 |
INTRODUCTION |
In the adult, angiogenesis physiologically occurs in the uterus
and ovary during the reproductive cycle and pregnancy under physiological conditions (1, 2). Increased uterine vascular permeability and angiogenesis are two hallmarks of successful implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor
(VEGF)1 (3, 4), which exists
in multiple isoforms. VEGF signals via two transmembrane tyrosine
kinase receptors, VEGF receptors 1 and 2 (5-11). We have
previously shown that VEGF isoforms and VEGF receptors are expressed in
the uterus during early pregnancy in a spatiotemporal manner (1),
suggesting that VEGF plays an important role in uterine vascular
permeability and angiogenesis required for implantation and
decidualization. Because the uterus is a primary target for estrogen
and progesterone (P4), which profoundly influence uterine
function prior to and during implantation, it is thought that steroid
hormones modulate the uterine angiogenic status via the VEGF system.
Indeed, our recent studies have shown that estrogen and P4
have different effects in vivo; estrogen promotes uterine
vascular permeability but profoundly inhibits angiogenesis, whereas
P4 stimulates angiogenesis with little effect on vascular
permeability. These effects of estrogen and P4 are mediated
by differential spatiotemporal expression of proangiogenic factors in
the uterus (12).
VEGF effects are complemented and coordinated by another class of
angiogenic factors, the angiopoietins that act via the tyrosine kinase
receptor Tie2 (13). We have recently shown that although VEGF signaling
primarily regulates uterine vascular permeability and angiogenesis
prior to the attachment phase of the implantation process, VEGF in
conjunction with the angiopoietin system directs angiogenesis during
uterine decidualization following implantation (14). Furthermore, our
results provide evidence that although ovarian steroid hormones
influence uterine vascular permeability and angiogenesis during the
preimplantation period, cyclooxygenase-2 (COX-2)-derived
prostaglandins direct these events during implantation and
decidualization by differentially regulating VEGF and angiopoietin signaling. However, the mechanisms by which steroid hormones and prostaglandins differentially regulate uterine angiogenesis during early pregnancy remain unresolved.
Oxygen homeostasis is essential for cell survival and is primarily
mediated by hypoxia-inducible factors (HIFs), which are intimately
associated with vascular events (15-18) and induce Vegf gene transcription by binding to the hypoxia response element in the
Vegf promoter (15, 19-22). Several HIFs have been
identified that all function as heterodimeric transcription factors
consisting of
- and
-subunits. These subunits belong to the basic
helix-loop-helix-PAS protein superfamily (15, 18).
HIF-1
was first cloned in humans followed by its cloning
in mice and rats (23-26). Subsequently, HIF-2
and
HIF-3
, which have a high sequence homology to
HIF-1
, were cloned in mice, rats, and humans (26-28).
HIF-2
is also known as endothelial PAS domain protein-1,
HIF-1
-like factor, HIF-related factor, or MOP2 (member
of the PAS superfamily 2) (15).
HIF-
subunits are identical to the aryl hydrocarbon nuclear
translocators (ARNTs). The ARNT family consists of ARNT1, ARNT2, and
ARNT3. ARNT3 is also known as BMAL1 (brain and
muscle ARNT-like
protein-1) (15). HIF-
subunits can heterodimerize with
the ARNT family members without specificity for their dimerization
partners (15).
HIF-1
is expressed in most human and rodent tissues (25, 29). Levels
of the HIF-1
protein are primarily regulated by protein
stabilization under hypoxic conditions, whereas its rapid degradation
occurs under normoxic conditions via an ubiquitination mechanism (15,
30-33). Normally, the expression of HIF-1
is ubiquitous, whereas
that of HIF-2
and HIF-3
shows a more restricted expression
pattern. There is evidence that HIF-2
mRNA expression is much
higher under normoxic conditions and that its expression correlates
with that of VEGF (34). The expression patterns of ARNT1, ARNT2, and
ARNT3 in general resemble those of HIF-1
, HIF-2
, and HIF-3
(15). Mice deficient in ARNT1, HIF-1
, or HIF-2
die at
midgestational stage because of vascular defects primarily involving
the embryonic and extraembryonic vasculature (15, 19, 20, 35-38). In
contrast, mice deficient in ARNT2 or ARNT3 do not exhibit any vascular
abnormalities (39, 40). These results suggest that VEGF expression is
primarily regulated by HIF-1
, HIF-2
, and ARNT1 but not ARNT2 or
ARNT3 during embryonic development. However, there is very limited
information regarding the relationship between HIFs, ARNTs, and VEGF in
the adult normal uterus during early pregnancy, although the role of
HIFs in regulating VEGF and thus angiogenesis in tumor tissues has
clearly been documented (41, 42). In the present study, we examined the
temporal and cell-specific expression of HIFs and ARNTs in parallel
with the expression of VEGF in the uterus during the peri-implantation period and under steroid hormonal regulation. We observed that expression of HIFs and ARNTs does not spatiotemporally correlate with
the expression of VEGF in the uterus during the preimplantation period
(days 1-4 of pregnancy). In contrast, the expression of these
transcription factors follows the localization of VEGF expression in
the uterus with increasing angiogenesis during the postimplantation period (days 5-8). The disparate pattern of HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests that they have different roles in addition to the regulation of angiogenesis in the uterus during the peri-implantation period. We also observed that although HIF-1
is primarily regulated by P4 in the mouse uterus,
estrogen transiently regulates HIF-2
.
 |
MATERIALS AND METHODS |
Mice and Treatments--
Adult CD-1 mice were purchased from the
Charles Rivers Laboratory (Raleigh, NC). Females were mated with
fertile males of the same strain to induce pregnancy (day 1 = vaginal plug). Estrogen receptor-
(ER
)-deficient mice
(129/J/C57BL/6J) and progesterone receptor (PR)-deficient mice
(129SvEv/C57BL/6) were generated as previously described (43, 44) and
were kindly provided by Dennis Lubahn (University of Missouri,
Columbia, MO) and Bert O'Malley (Baylor College of Medicine, Houston,
TX), respectively, for establishing our colonies. PCR analysis of the
genomic DNA determined the genotypes. All of the mice were housed in
our Animal Care Facilities according to the National Institutes of
Health and institutional guidelines for laboratory animals.
To examine the effects of estrogen and/or P4 on uterine
gene expression, ovariectomized mice were injected with sesame oil (0.1 ml/mouse), estradiol-17
(E2) (100 ng/mouse),
P4 (2 mg/mouse), or E2 plus P4. At
termination of the treatments, uteri were processed for subsequent
analysis. The steroids were dissolved in sesame oil and injected subcutaneously.
Probes--
The cDNA clones for Vegf and
ribosomal protein L7 (rpL7) have previously been
described (1, 45). Peter Carmeliet (Flanders Inter-University
Institute, Leuveen, Belgium) kindly provided a cDNA clone for the
mouse HIF-1
. A 192-bp HIF-1
was subcloned into a pGEM3ZF(+) vector at the EcoRI site.
Mouse-specific HIF-3
and ARNT1 cDNAs were
gifts from Chris Bradfield (University of Wisconsin, Madison, WI).
Partial cDNAs for mouse HIF-2
, ARNT2, and
ARNT3 were generated by reverse transcription-PCR cloning with specific primers. For Northern hybridization, antisense
32P-labeled cRNA probes were generated using T7
polymerase. For in situ hybridization, sense and antisense
35S-labeled cRNA probes were generated using Sp6
and T7 polymerases, respectively. Probes had specific
activities of about 2 × 109 dpm/µg.
Northern Hybridization--
For Northern hybridization,
poly(A)+ RNA (2.0 µg) was denatured and separated by
formaldehyde/agarose gel electrophoresis, transferred to nylon
membranes, and UV cross-linked. Northern blots were prehybridized,
hybridized, and washed as previously described by us (1, 45).
Quantification of hybridized bands was analyzed by densitometric scanning.
In Situ Hybridization--
In situ hybridization was
performed as previously described by us (1, 45). In brief, frozen
sections (10 µm) were mounted onto poly-L-lysine-coated
slides and fixed in cold 4% paraformaldehyde in phosphate-buffered
saline. The sections were prehybridized and hybridized at 45 °C for
4 h in 50% formamide hybridization buffer containing the
35S-labeled antisense or sense cRNA probes. RNase
A-resistant hybrids were detected by autoradiography. The sections were
post-stained with eosin and hematoxylin.
Immunohistochemical Localization--
Frozen sections (10 µm
thick) were mounted onto poly-L-lysine-coated slides and
stored at
80 °C until used. The sections were fixed in 4%
formaldehyde in phosphate-buffered saline (pH 7.4) for 10 min at room
temperature followed by washing in Tris-buffered saline (pH 7.4) for 5 min twice. Immunolocalization of HIF-1
was performed as previously
described with some modifications (46, 47). In brief, the sections were
incubated with chicken polyclonal anti-HIF-1
antibodies (1:50)
overnight at 4 °C followed by washing in phosphate-buffered saline
(46, 47). A peroxidase-conjugated rabbit anti-chicken IgY antibody
(1:100; Pierce) was added onto the sections, and the sections were
incubated for 45 min at room temperature. Immunolocalization for ARNT1
was performed as previously described (48). In brief, the sections were
incubated with a rabbit polyclonal anti-ARNT1 antibody (1:250; Affinity
BioReagents, Neshanic Station, NJ) overnight at 4 °C. Immunostaining
was performed using a Histostain-SP kit (Zymed Laboratories
Inc.). After immunostaining, the sections were counterstained
with 0.5% Fast Green. The red color indicated the site of positive staining.
Cell Culture, Transfection, and Luciferase Assays--
AN3CA
uterine carcinoma cells were grown in Dulbecco's modified Eagle's
medium (Cellgro), whereas L929 cells were cultured in Joklik's
modified Eagle's medium supplemented with 10% fetal bovine serum
(Atlanta Biologicals), L-glutamine (2 mM),
penicillin (100 units/ml), and streptomycin (100 µg/ml) in a 5%
CO2 atmosphere. In addition, amphotericin B (250 ng/ml) was
added to the medium for culturing L929 cells. AN3CA cells (7.5 × 105) and L929 cells (3.5 × 105) were
co-transfected with the following vectors (all at 0.6 µg/ml): CMV hPR-A and/or CMV hPR-B or
pcDNA3 (control; Invitrogen), and different combinations
of the luciferase constructs, pHXN1a-Luc (HIF-1
, exon 1.2 upstream region, 1.5 kb) (41),
pH1030-Luc (HIF-1
, exon I.1 upstream region,
1.0 kb) (49), PRE/GRE-elb-Luc (50), or pGL3-basic
(Promega) using LipofectAMINE at a DNA:lipid ratio of 1:3.5 in Opti-MEM
(Invitrogen) for 4 h (41, 51). The CMV hPR-A and
CMV hPR-B expression vectors were kindly provided by Dean
Edwards (University of Colorado, Boulder, CO), whereas pH1030-Luc and PRE-Luc constructs were generously
provided by Roland Wenger (Carl-Ludwig Institute of Physiology,
University of Leipzig, Leipzig) and Nancy Weigel (Baylor College of
Medicine, Houston, TX), respectively.
All transfection was normalized to a total of 2.0 µg/ml with
pcDNA3. The transfection mixture was replaced with complete
medium containing the vehicle (1% ethanol) or P4 (1 µM). After 48 h, the cells were harvested in 1×
luciferase lysis buffer. Relative light units from firefly luciferase
activity were determined using a luminometer (Mono Light 2010) and
normalized to the relative light units from Renilla
luciferase using a dual luciferase kit (Promega).
 |
RESULTS |
Vegf, HIFs, and ARNTs Are Differentially Expressed in the
Peri-implantation Uterus--
The objective of these experiments was
to examine whether Vegf expression is co-localized with that
of HIFs and ARNTs during the pre- and post-implantation periods. As
previously shown by us (1), Vegf mRNA expression was
restricted to the luminal epithelium on day 1 of pregnancy when the
uterus is under the influence of preovulatory estrogen, whereas on day
4 of pregnancy, this expression became primarily localized in the
stroma under the influence of rising P4 levels fortified
with a small amount of estrogen (Fig.
1A). However, the mechanism by
which steroid hormones influence Vegf expression is not
fully understood. Because HIFs are known to regulate Vegf
expression associated with angiogenesis, we examined the expression of
HIFs and their partners ARNTs in the uterus to
determine whether HIFs play any role in uterine Vegf
expression. We observed that the expression of HIFs
(HIF-1
, -2
, and -3
) was very
low to undetectable in the uterus on day 1 of pregnancy. However,
HIF-1
was distinctly expressed in the luminal epithelium
on day 4 of pregnancy as opposed to the expression of Vegf
in the stroma. Interestingly, distinct but patchy expression of
HIF-2
was noted in the stroma, whereas the expression of
HIF-3
was undetectable (Fig. 1A).

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Fig. 1.
Expression of Vegf,
HIFs
(1 -3 ), and
ARNTs (1-3) in the preimplantation mouse uterus.
A and B, in situ hybridization.
Representative dark field photomicrographs of longitudinal uterine
sections on days 1 and 4 showing Vegf, HIFs, and
ARNT expression at 100×. C,
immunohistochemistry. Longitudinal uterine sections on day 4 morning
and afternoon and sections of brain (control) were used for HIF-1 or
ARNT1 immunostaining (100×). le, luminal epithelium;
ge, glandular epithelium; s, stroma;
myo, myometrium.
|
|
Because HIFs must heterodimerize with ARNTs for transcriptional
activation of Vegf (17), we next examined the expression of
ARNTs in the uterus on similar days of pregnancy. To our
surprise, we observed that all three ARNTs
(ARNT1, ARNT2, and ARNT3) were expressed at very low to undetectable levels on these days of pregnancy, except ARNT1, which was expressed at a low to
modest level both in the luminal epithelium and stroma on day 4 of
pregnancy (Fig. 1B). On the other hand, the stromal
expression of HIF-2
that correlates with ARNT1
on day 4 of pregnancy suggests that HIF-2
regulates Vegf
transcription after heterodimerization with ARNT1. We next asked
whether the localization of these proteins follows the same pattern as
their respective mRNAs. Surprisingly, immunoreactive HIF-1
and
ARNT1 were primarily localized to the uterine epithelium on day 4 of
pregnancy, suggesting that HIF-1
effects are probably restricted to
the epithelium at this time (Fig. 1C). The unavailability of
suitable antibodies to other HIFs and ARNTs has precluded us from
determining the localization of these proteins in the uterus.
Nonetheless, the mRNA localization of HIF-2
in the
stroma in the presence of little or no expression of ARNT2
and ARNT3 and a very low level of ARNT1
expression with restricted localization of its protein in the
epithelium raises questions regarding a role for HIF-2
in stromal
Vegf expression on day 4 of pregnancy. It is possible that a
yet unidentified ARNT isoform is expressed in the stroma at this time.
Nonetheless, the localization of both the mRNA and protein for
HIF-1
and ARNT1 in the epithelium on day 4 of pregnancy suggests
that HIF-1
has a different role in the uterus, because
Vegf is expressed in the stroma but not in the epithelium at
this time.
There are increases in Vegf expression and angiogenesis in
the uterus at the site of the blastocyst as implantation progresses. Therefore, we compared the expression of Vegf with those of
HIFs and ARNTs during the postimplantation
period, particularly on days 5 and 8 of pregnancy. These 2 days were
chosen because day 5 represents a very early stage of implantation that
correlates with initiation of the decidualization process, whereas day
8 represents a late phase of the implantation process when decidual growth is maximal. As previously observed (1), Vegf
expression is more localized to the luminal epithelium and stroma
surrounding the implanting blastocyst on day 5 of pregnancy. The
expression further increases in the stromal decidua on day 8 (Fig.
2A). With respect to HIFs,
both the luminal epithelium and stroma exhibited HIF-1
expression similar to that of Vegf, whereas
HIF-2
expression was restricted to only stromal cells
surrounding the blastocyst on day 5. In contrast, the expression of
HIF-3
was very low without any cell-specific
localization. On day 8, HIF-1
expression showed further
increases in the decidual bed, but the most robust expression was noted
for HIF-2
. The expression of HIF-3
was
again very low and diffuse. All three HIFs showed expression
in the developing embryo. The cell-specific accumulation of
HIF-1
and HIF-2
mRNAs closely
correlated with the levels determined by Northern hybridization of
whole uterine RNA samples (Fig. 3).

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Fig. 2.
In situ hybridization of
Vegf, HIFs
(1 -3 ), and
ARNTs (1-3) in the postimplantation mouse
uterus. A and B, dark field photomicrographs
of representative cross-sections of implantation sites on days 5 and 8 of pregnancy are shown at 100×. m, mesometrial site;
am, antimesometrial site. The arrows indicate the
locations of embryos.
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Fig. 3.
Northern blot detection of
HIF-1 and HIF-2 mRNAs in
the mouse uterus during early pregnancy. Poly(A)+ RNA
samples (2 µg) isolated of uteri on the indicated days of pregnancy
were separated by formaldehyde-agarose gel electrophoresis, transferred
to nylon membranes, UV-cross-linked, and hybridized to specific
32P-cRNA probes. The same blots were stripped and
rehybridized to an rPL7 (a housekeeping gene) probe to
confirm the integrity of RNA samples.
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|
When expression patterns of HIFs were compared with those of
ARNTs, we observed that ARNT1 and
ARNT3 showed similar expression patterns in the stroma as
that of HIF-1
and HIF-2
on day 5, but the
expression of ARNT2 was primarily restricted to the luminal epithelium (Fig. 2B). These results suggest that HIF-1
and HIF-2
can partner with ARNT1 or ARNT3 in the stroma, but only
HIF-1
can partner with ARNT2 in the epithelium on day 5. On day 8 of pregnancy, the localization of ARNTs was similar to that of
HIF-1
and HIF-2
, but the expression
intensity was low to modest in the decidual bed. The expression of
ARNTs in the developing embryo was similar to that of
HIFs. No specific localization of HIF and ARNT mRNAs was detected after hybridization of uterine
sections with sense probe (Fig. 4).
Collectively, these results suggest that ARNT1 and ARNT3 are perhaps
the major partners of HIF-1
and HIF-2
in the uterine stroma that
is operative for the Vegf expression during the
postimplantation period.

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Fig. 4.
Absence of localization (negative controls)
of HIFs
(1 -3 ) and
ARNTs (1-3) mRNAs in uterine sections hybridized
with the sense probes. Dark field photomicrographs of uterine
sections on representative days of pregnancy hybridized with the sense
probes show the absence of specific hybridization signals as compared
with similar sections hybridized with the antisense probes that
exhibited distinct and specific signals.
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|
Uterine Expression of HIF-1
and HIF-2
Is Regulated by
Progesterone and Estrogen--
Our observations of uterine expression
of HIF-1
, HIF-2
, and Vegf on day
1 and day 4 of pregnancy suggested that these genes are regulated by
ovarian estrogen and P4. Therefore, we further examined the
expression of these genes in a more defined system, i.e. in
ovariectomized mice after steroid hormone treatment. Northern blot
analysis showed that the levels of HIF-1
mRNA
increased by about 3.5 times within 6 h of an E2
injection and that the levels peaked at 12 h followed by a
decrease by 24 h. In contrast, the levels of HIF-1
mRNA showed gradual increase from 2 h after an injection of
P4 showing a peak at 24 h (Fig.
5). When P4 treatment was
combined with E2, the response was advanced exhibiting peak levels at 6 h followed by a decline at 12 h. With respect to
HIF-2
, we observed that the expression of this gene is
primarily regulated by E2 in a transient manner reaching
maximal levels at 4 h. In contrast, P4 was not very
effective in influencing this gene. A combined treatment with
E2 and P4 showed an expression pattern similar
to that of E2 alone but at lower levels (Fig. 5). When the
levels of Vegf mRNA was compared with those of
HIF-1
and HIF-2
, we observed that the
accumulation of Vegf mRNA was very rapid peaking at
2 h of an E2 injection followed by a sharp decline thereafter. In contrast, the levels of this mRNA showed a gradual increase from 1 h after an injection of P4 showing a
peak at 24 h. A co-injection of E2 with P4
increased the levels of Vegf mRNA by 2 h similar to
that observed for E2 alone (Fig. 5). These results again
suggest that Vegf, HIF-1
, and
HIF-2
are not always coordinately expressed.

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Fig. 5.
Northern blot detection of Vegf,
HIF-2 , and HIF-1 mRNAs in the
ovariectomized mouse uterus after steroid treatments.
Poly(A)+ RNA samples (2 µg) isolated of uteri after
steroid hormone treatments at indicated times were separated by
formaldehyde-agarose gel electrophoresis, transferred to nylon
membranes, UV-cross-linked, and hybridized to specific
32P-cRNA probes. The same blots were stripped and
rehybridized to an rPL7 (a housekeeping gene) probe to
confirm integrity of RNA samples.
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|
However, Northern analysis gives no indication of the uterine cell
types involved, and the levels of whole uterine mRNAs by Northern
hybridization may have limited value because of the dilution effects
resulting from heterogeneous uterine cell types that undergo dynamic
changes during pregnancy and under steroid hormonal stimulation. For
example, the luminal epithelium represents only 5-10%, the stroma
30-35%, and the myometrium 60% of the major uterine cell types.
Thus, even a 50% or greater increase in the expression level in the
epithelium may not be reflected when the expression level is measured
in whole uterine extracts (52). Therefore, in situ
hybridization was performed to examine the cell-specific expression of
Vegf, HIF-1
, and HIF-2
in uteri
of ovariectomized wild-type mice treated with E2 or
P4 at various time points (Fig. 6). We observed that Vegf
expression was low in ovariectomized uteri treated with oil (control)
and that expression was localized to stromal cells both at 6 and
24 h. However, the expression showed a prominent increase in
stromal cells at 6 h of an E2 injection. By 24 h
the expression became localized to epithelial cells. In contrast, the
expression of Vegf was always localized to stromal cells
both at 6 and 24 h after P4 treatment (Fig.
6A). When these patterns of Vegf expression were
compared with those of HIF-1
, we observed that
HIF-1
expression did not follow the expression pattern of
Vegf (Fig. 6, A and B). The expression
of HIF-1
is always restricted to the uterine epithelium
(Fig. 6B). For example, the expression was relatively low in
ovariectomized uterine epithelium after an oil injection either at 6 or
24 h. The epithelial expression showed an increase at 6 h of
an E2 injection but dramatically declined by 24 h. In
contrast, a P4 injection increased the epithelial expression of HIF-1
at 6 h and more prominently at
24 h. These results suggest that E2 modestly and
transiently influences HIF-1
expression in the epithelium
as opposed to P4, which induces this gene in a more robust
and sustained manner, suggesting a primary role of P4 in
regulating HIF-1
expression in the mouse uterus. On the
other hand, HIF-2
expression is primarily restricted to the stroma and is clearly up-regulated by E2 in
a transient manner and follows the pattern of Vegf
expression at this time point (Fig. 6C). To our knowledge,
this is the first demonstration of the regulation of HIFs by ovarian
steroids in a target tissue.

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Fig. 6.
In situ hybridization of
Vegf, HIF-1 , and
HIF-2 mRNAs in ovariectomized mouse uteri after
steroid hormone treatments. Representative dark field
photomicrographs of longitudinal uterine sections are shown at 100×.
Ovariectomized mice were treated with oil (vehicle control),
E2, or P4 and sacrificed at the indicated
times. A, Vegf. B,
HIF-1 . C, HIF-2 .
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Our next goal was to examine whether P4 and estrogen
regulation of HIF-1
is mediated via their cognate nuclear
receptors. We employed mice lacking the nuclear PR or the nuclear ER
to further define the mechanism of steroidal regulation of
HIF-1
. The induction of HIF-1
that we
observed in P4-treated wild-type mice was virtually
abolished in PR(
/
) mice (Fig.
7). For example, the expression of
HIF-1
was very low in intact PR(
/
) mice or in ovariectomized PR(
/
) mice treated with either
E2 or P4. In contrast, P4 showed an
increased expression of HIF-1
in ER
(
/
) mice. An injection of E2 also increased the accumulation of
HIF-1
in ER
(
/
) ovariectomized mice.
These results clearly suggest that this gene is primarily under the
influence of P4 in the mouse uterus and requires the
activation of PR. However, an effect of estrogen in uterine induction
of HIF-1
could be mediated independently of ER
.

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Fig. 7.
In situ hybridization of
HIF-1 in PR( / ) and
ER ( / ) mice uteri after steroid hormone
treatments. Ovariectomized (OVX) mice were treated with
oil, E2, or P4 and killed 24 h later along
with intact mice without any treatment. Representative dark field
photomicrographs of longitudinal uterine sections are shown at
100×.
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|
Finally, we asked whether this P4 regulation of HIF-1
was directly transcriptionally regulated, because, although two
half-site progesterone/glucocorticoid-responsive element (PRE/GRE) and
eight half-site PRE/GRE-like elements were located in the promoter
region of the exon I.2, six half-site PRE/GRE-like elements were
located in the exon I.1 promoter region of the mouse
HIF-1
gene (Transcriptional element search:
www.cbil.upenn.edu/tess). Expression of these two mouse
HIF-1
mRNA transcripts is regulated by distinct
promoters rather than by differential splicing. This results in two
distinct mRNA isoforms differing in the composition of their
5'-untranslated regions. Furthermore, there is evidence that
HIF-1
exon I.1 exhibits tissue-specific features with
modest activity, whereas the exon I.2 promoter resembles a housekeeping
type promoter with higher activity (41, 49).
Using a uterine cell line (AN3CA) and a fibroblast cell line (L929), we
observed that although P4 up-regulated PRE-luciferase activity in these cell lines expressing PRA or PRB, similar treatment with P4 did not show any heightened HIF-1
-Luc activity
in these cell lines after co-transfection with PRA or PRB (Fig.
8). HIF-1
-Luc constructs
were functional, because basal levels in L929 cells were markedly
higher than those observed with the pGL3 basic (control) construct (data not shown). This latter observation is similar to one
that has been previously reported (41). The results suggest that
steroidal regulation of HIF-1
is more complex compared with other
PR-regulated genes.

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Fig. 8.
Effect of progesterone on
HIF-1 transcriptional activity in AN3CA and
L929 cells expressing PRs. AN3CA and L929 cells were
co-transfected with CMV hPR-A, CMV hPR-B, or
pcDNA3 (control) expression vectors and different
combinations of pHXN1 -Luc, pH1030-Luc,
PRE/GRE-elb-Luc, and pGL3-basic constructs using
LipofectAMINE for 4 h. The transfected cells were treated with the
vehicle (1% ethanol) or P4 (1 µM). After
48 h, the cells were harvested, and dual luciferase assays were
performed as described under "Materials and Methods." The data are
presented as fold activation relative to vehicle-treated cells and
represent the means from three independent transfections (means ± S.E.).
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|
 |
DISCUSSION |
A number of target genes involved in angiogenesis, erythropoiesis,
and glycolysis are activated by HIFs, particularly HIF-1
(15). The
most well known and potent stimulus for the induction of HIFs is
hypoxia (15, 18, 41, 49). This would then suggest that dissimilar
partial oxygen pressures in different uterine tissue compartments are
likely to exist (53), thus regulating HIF expression
differentially. Because the uterine epithelium is devoid of any blood
vessels and these cells are polarized and separated from the stroma by
a basement membrane, it seems reasonable to assume that the epithelium
is more hypoxic than the stroma and myometrium. Thus, our observation
of HIF-1
expression in the epithelium as opposed to the
induction of HIF-2
in the stroma on day 4 of pregnancy is
interesting and suggests that differential regulation and functions of
HIFs in the uterus. Because one of the major roles of HIFs is to
maintain oxygen homeostasis, we could surmise that HIF-1
in the
epithelium is meant to fulfill this function. In contrast, the
expression of HIF-2
in the stroma could be involved in the induction
of Vegf required for angiogenesis in this compartment.
However, it is also possible that these genes are regulated in the
uterus under the influence of ovarian steroids, because the uterus is
the major target for P4 and estrogen. The very low level of
expression of HIFs in the estrogenized uterus on day 1 of
pregnancy suggests that estrogen has a limited role in regulating HIF levels. Alternatively, estrogen may have a transient
role in influencing HIF expression, which was not detected
on the morning of day 1 of pregnancy several hours after the
preovulatory estrogen surge. In contrast, the expression of
HIF-1
in the epithelium and of HIF-2
in the
stroma on day 4 of pregnancy with rising P4 levels
superimposed by a small amount of estrogen is suggestive of
differential regulation of these two genes in two different tissue
compartments. It is known that under this condition on day 4 of
pregnancy, epithelial cells undergo differentiation, and stromal cells
exhibit heightened proliferation in the mouse uterus (54). This is not
surprising, because nuclear receptors for progesterone (PR) and
estrogen (ER) are expressed in the epithelium and stroma at this time
(55). Whether this effect of P4 and estrogen on
HIF induction is direct or indirect is not clearly understood. It is possible that uterine compartments become more hypoxic under P4 influence than under estrogen when the
uterus is more perfused. However, the ovariectomized uterus in the
absence of steroid hormones is likely to be more hypoxic. Thus, a low level of induction of HIF-1
in the uterus under such a
condition suggests that partial oxygen pressure is not the major
inducer of HIFs in the uterus. In contrast, the low to undetectable
expression of HIFs in the estrogenized and well perfused day
1 pregnant uterus suggests that a less hypoxic condition could be
involved in regulating HIF expression. Alternatively,
HIF expression may not be very responsive to estrogen.
However, a modest increase in HIF-1
expression and a more
robust expression of HIF-2
in the ovariectomized uterus 6 h after an injection of estrogen suggests that this steroid preferentially influences the regulation of HIF-2
in the
uterus. Because estrogen induces vascular permeability but inhibits
angiogenesis in the mouse uterus, the coordinate expression of
HIF-2
and Vegf in the stroma at 6 h after
an estrogen injection suggests that HIF-2
could influence VEGF
expression presumed to participate in vascular permeability (12).
A robust induction of HIF-1
in the wild-type uterus by
P4 but its failure to induce such an induction in
PR(
/
) uteri clearly suggests that the P4
regulation of this gene is mediated by PR. This is consistent with the
induction of HIF-1
by P4 in uteri of
ER
(
/
) mice with intact PR. However, the mechanism of
P4 induction of HIF-1
in the uterus is not
clearly understood. Our failure to observe P4 activation of
HIF-1
-Luc activity in cell lines expressing PRA or PRB, despite the
presence of PRE-like elements in the HIF-1
promoter,
suggests that the regulation of this gene in the uterus is more complex
and may require a set of activators that were not available in our
in vitro systems. It could be argued that the expression of
both PRA and PRB is required for P4 to induce HIF-1
expression. However, cells expressing both PRA and PRB also failed to
respond to P4 in inducing HIF-1
(data not shown). A
transient increase in HIF-1
expression in ovariectomized
wild-type mice and a modest increase in ER
(
/
) uteri
by estrogen indicate that estrogen may influence this gene independent
of ER
. Whether ER
has any role in this induction is not known,
although the levels of ER
are very low in the
ER
(
/
) uterus (48). In this respect, there is evidence
that estrogen can influence the expression of several genes in the
uterus independent of both ER
and ER
(56, 57). Future studies
will determine whether transient uterine HIF-2
expression
by estrogen is mediated via classical ERs or whether this effect is
independent of such receptors.
Although HIF-1
in the uterine epithelium during the preimplantation
period and in ovariectomized moue uterus is responsive to
P4 regulation, the function of HIF-1
in the epithelium
is far from being elucidated. The presence of ARNT1 protein in a location similar to that of HIF-1
suggests that heterodimerization between these two partners is possible to influence specific functions in the epithelium. Because angiogenesis is absent in the uterine epithelium, we speculate that HIF-1
has different functions in this
tissue compartment. Glucose transporter-1
(GLUT-1) is also an HIF-1
-responsive gene (15, 58-60)
and is expressed in the uterine epithelium on day 4 of pregnancy under
the influence of P4. Thus, it is possible that HIF-1
in
the uterine epithelium influences glucose transport across the
epithelium. However, other functions of HIF-1
in the uterine
epithelium cannot be ruled out. For example, HIF-1
has been shown to
play important roles in developing embryos (53, 61). Thus, these
results indicate that HIF-1
is a P4-regulated
uterine epithelial responsive gene with a function not associated with
Vegf expression. On the other hand, the presence of
HIF-2
in the stroma together with ARNT1 on day
4 of pregnancy under P4 dominance could be associated with Vegf induction for vascular permeability and subsequent
angiogenesis in this tissue compartment. This is a very interesting
observation because P4 and E2 show differential
regulation of HIF-1
and HIF-2
in the uterus
depending on the cell types. Whether this differential regulation is
mediated by epithelial-mesenchymal cross-talk is not known. However,
there are numerous examples of epithelial-mesenchymal interactions in
inducing gene expression and mediating important uterine functions with
respect to P4 and estrogen effects (reviewed in Ref.
62).
Heightened angiogenesis with increasing levels of Vegf in
the decidualizing stroma during the postimplantation period has been
associated with COX-2 derived prostaglandins. However, it is not yet
known whether HIFs have any role in this event during this time. There
is evidence that hypoxia can induce COX-2 (63). However, whether HIFs
are capable of inducing COX-2 in the uterus is not known. Nonetheless,
expression of Cox-2 in the decidual sites, similar to that
of HIF-1
and HIF-2
as well as their
partners ARNT1 and ARNT3, suggests a correlation
between COX-2 and HIFs with respect to Vegf induction (64).
It is also interesting to note the switching of HIF-1
expression from the epithelium to the stroma during the
postimplantation period when the uterus is still under the predominant
influence of P4. The developing embryo could influence
HIF-1
expression in the decidua. However, during
decidualization the heightened expression of HIF-2
is indicative of a preferential role for this HIF isoform instead of
HIF-1
. The question still remains of how HIF-1
becomes more dominant in the epithelium during the preimplantation period, whereas
HIF-2
is more prominent in the stroma during the postimplantation period, although elevated P4 levels are characteristic of
both the phases. In conclusion, the results of the present
investigation show that HIFs are differentially expressed in the uterus
depending on the stage of implantation and cell types involved,
implicating differential roles of HIFs in the epithelial and stromal
compartments of the uterus.