Expression of Hypoxia-inducible Factors in the Peri-implantation Mouse Uterus Is Regulated in a Cell-specific and Ovarian Steroid Hormone-dependent Manner

EVIDENCE FOR DIFFERENTIAL FUNCTION OF HIFs DURING EARLY PREGNANCY*

Takiko DaikokuDagger §, Hiromichi Matsumoto, Rajnish A. Gupta||, Sanjoy K. DasDagger §, Max Gassmann**, Raymond N. DuBois||, and Sudhansu K. DeyDagger §DaggerDagger

From the Departments of Dagger  Pediatrics, § Cell and Developmental Biology, and || Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2678, the  Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan, and the ** Institute of Veterinary Physiology, University of Zürich, Winterthurerstrasse 260, Zürich CH-8057, Switzerland

Received for publication, November 7, 2002, and in revised form, December 6, 2002

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIFalpha isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-beta ) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1alpha expression, estrogen transiently stimulates that of HIF-2alpha .

    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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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 alpha - and beta -subunits. These subunits belong to the basic helix-loop-helix-PAS protein superfamily (15, 18). HIF-1alpha was first cloned in humans followed by its cloning in mice and rats (23-26). Subsequently, HIF-2alpha and HIF-3alpha , which have a high sequence homology to HIF-1alpha , were cloned in mice, rats, and humans (26-28). HIF-2alpha is also known as endothelial PAS domain protein-1, HIF-1alpha -like factor, HIF-related factor, or MOP2 (member of the PAS superfamily 2) (15). HIF-beta 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-alpha subunits can heterodimerize with the ARNT family members without specificity for their dimerization partners (15).

HIF-1alpha is expressed in most human and rodent tissues (25, 29). Levels of the HIF-1alpha 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-1alpha is ubiquitous, whereas that of HIF-2alpha and HIF-3alpha shows a more restricted expression pattern. There is evidence that HIF-2alpha 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-1alpha , HIF-2alpha , and HIF-3alpha (15). Mice deficient in ARNT1, HIF-1alpha , or HIF-2alpha 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-1alpha , HIF-2alpha , 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-1alpha is primarily regulated by P4 in the mouse uterus, estrogen transiently regulates HIF-2alpha .

    MATERIALS AND METHODS
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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-alpha (ERalpha )-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-17beta (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-1alpha . A 192-bp HIF-1alpha was subcloned into a pGEM3ZF(+) vector at the EcoRI site. Mouse-specific HIF-3alpha and ARNT1 cDNAs were gifts from Chris Bradfield (University of Wisconsin, Madison, WI). Partial cDNAs for mouse HIF-2alpha , 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-1alpha was performed as previously described with some modifications (46, 47). In brief, the sections were incubated with chicken polyclonal anti-HIF-1alpha 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-1alpha , exon 1.2 upstream region, 1.5 kb) (41), pH1030-Luc (HIF-1alpha , 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).

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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-1alpha , -2alpha , and -3alpha ) was very low to undetectable in the uterus on day 1 of pregnancy. However, HIF-1alpha 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-2alpha was noted in the stroma, whereas the expression of HIF-3alpha was undetectable (Fig. 1A).


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Fig. 1.   Expression of Vegf, HIFs (1alpha -3alpha ), 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-1alpha 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-2alpha that correlates with ARNT1 on day 4 of pregnancy suggests that HIF-2alpha 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-1alpha and ARNT1 were primarily localized to the uterine epithelium on day 4 of pregnancy, suggesting that HIF-1alpha 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-2alpha 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-2alpha 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-1alpha and ARNT1 in the epithelium on day 4 of pregnancy suggests that HIF-1alpha 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-1alpha expression similar to that of Vegf, whereas HIF-2alpha expression was restricted to only stromal cells surrounding the blastocyst on day 5. In contrast, the expression of HIF-3alpha was very low without any cell-specific localization. On day 8, HIF-1alpha expression showed further increases in the decidual bed, but the most robust expression was noted for HIF-2alpha . The expression of HIF-3alpha was again very low and diffuse. All three HIFs showed expression in the developing embryo. The cell-specific accumulation of HIF-1alpha and HIF-2alpha 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 (1alpha -3alpha ), 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-1alpha and HIF-2alpha 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.

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-1alpha and HIF-2alpha on day 5, but the expression of ARNT2 was primarily restricted to the luminal epithelium (Fig. 2B). These results suggest that HIF-1alpha and HIF-2alpha can partner with ARNT1 or ARNT3 in the stroma, but only HIF-1alpha 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-1alpha and HIF-2alpha , 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-1alpha and HIF-2alpha 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 (1alpha -3alpha ) 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.

Uterine Expression of HIF-1alpha and HIF-2alpha Is Regulated by Progesterone and Estrogen-- Our observations of uterine expression of HIF-1alpha , HIF-2alpha , 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-1alpha 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-1alpha 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-2alpha , 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-1alpha and HIF-2alpha , 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-1alpha , and HIF-2alpha are not always coordinately expressed.


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Fig. 5.   Northern blot detection of Vegf, HIF-2alpha , and HIF-1alpha 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.

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-1alpha , and HIF-2alpha 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-1alpha , we observed that HIF-1alpha expression did not follow the expression pattern of Vegf (Fig. 6, A and B). The expression of HIF-1alpha 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-1alpha at 6 h and more prominently at 24 h. These results suggest that E2 modestly and transiently influences HIF-1alpha 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-1alpha expression in the mouse uterus. On the other hand, HIF-2alpha 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-1alpha , and HIF-2alpha 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-1alpha . C, HIF-2alpha .

Our next goal was to examine whether P4 and estrogen regulation of HIF-1alpha is mediated via their cognate nuclear receptors. We employed mice lacking the nuclear PR or the nuclear ERalpha to further define the mechanism of steroidal regulation of HIF-1alpha . The induction of HIF-1alpha that we observed in P4-treated wild-type mice was virtually abolished in PR(-/-) mice (Fig. 7). For example, the expression of HIF-1alpha 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-1alpha in ERalpha (-/-) mice. An injection of E2 also increased the accumulation of HIF-1alpha in ERalpha (-/-) 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-1alpha could be mediated independently of ERalpha .


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Fig. 7.   In situ hybridization of HIF-1alpha in PR(-/-) and ERalpha (-/-) 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×.

Finally, we asked whether this P4 regulation of HIF-1alpha 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-1alpha gene (Transcriptional element search: www.cbil.upenn.edu/tess). Expression of these two mouse HIF-1alpha 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-1alpha 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-1alpha -Luc activity in these cell lines after co-transfection with PRA or PRB (Fig. 8). HIF-1alpha -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-1alpha is more complex compared with other PR-regulated genes.


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Fig. 8.   Effect of progesterone on HIF-1alpha 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 pHXN1alpha -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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ABSTRACT
INTRODUCTION
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REFERENCES

A number of target genes involved in angiogenesis, erythropoiesis, and glycolysis are activated by HIFs, particularly HIF-1alpha (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-1alpha expression in the epithelium as opposed to the induction of HIF-2alpha 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-1alpha in the epithelium is meant to fulfill this function. In contrast, the expression of HIF-2alpha 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-1alpha in the epithelium and of HIF-2alpha 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-1alpha 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-1alpha expression and a more robust expression of HIF-2alpha in the ovariectomized uterus 6 h after an injection of estrogen suggests that this steroid preferentially influences the regulation of HIF-2alpha in the uterus. Because estrogen induces vascular permeability but inhibits angiogenesis in the mouse uterus, the coordinate expression of HIF-2alpha and Vegf in the stroma at 6 h after an estrogen injection suggests that HIF-2alpha could influence VEGF expression presumed to participate in vascular permeability (12).

A robust induction of HIF-1alpha 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-1alpha by P4 in uteri of ERalpha (-/-) mice with intact PR. However, the mechanism of P4 induction of HIF-1alpha in the uterus is not clearly understood. Our failure to observe P4 activation of HIF-1alpha -Luc activity in cell lines expressing PRA or PRB, despite the presence of PRE-like elements in the HIF-1alpha 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-1alpha expression. However, cells expressing both PRA and PRB also failed to respond to P4 in inducing HIF-1alpha (data not shown). A transient increase in HIF-1alpha expression in ovariectomized wild-type mice and a modest increase in ERalpha (-/-) uteri by estrogen indicate that estrogen may influence this gene independent of ERalpha . Whether ERbeta has any role in this induction is not known, although the levels of ERbeta are very low in the ERalpha (-/-) uterus (48). In this respect, there is evidence that estrogen can influence the expression of several genes in the uterus independent of both ERalpha and ERbeta (56, 57). Future studies will determine whether transient uterine HIF-2alpha expression by estrogen is mediated via classical ERs or whether this effect is independent of such receptors.

Although HIF-1alpha in the uterine epithelium during the preimplantation period and in ovariectomized moue uterus is responsive to P4 regulation, the function of HIF-1alpha in the epithelium is far from being elucidated. The presence of ARNT1 protein in a location similar to that of HIF-1alpha 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-1alpha has different functions in this tissue compartment. Glucose transporter-1 (GLUT-1) is also an HIF-1alpha -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-1alpha in the uterine epithelium influences glucose transport across the epithelium. However, other functions of HIF-1alpha in the uterine epithelium cannot be ruled out. For example, HIF-1alpha has been shown to play important roles in developing embryos (53, 61). Thus, these results indicate that HIF-1alpha is a P4-regulated uterine epithelial responsive gene with a function not associated with Vegf expression. On the other hand, the presence of HIF-2alpha 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-1alpha and HIF-2alpha 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-1alpha and HIF-2alpha 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-1alpha 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-1alpha expression in the decidua. However, during decidualization the heightened expression of HIF-2alpha is indicative of a preferential role for this HIF isoform instead of HIF-1alpha . The question still remains of how HIF-1alpha becomes more dominant in the epithelium during the preimplantation period, whereas HIF-2alpha 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.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HD37830 (to S. K. Das) and HD12304 and HD33994 (to S. K. Dey).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger Recipient of a Merit Award from the NICHD, National Institutes of Health. To whom correspondence should be addressed. E-mail: sk.dey@vanderbilt.edu.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211390200

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; P4, progesterone; COX-2, cyclooxygenase-2; HIF, hypoxia-inducible factor; ARNT, aryl hydrocarbon nuclear translocator; ER, estrogen receptor; PR, progesterone receptor; E2, estradiol-17beta ; PRE/GRE, progesterone/glucocorticoide-responsive element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Halder, J. B., Zhao, X., Soker, S., Paria, B. C., Klagsbrun, M., Das, S. K., and Dey, S. K. (2000) Genesis 26, 213-224[CrossRef][Medline] [Order article via Infotrieve]
2. Folkman, J. (1995) Nat. Med. 1, 27-31[Medline] [Order article via Infotrieve]
3. Dvorak, H. F., Nagy, J. A., Feng, D., Brown, L. F., and Dvorak, A. M. (1999) in Current Topics in Microbiology and Immunology: Vascular Growth Factors and Angiogenesis (Claesson-Welsh, L., ed), Vol. 237 , pp. 97-132, Springer-Verlag, Berlin
4. Ferrara, N. (1996) Eur. J. Cancer 32A, 2413-2422[CrossRef]
5. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P., Risau, W., and Ullrich, A. (1993) Cell 72, 835-846[Medline] [Order article via Infotrieve]
6. Peters, K. G., De, Vries, C., and Williams, L. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8915-8919[Abstract]
7. Quinn, T. P., Peters, K. G., De, Vries, C., Ferrara, N., and Williams, L. T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7533-7537[Abstract/Free Full Text]
8. Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H., and Sato, M. (1990) Oncogene 5, 519-524[Medline] [Order article via Infotrieve]
9. Kitsukawa, T., Shimizu, M., Sanbo, M., Hirata, T., Taniguchi, M., Bekku, Y., Yagi, T., and Fujisawa, H. (1997) Neuron 19, 995-1005[Medline] [Order article via Infotrieve]
10. Soker, S., Gollamudi-Payne, S., Fidder, H., Charmahelli, H., and Klagsbrun, M. (1997) J. Biol. Chem. 272, 31582-31588[Abstract/Free Full Text]
11. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., and Klagsbrun, M. (1998) Cell 92, 735-745[Medline] [Order article via Infotrieve]
12. Ma, W., Tan, J., Matsumoto, H., Robert, B., Abrahamson, D. R., Das, S. K., and Dey, S. K. (2001) Mol. Endocrinol. 15, 1983-1992[Abstract/Free Full Text]
13. Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S., Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J., Aldrich, T. H., Papadopoulos, N., Daly, T. J., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1997) Science 277, 55-60[Abstract/Free Full Text]
14. Matsumoto, H., Ma, W. G., Daikoku, T., Zhao, X., Paria, B. C., Das, S. K., Trzaskos, J. M., and Dey, S. K. (2002) J. Biol. Chem. 277, 29260-29267[Abstract/Free Full Text]
15. Semenza, G. L. (2000) J. Appl. Physiol. 88, 1474-1480[Abstract/Free Full Text]
16. Semenza, G. L. (2000) Genes Dev. 14, 1983-1991[Free Full Text]
17. Hofer, T., Wenger, R. H., and Gassmann, M. (2002) Eur. J. Physiol. 443, 503-507[CrossRef][Medline] [Order article via Infotrieve]
18. Wenger, R. H. (2002) FASEB J. 16, 1151-1162[Abstract/Free Full Text]
19. Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger, R. H., Gassmann, M., Gearhart, J. D., Lawler, A. M., Yu, A. Y., and Semenza, G. L. (1998) Genes Dev. 12, 149-162[Abstract/Free Full Text]
20. Ryan, H. E., Lo, J., and Johnson, R. S. (1998) EMBO J. 17, 3005-3015[Abstract/Free Full Text]
21. Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., Keshert, E., and Keshet, E. (1998) Nature 394, 485-490[CrossRef][Medline] [Order article via Infotrieve]
22. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. (1996) Mol. Cell. Biol. 16, 4604-4613[Abstract]
23. Kietzmann, T., Knabe, W., and Schmidt-Kastner, R. (2001) Eur. Arch. Psychiatry Clin. Neurosci. 251, 170-178[CrossRef][Medline] [Order article via Infotrieve]
24. Wang, G. L., and Semenza, G. L. (1995) J. Biol. Chem. 270, 1230-1237[Abstract/Free Full Text]
25. Wenger, R. H., Rolfs, A., Marti, H. H., Guenet, J. L., and Gassmann, M. (1996) Biochem. Biophys. Res. Commun. 223, 54-59[CrossRef][Medline] [Order article via Infotrieve]
26. Kietzmann, T., Cornesse, Y., Brechtel, K., Modaressi, S., and Jungermann, K. (2001) Biochem. J. 354, 531-537[CrossRef][Medline] [Order article via Infotrieve]
27. Hogenesch, J. B., Chan, W. K., Jackiw, V. H., Brown, R. C., Gu, Y. Z., Pray-Grant, M., Perdew, G. H., and Bradfield, C. A. (1997) J. Biol. Chem. 272, 8581-8593[Abstract/Free Full Text]
28. Gu, Y. Z., Moran, S. M., Hogenesch, J. B., Wartman, L., and Bradfield, C. A. (1998) Gene Expr. 7, 205-213[Medline] [Order article via Infotrieve]
29. Wiener, C. M., Booth, G., and Semenza, G. L. (1996) Biochem. Biophys. Res. Commun. 225, 485-488[CrossRef][Medline] [Order article via Infotrieve]
30. Jewell, U. R., Kvietikova, I., Scheid, A., Bauer, C., Wenger, R. H., and Gassmann, M. (2001) FASEB J. 15, 1312-1314[Abstract/Free Full Text]
31. Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7987-7992[Abstract/Free Full Text]
32. Kallio, P. J., Wilson, W. J., O'Brien, S., Makino, Y., and Poellinger, L. (1999) J. Biol. Chem. 274, 6519-6525[Abstract/Free Full Text]
33. Salceda, S., and Caro, J. (1997) J. Biol. Chem. 272, 22642-22647[Abstract/Free Full Text]
34. Ema, M., Taya, S., Yokotani, N., Sogawa, K., Matsuda, Y., and Fujii-Kuriyama, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4273-4278[Abstract/Free Full Text]
35. Maltepe, E., Schmidt, J. V., Baunoch, D., Bradfield, C. A., and Simon, M. C. (1997) Nature 386, 403-407[CrossRef][Medline] [Order article via Infotrieve]
36. Adelman, D. M., Gertsenstein, M., Nagy, A., Simon, M. C., and Maltepe, E. (2000) Genes Dev. 14, 3191-3203[Abstract/Free Full Text]
37. Kozak, K. R., Abbott, B., and Hankinson, O. (1997) Dev. Biol. 191, 297-305[CrossRef][Medline] [Order article via Infotrieve]
38. Peng, J., Zhang, L., Drysdale, L., and Fong, G. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8386-8391[Abstract/Free Full Text]
39. Hosoya, T., Oda, Y., Takahashi, S., Morita, M., Kawauchi, S., Ema, M., Yamamoto, M., and Fujii-Kuriyama, Y. (2001) Genes Cells 6, 361-374[Abstract/Free Full Text]
40. Cowden, K. D., and Simon, M. C. (2002) Biochem. Biophys. Res. Commun. 290, 1228-1236[CrossRef][Medline] [Order article via Infotrieve]
41. Wenger, R. H., Rolfs, A., Spielmann, P., Zimmermann, D. R., and Gassmann, M. (1998) Blood 91, 3471-3480[Abstract/Free Full Text]
42. Zhong, H., De, Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D., Buechler, P., Isaacs, W. B., Semenza, G. L., and Simons, J. W. (1999) Cancer Res. 59, 5830-5835[Abstract/Free Full Text]
43. Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S., and Smithies, O. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11162-11166[Abstract]
44. Lydon, J. P., DeMayo, F. J., Funk, C. R., Mani, S. K., Hughes, A. R., Montgomery, C. A. Jr., Shyamala, G., Connelly, O. M., and O'malley, B. W. (1995) Genes Dev. 9, 2266-2278[Abstract]
45. Chakraborty, I., Das, S. K., and Dey, S. K. (1995) J. Endocrinol. 147, 339-352[Abstract]
46. Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J., Spielmann, P., Wenger, R. H., and Gassmann, M. (1999) FASEB J. 13, 81-88[Abstract/Free Full Text]
47. Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A., Bauer, C., Gassmann, M., and Candinas, D. (2001) FASEB J. 15, 2445-2453[Abstract/Free Full Text]
48. Paria, B. C., Zhao, X., Das, S. K., Dey, S. K., and Yoshinaga, K. (1999) Dev. Biol. 208, 488-501[CrossRef][Medline] [Order article via Infotrieve]
49. Wenger, R. H., Rolfs, A., Kvietikova, I., Spielmann, P., Zimmermann, D., and Gassmann, M. (1997) Eur. J. Biochem. 246, 155-165[Abstract]
50. Nawaz, Z., Lonard, D. M., Smith, C. L., Lev-Lehman, E., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1999) Mol. Cell. Biol. 19, 1182-1189[Abstract/Free Full Text]
51. Lim, H., Gupta, R. A., Ma, W. G., Paria, B. C., Moller, D. E., Morrow, J. D., DuBois, R. N., Trzaskos, J. M., and Dey, S. K. (1999) Genes Dev. 13, 1561-1574[Abstract/Free Full Text]
52. Finn, C. A., and Porter, D. G. (1975) The Uterus: Cells and Tissues of the Endometrium , Vol. 1 , p. 18, Publishing Sciences Group, Inc., Acton, MA
53. Gassmann, M., Fandrey, J., Bichet, S., Wartenberg, M., Marti, H. H., Bauer, C., Wenger, R. H., and Acker, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2867-2872[Abstract/Free Full Text]
54. Huet-Hudson, Y. M., Andrews, G. K., and Dey, S. K. (1989) Endocrinology 125, 1683-1690[Abstract]
55. Tan, J, Paria, B. C., Dey, S. K., and Das, S. K. (1999) Endocrinology 140, 5310-5321[Abstract/Free Full Text]
56. Das, S. K., Tan, J., Shefali, R., Jyotsnabaran, H., Paria, B. C., and Dey, S. K. (2000) J. Biol. Chem. 275, 28834-28842[Abstract/Free Full Text]
57. Das, S. K., Taylor, J. A., Korach, K. S., Paria, B. C., and Dey, S. K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12786-12791[Abstract/Free Full Text]
58. Turkay Korgun, E., Demir, R., Hammer, A., Dohr, G., Desoye, G., Skofitsch, G., and Hahn, T. (2001) Biol. Reprod. 65, 1364-1370[Abstract/Free Full Text]
59. Welch, R. D., and Gorski, J. (1999) Endocrinology 140, 3602-3608[Abstract/Free Full Text]
60. Elson, D. A., Ryan, H. E., Snow, J. W., Johnson, R., and Arbeit, J. M. (2000) Cancer Res. 60, 6189-6195[Abstract/Free Full Text]
61. Caniggia, I., Mostachfi, H., Winter, J., Gassmann, M., Lye, S. J., Kuliszewski, M., and Post, M. (2000) J. Clin. Invest. 105, 577-587[Abstract/Free Full Text]
62. Matsumoto, H., Zhao, X., Das, S. K., Hogan, B. L., and Dey, S. K. (2002) Dev. Biol. 245, 280-290[CrossRef][Medline] [Order article via Infotrieve]
63. Bonazzi, A., Mastyugin, V., Mieyal, P. A., Dunn, M. W., and Laniado-Schwartzman, M. (2000) J. Biol. Chem. 275, 2837-2844[Abstract/Free Full Text]
64. Chakraborty, I., Das, S. K., Wang, J., and Dey, S. K. (1996) J. Mol. Endocrinol. 16, 107-122[Abstract]


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