Regulation of Tissue Factor Gene Expression In Human Endometrium by Transcription Factors Sp1 and Sp3
Graciela Krikun,
Frederick Schatz,
Nigel Mackman,
Seth Guller,
Rita Demopoulos and
Charles J. Lockwood
The Departments of Obstetrics and Gynecology (G.K., F.S., S.G.,
R.D., C.J.L.), Biochemistry (S.G.), and Pathology (R.D.) New York
University School of Medicine New York, New York 10016
The Scripps Research Institute (N.M.) Departments of
Immunology & Vascular Biology La Jolla, California 92037
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ABSTRACT
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Prior studies indicate that tissue factor (TF),
the primary cellular initiator of hemostasis, is persistently
up-regulated in human endometrial stromal cells (HESCs) undergoing
progestin-induced decidualization in vivo and in
vitro. The mechanism underlying progestin enhancement of TF mRNA
and protein levels in these cells involves transcriptional activation
of the TF gene. Transient transfections of HESCs with the truncated TF
promoters driving the luciferase reporter gene have demonstrated that
the region spanning -111 to +14 bp retained differential
progestin-enhancing effects. We now demonstrate that RU486 displays
inhibitory effects on the progestin-induced TF promoter activity,
confirming the involvement of the progesterone receptor. Since the TF
minimal promoter (pTF 111 spanning -111 to +14 bp) contains three
overlapping Sp1 and three Egr-1 sites, the present study determined
whether Sp1 and/or Egr-1 were required for progestin-regulated TF
expression. The results indicate that the three Sp1 sites are primarily
responsible for both the constitutive and progestational activity of
the pTF 111 promoter, whereas the Egr-1 sites have only a minor
involvement in both activities. Overexpression of the Sp1 protein
resulted in greater than a 6-fold induction in TF promoter activity. In
contrast, no enhancement was observed when the Sp3 protein was
overexpressed. The concomitant overexpression of Sp1 and Sp3
demonstrated that Sp3 completely blocked the induction of TF promoter
activity by Sp1. Moreover, the addition of 10
nM mithramycin, a concentration that inhibits
Sp1 binding to target DNA, blocked the progestational induction of TF
mRNA expression. Immunohistochemical studies demonstrated increased Sp1
levels in perivascular stromal cells in secretory phase compared with
proliferative phase endometria. In contrast, Sp3 expression was greatly
decreased in stromal cells of secretory, compared with proliferative
phase tissues. The levels of Egr-1 were low in both proliferative and
secretory endometria. Immunocytochemistry of E2
vs. E2 + medroxyprogesterone
acetate-treated HESCs demonstrated a dramatic reduction in
Sp3 expression after progestin treatment, and Northern blots
demonstrated progestational increases in Sp1 and reduction in Sp3 mRNA
expression compared with controls. Taken together, our results
demonstrate that progestin enhancement of TF gene expression in HESCs
is mediated principally by Sp1. We propose that progestins
regulate HESC TF gene expression in vivo by altering the
ratio of Sp1 to Sp3 nuclear factors.
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INTRODUCTION
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Tissue factor (TF), the cell membrane-bound glycoprotein
responsible for the initiation of hemostasis, is synthesized in several
cell types (1, 2, 3). Our prior studies demonstrated that TF is also
expressed in human endometrial stromal cells (HESCs) where its
expression is greatly enhanced by progestins in vitro and
in vivo (4, 5). In contrast to the immediate and transient
induction of TF expression in endothelial, epithelial, monocytic, and
fibroblastic cells (6, 7, 8, 9), the induction of TF in HESCs by progestins
is delayed and prolonged. Subsequent studies in our laboratory
demonstrated that progestational up-regulation of TF in HESCs occurs at
the transcriptional level (10). Moreover, transfection with truncated
TF promoter constructs showed that the region -111 to +14 bp retained
both basal and differential progestin effects (10).
The -111 to +14 region contains three overlapping Sp1/Egr-1 sites
(Fig. 1
). Sp1 is a nuclear activator protein that stimulates eukaryotic
transcriptional initiation by aiding in the formation of a functional
preinitiation complex consisting of RNA polymerase II,
activator-proteins, and the target DNA (11, 12, 13). Activation is further
enhanced if multiple Sp1 binding sites are present (14, 15). Four
members of the Sp family of proteins have thus far been identified.
They include Sp1, Sp2, Sp3, and Sp4 (16, 17, 18, 19). Sp3 and Sp4 proteins bind
with similar affinities to the same consensus DNA site as Sp1 (19, 20, 21).
Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 is expressed
predominantly in the brain (22, 17). Interestingly, Sp3 generally
functions as a competitive repressor of Sp1-induced transcription (17).
In addition to competition for binding sites among the Sp family of
proteins, other zinc-binding proteins, such as Egr-1, bind to GC-rich
regions and can partially interfere with Sp binding (23, 24). The Egr-1
transcription factor, also known as NGFI-A, Zif 268, Krox24, and TIS8
(25, 26), is involved in cell proliferation and differentiation (27).
It is a nuclear phosphoprotein that is rapidly and transiently induced
by serum and growth factors and binds to a specific DNA sequence in a
zinc-dependent manner (25).

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Figure 1. TF Promoter Constructs
The minimal TF promoter spanning the region -111 to + 14 bp (pTF 111)
was altered as described in Materials and Methods. The
resulting promoters contained critical mutations in the three Sp1 sites
(pTF Sp1m), the three Egr-1 sites (pTF Egr-1m),
and the six overlapping Sp1/Egr-1 (pTF Sp1/Egr-1m) sites as
denoted by the bold letters.
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Sp1 and/or Egr-1 have been shown to regulate the immediate and
transient expression of TF in various systems. In HeLa cells, Sp1 is
constitutively expressed, whereas Egr-1 was induced in response to
phorbol myristate acetate (PMA) or serum (28). Shear stress-induced TF
expression by human umbilical vein endothelial cells and human aortic
endothelial cells requires Sp1 and Egr-1 (29, 30). Hypoxia enhances
transcription and expression of TF in lung via Egr-1 (31). The current
study determined the role of the three Sp1 and the three Egr-1 sites
contained within the region -111 to +14 bp of the TF gene in
progestin-induced transcription in HESCs.
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RESULTS
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RU486 Blocks Progestin-Enhanced TF Gene Transcription
The antiprogestin RU486 was used to assess the requirement
for the progesterone receptor (PR) in mediating progestin enhancement
of TF transcription. Figure 2
demonstrates that RU486
abolished progestin-enhanced transcription of both the pTF 278 and
pTF 111 promoter constructs. Although RU486 blocks the action of
progesterone and glucocorticoid receptors, we have previously shown
that glucocorticoids have no effect on the induction of TF expression
(32). Therefore, these effects of RU486 confirm the involvement of the
PR in our system.

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Figure 2. RU486 Blocks MPA Transcriptional Activation of TF
Cultured HESCs were treated with E2, E2 + MPA,
E2 + RU486 (RU), or E2 + MPA + RU as described
in Materials and Methods. Transfections were carried out
with two TF promoter constructs spanning the regions -278 to +14 bp
(P-278) and -111 to +14 bp (P-111). Firefly luciferase activity
derived from the TF promoter was normalized for transfection efficiency
by comparison with Renilla luciferase activity derived from the
cotransfected Renilla vector. (n = 6, triplicates from two
separate experiments). *, P < 0.05 comparing
E2 + MPA vs. E2.
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Role of Sp1 and Egr-1 in Progestin Enhancement of TF Gene
Transcription
To investigate the role of Sp1 and Egr-1 in mediating
progestin-enhanced TF transcription, HESCs were transfected with the
luciferase-linked minimal wild-type promoter construct (pTF 111) or
with promoter constructs in which either the three Sp1 sites (pTF
Sp1-m), the three Egr-1 (pTF Egr1-m), or all six Sp1 and Egr-1 sites
(pTF Sp1/Egr1-m) were mutated. The effect of hormone treatment as well
as a schematic of the various promoter constructs that were used is
depicted in Fig. 3
. This figure
demonstrates that promoter activity was significantly enhanced by
treatment with E2 + medroxyprogesterone acetate
(MPA) compared with E2 for both pTF 111 and pTF
Egr1-m. In addition, the activities of pTF Sp1-m and pTF Sp1/Egr1-m
were significantly reduced compared with the activity of pTF 111 after
incubation with E2 or E2 +
MPA (P < 0.0001 in all cases). By contrast, the
promoter activity observed with pTF Egr1-m did not differ significantly
from that observed with pTF 111 for either E2 or
E2 + MPA. These results indicate that in the
presence or absence of progestin, mutating the Egr-1 sites alone does
not affect the transcriptional activity of the TF promoter. Conversely,
the rates of transcription are greatly diminished by mutating the Sp1
sites, demonstrating the requirement for the latter. When the Sp1 sites
are mutated, both the constitutive and the progestational induced
transcription rates are decreased. Nevertheless, a trend toward higher
rates in the presence of progestins is evident. This trend, as well as
the overall constitutive rates for construct pTF Sp1-m, are
significantly decreased when all the Sp1 and Egr-1 binding sites are
mutated in the pTF Sp1/Egr1-m construct (P < 0.03 for
E2 and P < 0.02 for
E2 + MPA). This result likely reflects the small
binding affinity of Sp1 for Egr-1 sites as has been previously
described (23, 24). In summary, Fig. 3
indicates that the three Sp1
sites are primarily responsible for the constitutive and progestational
activity of the pTF 111 promoter, whereas the Egr-1 sites have a minor
involvement in both activities.

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Figure 3. Transfection of Mutant TF Promoters
Cultured HESCs were treated with E2 or E2 + MPA
as described in Materials and Methods.
Transfections were carried out with the minimal TF promoter -111 to
+14 bp (pTF111) and with promoters mutated in the three Sp1 (pTFSp1-m),
the three Egr-1 (pTFEgr1-m), or all six Sp1 and Egr-1 (pTFSp1/Egr1-m).
A schematic of these is shown on the
left. Levels of luciferase activity were determined and
corrected for transfection efficiency as described in Materials
and Methods (n = 9 triplicates from three separate
experiments). *, P < 0.0001 comparing
E2 vs. E2 + MPA for pTF111 and
**, P < 0.002 comparing E2
vs. E2 + MPA for pTFEgr1-m.
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The promoter activity for construct pTF Sp1/Egr1-m was
significantly above that found in control cultures transfected with a
promoterless PGL2Basic vector (1.56 ± 0.18 vs.
0.66 ± 0.17 for E2, P <
0.02; and 1.78 ± 0.32 vs. 0.66 ± 0.23 for
E2 + MPA, P < 0.01).
Overexpression of Sp1 but not Sp3 Induces TF Promoter Activity
Figure 4
demonstrates that
overexpression of the transcription factor Sp1 increases TF promoter
activity greater than 6-fold. In contrast, no increase in promoter
activity is observed by Sp3 overexpression. Moreover, simultaneous
coexpression of Sp1 and Sp3 abolishes the induction observed with Sp1
overexpression alone. These data demonstrate that the balance between
levels of Sp1 and Sp3 regulates TF gene expression in HESCs.

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Figure 4. Overexpression of the Transcription Factor Sp1
and/or Sp3
HESCs were cultured and treated in control medium and transfected with
pTF 111 (TF) ± the Sp1, the Sp3, or both overexpressing vectors,
as described in Materials and Methods (n = 3
triplicates from a typical experiment). *, P <
0.002 compared with HESCs transfected with pTF 111 alone.
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Effect of Mithramycin on Progestin Induction of TF mRNA
Expression
To further investigate the role of Sp1 on the regulation of TF
expression, control, E2, or
E2+MPA-treated HESCs were incubated with or
without 10 nM mithramycin, an inhibitor of Sp1 binding to
its corresponding GC box. As can be seen in Fig. 5
, mithramycin abrogated the
progestational induction of TF mRNA levels, further supporting the role
of Sp1 in enhanced TF expression by HESCs.

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Figure 5. Effect of Mithramycin on Progestin Induction of TF
mRNA Expression
Control (C), E2 (E)-, or E2 + MPA (P)-treated
HESCs were incubated with and without 10 nM mithramycin
(Mit) as described in Materials and Methods. Northern
blots were performed for TF and loading efficiencies assessed with
glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
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Localization of Sp1, Sp3, and Egr-1 in Cycling
Endometria
Immunohistochemical staining for Sp1, Sp3, and Egr-1 was conducted
on six proliferative phase and six secretory phase endometria. Three
areas per slide were evaluated by two independent observers (G.K. and
F.S.). Figure 6
reveals that both Sp1 and
Sp3 are expressed throughout the menstrual cycle in stromal and
endothelial cells. Interestingly, an increase in Sp1 staining was
observed in secretory-compared with proliferative-phase stromal cells
(mean nuclear staining of 77% secretory vs. 38%
proliferative; P < 0.01). Particularly intense
immunostaining for Sp1 was observed in the nuclei, and weaker staining
was observed in the cytoplasm of perivascular stroma surrounding
clusters of spiral arterioles. These are precisely the sites where both
decidualization and enhanced TF expression commence (4, 5). In
contrast, immunostaining for the Sp1-competitor, Sp3, was decreased in
secretory vs. proliferative phase tissues (mean nuclear
staining of 31% secretory vs. 71% proliferative;
P < 0.01). Unlike the results for Sp1 and Sp3,
staining for Egr-1 was of a lower magnitude, unchanged by hormone
treatment and limited to a much smaller population of stromal cells in
all tissues studied. No staining was observed for Sp4 (not shown).

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Figure 6. Immunohistochemistry of Sp1 and Sp3 in Human
Endometrium
Immunohistochemical staining for Sp1and Sp3 in proliferative and
secretory phase human endometria were carried out in paraffin-fixed
sections as described in Materials and
Methods. Detailed perivascular areas for Sp1 and Sp3
staining in a secretory endometrium are shown as insets on
the upper left corner. Antigens are identified by
brown peroxidase staining (x400).
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Localization of Sp1 and Sp3 in Cultured HESCs
Immunocytochemical staining was used to observe the direct effects
of progestins on the expression of Sp1 and Sp3 in primary cultured
HESCs (Fig. 7
). The in
vitro expression of Sp1 showed small increases with progestin
treatment compared with E2-treated controls. More
dramatically, progestin treatment greatly decreased the expression of
the Sp3 nuclear factor in these cells. As with the immunohistochemical
studies, cultured HESCs showed low levels of Egr-1 staining that were
above background and were not affected by progestin treatment (not
shown).

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Figure 7. Immunocytochemistry of Sp1 and Sp3 in Primary
Cultured HESCs
Immunocytochemical staining for Sp1 and Sp3 in E2- and
E2 + MPA-treated HESCs was carried out as described in
Materials and Methods. Antigens are identified by
brown peroxidase staining (x200).
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Sp1 and Sp3 Northern Blots
Figure 8
displays the results of
densitometric analysis of five separate blots and demonstrates
statistically significant differences in steady state Sp1 and Sp3 mRNA
levels in E2 compared with
E2 + MPA-treated HESCs. Thus, progestin treatment
of the cells resulted in a 2-fold enhancement of Sp1 mRNA levels and a
2-fold decrease in Sp3 mRNA levels. Progestin treatment had no
significant effect on Egr-1 mRNA levels (data not shown).

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Figure 8. Northern Blot Analysis of Sp1 and Sp3 in Primary
Cultured HESCs
Densitometric analysis from five separate Northern blots depicting the
relative intensity of Sp1 or Sp3 adjusted to glyceraldehyde 3-phosphate
dehydrogenase (GAP) from HESCs treated with E2 or
E2 + MPA. *, P < 0.05.
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DISCUSSION
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The human endometrium undergoes a series of morphological and
biochemical changes involving proliferation (proliferative phase),
differentiation, and secretion (secretory phase). The latter
facilitates blastocyst implantation and invasion, which normally occurs
in the absence of potentially catastrophic hemorrhage. These events
are, to a large extent, under the control of the ovarian steroids,
estrogen and progesterone. Paradoxically, in the absence of
implantation, the endometrium must provide a milieu to facilitate the
hemorrhagic bleeding associated with menstruation. Prior in
vivo and in vitro studies have demonstrated that
decidualization is associated with enhanced HESC TF expression while
progestin withdrawal is associated with reduced TF levels (4, 5, 32).
Recently, we have shown that progestin-enhanced TF expression in HESCs
is transcriptionally regulated and requires a region of the TF promoter
-111 to +14 bp upstream of the transcription start site. This region
of the TF promoter contains three overlapping Sp1/Egr-1 sites. The
present study demonstrates that in HESCs, the three Sp1 sites are
primarily responsible for the constitutive and progestational activity
of the pTF 111 promoter, whereas the Egr-1 sites have a minor
involvement in both activities. Furthermore, our finding that Sp1
expression is enhanced while Sp3 expression is decreased in
perivascular stromal cells from secretory-phase endometrium suggests
that Sp1/Sp3 ratios are regulated by progestins during the menstrual
cycle. An in vitro model utilizing primary cultured HESCs
supports these findings. In this system we observed that progestins
increased Sp1 and decreased Sp3 mRNA and protein levels. Furthermore,
we noted that the Sp1-DNA binding inhibitor, mithramycin, abolished
progestational-induction of TF mRNA, further supporting the role of Sp1
on TF gene expression. Interestingly, we observed enhanced perivascular
staining for Sp1 in secretory endometria paralleling the expression of
TF (4, 5). While Sp1 staining was primarily nuclear, some cytoplasmic
staining was observed. This finding suggests that Sp1 protein is being
rapidly synthesized in these regions.
There are growing examples of the reciprocal regulation of target genes
by Sp3 and Sp1 (17, 33, 34, 35, 36). In these cases, Sp3 has been shown to
inhibit Sp1 binding, resulting in altered gene expression. For example,
the mitogenic effects of thrombin are mediated by the human thrombin
receptor gene (34). Cotransfection with an Sp1 expression vector
significantly augmented human thrombin receptor promoter activity,
while cotransfection with Sp1 and Sp3 inhibited Sp1-mediated activation
(34). Interestingly, a recent report by Hata and colleagues (35)
demonstrated that the kinase domain receptor promoter activity in
endothelial cells was partially regulated by variations in the Sp1/Sp3
ratio. Overexpression of Sp1 protein increased kinase domain receptor
promoter activity 3-fold, whereas simultaneous coexpression of Sp3
attenuated this response. This Sp3 antagonism of Sp1-mediated
transcription has also been reported for activation of the human
papillomavirus type 16 promoter during epithelial cell differentiation,
as well as in epithelial-specific cellular genes encoding keratin 18
and E-cadherin (36). Our results suggest that progestin plays a
critical role in altering the ratio of Sp1/Sp3 expression and that
this, in turn, controls the expression of TF in HESCs.
Although expression of Egr-1 protein in cycling endometrium was low,
mutation of the three Egr-1 sites together with the three Sp1 sites in
the TF promoter indicated that these sites played an additional role in
the basal and the progestational induction of TF expression in HESCs.
Egr-1 is the prototypic member of a family of immediate-early genes
including Egr-2, -3, and -4, each of which contains similar DNA-binding
domains (37). Like the Sp family of proteins, Egr proteins are involved
in differentiation and mitogenesis (37, 38). In addition to binding to
their own sites, Sp proteins can also bind to the Egr-1 sites (23, 24).
Figure 3
shows that mutation of the three Sp sites in pTF 111 greatly
reduces the basal transcriptional activity of this promoter. The
residual progestin effects that are still apparent likely reflect Sp1
binding to the three remaining Egr-1 sites. The minimal expression of
Egr-1 protein in HESCs throughout the cycle also suggests that Egr-1
does not effectively compete with Sp1. Taken together, our results
provide a mechanism to account for progestin-enhanced TF expression in
HESCs through the interaction of Sp nuclear proteins and through a
dramatic alteration in the levels of Sp1 and Sp3 nuclear factors in
proliferative vs. secretory endometria.
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MATERIALS AND METHODS
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Preparation of Primary Endometrial Stromal Cell Cultures
Experiments in which Northern blot or immunocytochemical
analysis were to be conducted used freshly isolated stromal cells grown
to confluence in a basal medium as previously described (4). The
experimental period was initiated by the addition of
10-8 M estradiol
(E2) ± 10-7
M MPA. The medium was changed every 24 days. The cells
were treated for 10 days.
Promoter Constructs
The TF promoter-plasmid construct containing the 5'-flanking
sequence of the TF promoters spanning -278 to +14 bp (pTF 278) or
-111 to +14 bp (pTF 111) was cloned into the luciferase reporter
vector pGL2 (Promega Corp., Madison, WI) as described (6).
Site-directed mutagenesis of the three Sp1 sites, the three Egr-1
sites, or all six Sp1 and Egr-1 sites were performed as described (28)
(Fig. 1
). The Sp1 and Sp3 expression vectors (pSp1 and pSp3)
were generously provided by Jonathan M. Horowitz (North Carolina State
University, Raleigh, NC) and were constructed as previously described
(39).
Transient Transfections
Primary HESCs were seeded at a density of 2 x
105 cells/3-cm polystyrene well and allowed to
attach and proliferate to approximately 60% confluence for 24 h.
HESCs were then treated with E2
(10-8 M), E2 +
MPA (10-7 M),
E2 + RU486 (10-6
M), or E2 + MPA + RU486 or vehicle
control for 6 days. RU486 was provided by Dr. Indrani Bagchi
(Population Council, New York, NY). Subsequent to this treatment, the
cultures were transiently transfected for 4 h with 1 µg of the
various TF promoters or with the promoterless pGL2 basic vector
(Promega Corp.) utilizing the lipofectamine-Plus reagent
(Life Technologies, Inc., Gaithersburg, MD). Experiments in which
Sp1 and/or Sp3 were overexpressed were carried out with 1 µg pTF 111
in conjunction with 0.2 µg of either pSp1, pSp3, or both. The cells
were cotransfected with 0.1 µg of a Renilla-luciferase reporter
vector (Promega Corp.) to provide an internal control of
transfection efficiency. The medium was then changed back to basal
medium containing either control, E2, or
E2 + MPA for an additional 48 h. At this
time cells were lysed in passive lysis buffer according to the
manufacturer (Promega Corp.) and stored at -80 C.
Transfection Reporter Assays
Luciferase activity was monitored with a luminometer using the
Dual Luciferase Reporter Assay System (Promega Corp.) to
sequentially assay for TF promoter-dependent firefly luciferase and
Renilla luciferase from the internal control.
Immunohistochemistry and Immunocytochemisty
Immunohistochemistry was performed in paraffin-fixed
sections of endometria. Immunocytochemistry was conducted on HESCs
grown to confluence in tissue culture chamber slides (Nunc Inc.,
Naperville, IL) and treated with hormones as described above. The cells
were fixed in a 4% parformaldehyde solution for 10 min. Peroxidase
staining was conducted with the ABC elite kit from Vector Laboratories, Inc. (Burlingame, CA) as described (5). Rabbit
polyclonal antibodies to Sp1 (Accurate Surgical & Scientific Corp., Westbury, NY), as well as to Sp3, Sp4, and Egr-1
(Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) were used
at a concentration of 0.2 µg/ml.
Northern Blots
Northern blots were carried out as previously described (4, 5).
The Sp1 cDNA probe was a kind gift from Dr. C. Kingsley. The Sp3 cDNA
probe was purchased from the ATCC (Manassas, VA). The TF
cDNA probe was previously described (4, 5). Mithramycin
(Sigma, St. Louis, MO) treatment of cells was carried out
12 h before RNA isolation.
Statistical Analysis
All data were analyzed with a Mann-Whitney Rank Sum Test, Sigma
Stat computer program (Jandell Scientific, San Rafael, CA). A
P value < 0.05 was considered significant.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Graciela Krikun, New York University School of Medicine, Tisch Hospital, Room 533, 550 First Avenue, New York, New York 10016.
This work was supported in part by NIH Grant RO1 HL-3393703
(C.J.L.).
Received for publication April 29, 1999.
Revision received December 2, 1999.
Accepted for publication December 10, 1999.
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