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Department of Reproductive Biology (F.L., R.G.-B.), Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico 14000; Department of Reproductive Biology (A.E.L.), Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico 09340; Facultad de Química (G.A.G.), Universidad Nacional Autónoma de México, Mexico City, Mexico 04510; School of Medicine (G.P.-P.), Universidad Nacional Autónoma de México, Mexico City General Hospital, Mexico 06726; and Department of Molecular and Cellular Biology (K.J.J., K.M.C., R.D., C.L.S., A.J.C.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Fernando Larrea, M.D., Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Quiroga No. 15, México D. F., C. P. 14000, México. E-mail: larrea{at}conacyt.mx
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. In this study, we have examined both ER- and
PR-mediated transcriptional activity of a number of A-ring chemically
reduced derivatives of norethisterone and Gestodene.
Double bond hydrogenation decreased the transcriptional potency of
norethisterone and Gestodene through both PR isoforms with
a 100- to 1,000-fold difference, respectively. In terms of the effects
of norethisterone and Gestodene and their corresponding
5-dihydro (5-norethisterone and 5-Gestodene), or
3,5-tetrahydro or 3ß,5-tetrahydro derivatives
(3,5-norethisterone/3,5-Gestodene and
3ß,5-norethisterone/3ß,5-Gestodene,
respectively) on estrogen-mediated transcriptional regulation, the
3ß,5-tetrahydro derivatives of both norethisterone and
Gestodene showed the highest induction when HeLa cells
were transiently transfected with an expression vector for ER. This
activity could be inhibited with tamoxifen. These compounds did not
activate gene transcription via ERß, and none of them showed
antagonistic activities through either ER subtype. The
3ß,5-tetrahydro derivatives of both norethisterone and
Gestodene were active in other cells in addition to HeLa
cells and activated reporter expression through the oxytocin promoter.
In summary, two ER selective agonists have been identified. These
compounds, with ER vs. ERß selective agonist
activity, may be useful in evaluating the distinct role of these
receptors as well as in providing useful insights into ER
action. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Although the biological
significance of the existence of two ER subtypes is still unclear, the
relative homology (60%) between their respective ligand binding
domains (4) raises the possibility of the existence of
natural or synthetic ligands with unique binding characteristics for
ER and ERß subtypes. Thus, the differential expression and unique
ligand binding specificities of the ER subtypes could provide an
explanation for the pleotropic actions of estrogens in many target
tissues (5, 6). We, and others, have previously
demonstrated that 19-nor progestins are bio-transformed into several
metabolites that exhibit altered hormone properties in target tissues
(7, 8, 9, 10). A-ring reduction of 19-nor T derivatives such as
norethisterone (NET), Gestodene (GSD), and levonorgestrel
(LNG) to their corresponding 5-dihydro and 3ß,5-tetrahydro
metabolites significantly reduces their progestational activity.
Although the dihydro-reduced metabolites bind mainly to PRs, the
tetrahydro-reduced metabolites lose their progestational activity and
demonstrate significant binding affinity for the ER with in
vivo estrogenic effects (9, 10, 11, 12, 13). These observations
support the idea that a given steroid can induce selective and even
opposing effects in a variety of organs and tissues depending on its
metabolic fate, the availability of steroid receptors, and the presence
of different subsets of available steroid-responsive promoters and
cofactors.
In the present study, we have evaluated the estrogenic activities of
NET and GSD and their metabolites using transient transfections in HeLa
and CHO cells with an estrogen response element driven chloramphenicol
acetyltransferase (CAT) reporter or the oxytocin luciferase reporter
and expression vectors for either ER or ERß. The results
demonstrated that at low concentrations the 3ß,5-tetrahydro
derivatives of both NET and GSD (3ß,5-NET and 3ß,5-GSD,
respectively) selectively activate ER, whereas a weak ERß
agonistic activity was observed only with the 3ß,5-NET derivative
at very high concentrations. It appears, therefore, that there are some
unique features in the structure of these compounds that promote
specific binding to ER and transactivation via this receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ethynyl-17ß-hydroxy-4-estren-3-one)
and GSD (13ß-ethyl-17-ethynil-17ß-hydroxy-4,15-gonadien-3-one)
were kindly provided by Schering AG Mexicana, S.A. (Mexico
City) and Schering AG (Berlin, Germany), respectively.
Synthesis of the corresponding 5-dihydro (5-NET and 5-GSD),
and the 3,5- (3,5-NET and 3,5-GSD) and 3ß,5-
(3ß,5-NET and 3ß,5-GSD) tetrahydro derivatives, including the
description of their corresponding physical and spectroscopic constants
has been previously described (12, 13).
Plasmids
The pLEN-hPRA was constructed by
inserting the full-length human progesterone receptor
(PRA) cDNA into the BamHI site of the
pLEN mammalian expression vector (14). The
pLEN-hPRB was generated by inserting the
full-length human PRB cDNA into the
BamHI site of the pLEN vector. The expression vectors for
human ER and ERß (pCMV5-hER and
pCMV5-hERß) containing the coding sequence of
the ER and ERß were kindly provided by Drs. B. S.
Katzenellenbogen, University of Illinois (Urbana, IL) and J.-Å.
Gustafsson, Karolinska Institute (Huddinge, Sweden), respectively. The
estrogen responsive reporter plasmid (ERE-E1b-CAT) contains a fragment
of the vitellogenin A2 gene promoter (positions -331 to -87) upstream
of the adenovirus E1b TATA box fused to the chloramphenicol
acetyltransferase (CAT) gene (15). The progesterone
responsive reporter plasmid (PRE-E1b-CAT) was used as a reporter for
PRA and PRB (16).
The oxytocin reporter, pROLUC, has been previously described
(17).
Transfections and reporter assays
HeLa and CHO cells were plated the day before
transfections, at a density of 3.0 x 105
cells/well/6-well plate, in DMEM without phenol red (DMEM-HG), which
was supplemented with 5% stripped FBS and 100 U/ml of penicillin and
100 µg/ml streptomycin; and incubated in 5%
CO2 at 37 C. The next day, the HeLa or CHO cells
were visualized on a microscope to verify that the cell density was
30–50% confluent. Transfections were performed in triplicate using
SuperFect (QIAGEN Inc., Valencia, CA) or Lipofectamine
(Life Technologies, Inc., Gaithersburg, MD) according to
the protocol provided by the manufacturer. Briefly: serum-free media
(0.1 ml) was aliquoted and DNA added (1 µg of the reporter gene
plasmid and 0.025–0.5 µg of the expression vector depending upon
whether ER or PR assays were being performed), after vortexing, 10 µl
of SuperFect reagent was added and vortexed for 10 sec. Following
incubation at room temperature for 5–10 min, 0.6 ml of supplemented
DMEM-HG was added to each sample. The medium containing the
transfection complexes was added to the cell monolayer, which had
previously been rinsed with PBS. The plates were incubated for 3 h
at 37 C in 5% CO2. After incubation, the plates
containing the transfection complexes were rinsed with PBS and 3 ml of
supplemented DMEM-HG was added to each well. Lipofectamine
transfections were performed as previously described (18).
Twenty-four hours later, the medium was replaced with medium containing
the compounds of interest at various concentrations
(10-12–10-6
M). Dimethyl sulfoxide or ethanol was used as vehicle. CAT
activity using 5 µg of protein, 10 µg of butyryl coenzyme-A
(Sigma MO), 2 x 105 cpm of
xylene-extracted [3H]chloramphenicol in 0.25
M Tris-HCl, pH 8.0, was assayed as previously described
(19, 20, 21). For the luciferase assays, 48 h after
transfection the cells were rinsed 1x with PBS without
Ca2+ or Mg2+. After
aspiration 600 µl of 1x Passive Lysis Buffer (Promega Corp., Madison, WI) was added to each well of a six-well plate.
The plates were incubated at room temperature with rocking/shaking
until cells lysed. The cell lysates were transferred to a 1.5-ml
microcentrifuge tube and centrifuged for 2 min at 4 C to form clear
lysates. Aliquots of cell lysates (20 µl) were transferred to 12
x 75 mm polystyrene tubes (Sarstedt, Newton NC) suitable for use with
a luminometer. Reagents used were appropriate for the dual-luciferase
assay (Promega Corp., Madison, WI). Samples were read on a
Monolight 3010 luminometer (PharMingen, San Diego, CA).
Statistical significance was determined using two tailed t
test.
Receptor binding studies
The relative receptor binding affinities were determined
as described by Smith and Kreutner (22). Briefly: an
adenovirus-mediated DNA transfer procedure (22, 23) was
used to transfect COS-1 cells with 3 µg of ER expression vector
(pCMV5-hER or
pCMV5-hERß). Twenty-four hours later, cells
were harvested and whole cell extracts were prepared in TESH (10
mM Tris, pH 7.7, containing 1 mM EDTA, 0.1%
monothioglycerol and 0.4 M NaCl). Cell extracts were
incubated with 1 pmol [3H]E2 and increasing
concentrations (0.005–5000 pmol) of either E2 or the synthetic test
compounds for 3 h on ice. Free steroid was separated from
receptor-bound steroid by adsorption to hydroxyapatite Bio-Gel HTP gel
(Bio-Rad Laboratories, Inc., Hercules, CA). The amount of
ER-bound [3H]E2 was quantified by scintillation
counting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. All compounds are
19-nor T derivatives. The 5-dihydro and 3,5- and
3ß,5-tetrahydro derivatives of NET and GSD, respectively were
prepared by chemical double bond hydrogenation of the A-ring as
described in Materials and Methods.
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shows the effects on PR
transcriptional activity of NET and GSD and their corresponding A-ring
reduced derivatives in HeLa cells transfected with
PRA (Fig. 2, A and B) or
PRB (Fig. 2, C and D) expression vectors. Cells
were transfected with an expression plasmid for either
PRA or PRB with a
progesterone (P4)-responsive reporter gene and
treated with a 10-8 M concentration
of each steroid, including P4. As shown in Fig. 2, both NET and GSD behaved as full PR agonists. GSD is a more potent
transcriptional activator of both PRA and
PRB than either NET or P4
(Table 1). As expected by the previously
reported relative binding affinities to PR (24), double
bond hydrogenation resulted in a significant reduction in the
transcriptional potency of NET and GSD. The 3,5- and
3ß,5-tetrahydro derivatives of NET and GSD gave the lowest
transactivation through both PRs compared with the other compounds. The
3,5- and 3ß,5-tetrahydro derivatives of NET and GSD have
approximately 500- to 1,000-fold difference in transcriptional potency,
as judged by their respective EC50 values (Table 1), obtained in dose-response experiments (data not shown), when
compared with that obtained with their corresponding nonreduced parent
compounds.
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and ERß. In these studies, the effects of GSD, NET, and
their corresponding dihydro and tetrahydro derivatives on
receptor-mediated reporter gene transcription were compared with those
of E2. As shown in Fig. 3, out of the
eight synthetic steroids tested, both 3ß,5-NET and 3ß,5-GSD
showed the highest induction of ER-mediated reporter gene transcription
when HeLa cells were transiently transfected with the ER and the
ERE-E1b-CAT reporter (Fig. 3, A and B). As depicted, these two
compounds 3ß,5-NET and 3ß,5-GSD at a concentration of
10-8 M exhibited a similar
transcriptional activity to that observed with the same dose of E2.
Interestingly, a similar concentration (10-8
M) of these compounds showed no stimulation of
gene transcription through ERß (Fig. 3, C and D). Similar results
were obtained with the 5-dihydro and the 3,5-tetrahydro
derivatives of NET and GSD, respectively, but with significantly lower
potency for ER. At all doses used (10-12
M to 10-6
M), unmodified NET and GSD led to the lowest
levels of induction of ER subtype-mediated reporter gene transcription,
compared with the derivatized compounds (data not shown).
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, A and B, shows a comparison of
the agonist activity at various doses
(10-12–10- 6
M) of 3ß,5-NET and 3ß,5-GSD on reporter gene
transcription through either the ER or ERß. As depicted in Fig. 4A, 3
ß,5-NET resulted in ER selective transactivation of
reporter gene expression with an EC50 value that
was 100-fold higher than that obtained with E2. As can also be observed
in Fig. 4A, 3ß,5-NET, at the doses of
10-12–10-7
M, was unable to stimulate ERß-mediated reporter gene
transcription. Only at the highest concentration tested of this
compound (10-6 M) was ERß able to
stimulate reporter CAT activity. Similar results were obtained on
transcriptional activation through the ER and ERß with
3ß,5-GSD (Fig. 4B). However, 3ß,5-GSD did not activate
transcription through ERß even when used at the highest concentration
tested (Fig. 4B). Therefore, 3ß,5-NET and 3ß,5-GSD have
increased potency and efficacy for activation of ER-dependent gene
expression relative to ERß, and these compounds are therefore ER
selective agonists.
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-selective ligands were shown to be
potent ERß antagonists, we investigated whether the NET and GSD
derivatives act as antagonists, particularly through ERß (26, 27). HeLa cells were transfected with expression vectors for
ER or ERß together with the estrogen-responsive reporter gene, and
treated with E2 (10-9 M) in the
presence or absence of increasing concentrations
(10-8–10-6
M) of NET, or GSD and their corresponding A-ring reduced
derivatives, or the antiestrogen 4-hydroxytamoxifen
(10-7 M). As shown in Fig. 5, with the exception of
4-hydroxytamoxifen, none of the NET (Fig. 5C) or GSD (Fig. 5D)
derivatives, at various concentrations (10-6
–10-8 M), significantly inhibited
ERß-induced reporter gene activation. The increased activities that
were observed in Fig. 5D were minor and not reproducible. No inhibition
of ER activity was observed, except with 4-hydroxytamoxifen (Fig. 5, A and B), but this is not unexpected considering the relatively poor
binding affinity of 3ß,5-NET and 3ß,5-GSD in comparison to
estradiol, and the ER agonist activity of these ligands. The
antiestrogen 4-hydroxytamoxifen was used to ensure that 3ß,5-NET
and 3ß,5-GSD were stimulating target gene expression via ER. As
shown in Fig. 5E, 4-hydroxytamoxifen effectively inhibited the ability
of both tetrahydro derivatives to stimulate ER transcriptional
activity.
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-tetrahydro
derivatives of NET and GSD, respectively, were determined in extracts
of COS-1 cells transfected with expression vectors for either ER or
ERß. The numbers represent relative affinities for ER in
comparison to E2, which is set at 100. As shown in Fig. 6A, both compounds showed the ability to
displace bound [3H]E2 from the ER with an
RBA of 1.18 and 5.23 for the 3ß,5-NET and 3ß,5-GSD,
respectively. In contrast, these compounds behaved as weak competitors
for ERß binding sites (Fig. 6B), as judged by their corresponding
RBAs (0.00418 and 0.039 for 3ß,5NET and 3ß,5GSD,
respectively).
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-NET and 3ß,5-GSD
activation of ER-NET and 3ß,5-GSD would
activate a natural estrogen responsive promoter selectively through
ER, we transfected the oxytocin promoter luciferase reporter
(pROLUC) into HeLa cells with either the ER or ERß expression
vectors and subsequently treated with the indicated compounds (Fig. 7, A and B). Both 3ß,5-NET and
3ß,5-GSD activated reporter expression from the oxytocin promoter
through ER but not ERß, whereas the parent compounds NET and GSD
had very little effect. Thus, both 3ß,5-NET and 3ß,5-GSD
could selectively activate a naturally occurring estrogen responsive
promoter. To investigate the cell specificity of the estrogenic effects
of 3ß,5-NET and 3ß,5-GSD, we transfected the ERE-E1b-CAT
reporter into CHO cells along with an expression vector for ER (Fig. 7C). Activation of reporter activity by ER was observed with both
3ß,5-NET and 3ß,5-GSD, but not to the same level as E2;
however, they were statistically significant with P <
0.05. Both compounds appear to display lower activities in CHO cells
than in HeLa cells. This lower activity suggests the relative agonist
activity of these compounds may vary depending on cell type.
Importantly neither compound stimulated ERß transactivation in CHO
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tetrahydro
derivatives (9). In addition, this study clearly shows the
specific activation of ER-dependent reporter gene expression by both
3ß,5-NET and 3ß,5-GSD with little if any activation through
other receptors tested. Activation of ER by 3ß,5-NET and
3ß,5-GSD was observed in both HeLa and CHO cells. In addition,
these compounds are capable of activating the oxytocin promoter, a
natural estrogen responsive promoter. Therefore, we have demonstrated
that these compounds are ER selective in two different cell lines
and on simple and complex natural promoters. This is an important
consideration because the relative agonist activity of many ER ligands
(e.g. selective ER modulators) varies by cell and promoter
type. More detailed analyses will be required to gain a broader
understanding of the extent of this specificity. Overall, the results
are of biological importance in the sense that although a second ER
(ERß) has been identified, its relevance in estrogen endocrinology is
still unclear and compounds such as 3ß,5-NET and 3ß,5-GSD
will enable selective in vivo studies of only ER
function.
The calculated RBA values from the slopes generated for ER were very
similar to those obtained in previous studies using rat uterus cytosol
that involved mainly the ER (24). This observation,
together with previously published data on the ability of NET and
3ß,5-NET to suppress pituitary LH release, including their
estrogen-like effects upon the endometrium in castrated female rats
(9, 25), supports the selective binding of the
3ß,5-NET to ER and agrees with the relative distribution of
ER mRNA in different rat and mouse tissues (5, 6, 28, 29). The ER mRNA is highly expressed in pituitary, uterus,
testis, epididymis and kidney, and 3ß,5-NET estrogenic responses
would be expected in these tissues. Furthermore, these compounds may
serve as useful tools to discriminate ER from ERß functions in
tissues (e.g. ovary) that express both receptors. Although
our findings suggest that 3ß,5-tetrahydro derivatives have
receptor-selective actions, a detailed evaluation of the biological
effects of these selective ER agonists in the ER or ERß
knockout mouse models is, of course, also of relevant interest.
The ability to have preferential ligand selectivity for ER
vs. ERß receptors, as in the case of this study, could
help in the process of identifying additional synthetic or naturally
occurring steroids with different relative affinities for both ER
subtypes. In this regard, it is known that only those C-19 steroids
with a hydroxyl group at C-3 and C-17 have significant affinity for
both ER subtypes (5). In addition, as shown herein, the
relative spatial orientation of the A-ring with respect to the B-ring,
as in the case of 5-reduction and hydroxylation at C-3 of NET and
GSD, should also be considered as important structural characteristics
for ER ligand recognition. Whether other alterations in ligand
structure, besides those occurring in the A-ring, such as substitutions
at C-17 in the position or the absence of the C-19 methyl angular
(30) in NET and GSD, are important in selective binding to
ER deserves further investigation.
Interestingly, these observations are consistent with previous
computer-based quantitative structure-activity relationship studies of
ligand-receptor interactions (31). In these studies,
comparative molecular field analysis of both ERs revealed that they are
sensitive to adding steric bulk at the 17-position on the steroid
ring, suggesting that substitutions in this region will enhance the
binding affinity more for ER rather than ERß, which is consistent
with the experimental findings reported in this study. In addition, the
molecular changes elicited by the 19-nor substitution might increase
the mobility and electronic density of the A-ring allowing the
alignment of the 3ß-hydroxy group and the A-ring hydrogen atoms
resembling an estrogen-like environment. There have been several other
examples of compounds with differences in the transcriptional
activities through both ER subtypes (26, 27). Recently,
two novel ligands for ER and ERß have been described
(26). One, a nonsteroidal triaryl-substituted pyrazole,
with a 120-fold agonist potency preference for ER and the other, a
cis-diethyl-substituted tetrahydrochrysene prepared as a
fluorescent ER ligand, is an agonist on ER but a complete antagonist
for ERß. In addition, metabolites of methoxychlor were also shown to
have similar properties, i.e. ER agonist and ERß
activities (27). Similarly, Kuiper et al.
(5), reported ER and ERß relative binding affinities
for a number of estrogen derivatives, as well as nonsteroidal
phytoestrogens, such as genistein. In addition, these authors also
presented evidence that A-ring reduced natural androgens are more ERß
selective, which may indicate, as described above, the importance of
C-17 substitutions and/or the absence of the C-19 methyl angular in
ligand-ER interactions. Relative to these previous reports,
3ß,5-NET and 3ß,5-GSD produce better discrimination in
relative binding affinity for ER and ERß than other compounds
examined and this should facilitate studies on the distinct biological
roles of both ER subtypes.
Estrogens are known to have important effects on the reproductive
system, and targeted disruption of the ER gene results in sterility
in both male and female mice (32). The ER knockout mice
developed normally but were infertile and did not respond to estradiol.
These data, together with other observations in the male mice
(33), indicate that ERß alone does not appear to be
capable of maintaining normal reproductive function in the ER
knockout mice. Although a complementary role between these two ER
subtypes cannot be discarded, it is obvious the importance of ER in
the overall actions of estrogens. In this regard, we have shown
(34) the ability of NET to significantly suppress serum LH
levels in castrated subjects with testicular feminization syndrome, and
the effect of NET and its 3ß,5-tetrahydro reduced metabolite on LH
suppression in the long-term ovariectomized female rat (9, 25, 35). Because these conditions are characterized by the absence
of androgen action and estrogen-dependent PRs in both testicular
feminization syndrome and ovariectomized rats, the LH suppressing
activity of NET was probably due to its interaction with
hypothalamic-pituitary ERs. Furthermore, administration of
3ß,5-tetrahydro derivatives of NET and GSD is capable of restoring
both the content of pituitary PR in the ovariectomized female rat
(12, 13) and inducing male sexual behavior when given
chronically in combination with 5-dihydrotestosterone to castrated
male rats (36), indicating their intrinsic estrogenic
activities. These observations suggest that ER is involved at the
hypothalamic-pituitary unit in terms of gonadotropin regulation as well
as in other brain areas controlling sexual behavior. In as much as the
clinical dimension of the availability of specific ER subtype ligands
has yet to be determined, it is envisioned that identification of
compounds with preferential selectivity for ER subtypes would be
valuable for the development of new potential therapeutic approaches
based on receptor-selective hormonal actions and provide an important
tool to examine ER and ERß specific cellular and molecular
functions.
Overall, the data presented demonstrate that the tetrahydro derivatives
of NET and GSD bind and selectively activate gene transcription via
ER. These compounds, in addition to their scientific and therapeutic
implications, may also help to identify and differentiate structural
features in natural and synthetic ligands responsible for selective
binding to ER and ERß.
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Acknowledgments |
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Footnotes |
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Abbreviations: AF, Activation functions; CAT, chloramphenicol acetyltransferase; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DMEM-HG, DMEM without phenol red; ERE, estrogen response element; GSD, Gestodene; LNG, levonorgestrel; LUC, luciferase; NET, norethisterone; P4, progesterone; PRE, progesterone response element; RBA, relative binding affinities.
Received November 14, 2000.
Accepted for publication May 31, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 (ER) and estrogen receptor-ß (ERß)
messenger ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 or
estrogen receptor-ß. Endocrinology 140:800–804 and ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
and ß subtypes. Endocrinology 138:4022–4025This article has been cited by other articles:
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