Ligand-Activated Progesterone Receptor Isoform hPR-A Is a Stronger Transactivator Than hPR-B for the Expression of IGFBP-1 (Insulin-Like Growth Factor Binding Protein-1) in Human Endometrial Stromal Cells
Jiaguo Gao,
James Mazella,
Meiyi Tang and
Linda Tseng
Department of Obstetrics/Gynecology and Reproductive Medicine
School of Medicine State University of New York at Stony Brook
Stony Brook, New York 11794
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ABSTRACT
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In human endometrium, the levels of progesterone
receptor (PR) isoforms hPR-A and hPR-B are differentially regulated
during the reproductive cycle. Progesterone significantly increases the
content of hPR-A, the predominant isoform in decidualized stromal cells
(1). The purpose of this study was to determine the capacity of hPR-A
and hPR-B to transactivate the progestin-dependent target gene in human
endometrial stromal cells. We examined the effect of cotransfection of
hPR-A or hPR-B on the expression of the human insulin-like growth
factor binding protein-1 (IGFBP-1) in endometrial stromal cells. The
primary culture of human endometrial stromal cells was transfected with
the hPR-A or hPR-B expression vector and the IGFBP-1 promoter
construct p275CAT, which contains two functional progesterone response
elements (PRE1 and PRE2) in decidualized stromal cells.
Medroxyprogesterone acetate (MPA) increased the promoter activities
ranging from 1.2- to 27-fold in cells cotransfected with hPR-A or hPR-B
in eight endometrial specimens. The promoter activity increased by the
hPR-A was significantly higher than hPR-B (15 ± 8 vs.
4 ± 2, mean ± SD; n = 8,
P < 0.005). Site-specific mutation showed that the
induced activity by hPR-A was mediated through the PRE1 and PRE2 sites.
Addition of hPR-B reduced the effect of hPR-A. The high transactivation
capacity of hPR-A was also activated by other ligands, progesterone,
Org 2058, and norethindrone. These observations indicate that hPR-A is
a stronger transactivator than hPR-B for the IGFBP-1 promoter in
endometrial stromal cells.
Previous studies have shown the progestin- dependent production of
IGFBP-1 correlates with its mRNA levels and transcription rate. Thus,
we have determined the effect of hPR-A and hPR-B on the production of
IGFBP-1 in stromal cells treated with MPA. The production rate in cells
uniformly infected with AdPRA (recombinant Ad5- directed PR expression
system) was significantly higher (P < 0.001) than the
rate in uninfected cells and in cells infected with AdPRB or AdCMV (the
Ad5 viral expression vector). This result, in concert with the promoter
analysis, provides evidence that hPR-A is a strong inducer for the
chromosomal IGFBP-1 gene in endometrial stromal cells.
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INTRODUCTION
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Ligand-activated progesterone receptor (PR) mediates the majority
of progestational effects in endometrium, although nongenomic
progesterone action has been implicated. Human endometrium contains
both isoforms of PR, hPR-A and hPR-B. Steroid hormones regulate the
content of these two isoforms differentially in two types of
endometrial cells (1, 2). In glandular cells, progestin reduces both
hPR-A and hPR-B, whereas in stromal cells, it increases the content of
both isoforms in cells treated with progestin for 15 days. Prolonged
exposure of stromal cells to progestin significantly increases the
content of hPR-A (1), the major isoform in decidualized endometrial
stromal cells. Thus, it is generally believed that the majority of the
progestational effects and antiprogestin blockade are mediated through
hPR-A in endometrial decidual cells. This concept remains to be
verified.
Progesterone-regulated gene expression in endometrial cells has
been extensively studied in many laboratories. However, information
about PR isoform-specific gene regulation is limited in this target
tissue. We believe that hPR-A is active and perhaps more active than
hPR-B on those genes that are extensively expressed in decidual cells.
However, in several endocrine-sensitive breast cancer cell lines, hPR-B
is a stronger transactivator than hPR-A. In addition, hPR-A acts as a
dominant repressor of hPR-B on the progesterone response element (PRE)
linked-reporter constructs or mouse mammary tumor virus (MMTV) (3, 4)
as well as the endogenous target gene (5). Although hPR-A is not a
universal repressor (6), additional information showed that the
repression is through the structure-specific interaction with the
consensus PRE (7, 8). These findings raise the question of the
functional aspects of hPR-A in endometrial stromal/decidual cells. The
purpose of this study was to determine the transactivation capacity of
hPR-A in endometrial stromal cells by using a progestin-induced gene,
human insulin-like growth factor binding protein-1 (IGFBP-1).
The IGFBP-1 gene is activated by progestin, and its gene product
is the major secretory protein in decidualized endometrial stromal
cells (9, 10, 11, 12, 13). Previous studies have shown that cis-elements
PRE1 and PRE2 in the IGFBP-1 promoter are the sites responsible to
increase the promoter activity in decidualized endometrial stromal
cells (13). However, it is not clear whether the activation is mediated
from the predominant hPR-A or from the less dominant hPR-B or both. To
determine the capacity of transactivation by the PR isoforms, we have
measured the effect of cotransfection of hPR-A or hPR-B expression
vector on the IGFBP-1 promoter activity in undifferentiated stromal
cells in which PRE1/PRE2 have little activity (13). In addition, the
production of IGFBP-1 was measured in stromal cells uniformly
transfected with AdPRA or AdPRB. Our results indicate that hPR-A is a
stronger transactivator than hPR-B to increase the promoter activity
in transient transfection assay and production of IGFBP-1 from
the endogenous IGFBP-1 gene in endometrial stromal cells.
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RESULTS
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hPR-A Is a Stronger Activator Than hPR-B For The IGFBP-1 Promoter
Activity in Human Endometrial Stromal Cells
The transactivation capacity of hPR-A and hPR-B on the
IGFBP-1 promoter was determined using the promoter construct p275CAT
(-275 to +68 bp, Fig. 1
) for most of the
experiments presented in this paper (Table 1
and Figs. 2 to 5


). Only three experiments were
carried out using p246CAT and p1.2CAT (Table 1
). The promoter activity
was low in cells not treated with MPA (
Figs. 25


) in agreement with
the previous observation (13). MPA had no significant effect on the
promoter activity in cells cotransfected with the empty vector (Fig. 4
, lane 1). MPA increased the promoter activity derived from p246CAT
(n = 2), p275CAT (n = 5), and p1.2CAT (n = 1). The
degree of activation varied from 1.2- to 27-fold compared with cells
not exposed to MPA (Table 1
) and had no clear correlation with the
stage of menstrual cycle nor with the length of the promoter. However,
the capacity of transactivation by hPR-A was significantly higher than
that of hPR-B determined in eight specimens (Table 1
; 15-
vs. 4-fold increase respectively, n = 8, mean ±
SD p < 0.005).

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Figure 1. IGFBP-1 Promoter Construct
The location of the functional PRE1 (-193 to -179 bp) and PRE2 (-102
to -88 bp) sites are shown in the diagram. Also shown are the
sequences of wild-type PRE1 and PRE2, and the nucleotides mutated for
the construction of mutants p275PRE1.2 m, p275PRE1m, and p275PRE2m.
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Table 1. Activation of IGFBP-1 Promoter Activity
Cotransfected with hPR-A or hPR-B in Human Endometrial Stromal
Cells
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Figure 2. Promoter Activity of IGFBP-1 (2A) and MMTV (2B)
Induced by hPR-A or hPR-B in Human Endometrial Stromal Cells Treated
with MPA
Eight micrograms of plasmid p275CAT or pMMTV-CAT, 1 µg of hPR-A
or hPR-B, and 1 µg of pRSV-luc were transfected into human stromal
cells (early secretory phase) and then cultured with or without 0.1
µM MPA for 2 days. CAT activities were normalized to the luciferase
activities determined in the same cell lysate. Results are mean ±
SD of triplicate dishes. Promoter activity in cells treated
with MPA was significantly higher than untreated cells, MPA+
vs. MPA-, P < 0.0001. The difference
between hPR-A vs. hPR-B in MPA+ samples was also significant
(Fig. 2A , P < 0.0001 and Fig. 2B , P <
0.0006).
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Figure 3. Effect of hPR-A on the IGFBP-1 Promoter Mutant
Constructs of PRE1 and PRE2
Stromal cells (early-midsecretory) were transfected with p275CAT,
p275PRE1m, p275PRE2m, or p275PRE1,2 m and cotransfected with hPR-A and
pRSV-Luc and then treated without or with MPA for 2 days. Results are
means ± SD of triplicate dishes. Reductions of
the promoter activity by p275 PRE1m, p275 PRE2m, and p275 PRE1.2 m were
significant, P < 0.01, 0.001, and 0.0002.
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Figure 4. Induction of the IGFBP-1 Promoter Activity by
Various Doses of hPR-A, hPR-B, and Mixtures of hPR-A and hPR-B in
Endometrial Stromal Cells
Stromal cells (early secretory phase) were transfected with p275CAT (8
µg), various concentrations of hPR-A, hPR-B, mixtures of hPR-A/hPR-B,
or hPRB-DBDcys, and pRSV-Luc. The vector (plasmid without PR insert)
was included in control sample (2 µg) and in samples transfected with
PR constructs to yield 2 µg DNA. The promoter activities (normalized
to pRSVluc) were measured in cells after 2 days treatment with or
without MPA. Results are mean ± SD of triplicate
dishes.
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Figure 5. Effects of MPA, Progesterone (P), ORG2058 (ORG),
Norethindrone (NOR), RU-486 (Ru) and Cortisol (F) on the IGFBP-1
Promoter Activity Induced by hPR-A or hPR-B in Endometrial Stromal
Cells
Stromal cells (midsecretory phase) were transfected with p275CAT,
hPR-A, or hPR-B and pRSV-luc. Cells were treated with 0.1
µM of steroids and cultured for 2 days. Results are
mean ± SD of triplicate dishes. Promoter
activity was significantly higher in cells cotransfected with hPR-A
than hPR-B when treated with MPA, P, ORG, or Nor, P
< 0.002.
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Differential Effects of hPR-A and hPR-B on the Promoter Activity of
IGFBP-1 and MMTV in Endometrial Stromal Cells
To demonstrate that the relative capacity of transactivation
by hPR-A or hPR-B is target gene specific, the promoters of IGFBP-1 and
MMTV were separately transfected into stromal cells. The promoter
activity derived from p275CAT cotransfected with hPR-A was
significantly higher than that of hPR-B (Fig. 2A
; P <
0.001). In contrast, we found that hPR-B was a stronger transactivator
than hPR-A for the promoter of MMTV in endometrial stromal cells (Fig. 2B
), which is similar to data observed in cancer cell lines (4). This
result shows that the ligand-activated PR isoforms display a spectrum
of transactivation capacity in different target genes in endometrial
stromal cells. In addition, we found that the promoter activities
derived from p275CAT were similarly induced by hPR-A or hPR-B in HeLa
or HepG2 cells (data not shown). This result showed that the capacity
of PR transactivation on the target gene also varies in different types
of cells.
PRE1 and PRE2 Sites in the IGFBP-1 Promoter Mediate the hPR-A
Function
The PRE1 and PRE2 sites are located in the proximal promoter
region shown in Fig. 1
. Previously, we have shown that PRE1 and PRE2
are functional in decidualized stromal cells (13). To determine whether
the action of hPR-A is mediated through these two sites, stromal cells
were transfected with hPR-A and p275CAT and three PRE mutants,
respectively (Fig. 1
). Figure 3
shows the
promoter activities derived from the wild-type plasmid p275CAT and
three mutants, mutation of PRE1, PRE2, or both. p275PRE1m or p275PRE2m
reduced the promoter activity to 75% and 45%, respectively, of
the p275CAT (P < 0.05 and <0.001). p275PRE1,2 m,
double mutation at PRE1 and PRE2 sites, decreased the promoter
activity to 15% of the wild type (P < 0.0001) (Fig. 3
). These findings indicate that the transactivation of hPR-A is
mediated by both PRE1 and PRE2 sites in the IGFBP-1 promoter.
Transactivation of the IGFBP-1 Promoter by hPR-A Is Dose
Dependent
To test whether differential transactivation of hPR-A and hPR-B on
the IGFBP-1 promoter is caused by the concentration of PR-A or PR-B,
stromal cells were transfected with p275CAT and cotransfected with
various amounts of hPR-A or hPR-B expression vector. The promoter
activity increased by 2.5-, 8-, and 20-fold at concentrations of 0.1,
0.3, and 1 µg hPR-A, respectively (Fig. 4
, lanes 14). The response to hPR-B was
also dose dependent at the same concentrations (1.5-, 3-, and 4-fold
increase, lanes 57, respectively). At a comparable dose, the potency
of hPR-A was always stronger than hPR-B. No self-squelching was shown
within the range tested.
hPR-B Inhibits the Effect of PR-A and Repression Does Not Require
the DNA Binding Domain
To test whether hPR-B would interfere with the action of hPR-A,
cells were transfected with hPR-A alone or a mixture of hPR-B and
hPR-A. Addition of hPR-B reduced the activity by 50% and 60% at the
ratios of hPR-B/hPR-A 0.3 and 1, respectively (Fig. 4
, lanes 8 and 9).
These results suggest that hPR-B acts as a repressor to quench the
transactivation of hPR-A. Cotransfection with hPRB-DBDcys, which
produces a mutant hPR-B unable to bind to the DNA, did not reverse the
inhibition (lane 10). The result indicates that inhibition does not
require DNA binding. Data suggest that the inhibition is mediated by
protein-protein interaction, although the exact nature of the
interaction is not clear and requires additional study.
Various Ligands Do Not Change the Relative Capacity of
Transactivation of hPR-A and hPR-B
To test whether the high capacity of hPR-A is ligand specific, the
promoter activity was determined in cells treated with MPA,
progesterone (P), or the 19 nor-progestins: Org2058 and norethindrone.
Although the degree of activation varied among different progestins (5-
to 8-fold), hPR-A was always more potent than hPR-B (Fig. 5
). These results show that the high
capacity of transactivation from hPR-A is not caused by a specific
ligand. The antiprogestin, RU-486, caused a moderate increase in cells
co-transfected with hPR-A or hPR-B (1.8- or 1.6-fold increase).
Cortisol had no effect on the promoter activity.
hPR-A Is a Strong Inducer For the Production of IGBFP-1 in
Endometrial Stromal Cells
The findings described in the previous sections were demonstrated
in transient transfection experiments. It is important to provide
evidence whether hPR-A is indeed a strong transactivator for the
endogenous IGFBP-1 gene in stromal cells. Previous studies have shown
that rate of production and mRNA levels correlate with the rate of
transcription of the IGFBP-1 gene (9, 10). Thus, we have determined the
effect of hPR-A and hPR-B on the induction of the endogenous
IGFBP-1 gene in stromal cells. Such experiment requires a high
efficiency of transfection to achieve a uniform gene transfer. To
increase the efficacy, Ad5-recombinant gene transfer technique was used
for this study. Adßgal infection was used as a positive control (Fig. 6D
). Stromal cells remained viable after
infection with various Ad5-recombinant constructs. Immunohistochemical
localization showed that approximately 95% of the stromal cells
expressed hPR-A and hPR-B with a similar intensity of staining (Fig. 6
, B and C) in the nuclei when the cells were infected with equal amount
of AdPR-A and AdPR-B, respectively (five viral particles per cell).
Twelve days after infection, the intensity was reduced approximately
50%. In stromal cells with no viral infection or infected with the
vector AdCMV, no detectable PR was found under the same experimental
condition (Fig. 6A
), indicating that the endogenous hPR is relatively
low compared with the cells infected with AdPRs.

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Figure 6. Immunohistochemistry Localization of hPR-A and
hPR-B in Endometrial Stromal Cells Transfected with AdCMV, AdPRA, and
AdPRB
Stromal cells isolated from a midsecretory endometrium were treated
with 0.1 µM MPA and infected with AdCMV, AdPRA, and
AdPRB, and Adßgal (10 particles per cell) for two days, respectively.
Cells were processed for immunohistochemical staining in A, B, and C
and ß-gal enzyme reaction in D as described in detail in
Materials and Methods (A, phase contrast; B, C, and D,
bright field).
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The effect of overexpression of hPR-A or hPR-B on the endogenous
IGFBP-1 gene was determined by measuring the amount of IGFBP-1
accumulated in medium by enzyme-linked immunosorbent assay (ELISA) (9).
Various concentrations of viral particles (1, 5, 10, 20, and 50
particles/cell) were infected into cells to determine whether Ad5
infection would impair the production of IGFBP-1. We found that cells
infected with >20 particles per cell reduced the production rate.
Figure 7
shows the production of IGFBP-1
in cells infected with five viral particles per cell and then treated
with MPA. IGFBP-1 was undetectable by ELISA in long-term cell culture
(11 days) without MPA treatment in agreement with previous observation
(9). In cells treated with MPA, the production rate was minimal on the
first 6 days of culture, which is similar to the results published
previously (9). The rate was significantly increased after 7 days MPA
treatment (Fig. 7
) with no difference between uninfected cells and
cells infected with AdCMV (0.04, 0.25, and 4.4; 0.05, 0.45, and 3.2
µg/1 x 106 cells/day on days 7, 9, and
11, respectively). This result indicates that infection of the Ad5
vector (five particles per cell) did not appreciably alter the
MPA-induced production of IGFBP-1. The production rates in
cells infected with AdPRB (five particles per cell) were also similar
to the rates in uninfected cells. The production rates in cells
infected with AdPRA (five particles per cell) were 0.07, 2.2 to 11
µg/1 x 106 cells/day on days 7, 9, and
11, respectively, which are significantly higher than that of
uninfected cells or Ad5 or Ad5-hPR-B infected cells (P
< 0.01, day 7; P < 0.001, days 9 and 11). These
results indicate that hPR-A, not hPR-B, is a strong transactivator for
the induction of IGFBP-1 production.

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Figure 7. Production Rate of IGFBP-1 in Endometrial Stromal
Cells
Stromal cells isolated from a midsecretory endometrium were infected
with AdCMV, AdPRA, and AdPRB, respectively, and then cultured with 0.1
µM MPA for 11 days. IGFBP-1 in the media was
measured by ELISA every other day. The amount of IGFBP-1 in media
collected before day 6 was below the detection limit of ELISA. The
production rate on days 7, 9, and 11 are shown. Data represent
triplicate determination, mean ± SD. Production
rates on day 7 were 0.043, 0.049, 0.071, and 0.031 µg/1 x
106 cells per day in control (cells not infected by Ad) and
cells infected with AdCMV, AdPRA, and AdPRB, respectively. Day 7, AdPRA
vs AdPRB (P < 0.01); days 9 and 11, AdPRA
vs. control, AdCMV, and AdPRB (P <
0.001).
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DISCUSSION
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The promoter activity of the IGFBP-1 gene has been studied in
endometrial stromal cells (10, 11, 12, 13, 14, 15, 16) as well as in HepG2 cells (17, 18).
The transactivators that interact with the functional
cis-elements in the IGFBP-1 promoter are different in these
two types of cells (10, 11, 12, 13, 14, 15, 16, 17, 18). For example, distal promoter SP1 sites are
extremely active in decidualized stromal cells which account for >95%
of the total induction (12, 15, 16), but they are silent in HepG2 cells
(15, 16, 17). In the proximal promoter region, CCAAT (Fig. 1
) represses the
activity in endometrial cells (14) but it activates the promoter in
HepG2 cells by HNF1, a liver factor not expressed in stromal cells
(14). More importantly, progestin and its receptor are essential to
activate the endometrial/decidual IGFBP-1 expression (10) whereas
in HepG2 cells, glucocorticoids activate its expression (17, 18).
In this study, we have demonstrated that ligand-activated hPR-A is a
stronger transactivator than hPR-B to increase the promoter activity as
well as the induction of endogenous IGFBP-1 gene in endometrial stromal
cells. The promoter analyses, using transient transfection of a short
promoter fragment, have shown that active sites, PRE1/2, mediate the PR
activation. Also, the response is dose dependent and can be detected
after 2 days incubation with MPA. These data provide evidence that
transactivation of PR is directly interacting on the IGFBP-1 promoter
although indirect effect is also probable. These data imply that the
high production of IGFBP-1 (Fig. 7
), although it was slow to rise, is
mediated by interaction of hPR-A with the PRE1 and PRE2 sites.
The delayed effect shown in Fig. 7
is likely caused by the multiple
regulatory elements that prevent an immediate response. For example, we
have shown that CCAAT (-82 to -52 bp) represses the promoter activity
mediated by NF-Ys which was down-regulated by MPA (14). The overall
production of IGFBP-1 is likely resulted from the balance between
activation and repression at transcription and posttranscription levels
(9, 10, 11, 12, 13, 14, 15).
Although overexpression of hPR-A or hPR-B was used in the present
study, we consider that the results are physiologically relevant since
the study was carried out in a primary culture system that depicts
decidualization in vivo (16). Also, no squelching was
observed (Fig. 4
). In Fig. 4
, we found that hPR-B reduced the hPR-A
activated promoter activity (lanes 57, 8, and 9). These results
coincide with the low production rate in the first 6 days of culture
with MPA and in undifferentiated stromal cells (9) since they contain
an equivalent amount of hPR-A and hPR-B (1). The high promoter activity
and production rate activated by PRA (Fig. 3
, lanes 24, and Fig. 7
)
correlate with the exponential increase of the IGFBP-1 production and
mRNA level in decidualized stromal cells (9, 10) where hPR-A is
predominant (1, 2). Taken together, the present findings illustrate, at
least in part, the induction mode of IGFBP-1 gene in endometrial
stromal/decidual cells, i.e. activation of the endometrial
cell IGFBP-1 gene is progestin dependent and mediated by the content of
the PR isoforms at different stages of stromal cell decidualization.
Other factors, such as Sp3 and CCAAT binding proteins, also regulate
the transcription (12, 13, 14, 15, 16), which may be either dependent or
independent on the progestin regulation.
In comparison, hPR-B transactivates the pMMTV-CAT stronger than that of
hPR-A in stromal cells (Fig. 2B
). Our observations, together with data
obtained in cancer cell lines (3, 4, 5, 6, 7, 8), indicate that the two PR isoforms
can have dissimilar responses to different promoters. In addition, we
have shown that hPR-B acts as a transdominant repressor of hPR-A in
contrast to the findings in cancer cell lines (3, 4, 5). Since the
repressor "domain" resides in both isoforms (6, 7, 8), the repressive
effects of the two isoforms appear to be also relative depending on the
target genes and cell context.
At present, the molecular mechanism of the high transactivation
capacity of hPR-A for the IGFBP-1 promoter is unclear. It may be due to
the differences in binding capacity or affinity of hPR-A/hPR-B to
PRE1/PRE2 sites. However, our binding analysis ruled out this
possibility. It is likely due to its interaction with the adjacent
sequences of PRE1 and PRE2 or its interaction with other factors
associated with these two regions in stromal cells.
The high capacity of hPR-A to transactivate the IGFBP-1 gene suggests
that endogenous hPR-A in decidual cells may be also active on the other
genes expressed in endometrial/decidual cells. Previous studies have
shown that progestin induces aromatase, fibronectin, IGFs, PRL/PRL-R,
hPRs, and IGFBP-1 to a moderate degree in the predecidual cells. Among
these genes, only hPR-A, PRL, PRL-R, IGFBP-1, and IGF-II are
extensively induced in decidual cells (1, 9, 10, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22). The
continuous induction of these genes could be associated with the high
transactivation capacity of hPR-A directly or indirectly acting on the
target genes. Interestingly, preliminary data showed that hPR-A is also
a strong transactivator for the production of PRL (our
unpublished observation). Further study is needed to clarify the PR
isoform specific for gene activation in this system.
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MATERIALS AND METHODS
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Cell Culture
Human endometrial specimens were obtained from premenopausal
women (35 to 50 yr old) who had undergone hysterectomies for a variety
of medical reasons but not related to any abnormality of the
endometrium. Permission for using these human specimens was approved by
the Human Subject Committee of our institution in accordance with US
Department of Health regulations. Endometrial stromal cells were
digested from tissue fragments with collagenase, isolated (purity
>95%), and cultured in Petri dishes, as described previously (22).
Stromal cells (1 x 106 cells per dish) were
transfected with a total of 10 µg plasmid constructs, 8 µg IGFBP-1
promoter, 0.1 to 1 µg hPR-A or hPR-B expression vectors, and 1 µg
pRSV-Luc by a modified calcium phosphate precipitation method (23).
Plasmid Constructs and Transfection Assay
IGFBP-1 promoter-reporter constructs p275CAT and p1.2CAT [the
promoter regions between -275 to +68 bp and -1.2 kb to +68 linked to
the chloramphenicol acetyl transferase (CAT) reporter gene] were
constructed previously (Fig. 1
and Ref. 13). Both constructs contain
the PRE1 and PRE2 sites, which are located at -193 to -179 bp and
-102 to -88 bp of the IGFBP-1 promoter, respectively. Mutants
(p275PRE1.2 m, p275PRE1m, and p275PRE2m) were constructed previously by
double or single mutation at core sequence of the PRE1 and/or PRE2 site
(Fig. 1
). hPR-A and hPR-B expression vectors were a gift from Dr. P.
Chambon (24). hPRB-DBDcys, a construct of PR-B
DNA binding mutation, was provided by Dr. K. Horwitz (25). pMMTV-CAT
(containing two sets of consensus PRE) and pRSV-luc were also used
(26, 27, 28). Plasmids were purified twice by CsCl/ethidium bromide
gradient centrifugation before use. After transfection, cells were
cultured without or with MPA for 2 days and then harvested in 0.15 ml
lysis buffer (Promega Corp., Madison, WI). The promoter
activity (CAT) was measured in a reaction mixture of cell lysis, 0.1
µCi [3H]acetyl coenzyme A (NEN Life Science Products, Boston, MA) and chloramphenicol at 37 C in 0.2
ml buffer solution containing 0.12 M Tris, pH 6.8. The
reaction was stopped by adding 25 µl of 2 M perchloric
acid and counted in 3 ml of ScintiLene (Fisher Scientific,
Pittsburgh, PA) by a Beckman Coulter, Inc. scintillation
counter. The CAT activity was normalized to luciferase activity derived
from pRSV-Luc cotransfected with the promoter construct. The Luc
activity was measured by Luminometer (Fluoroskan Ascent, Fl, Labsystem
OY, Helsinki, Finland). Each experimental design was confirmed in at
least two independent experiments. Representative results are
summarized in the present report. Data are presented as mean ±
SD calculated from triplicate dishes.
Construction and Infection of AdPRA and AdPRB and IGFBP-1
Assay
Recombinant adenoviral transfer vector containing hPR-A or hPR-B
under the control of the cytomegalovirus (CMV) was constructed
according to the established procedures (29, 30, 31). A
replication-defective human adenovirus, Ad5 [lacking E1A and E1B,
replication incompetent in normal mammalian cells (29, 30)], was used
for the construction of AdPRA and AdPRB. Briefly, hPR-A and hPR-B cDNAs
were separately cloned into plasmid [pACsk2CMV5 (31)], containing
CMV-1 promoter and adenovirus sequences to yield pACsk2CMV-PRA, and
pACsk2CMV-PRB, respectively. The recombinant plasmids and pJM17 (29)
were transfected into human embryo kidney cells (293 cells) by
lipofectamine (Life Technologies, Inc., Gaithersburg,
MD). The two plasmids were recombined at the overlapping viral
DNA sequence to yield a recombinant packageable viral genome. Positive
viral plaques with hPR-A or hPR-B insert were identified and then
propagated to produce large quantities of the recombinant AdPRA and
AdPRB, respectively. Titered viral stocks were used to infect the
endometrial stromal cells. Cells (1 x 106
cells per dish) were incubated with recombinant viral particles (1, 5,
10, or 50 particles per cell) to achieve an optimal infection. Ninety
five percent of the cell population was infected by Ad5 viral particle
as shown in Fig. 6
.
To identify recombinant PR, stromal cells infected with AdPRA and AdPRB
were incubated with MPA 2 to 12 days. Cells were then processed for
immunohistochemistry using Mab hPRa3 provided by Dr. P. Satyaswaroop
(32) and the Histostain SP-kit detection system (Zymed Laboratories, Inc. South San Francisco, CA) described previously
(1). Cells infected with Adßgal were stained with substrate X-gal
solution (United States Biochemical Corp., Cleveland, OH).
The IGFBP-1 in culture medium was measured by ELISA (9) from stromal
cells infected with equal amount of viral particles, Ad5, AdPRA, and
AdPRB, respectively.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to the late Dr. Satyaswaroop who provided us the
Mab hPRa3 to identify the PR in stromal cells. We thank the clinicians
in the Department of Obstetrics/Gynecology and Reproductive Medicine
and Department of Pathology and SUNY-Stony Brook for providing us with
viable endometrial specimens.
 |
FOOTNOTES
|
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Address requests for reprints to: Linda Tseng, Ph.D, Department of Obstetrics/Gynecology and Reproductive Medicine, State University of New York at Stony Brook School of Medicine, Stony Brook, New York 11794.
Supported by NIH Grant HD-19247.
Received for publication May 25, 2000.
Revision received August 15, 2000.
Accepted for publication August 23, 2000.
 |
REFERENCES
|
---|
-
Tseng L, Zhu HH 1999 Regulation of progesterone receptor
messenger ribonucleic acid by progestin in human endometrial stromal
cells. Biol Reprod 57:13601366[Abstract]
-
Mote PA, Balleine RL, McGowan EM, Clarke CL 1999 Colocalization of progesterone receptors A and B by dual
immunofluorescent histochemistry in human endometrium during the
menstrual cycle. J Clin Endocrinol Metab 84:29632971[Abstract/Free Full Text]
-
Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate
transcription without binding to progesterone response elements and are
dominantly inhibited by A-receptors. Mol Endocrinol 7:12561265[Abstract]
-
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, OMalley BW,
McDonnell DP 1993 Human progesterone receptor A form is a cell- and
promoter-specific repressor of human progesterone receptor B function.
Mol Endocrinol 7:12441255[Abstract]
-
McGowan EM, Clarke CL 1999 Effect of overexpression of
progesterone receptor A on endogenous progestin-sensitive endpoints in
breast cancer cells. Mol Endocrinol 13:16571671[Abstract/Free Full Text]
-
Giangrande PH, Pollio G, McDonnell DP 1997 Mapping and
characterization of the functional domains responsible for the
differential activity of the A and B isoforms of the human progesterone
receptor. J Biol Chem 272:3288932900[Abstract/Free Full Text]
-
Hovland AR, Powell RL, Takimoto GS, Tung L, Horwitz KB 1998 An N-terminal inhibitory function, IF, suppresses transcription by the
A-isoform but not the B-isoform of human progesterone receptors. J
Biol Chem 273:54555460[Abstract/Free Full Text]
-
Huse B, Verca SB, Matthey P, Rusconi S 1998 Definition of a
negative modulation domain in the human progesterone receptor. Mol
Endocrinol 12:13341342[Abstract/Free Full Text]
-
Bell SC, Jackson JA, Ashmore J, Zhu HH, Tseng L 1991 Regulation of insulin-like growth factor-binding protein-1 synthesis
and secretion by progestin and relaxin in long term cultures of human
endometrial stromal cells. J Clin Endocrinol Metab 72:10141024[Abstract]
-
Tseng L, Gao JG, Chen R, Zhu HH, Mazella J, Powell DR 1992 Effect of progestin, antiprogestin, and relaxin on the accumulation of
prolactin and insulin-like growth factor-binding protein-1 messenger
ribonucleic acid in human endometrial stromal cells. Biol Reprod 47:441450[Abstract]
-
Gao J, Mazella J, Tseng L 1995 Activation of human
insulin-like growth factor binding protein-1 gene promoter by a distal
regulatory sequence in a human endometrial adenocarcinoma cell line.
Mol Endocrinol 9:14051412[Abstract]
-
Tseng L, Zhu HH, Gao JG 1998 Human IGF BP-1 gene activation in
human endometrium. In: Takano K, Hisuka N, Takahhashi SI (eds)
Insulin-Like Growth Factor Binding Protein-1 Gene Activation in Human
Endometrium. Proceeding of the 4th International Symposium of IGFs,
October 2124, 1987, Tokyo, Japan. Elsevier, New York, pp 3947
-
Gao J, Mazella J, Suwanichkul A, Powell DR, Tseng L 1999 Activation of the insulin-like growth factor binding protein-1 promoter
by progesterone receptor in decidualized human endometrial stromal
cells. Mol Cell Endocrinol 153:1117[CrossRef][Medline]
-
Gao J, Mazella J, Tseng L 2000 Partial characterization of the
CCAAT box in the promoter of the hIGFBP-1 gene: interaction with
negatively acting transcription factors in decidualized human
endometrial stromal cells, Mol Cell Endocrinol 159:1117
-
Gao JG, Tseng L 1996 Distal Sp3 binding sites in the hIGFBP-1
gene promoter suppress the transcriptional repression in decidualized
human endometrial stromal cells: Identification of a novel Sp3 form in
decidual cells. Mol Endocrinol 10:613621[Abstract]
-
Gao JG, Mazella J, Powell DR, Tseng L 1994 Identification of a
distal regulatory sequence of the human IGFBP-1 gene promoter and
regulation by the progesterone receptor in a human endometrial
adenocarcinoma cell line. DNA Cell Biol 13:829837[Medline]
-
Powell DR, Lee PD, Suwanichkul A 1993 Multihormonal regulation
of IGFBP-1 promoter activity. Adv Exp Med Biol 343:205214[Medline]
-
Suwanichkul A Allander SV Morris SL Powell DR 1994 Glucocorticoids, insulin regulate expression of the human gene for
insulin-like growth factor-binding protein- 1 through proximal promoter
elements. J Biol Chem 269:3083530841[Abstract/Free Full Text]
-
Lane B, Oxberry W, Mazella J, Tseng L 1994 Decidualization of
human endometrial stromal cells in vitro: effects of
progestin and relaxin on the ultrastructure and production of decidual
secretory proteins. Hum Reprod 9:25926[Abstract]
-
Tseng L 1996 Endometrial cells gene activation during
implantation, early pregnancy. J Reprod Med 5:4551
-
Tseng L, Zhu HH 1998 Progestin, estrogen, and IGF-1 stimulate
the prolactin receptor mRNA in human endometrial cells. J Soc Gynecol
Invest 5:149155[CrossRef][Medline]
-
Zhu HH, Huang JR, Mazella J, Rosenberg M, Tseng L 1990 Differential effects of progestin and relaxin on the synthesis and
secretion of immunoreactive prolactin in long term culture of human
endometrial stromal cells. J Clin Endocrinol Metab 71:889899[Abstract]
-
Chen C, Okayama H 1987 High-efficiency transformation of
mammalian cells by plasmid DNA. Mol Cell Biol 7:27452752[Medline]
-
Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonistic and antagonistic activities of RU486 on the functions of
the human progesterone receptor. EMBO J 9:39233932[Abstract]
-
Takimoto GS, Tasset DM, Eppert AC, Horwitz KB 1992 Hormone-induced progesterone receptor phosphorylation consists of
sequential DNA-independent and DNA-dependent stages: analysis with zinc
finger mutants and the progesterone antagonist ZK98299. Proc Natl Acad
Sci USA 89:30503054[Abstract]
-
de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 7:725737[Medline]
-
Cato AC, Miksicek R, Schutz G, Arnemann J, Beato M 1986 The
hormone regulatory element of mouse mammary tumour virus mediates
progesterone induction. EMBO J 5:22372240[Abstract]
-
Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH The 1982 Rous sarcoma virus long terminal repeat is a strong promoter when
introduced into a variety of eukaryotic cells by DNA-mediated
transfection. Proc Natl Acad Sci USA 79:67776781[Abstract]
-
McGrory WJ, Bautista DS, Graham FL 1988 A simple technique for
the rescue of early region I mutations into infectious human adenovirus
type 5. Virology 163:614617[Medline]
-
Gluzman Y, Herwig R, Solnick D 1982 Helper-free adenovirus
type-5 vectors. In: Gluzaman Y (ed) Eukaryotic Viral Vectors. Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 187192
-
Alcorn JL, Gao E, Chen Q, Smith ME, Gerard RD, Mendelson CR 1993 Genomic elements involved in transcriptional regulation of the
rabbit surfactant protein-A gene. Mol Endocrinol 7:10721085[Abstract]
-
Clarke CL, Zano RJ, Mortel R, Satyaswaroop PG 1987 Monoclonal
antibodies to human progesterone receptor: characterization by
biochemical and immunohistochemical techniques. Endocrinology 121:11231132[Abstract]