of 0.1 nM 17-estradiol for 24 h prior to
isolation of nuclear RNA. Cells were also treated with 10 nM ORG 2058 (lane 7), 100 nM RU 38486 (lane
8), or their combination (lane 9). RNA fragments
protected by the PR(+464,+742), PR(+814,+1194), and
36B4 antisense RNA probes were visualized by S1 nuclease protection
assay. Panel C, PR B promoter-specific RNA (open
bars) and total PR RNA (hatched bars) concentrations were
measured by densitometry. The data are expressed as a percentage of
control, corrected for 36B4 RNA
concentration.
Figure 1:
Time course of
progestin effect on PR gene transcription rate. T-47D cells were
passaged twice in RPMI 1640 + 5% charcoal treated fetal calf serum
(SFCS), then changed to RPMI 1640 + 1% SFCS 24 h before treatment.
The cells were treated with either 10 nM ORG 2058 (+) or
vehicle(-) for the indicated times. PR, PRLR, and -tubulin
gene transcription rates were estimated in duplicate using the nuclear
run-on technique. The plasmid pUC12 was used as a negative control. The
effect of ORG 2058 on PR gene transcription rate was quantified by
densitometry and is shown as a percentage of control, corrected for
-tubulin transcription rate. The data between 0 and 9 h are
representative of four to five experiments. The 28-h time point
represents a single determination in
duplicate.
A detailed analysis of the long term effects of the progestin on PR transcription rate revealed that inhibition of >60% was apparent within 1 h, had reached 72% at 9 h of treatment (Fig. 1), and was sustained to 28 h. Exposure to estradiol significantly increased PR transcription rate (171 ± 24% (mean ± S.D.), n = 3, p = 0.007, Fig. 2), and this effect was detectable within 1 h of treatment. Progestin was able to abrogate the estradiol-mediated increase in PR transcription rate to levels that were below control (56 ± 38% (mean ± range), n = 2) but not as low as progestin treatment alone (25 ± 3% (mean ± S.D.), n = 3) (Fig. 2).
Figure 2:
Effect of estrogen and progestin
co-treatment on PR gene transcription. T-47D cells cultured as
described in Fig. 1were treated with 10 nM 17-estradiol, 10 nM ORG 2058, their combination, or
vehicle for 1 or 3 h prior to measurement of PR, PRLR, and
-tubulin gene transcription rates by the nuclear run-on technique.
The effects of these agents on PR gene transcription rate was the same
at 1 h and 3 h, so the data were quantified by densitometry, 1-h and
3-h data pooled, and the results described in panel B as a
percentage of control, corrected for
-tubulin gene transcription
rate. Data are expressed as the mean ± S.D. (E2) of 3
or range (ORG, ORG+E2) of two experiments. p value, E2 versus control,
0.007.
The basal CAT activity arising from both PR promoter
constructs was similar, as measured by the ratio of CAT activity
derived from PR-B and PR-A in vehicle-treated samples (A/B ratio 0.91
± 0.55 (mean ± S.D.), n = 3, data not
shown). E2 treatment caused a statistically significant increase in
PR-B-CAT activity (411 ± 182% control (mean ± S.D.), n = 4, p = 0.014) but had little or no
effect on PR-A-CAT activity (90 ± 36% control (mean ±
S.D.), n = 3) (Fig. 3) at any plasmid
concentration used (data not shown). The E2-mediated increase in
PR-B-CAT activity was abolished upon co-transfection of PR-A-CAT (data
not shown), in agreement with PR-A inhibition of ER activity on other
estrogen-responsive sequences(19, 20, 21) .
There was little or no effect of the steroids used on transfection
efficiency or -galactosidase activity in T-47D cells (data not
shown). There was no effect of the steroids used on CAT activity of the
pBLCAT8+ vector alone (data not shown).
Figure 3:
Estradiol and ORG 2058 effects on PR
promoter activity in T-47D cells. The PR A (hatched bars) and
PR B (open bars) promoters linked to CAT were transiently
transfected with the -galactosidase expression vector pCH110 and a
plasmid encoding the human estrogen receptor (pAER) into T-47D cells.
Twenty-four hours later, cells were treated with 17
-estradiol (10
nM), ORG 2058 (10 nM), their combination, or vehicle
and harvested 44 h thereafter for measurement of CAT and
-galactosidase activity. CAT activity is expressed as a percentage
of control and is corrected for
-galactosidase activity. The data
shown are the mean ± S.D. of four (PR B promoter) or three (PR A
promoter) experiments. CAT activity in E2-treated versus control PR B samples: p =
0.014.
These data obtained in T-47D cells were at odds with the demonstration in HeLa cells that both promoter constructs were inducible by estrogen (12) . Therefore, promoter constructs were co-expressed with ER in CHO and HeLa cells and the effect of E2 treatment measured. The basal expression of PR-A promoter was greater than PR-B promoter in CHO cells, measured as indicated above (A/B ratio 4.17 ± 0.08, n = 3, data not shown) and E2 treatment increased activity of both promoters; E2 treatment caused a significant increase in PR-B-CAT activity (858 ± 284% (mean ± S.D.), p = 0.002, n = 4) and a modest induction of PR-A-CAT (140 ± 58% (mean ± S.D.), p = 0.217, n = 4) (Fig. 4). The difference in the estrogen effect on A and B promoters was significant (p = 0.003). In HeLa cells, the basal activity of both promoters was similar and was increased by estrogen treatment (PR-B-CAT: 406 ± 48% control; PR-A-CAT: 227 ± 3% control (mean ± range), n = 2) (Fig. 4), in agreement with previous observations in HeLa cells(12) . Taken together, these data showed that the PR-B promoter was inducible by estrogen in all cells tested, but that there was a cell-specific effect of estrogen on the PR-A promoter, which was estrogen-induced in CHO and HeLa cells but unaffected by estrogen in progestin-responsive T-47D cells.
Figure 4:
Regulation of PR promoter activity in CHO
and HeLa cells. CHO and HeLa cells were transfected with either the PR
A or PR B promoter CAT construct, plus pAER and pCH110 as described
under ``Experimental Procedures.'' Transfected cells were
treated with 10 nM 17-estradiol or vehicle and harvested
40 h later to measure CAT and
-galactosidase activity. The effect
of estradiol on CAT activity from the two promoters in each cell line
is shown as a percentage of the vehicle-treated control. Results for
CHO cells are the mean ± S.D. of four separate experiments: p value, E2 versus control, 0.002. The HeLa results
are calculated from duplicate determinations and are representative of
two separate experiments.
In nuclease protection assays, probe B protected a 278-base pair fragment and probe T a 380-base pair fragment detected on acrylamide gel electrophoresis. Relative transcript expression was determined after correction for recovery using the internal standard 36B4. Promoter B-derived transcripts accounted for 63% (calculated using control samples after correction for differences in the specific activity of antisense probes) of total detected transcripts and marked estradiol stimulation of these transcripts was observed (Fig. 5B). The magnitude of the effect of estradiol on B-derived transcripts (227%) was greater than that on total transcripts (125%), indicating that the effect of estradiol was primarily on promoter B activity (Fig. 5C). In the absence of probes specific for promoter A-derived transcripts, it could not be determined directly whether there was also an effect of estrogen on promoter A. However, the magnitude of the estrogen effect on total transcripts and the proportion of promoter B-derived transcripts in the total suggested that effect on promoter A, if present, was likely to be minor.
For human PR there are a number of mechanisms giving rise to mRNA size heterogeneity, which have been shown to result in transcripts of similar sizes arising from the two promoters(50) . This makes it difficult to clearly distinguish promoter B- from promoter A-derived transcripts on Northern analysis and may explain the failure to note transcript specific effects of estrogen on PR mRNA when analyzed previously by this method(27, 29, 33, 51, 52) . The use of non-overlapping probes in nuclease protection assays circumvented these limitations and allowed the quantitation of promoter B-derived transcripts and description of the preferential effect of estrogen on these transcripts.
The pure anti-estrogen ICI 164384 had little or no effect on PR transcript expression, but totally abrogated the estradiol stimulation of promoter B-derived transcripts (Fig. 5), whereas the triphenylethylene anti-estrogen tamoxifen was estrogenic both alone and in combination with estradiol: the effect of tamoxifen was predominantly on promoter B-derived transcripts (162% control versus 118% control for all transcripts), in agreement with the estradiol effect.
Figure 6:
Concentration-dependent estradiol
regulation of PR protein expression. T-47Dsd cells were cultured in
RPMI + 5% SFCS (+1 µg/ml insulin) and changed to RPMI
+ 1% SFCS on day 4 after plating. On day 6 cells were treated with
the range of 17-estradiol concentrations shown or vehicle and
harvested 24 h later. Panel A, cytosols were prepared and PR A
and B proteins were visualized by immunoblot as described under
``Experimental Procedures.'' Panel B, PR A
(
) and B (
) were measured densitometrically and expressed
as arbitrary units. Panel C, the ratio of PR A to B was
calculated at each concentration of estradiol
used.
Estrogen augmentation of PR levels increased until 48 h after treatment, when a decrease in induction, due to an increase in PR A and B levels in untreated cells (not shown), was noted. The preferential effect of estrogen on PR B levels observed in Fig. 7was also noted in the time course and resulted in a decrease in PR A/B ratio over time. The estrogen effect on PR A/B ratio was rapid and essentially maximal 24 h after treatment (Fig. 7C). The PR A/B ratio began to recover after 72 h (Fig. 7C), due to the increase in PR levels in untreated cells noted above.
Figure 7:
Time
dependence of estradiol effect on PR protein expression in T-47Dsd
cells. T-47Dsd cells were cultured in RPMI + 5% SFCS (+1
µg/ml insulin) and changed to RPMI + 1% SFCS on day 3 after
plating. Cells were treated 1 day later with 1 nM 17-estradiol or vehicle and harvested at the times indicated. Panel A, cytosols were prepared and PR A and B proteins were
visualized by immunoblot as described under ``Experimental
Procedures.'' Panel B, PR A (
) and B (
)
were measured densitometrically at each treatment time and expression
is shown as a percentage of the time-matched vehicle-treated control. Panel C, PR A/B ratio in estradiol-treated samples is
expressed as a percentage of PR A/B in time-matched
controls.
Although the estrogen-mediated increase in PR levels was primarily through an increase in PR B, there was also an increase in PR A of more modest magnitude. This was despite the fact that transfection studies had shown no effect of E2 on promoter A and nuclease protection had shown a preferential E2 effect on promoter B-derived transcripts. The mechanism underlying the estrogen augmentation of PR A protein levels is not known, but may be due to minor estrogen stimulation of promoter A-derived transcripts, which would be difficult to detect clearly on nuclease protection, given that probes specific to promoter A-derived transcripts are not feasible. Increases in PR A may also be due to low concentrations of PR A arising from translation of promoter B-derived transcripts; although full-length human PR expression vectors transfected into HeLa or COS-1 cells express only PR B(55) , it is not known whether promoter B-derived transcripts arising from the endogenous PR gene in target cells such as T-47D breast cancer cells express only PR B or also some PR A. Plasmids encoding the full-length chicken PR express low concentrations of PR A in addition to PR B (55) .
The differential estradiol stimulation of the PR promoters observed in transfection experiments was unexpected and raised the question of whether such effects were operative in the endogenous PR gene. Nuclease protection assays were employed to examine the estrogen stimulation of promoter B in the endogenous gene; transcript levels were measured in nuclear RNA, in order to measure transcriptional events distinct from cellular events such as cytoplasmic mechanisms controlling mRNA stability. Promoter B-derived transcripts accounted for over half of total transcripts, and a marked estrogen-mediated increase in their level was noted. The percentage increase in promoter B-derived transcripts was greater than that observed for all transcripts, indicating that estrogen augmented PR mRNA levels primarily through an effect on promoter B and confirming the transfection results. Interestingly, tamoxifen, which has known estrogen agonist activity with respect to increasing PR concentration(58) , also preferentially increased the concentration of promoter B-derived transcripts, whereas the pure anti-estrogen ICI 164384 was ineffective.
The observations that estradiol preferentially increased promoter B-derived transcripts in human PR are in contrast with evidence that estrogen stimulation of rabbit PR gene expression is confined to an estrogen response element within the open reading frame spanning the translation start site for PR B (34) and corresponding to the position of promoter A in the human receptor. The data in the human also contrast with the demonstration that the more proximal of the two rat PR promoters is preferentially stimulated by estrogen(59) , which may be consistent with the observation that the rodent PR exists predominantly as the A form(51, 60) . Recent studies have demonstrated four additional weak estrogen-responsive regions within the rat PR gene, which in vitro confer or contribute to estrogen responsiveness of both proximal and distal rat PR promoters(61) . It is not known why estrogen control of PR expression should vary in the rabbit, rat, and human, particularly as there is extensive homology in the sequence of the PR gene in these species. Nevertheless, it is clear from this and previous studies (12) that the human PR promoter B is inducible by estrogen in all cells tested to date, and this study has shown that this is accompanied by increases in promoter B-derived transcripts and cellular levels of PR B protein. Clearly, control of PR expression by estrogen is complex and likely to be species-specific.
The differential effect of estradiol on promoter B-derived transcripts was supported by the observation that the PR B protein concentration was increased by estradiol to a greater extent than the PR A protein, leading to an alteration in the A/B ratio in favor of PR B. This supports previous observations that PR B levels declined more rapidly than PR A upon withdrawal of estrogen in endometrial carcinoma grown in nude mice (53) and suggests that in breast cancer cells and in endometrial carcinoma estradiol may stimulate PR expression by a common mechanism involving a preferential increase in the level of PR B. However, alterations in the PR A/B protein ratio may be confined to PR stimulation, as down-regulation of PR by progestins and other agents such as retinoic acid takes place without any effect on the relative concentrations of PR A and B(36) .
Taken together, the progestin effects on PR transcription rate, PR promoters and PR RNA showed that: 1) progestins decreased both basal and estradiol-stimulated PR gene expression; 2) progestins equally decreased promoter A- and B-derived transcripts and therefore abrogated estrogen action independent of estrogen stimulation of promoter B and suggesting that the progestin effect was not mediated through the same sequence(s) as the estrogen effect; and 3) the progestin effect was not mediated directly through sequences contained within the PR promoters. This discordance between the site of estrogen and progestin action on human PR contrasts with the demonstration that progestin inhibition of estrogenic effects on rabbit PR are mediated through the same short sequence within the PR gene(35) . However, it is consistent with the demonstration that constructs containing estrogen-responsive elements can still be inhibited by progestin when the progestin-responsive element is located as far as 2 kilobases upstream from the estrogen-responsive element(21) . More generally, it is also consistent with the emerging view that progestin inhibition of ER activity on estrogen-responsive sequences is mediated not by binding of PR to DNA but indirectly, by quenching of transcription factors required for ER activity(19, 20, 21, 22) . Such a mechanism would explain progestin abrogation of estrogen induction of PR gene transcription and promoter B-derived transcript expression. Although no quenching of estrogen effects on minimal PR promoters in transfection studies was observed in this study, the endogenous levels of PR in T47D cells may not have been sufficiently high to quench the activity of high levels of exogenously transfected PR promoters and ER.
The inhibitory activity of PR A has been explored in vitro, and there is little evidence to date of this activity in vivo. Furthermore, the issue is clearly complex, as the rodent uterus is progesterone-responsive despite the fact that rodent PR exists predominantly as the A form of the receptor (51, 60) and ratios of rodent PR A/B fluctuate little during the year, with levels of PR A always exceeding levels of PR B(60) . Nevertheless, in vivo studies in the mammalian uterus and the chick oviduct have suggested that estrogen augmentation of PR levels precedes the acquisition of progestin responsiveness(64, 65) ; the in vivo evidence, combined with the demonstration in this study of preferential estradiol stimulation of PR B, indicates that PR B may be an important mediator of the physiological effects of progestins in some progesterone-responsive tissues. In this regard, it is of interest that a subset of ER+PR+ breast tumors express very low levels of PR B and consequently very high ratios of PR A/B(66) .
This study has shown that estrogen and progestin, the major physiological modulators of human PR expression, independently control the synthesis of transcripts arising from the two PR promoters in human breast cancer cells. Estrogen preferentially increased the concentration of transcripts derived from promoter B, whereas progestin decreased all transcripts equally. Promoter-specific control by estradiol of PR gene expression was reflected at the cellular level by a selective increase in the concentration of the PR B protein and a consequent change in the ratio of PR A and B proteins. Such independent control of gene expression and the resulting flexibility in the control of the cellular PR proteins, which mediate the physiological actions of progestins, are likely to provide a rational framework within which the complexity of cell- and tissue-specific regulation of progestin responsiveness can be considered.