Variant Estrogen and Progesterone Receptor Messages in
Human Vascular Smooth Muscle
Yvonne K. Hodges, PhD; Jennifer K. Richer, PhD;
Kathryn B. Horwitz, PhD; Lawrence D. Horwitz, MD
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Background—Estrogens stimulate growth of breast or uterine cells but have the opposite effect on vascular smooth muscle
cells, in which they protect against coronary artery disease with or without concomitant administration of progesterone.
A possible cause of differences in hormone action is variable tissue-specific expression of hormone receptor. Therefore,
we analyzed the structure of estrogen receptors (ERs) and progesterone receptors (PRs) in human vascular
smooth muscle.
Methods and Results—RNA was isolated from human vascular smooth muscle, and the functional domains of ER-a and
PR were characterized by reverse transcriptase and polymerase chain reaction. Interestingly, in addition to wild-type
ER-a and PR, 5 variant ER-a and 2 variant PR transcripts were found. These variants contained precise deletions of
exons encoding regions of the hormone-binding domain. The PR transcripts lacked exon 4 (PRD4) and exon 6 (PRD6).
The ER-a transcripts were missing exon 4 (ERD4), exon 5 (ERD5), exon 6 (ERD6), exon 7 (ERD7), and exons 6 and
7, (ERD6,7). ER-b variants were also detected. The PR variants were functionally characterized, and PRD6 was found
to be a dominant-negative transcription inhibitor of wild-type receptors. Variant PR was present in premenopausal
women but absent in postmenopausal women.
Conclusions—Variant PR and ER transcripts are extensively expressed in human vascular smooth muscle. The complex
tissue-specific effects of sex hormones may be mediated by the expression of heterogeneous forms of their cognate
receptors. The presence of variant ERs and PRs may be of importance in altering the physiological effects of estrogens
or progestins in vascular smooth muscle. (Circulation. 1999;99:2688-2693.)
Key Words: genes n muscle, smooth n receptors n hormones n coronary disease
tors. There are 2 wild-type ERs, designated ER-a and
ER-b,13,14 and 2 wild-type PR isoforms, called PR-A and
PR-B.15,16 Additionally, mRNA exon deletion splice variants
may arise that encode altered receptor proteins that are
missing key functional domains. Variant forms of ER-a
arising from exon splicing errors have been observed in
breast cancers, meningiomas, and normal human mammary
tissue.17–19 Recently, Inoue et al20 reported 3 exon deletion
variants of ER in vascular smooth muscle cells isolated from
rat aorta. PR splice variants have been reported in breast
cancer.21,22
Variant receptors are capable of anomalous transcriptional
activities that may dominantly inhibit or enhance the effects
of wild-type receptors.23,24 In addition, some variant receptors
are constitutively active: they can mimic the transcriptional
effects of estrogens or progestins in the absence of hormone.
Finally, some variant receptor messages are likely to be
incapable of being translated into protein. If these are the
most prevalent types of receptor mRNA present, normal
receptor-mediated actions of estrogens or progestins may be
T
he incidence of coronary artery disease in postmenopausal women is markedly reduced by treatment with
exogenous estrogens.1 A possible mechanism of this effect is
inhibition by estrogens of vascular smooth muscle cell
growth and migration, a process critical to atherogenesis and
vascular restenosis.2– 4 Estrogen receptors (ERs) are ligandactivated transcription factors. Activated ER binds to estrogen response elements (EREs) in the promoters of target
genes, thereby regulating gene expression.5 Among the proteins regulated by estrogens in vascular smooth muscle cells
are the progesterone receptors (PRs).6
The effects of estrogens vary markedly in different tissues;
for example, estrogens stimulate growth of breast and uterine
cells7,8 but inhibit growth of vascular smooth muscle cells.2– 4
Progesterone exhibits antagonism to estrogen effects in the
uterus9,10 but enhances the effects of estrogens in vascular
smooth muscle.11,12 A possible explanation for the heterogeneity of estrogen or progesterone action in different targets is
that some tissues express variant forms of ER or PR that have
transcriptional effects different from the “wild-type” recep-
Received December 15, 1998; revision received February 8, 1999; accepted February 12, 1999.
From the Department of Medicine, Division of Cardiology (Y.K.H., L.D.H.) and Division of Endocrinology (J.K.R., K.B.H.), University of Colorado
Health Sciences Center, Denver, Colo.
Correspondence to Lawrence D. Horwitz, MD, Cardiology B130, University of Colorado Health Sciences Center, Denver, CO 80262. E-mail
[email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2688
Hodges et al
May 25, 1999
2689
mRNA for Wild-Type PR and PR Variants Are Expressed in Human Vascular
Smooth Muscle
Age, y
D4
D6
Wild-Type
Disease
Tissue
Premenopausal women
32
23
28
49
Normal
Aorta
38
,1
44
56
Normal
Saphenous vein
23
27
28
45
Normal
Coronary artery
29
34
34
32
Normal
Iliac artery
33
,1
,1
99
Normal
Coronary artery
59
,1
,1
99
Normal
Iliac artery
61
,1
8
92
Normal
Aorta
63
,1
,1
99
CAD
Mammary artery
63
,1
,9
90
Normal
Iliac artery
Postmenopausal women
Men
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19
,1
27
73
Normal
Aorta
41
,1
,1
99
Normal
Saphenous vein
42
21
22
57
Normal
Saphenous vein
50
25
32
43
CAD
Saphenous vein
63
24
25
51
CAD
Saphenous vein
69
,1
36
64
CAD
Coronary artery
CAD indicates coronary artery disease.
Amounts of wild-type PR and PR HBD deletion mutants D4 and D6 are expressed in percentages.
absent. Therefore, expression of variant ER or PR in a
particular tissue, such as vascular smooth muscle, could have
effects that differ markedly from those of the wild-type
receptors.
To analyze the expression patterns of wild-type and variant
ER and PR mRNAs, we used reverse transcriptase (RT) and
the polymerase chain reaction (PCR) in human vascular
smooth muscle. We identified several ER-a and ER-b variants. We also found 2 PR variants that we cloned and
sequenced to identify the exon deletions. One of the PR
variants, an exon 6 deletion mutant, is a dominant-negative
inhibitor.21 We propose that the complex, tissue-specific
effects of sex hormones may be mediated in part by the
expression, or lack thereof, of heterogeneous types of their
cognate receptors.
Methods
Tissue Culture
As outlined in the Table, normal and diseased coronary artery,
saphenous vein, aorta, mammary artery, and iliac artery were used.
The sections were placed into DMEM with 1% (vol/vol) antibioticantimycotic solution at pH 7.4 (DMEM1AA) (Sigma Chemical Co).
The fat and connective tissue were removed, each segment was cut
longitudinally, and the endothelial layer was mechanically denuded.
After a 10-minute incubation in Ca21- and Mg21-free HBSS (Gibco
BRL) containing 73 U/mL collagenase (Worthington), the inner
muscular layer was carefully stripped from the outer adventitial
layer, and the smooth muscle was incubated in DMEM1AA for 72
hours at 37°C.
RNA Isolation
A modified version of a single-step method of RNA isolation by acid
guanidine thiocyanate-phenol-chloroform extraction (RNA STAT-
60, Tel-Test B Inc) was used to purify the RNA. RNase-free DNase
I was used to remove DNA contamination from the RNA.
RT-PCR
The GeneAmp RNA PCR kit (Perkin-Elmer) was used with 0.5- to
1.0-mg samples of total RNA and 2.5 mg of murine leukemia virus
(MuLV) RT. The RNA was heated to 70°C for 5 minutes before
addition of the RT. The RT reaction was performed at 42°C for 60
minutes, then heated to 99°C for 5 minutes to destroy the enzyme.
PCR was performed with primers specific for the receptor domains
of interest. An initial cycle at 94°C for 1 minute was followed by 35
cycles at 94°C for 30 seconds, 60°C for 40 seconds, 72°C for 90
seconds, and a final extension cycle at 72°C for 7 minutes.
[32P]a-dCTP (DuPont NEN) was incorporated into the PCR reaction.
The products were resolved on 1.5% agarose gels.
Cloning
The PCR products were inserted into a plasmid vector with a TA
cloning kit (Invitrogen) immediately after the reamplified PCR
products were obtained. The ligation reaction was performed overnight at 15°C. Cloned amplified PCR underwent automated sequencing at Biotechnology Resource Center, Cornell University.
Primers
Oligonucleotide primers were designed for PCR amplification of
specific DNA fragments of ER and PR. A primer for b-actin served
as an RNA quantification control. The primers amplified specific
exons in the DNA binding domain (DBD) or hormone binding
domain (HBD) of ER-a (exons 1 to 4) and PR (exons 5 to 8). These
primers were able to detect deletions or amplifications in exons 2 to
7 for both ER-a and PR. The primer locations were as follows:
primers 1 and 2 (nucleotide [nt] 617 to 637, nt 1082 to 1103) for
ER-a DBD (487 bp); primers 3 and 4 (nt 974 to 994, nt 1787 to
1811) for ER-a HBD (840 bp); primers 5 and 6 (nt 2355 to 2375, nt
2675 to 2697) for PR DBD (345 bp); and primers 7 and 8 (nt 2619
to 2642, nt 3393 to 3411) for PR HBD (824 bp).
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Estrogen and Progesterone Receptors in Smooth Muscle
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Figure 1. Autoradiograph of [32P]a-dCTP–labeled RT-PCR of
RNA from saphenous vein from premenopausal woman. Amplified DBD for wild-type ER-a and PR (487 and 345 bp, respectively) and HBD for ER-a and PR (840 and 824 bp, respectively)
are shown with black arrows. Variant ER-a and PR fragments
are marked with white arrows. ER-a and PR variant bands were
cut out from the gel, cloned, and sequenced.
The primers used for ER-b amplified both the DBD and HBD (nt
174 to 303, nt 1354 to 1383), generating a fragment of 1212 bp. All
the primers were tested on ER and PR cDNA plasmid clones to
ensure that they generated the specific products targeted.
Construction of Exon Deletion Mutants
The cDNAs encoding the PR variants were created by PCR with
pSG5-hPR1 or -hPR2 (expression vectors for the B or A isoforms of
PR) as templates and oligonucleotides designed to selectively eliminate the desired exon. The validity of the constructs was confirmed
by sequencing.
Results
Identification of ER-a and PR Transcripts in
Human Vascular Smooth Muscle by RT-PCR
To analyze the ER-a and PR transcripts, primers were
designed to amplify the encoding mRNA segments of the
DBD or HBD of these receptors. RT-PCR was performed
with each primer pair, and the labeled products were resolved
on an agarose gel. A representative result from smooth
muscle tissue isolated from the saphenous vein of a premenopausal woman is shown in Figure 1. The black arrow in each
lane marks the predicted wild-type DBD or HBD band for
each receptor; all other bands are due to variant transcripts.
Note the presence of $2 variants in lanes 2 and 3 and 3
variants in lane 4. The variant bands marked by white arrows
were isolated, cloned, and sequenced.
The mRNAs of all steroid receptors, including ER-a and
PR, are assembled from 8 exons. Exon 1 encodes the
N-terminus; exons 2 and 3 the DBD; exon 4 the nuclear
location signal (NLS), hinge, and proximal region of the
HBD; and exons 5 to 8 the rest of the HBD. The predicted
protein structure of the variant ER-a, determined from
mRNA extracted from saphenous vein vascular smooth muscle cells, is shown in Figure 2 for ER-a compared with the
wild-type receptors. Variant ER-a mRNAs present in vascular smooth muscle cells include deletions of exon 4 (D4),
Figure 2. Wild-type and 5 ER-a variants. Wild-type ER-a mRNA
consists of 8 exons that encode the receptor, DBD, HBD, and
NLS (small rectangle between exons 3 and 4). Five ER-a variants include deletions of exon 4 (D4), exon 5 (D5), exon 6 (D6),
exon 7 (D7), and exons 6 and 7 (D6,7). mRNA abnormalities and
presence of premature stop signals resulting from frameshifts
allow deduction of structures of resultant proteins. Three variants (D5, D6, and D7) produce frameshifts that lead to missense
amino acids (black boxes) followed by premature truncation.
exon 5 (D5), exon 6 (D6), exon 7 (D7), and exons 6 and 7
(D6,7) (Figure 2). Three of these deletions (D5, D6, and D7)
produce a frameshift generating a missense protein (black
boxes) with an early termination codon, yielding truncated
receptors. Two others, the D4 and D6,7 deletions, do not
generate frameshifts. Instead, these ER-a variants do not
encode some amino acids in the proximal region (D4) or
central region (D6,7) of the HBD.
We identified 2 variant PR mRNAs in vascular smooth
muscle cells. These were cloned and sequenced (Figure 3).
One is an exon 4 deletion (D4). This modification removes
the NLS, hinge, and proximal HBD from the receptors, but
because no frameshift occurs, the remainder of the HBD
would be translated normally. The second PR variant is an
exon 6 (D6) deletion. This produces a frameshift followed by
the synthesis of 12 missense amino acids, ending in a
termination codon, yielding a receptor truncated in the middle
of the HBD.
Prevalence of PR Variants in Human Vascular
Smooth Muscle
Results of studies of PR variants in 15 subjects are shown in
the Table. The subjects included 5 premenopausal women, 4
postmenopausal women, and 6 men. The men ranged in age
from 19 to 69 years. The samples obtained from men
contained widely varying percentages of variant or wild-type
Figure 3. Wild-type PR and 2 PR variants showing 8 exons that
encompass the mRNA, DBD, HBD, and NLS (small rectangle
between exons 3 and 4). Two PR variants that were observed
have been cloned and sequenced. One PR variant is an exon 4
(D4) deletion. The D4 deletion removes the NLS, hinge, and a
portion of the HBD. No frameshift occurs, and the remainder of
the HBD is translated. The other PR variant, a D6 deletion, leads
to a frameshift, and the protein is truncated in the middle of
the HBD.
Hodges et al
May 25, 1999
2691
Discussion
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Figure 4. Autoradiograph of 32P-labeled RT-PCR (with primers
specific for ER-b) of RNA from saphenous vein from premenopausal woman. Black arrow indicates 1212-bp wild-type PCR
product. Splice deletion variants of ER-b and an insertion variant are indicated by white arrows.
PR. PR was present in all the samples from premenopausal
women, but a substantial percentage of the PR was of the
variant type. The samples from the 4 postmenopausal women
contained wild-type but very little or no variant PR. Therefore, in this small sampling of subjects, variant PR appears to
be very prevalent in premenopausal women but essentially
absent in postmenopausal women.
ER-b Variant Transcripts Are Present in Human
Vascular Smooth Muscle
In Figure 4, the vascular smooth muscle in a saphenous vein
segment removed from a premenopausal woman expresses
variant ER-b transcripts. In addition to the wild-type ER-b
bands (black arrows), several variant PCR products (open
arrows) are present. We found 1 variant band in the HBD that
was larger than the wild type. A similar variant was described
recently in rat ovary and prostate cells.25
Although premenopausal women and postmenopausal
women treated with estrogens have lower incidences of
coronary artery disease than men or postmenopausal women
not receiving hormone therapy,1 the genomic mechanisms by
which this protection is afforded are unknown. Estrogens
probably influence atherogenesis and vascular injury through
regulation of gene transcription in vascular smooth muscle or
endothelium. Estrogens modulate gene transcription by binding to the HBD of ERs.5 The activated DBD of the receptor
then binds to the ERE sequences of targeted genes, regulating
their transcription. There are 2 wild-type ERs: ER-a, which
plays an important role in breast and uterine cells, and ER-b,
which has a different tissue expression pattern.13,14 Wild-type
PRs are larger proteins than ERs but have the same general
structural organization as ERs, with 1 additional complexity:
there are 2 natural isoforms, the A and B receptors.15,16
There is functional ER in vascular smooth muscle,6,26 and
there is evidence in cell culture and animal models that
estrogens inhibit vascular smooth muscle cell growth and
migration.2– 4 Interestingly, this growth-inhibitory effect of
estrogens in vascular smooth muscle differs from the action
of estrogens in other tissues, particularly breast and uterus,
where estrogens enhance cell proliferation.7,8
Progestins oppose the growth-stimulatory effects of estrogens in uterus and enhance cell proliferation in breast.9,10 In
2 recent studies,11,12 a progestin in high, probably nonphysiological, dosages had growth-inhibitory effects similar to
those of estrogens in cultured human and rat vascular smooth
muscle. However, in a study in atherosclerotic monkeys,27 a
progestin, medroxyprogesterone (MPA), attenuated estrogeninduced coronary vasodilation. In a study in normal monkeys,
MPA prevented inhibition of coronary vasospasm by estradiol, but progesterone did not.28 Additional evidence of
possible adverse vascular effects of MPA is provided by a
recent clinical trial in which use of high-dose MPA in
conjunction with estrogen in postmenopausal women with
preexisting coronary artery disease increased the risk of
adverse cardiac events during the first year.29 Thus, the
interactions of progestins and estrogens in vascular smooth
muscle are complex and poorly understood. In different
tissues, there is marked heterogeneity of estrogen action and,
probably, variation in the interactions between estrogens and
progestins. One possible explanation for these diverse physiological effects is that the forms of the receptor proteins
expressed differ from organ to organ.
Variant ERs arising from exon splicing errors have been
reported in human breast cancers and normal human mammary tissue.17–19 Recently, Inoue et al20 reported the presence
of 3 ER-a variants in smooth muscle isolated from rat aorta.
Variant receptors may have anomalous transcriptional activities: some may dominantly inhibit or enhance the effects of
wild-type receptors. In addition, some variant receptors are
constitutively expressed and may not require binding of their
hormone ligand to produce their effects. Therefore, understanding the structural types and functional characteristics of
variant ERs and PRs in the vascular bed could lead to
valuable insights into the actions of estrogens and progestins
in these tissues. We examined human vascular smooth muscle
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Estrogen and Progesterone Receptors in Smooth Muscle
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to determine whether messages encoding ER-a variants were
present.
Using RT-PCR, we discovered the presence of 5 HBD
variants of ER-a in vascular smooth muscle: D4, D5, D6, D7,
and D6,7 exon deletions. The D4 variant is not transcriptionally active alone, nor does it alter the transcriptional activity
of wild-type ER-a.19 The D7 variant is not transcriptionally
active alone and does not alter wild-type ER-a transcription
in HeLa cells, but it exerts a dominant-negative effect on
wild-type ER-a transcription in yeast cells.24 The transcriptional effects of the D6 and D6,7 ER-a variants are unknown.
Three of these transcripts, the D5, D6, and D7 exon deletions,
produce frameshifts that encode missense amino acids, followed by premature truncation. The other 2, the D4 and D6,7,
are without frameshifts. The D5, D6, D7, and D6,7 variants
lack portions of the HBD and would be expected to be
constitutively active in the absence of hormone; this has been
confirmed for the D5 variant.17
Of considerable interest is our finding of PR splice variants
in human vascular smooth muscle. These included a D4 and
a D6 exon deletion variant. Because this is the first description of PR splice variants in any vascular tissue, we sequenced the mRNA. The D4 deletion removes the NLS, the
hinge, and a portion of the HBD. Because no frameshift
occurs, translation of the remainder of the HBD would be
expected. The D6 deletion leads to a frameshift, which
truncates the protein in the HBD. Richer et al21 recently
reported the functional characteristics of these PR variants.
The D4 has no transcriptional activity. The lack of an NLS
probably renders this variant unable to enter the nucleus to
compete with the activity of the wild-type PR. However, the
D6 variant, when overexpressed, markedly inhibits the activity of both isoforms of wild-type PR. Therefore, at least 1 PR
variant is capable of dominantly inhibiting the effects of
wild-type PR. Differences in transcriptional effects, which
are dependent on the type of PR expressed, could cause
differences in the effects of progestins in vascular tissue.
There is also evidence that PR can alter the function of
ER.30,31
Samples from postmenopausal women showed little or no
PR variant mRNA in the tissue samples (Table). Three of the
4 postmenopausal women were taking estrogen replacement
therapy with no progestin. It is possible that exogenous
estrogen induces expression of a proportionate wild-type PR
level above that found in premenopausal women and men.
Alternatively, postmenopausal women may produce only
wild-type mRNA regardless of the type of hormonal
stimulation.
We discovered by RT-PCR that messages encoding several
variants of ER-b were also present in vascular smooth
muscle. Because the entire structure of wild-type ER-b is not
known, we did not attempt to functionally characterize these
variants. It has recently been reported that in ER-a knockout
mice, estrogens inhibit vascular smooth muscle growth and
migration.32 Because ER-b was present in the blood vessels
of these mice, ER-b is probably transcriptionally active in
vascular tissue. The concomitant presence of variants of
ER-b could have important effects on the transcriptional
activity of wild-type ER-b and ER-a.
We conclude that there are exon deletion variants of ER-a,
ER-b, and PR in human vascular smooth muscle. Because
these variants may have entirely different transcriptional
effects than the wild-type receptors from which they are
derived, they are potentially of physiological importance. An
attractive concept is that some of the puzzling differences in
estrogen and progestin effects in different target tissues could
be explained by heterogeneous expression of their cognate
receptor variants. To further address the possible importance
of variant ERs or PRs, specific antibodies need to be
developed for each variant to determine whether and in what
quantities the variant mRNAs are translated.
Acknowledgments
This work was supported by funding from the American Heart
Association, Colorado Affiliate, and NIH grants HL-55291, DK48238, and CA-26869.
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Variant Estrogen and Progesterone Receptor Messages in Human Vascular Smooth
Muscle
Yvonne K. Hodges, Jennifer K. Richer, Kathryn B. Horwitz and Lawrence D. Horwitz
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Circulation. 1999;99:2688-2693
doi: 10.1161/01.CIR.99.20.2688
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