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BOR - Papers in Press, published online ahead of print November 24, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.035949
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BIOLOGY OF REPRODUCTION 72, 530–537 (2005)
DOI: 10.1095/biolreprod.104.035949
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of Estrogen Receptors-{alpha} and -ß in the Pregnant Ovine Uterine Artery Endothelial Cells In Vivo and In Vitro1

Wu Xiang Liao3, Ronald R. Magness4, and Dong-bao Chen2,3

Department of Reproductive Medicine,3 University of California San Diego, La Jolla, California 92093-0802 Perinatal Research Laboratories, Departments of Obstetrics and Gynecology,4 University of Wisconsin-Madison, Meriter Hospital, Madison, Wisconsin 53715


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen is recognized to be one of the driving forces in increases in uterine blood flow through both rapid and delayed actions via binding to its receptors, ER{alpha} and ERß at the uterine artery (UA) wall, and especially in UA endothelium (UAE). However, information regarding estrogen receptor (ER) expression in UAE is limited. This study was designed to test whether ERs are expressed in UAE in vivo, and if they are, whether these receptors are maintained in cultured UA endothelial cells (UAECs) in vitro. By using immunohistochemical and Western blot analyses, we clearly demonstrated ER{alpha} and ERß protein expression in pregnant (Days 120–130) sheep UA and UAE in vivo and as well as cultured UAECs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) amplified both ER{alpha} and ERß mRNAs in UA, UAE, and UAECs. Of interest, a truncated ERß (ERß2) variant due to a splicing deletion of exon 5 of the ERß gene was detected in these cells. Quantitative RT-PCR analysis revealed that ER{alpha} mRNA levels are ~8-fold (P < 0.01) higher than that of ERß in UAECs, indicating that ER{alpha} may play a more important role than ERß in the UAEC responses to estrogen. Fluorescence immunolabeling analysis showed that ER{alpha} is present in both nuclei and plasma membranes in UAECs, and the latter is also colocalized with caveolin-1. The membrane and nuclear ER{alpha} presumably participate in rapid and delayed responses, respectively, to estrogen on UAE. Taken together, our data demonstrated that UAE is a direct target of estrogen actions and that the UAEC culture model we established is suitable for dissecting estrogen actions on UAE.

caveolin-1, endothelium, ER{alpha}, ERß, estradiol receptor, mechanisms of hormone action, nitric oxide, pregnancy, uterine artery, uterine blood flow, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During normal pregnancy, uterine blood flow (UBF) increases substantially (up to 50- to 80-fold) to provide sufficient oxygen and nutrient supply for the growth of the developing fetus. Shortage of oxygen and nutrient supply due to insufficient UBF is associated with fetal intrauterine growth restriction and higher prenatal and neonatal morbidity, or even mortality [1, 2]. A pivotal role for estrogen in elevating UBF has been suggested for several decades based on numerous studies demonstrating 1) a concomitant increase in circulating estrogen levels and UBF during the follicular phase of the estrous cycle and during normal pregnancy in many species [3–7] and 2) a dramatic increase in UBF in ovariectomized and intact pregnant and nonpregnant sheep following a bolus injection of exogenous estrogen [1, 8–11]. A large body of evidence exists showing that estrogen-induced and pregnancy-associated rises in UBF are, to a great extent, mediated by enhanced endothelial production of the potent vasodilator nitric oxide (NO) [12–14] via increasing endothelial nitric oxide synthase (eNOS) protein expression [15–17], or by increasing eNOS activity [18–20], or both. However, the mechanisms underlying the estrogen-induced rise in UBF is still far from resolved.

Estrogen-induced rise in UBF has been postulated to be mediated by one or more specific estrogen receptors [1, 21]. In ovariectomized sheep, we have recently observed that estrogen-induced rise in UBF can be partially (~70%) inhibited by the pure estrogen receptor (ER) antagonist ICI 182,780 (Magness, et al., unpublished data). This result demonstrates that the estrogen-induced rise in UBF is, at least in part, mediated by one or more ER-dependent mechanisms. Recently, we have also reported that rapid activation of extracellular signal-regulated kinases might be a mechanism responsible for acute activation of eNOS by estrogen to produce NO in uterine artery endothelial cells (UAECs) in vitro. In that study, we showed that the membrane-impermeable estradiol-17ß (E2)-BSA conjugate mimics the response to E2. We also demonstrated the presence of plasma membrane E2-BSA binding sites, which suggests that the rapid activation of the eNOS-NO pathway by estrogen in UAECs might be mediated by ER localized on the plasma membrane [19]. Despite these important observations suggesting that estrogen is able to act directly on the uterine artery endothelium (UAE), direct in vivo evidence of ER expression in uterine artery (UA) is still lacking.

Classically, the biological functions of estrogen in target tissues are believed to be mediated by specific high-affinity nuclear ERs that function as ligand-activated transcription factors to regulate gene expression [22]. Two types of ERs have been identified so far, including the originally described ER{alpha} [23, 24], and ERß [25], which was discovered later. Because detailed information of ER{alpha} and ERß expression in UA is needed to advance our knowledge of the mechanisms underlying estrogen-induced and pregnancy-associated rises in UBF, in this study we investigated the expression of both ER{alpha} and ERß mRNAs and proteins in late-pregnant sheep UA and UAE in vivo, and in cultured UAECs in vitro. We demonstrate herein for the first time the expression of both ER{alpha} and ERß mRNAs and proteins in pregnant sheep UAECs both in vivo and in vitro. The implication of potential functional roles for ER{alpha} and ERß is also discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Mouse anti-ER{alpha} monoclonal antibody (mAb, AER320) which was raised against amino acids 495–595 of calf uterus ER{alpha} [26] was purchased from Lab Vision Corp. (Fremont, CA). Rabbit anti-ERß polyclonal antibody (pAb) against amino acids 55–70 of human ERß was from Affinity BioReagents (Golden, CO). Rabbit anti-caveolin-1 pAb was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ß-actin mAb was from Ambion, Inc. (Austin, TX). Cy2-conjugated AffiniPure Fab fragment rabbit anti-mouse immunoglobulin G (IgG; H+L), Cy3-conjugated AffiniPure Fab fragment goat anti-rabbit IgG (H+L), and mouse and rabbit ChromPure IgGs were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Fetal calf serum (FCS) and phosphate-free Dulbecco modified Eagle medium (DMEM) were from Life Technologies, Inc. (Grand Island, NY). Tissue culture plastic ware was from Corning (Corning, NY). Enhanced chemiluminescence kits were from Amersham (Arlington Heights, IL). Immobilon-p polyvinyl difluoride membrane was from Millipore (Bedford, MA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Animals and Tissue Sample Collection

Pregnant (Days 120–130) sheep used for this study were purchased from Nebeker Ranch (Lancaster, CA). The animal use protocol was approved by the University of California San Diego Animal Subjects Committee, and we followed the National Research Council's Guide for the Care and Use of Laboratory Animals throughout the study. On the day of autopsy, the ewes were injected with an overdose of sodium pentobarbital (25 mg/kg). UA segments and ovaries were harvested immediately after the animals had been killed and processed for fixing in 3.7% paraformaldehyde (tissue sectioning) or for snap-frozen in liquid nitrogen (total RNA isolation and protein extracts), and for mechanical isolation of UAE protein as described [27]. Ovary samples were prepared in parallel to serve as a positive control because of its high levels of ER{alpha} and ERß expression [28]. Fresh UAs were also used for isolation of endothelial cells as described below.

Cell Culture, Preparation of Total Cell and Tissue Protein Extracts, and Western Blot Analysis

UAECs were isolated from pregnant ewes (Days 120–130) under nonsurvival surgery by collagenase digestion, cultured in growth media (D-Val MEM with 20% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin), and propagated as previously described [27]. Frozen UAEC aliquots (passage 3) were thawed and plated in 10-cm dishes to grow to about 70%–90% confluence in growth media, and used for RNA extraction as described below. Total cell extracts were prepared as described previously [19]. SDS-PAGE and immunoblotting analysis was carried out as described previously [19], except bound antibodies were visualized by using the Chemi-Glow Chemiluminescent substrate (Alpha Innotech Corporation, San Leandro, CA). Digital images were captured by using the ChemiImager Imaging System (Alpha Innotech) with a high-resolution charge-coupled device camera and analyzed with the ChemiImager 4400 software (Alpha Innotech).

Immunohistochemical Staining

Ovine UA and ovary segments were collected from pregnant (Days 120–130) ewes immediately after they were killed and fixed with 3.7% paraformaldehyde, embedded in paraffin, and 6-µm sections were cut and mounted onto slides. Immunohistochemical analysis using antibodies against ER{alpha} (ER{alpha} mAb at 5 µg/ml) and ERß (ERß pAb at 10 µg/ml) was performed using the PicTure-Plus kit by Zymed Laboratories, Inc. (South San Francisco, CA) following the manufacturer's protocol. Corresponding concentrations of rabbit and mouse IgGs served as experimental, nonspecific binding controls.

RNA Extraction and Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Competitive RT-PCR

Total RNA was extracted from homogenates of whole UA, isolated UAE samples, and ovaries collected from pregnant (Days 120–130) ewes and from cultured UAECs (passage 4–5) by guanidium acid-isothiocyanate-phenol-chloroform methods using the Trizol reagent (Invitrogen, San Diego, CA), quantified by measuring absorbance at 260 and 280 nm, and stored at –80°C until an RT-PCR assay was performed. Oligonucleotides used for RT-PCR were custom ordered from Gene Link, Inc. (Hawthorne, NY). PCR primer pairs were designed from different exons of the genes of interest to discriminate PCR products that might arise from possible chromosome DNA contaminants. Specifically, they are derived from the cDNA clones positioned at the following nucleotides: 1291–1311 and 1732–1753 for ER{alpha} [29]; 862–882 and 1397–1417 for ERß RT-PCR, 1120–1140 and 1397–1417 for ERß quantitative RT-PCR [30], and 38– 56 and 370–388 for ribosomal protein L19 (L19; GenBank accession number AY158223). The steady state levels of target mRNAs encoding ER{alpha}, ERß, and L19 were analyzed by competitive RT-PCR. Prior to PCR reaction, each internal control DNA (~250 or 500 base pairs; bp) possessing target-specific primer pairs were generated by PCR, purified by the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and quantified by OD260/280. The extracted RNA samples (2 µg) were subjected to an RT reaction (20 µl) using 15 units of Cloned AMV Reverse Transcriptase (Invitrogen, Carlsbad, CA) with random hexamer (10 ng/µl), and deoxynucleotide triphosphate (dNTP; 1 mM) at 45°C for 60 min and then 70°C for 10 min. The resultant single-strand cDNA was bought to 100 µl of volume with diethyl pyrocarbonate-water for PCR. The linear portion of PCR amplification between each target cDNA and its corresponding internal control DNA was determined separately for all targets. Briefly, a fixed amount (20 ng) of cDNA derived from UAECs was mixed with a series of dilutions of the internal control DNA. The target and the internal control DNAs were coamplified by PCR using a specific primer set for each individual target. PCR was performed in a reaction mixture (25 µl) containing MgCl2 (1.5 mM), dNTP (0.2 mM), and 2.5 units of Platinum Taq DNA polymerase (Invitrogen) under the following conditions: denaturation at 94°C for 30 sec (L19, 28 cycles; ER{alpha}, 35 cycles; and ERß, 38–40 cycles); annealing for 30 sec at 60°C (L19), 62°C (ER{alpha}), or 65°C (ERß); and extension at 72°C for 30 sec. Aliquots of PCR products were electrophoresed on 2% agarose gel, stained with GelStar nucleic acid gel stain (BioWittaker Molecular Applications, Rockland, ME), and visualized under a UV light. Digital images were captured and analyzed by using the FluorChem Imaging Systems by Alpha Innotech Corporation. The PCR products were sequenced and analyzed using a DS-Gene 1.5 software (Accelrys, San Diego, CA). The relative integrated density of each band was multiplied by the absorbance of the surface area. Finally, the logarithm (log) transformation of the ratios of the densitometric readings of the amplified target cDNA and internal control DNA were plotted on the ordinate against the log concentrations of internal control DNA on the abscissa. The concentrations of target mRNAs of ER{alpha} and ERß and L19 in the UAECs were calculated when the log transformation of a ratio between target and internal control signals equaled zero.

Immunofluorescence Microscopy

All reactions were performed at room temperature. UAECs at passage 4–5 were plated sparsely on gelatin-coated glass coverslips in growth media. After culture for 1–2 days, the cells were briefly washed twice with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, and fixed for 20 min in a PBS solution containing 4% paraformaldehyde. Fixed cells were rinsed once with PBS and three times with PBS containing 50 mM glycine, and blocked and permeabilized with blocking reagents containing 1% gelatin, 1% BSA, and 0.15% saponin in PBS for 20 min. Cells were then successively incubated for 60 min with working reagent (1/2; PBS, 1/2; blocking reagents) containing, first, first antibodies of 1 µg/ml of anti-caveolin-1 pAb and 5 µg/ml of anti-ER{alpha} mAb (AER320) with the corresponding concentrations of rabbit and mouse IgGs serving as nonspecific binding controls; and second, Cy2-conjugated AffiniPure Fab fragment rabbit anti-mouse IgG (H+L) (15 µg/ml) and Cy3-conjugated AffiniPure Fab fragment goat anti-rabbit IgG (H+L) (1.25 µg/ml). Cells were washed three times (5 min each) with working reagents after each step of incubation. The coverslips were then mounted with ProLong Gold antifade reagent (Molecular Probes, Eugene, OR) and analyzed under an LSM510 laser scanning confocal microscope (Carl Zeiss, Inc., Thornwood, NY). A 0.4-µm interval was set to detect the signals for both ER{alpha} and caveolin-1 at each cross section.

Statistics

Data are shown as mean ± SEM. A Student t-test was used to compare the expression levels of ER{alpha} and ERß mRNAs. P < 0.05 was considered significantly different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemistry

UA tissue sections from pregnant (Days 120–130) ewes show intensive immunoreactive signals for both ER{alpha} and ERß proteins in the nuclei of the endothelial and smooth muscle cells (Fig. 1). Weak signals of ER{alpha} and ERß immunostaining in the cytoplasm were also detected, which indicates the possible presence of both ER{alpha} and ERß proteins in the cytoplasm of both UAE and UA smooth muscle (UASM) cells. However, the cytoplasm staining signals of ER{alpha} and ERß may be derived from plasma membrane staining because the immunohistochemical technique used in this experiment does not give a clear subcellular structure of cytoplasm and plasma membrane. As the positive controls, ovary tissue sections from the same ewes also showed abundant expression of both ER{alpha} and ERß protein in the granulosa cells, but not the oocyte (Fig. 1). Negative controls run in parallel with corresponding concentrations of rabbit and mouse IgGs served as nonspecific binding controls, which displayed very weak or no brown color staining (Fig. 1).



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  		FIG. 1. Immunohistochemical analysis of both ER{alpha} (A) and ERß (B) in UA of pregnant ewes. Ovary tissue from the same ewes was used as the positive control for ER{alpha} and ERß. Corresponding concentrations of rabbit and mouse IgGs served as nonspecific binding controls. UA, uterine artery; E, endothelium; SM, smooth muscle; GC, granulosa cells; O, oocyte. Bar = 100 µm

Immunoblotting Analysis

The homogenized tissue samples of UA, UAE, and ovarian and cultured UAECs were extracted for proteins and used for Western immunoblot analysis with specific antibodies against ER{alpha} and ERß proteins. Figure 2A shows a band of native ER{alpha} protein (67 kDa) [29] detected in UA, UAE, UAEC, and ovary extracts, indicating the presence of ER{alpha} in these tissue samples and cultured UAECs. When ERß antibody was used, several bands were detected in the extracts of these tissue samples or cells (Fig. 2B). One band detected with a molecular weight of 55 kDa was the native, full-length ERß protein [30]. Moreover, an additional band with a molecular weight of ~30 kDa was also detected in these samples. As shown in the RT-PCR results below, this band should be the N-terminal truncated ERß protein (Fig. 2D) due to a 139-bp splicing deletion of exon 5 (Fig. 3B). We have termed the 55 kDa band as ERß1 and the ~30 kDa band as ERß2 (Fig. 2B). We also detected additional bands (Fig. 2; indicated with *) with the specific anti-ERß antibody, which could be other variants of ERß (Fig. 2B). Collectively, these results indicate the presence of both ER{alpha} and ERß proteins in sheep UA, UAE, and cultured UAECs.



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  		FIG. 2. Western immunoblot analysis of ER{alpha} (A), ERß (B), and ß-actin (C) in the protein extracts of UA, UAE, UAEC, and ovary from pregnant ewes. D) A diagram representing the truncated form of ERß2 that results from the splicing deletion of exon 5 shown in Fig. 3B. The shadowed box represents the amino acid sequences encoded by different reading frame. UA, uterine artery; UAE, UA endothelium; UAEC, UA endothelial cell; aa, amino acid. Bands marked with * may indicate additional truncated forms of ERß



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  		FIG. 3. RT-PCR analysis of ER{alpha}, ERß, and the ribosomal protein L19 mRNAs in the cell of UA and UAE, and in UAECs and ovary of pregnant ewes. B) A diagram representing the ERß2 PCR product missing exon 5. M, DNA marker, UA, uterine artery; UAE, UA endothelium; UAEC, UA endothelial cell

RT-PCR Analysis of ER{alpha} and ERß mRNAs

A distinct band of 442 bp was amplified in all of the total RNA samples isolated from UA, UAE, UAECs, and ovary when ER{alpha}-specific primers were used (Fig. 3A). Similarly, a distinct band of 556 bp was also amplified in all the tissues and UAECs when ERß-specific primers were used (ERß1, Fig. 3A). DNA sequencing confirmed these RT-PCR products are indeed from ER{alpha} and ERß cDNA. An additional band of 417 bp was also amplified in all the tissues and UAECs when ERß primers were used (ERß2, Fig. 3A). DNA sequencing showed that this PCR product is from ERß cDNA, with a 139-bp deletion of exon 5 as reported previously [30] (Fig. 3B). Because this deletion is not in frame, it results in a premature termination of the translation of ERß protein, which should be only 324 amino acids (~30 kDa), as seen in the immunoblotting data (Fig. 2B). It is noteworthy that for amplification of ER{alpha}, 35 PCR cycles was sufficient. However, it required 38–40 PCR cycles to amplify a detectable ERß signal. These results indicate that ERß mRNA level could be lower than that of ER{alpha} in these samples.

Quantitative RT-PCR Analysis of ER{alpha} and ERß mRNA Levels in UAECs

For quantification of the steady state levels of ER{alpha} and ERß mRNA in UAECs, internal controls (250 bp or 500 bp) were amplified and used for competitive RT-PCR as described in Materials and Methods. As shown in Figure 4A, the internal control we used ranges from 3.3 x 10–3 to 3.3 x 10–5 pM for ER{alpha}, 10–4 to 3.3 x 10–6 pM for ERß, and from 3.3 x 10–1 to 3.3 x 10–3 pM for L19. When the log transformation values of the ratios between the target and internal control were plotted against the log of concentrations of internal controls, linear regression curves were obtained (Fig. 4B). The correlation coefficients (r2) were >0.98 (P < 0.005), indicating that PCR amplification was linear. The concentration of ER{alpha} is 1.42 x 10–4 ± 3.5 x 10–6 pM, the ERß concentration is 1.71 x 10–5 ± 5.5 x 10–7 pM, and that of L19 is 0.038 ± 0.002 pM in cultured UAECs (n = 3). Thus, the relative level of ER{alpha} mRNA normalized to that of L19 is 0.372 ± 9.2 x 10–3%, ERß is 0.0449 ± 1.4 x 10–3%, and the ER{alpha} mRNA level is 8.30 ± 0.21 times that of ERß (P < 0.001) in cultured UAECs. This result indicates that the level of ER{alpha} mRNA is significantly greater than that of ERß in UAECs, which potentially suggests a primary role of ER{alpha} in mediation of estrogen action in this specific type of endothelial cell.



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  		FIG. 4. Competitive RT-PCR analysis of ER{alpha}, ERß, and ribosomal protein L19 mRNAs in cultured uterine artery endothelial cells. A) Various concentrations of respective internal control (ic) were added to compete with the target molecules. B) Summary of the log-log transformation plotting of the ratio in PCR product signal intensity between each target and its corresponding internal control. Linear regressions were obtained for all ER{alpha}, ERß, and L19 competitive PCR reactions. The concentrations of ER{alpha}, ERß, and L19 target mRNAs in the UAECs were determined at which the log-log transformation of the ratio in PCR product signal intensity between each target and its corresponding internal control equaled zero

Double Immunofluorescence Confocal Microscopy

To detect the subcellular localization of ER{alpha} in UAECs, double immunofluorescence labeling was carried out using specific antibodies against ER{alpha} and caveolin-1 proteins. Figure 5 shows a representative cross section of 0.4 µm of the cultured UAECs observed under confocal microscopy, which shows both nuclear and membrane structures. Abundant ER{alpha} (green) is detected in the nuclei of UAECs (arrowheads). Caveolin-1 (red) is exclusively expressed at the plasma membranes of cultured UAECs (arrows). Of interest, abundant ER{alpha} was found colocalized with caveolin-1 at the plasma membranes of UAECs (arrows). Negative controls show neither red nor green fluorescence labeling (data not shown). These results indicate that ER{alpha} locates at both the nucleus and plasma membrane of UAECs. We also performed similar subcellular localization experiments by using the same ERß antibody for Western immunoblot analysis as shown in Figure 2 to colocalize ERß with caveolin-1. However, with the use of this specific antibody we failed to detect ERß subcellular colocalization with caveolin-1 (data not shown).



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  		FIG. 5. Double fluorescence confocal microscopy for subcellular localization of ER{alpha} and caveolin-1 in UAECs. Confocal microscopy analysis of the immunofluorescence labeled ER{alpha} (green) and caveolin-1 (red) shows significant ER{alpha} and caveolin-1 colocalization at the plasma membrane of UAEC (arrows). An abundant level of ER{alpha} is also detected in the nuclei of UAECs (arrowheads). The representative image shown was taken from one cross section of a UAEC. UAEC, uterine artery endothelial cells. Bar = 10 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Estrogen acts on a wide range of tissues and cell types and the biological functions of this hormone are mediated by both ER{alpha} and ERß [31]. Numerous studies have demonstrated that a variety of mammalian tissues and cell types including vascular blood vessels and endothelial cells express ER{alpha} and ERß [32, 33]. Pertinent to the current study, it has been reported that both ER{alpha} and ERß are expressed in mouse and rat aorta; rat tail artery; rat UA, including endothelial cells [34, 35]; baboon carotid artery [36]; fetal sheep intrapulmonary artery endothelial cells [37]; cultured human UAECs [38]; human arterial endothelial cells [39]; and human internal thoracic artery endothelia [39]. ER{alpha} has also been identified in human coronary artery [40], human umbilical vein endothelial cells [40, 41], and human and bovine aortic endothelial cells [41]. From the majority of reported data in the literature, it appears that the relative expression levels of ER{alpha} and ERß vary among different vascular beds, and are associated with physiological status of the cells/tissues tested. Thus, it is suggested that the expression levels of ER{alpha}, ERß, or both may be critical for the tissue/cell-specific responses of various vascular functions manifested by estrogen [32, 34].

It has long been believed that estrogen-induced vasodilatation during pregnancy is at least in part regulated through one or more ERs [1, 21]. However, the much-needed detailed information about the presence of estrogen receptors in UAE has not been reported. Recently, we demonstrated the possible existence of membrane-bound ER (ER{alpha}) in cultured UAECs [19]. In the present study, we investigated the expression of both ER{alpha} and ERß mRNAs and proteins in pregnant sheep UAE in vivo and in cultured UAECs in vitro. The subcellular localization of ER{alpha} in cultured UAECs was also studied. Our data clearly demonstrate for the first time the presence of both ER{alpha} and ERß proteins and mRNAs in UAE in pregnancy and in cultured UAECs, and also suggest that a cultured UAEC model established previously in our laboratory [19] is likely to be suitable for studying the UAE responses to estrogen. Because some recent studies have shown that certain vascular endothelial cells do not express ER{alpha} or ERß [42, 43], which suggests that either one or both are not evenly distributed throughout the vascular tree. Thus, our data confirms the very existence of both ER{alpha} and ERß in the uterine vascular bed.

Many variants of both ER{alpha} and ERß have been identified in various known ER-positive tissues [44–46]. In the vascular artery tree, except for the full-length ER{alpha} and ERß receptors, ER{alpha} variants have been detected in human and rat vascular smooth muscle cells [47–49], and ERß variants in human vascular smooth muscle cells [49]. The functional significance of these ER variants in the target tissues is not fully understood. However, recent data suggest these variants may suppress estrogen-dependent transcriptional activity that requires both ER{alpha} and ERß [48, 50, 51]. In the present study, our data derived from immunoblotting and RT-PCR as well as DNA sequencing analyses clearly demonstrate for the first time the presence of an ERß variant (ERß2) in the UAE and in cultured UAECs, as well as in the ovary from pregnant ewes. This variant resulted from the splicing deletion of exon 5 and, as a consequence, it is missing all the C-terminal amino acids afterward [30], and lacks most of the ligand-binding domain and C-terminal domain of ERß [46]. A previous functional study showed that this variant may serve as a dominant negative receptor that is able to block both ER{alpha} and ERß signaling pathways [50]. This variant was also found to be significantly associated with the progression of breast cancer and menopausal status [52]. Because the expression level of this variant is relatively high in UAE and UAECs, its potential dominant negative effect should be taken into consideration when studying UAEC responsiveness to estrogen. Furthermore, it would be of great interest to determine whether other known or novel variants are also present in the UAE and to functionally characterize these variants.

It is now well established that ER{alpha} and ERß signaling is complex, and each one initiates its own diverse, rapid nongenomic and genomic biological responses that are differentially dependent on the cell types, receptor density, and ligands being studied [33,53]. Thus, quantitative studies of ER{alpha} and ERß expression in the UA would be important for our understanding of the mechanism or mechanisms of estrogen actions in this specific vascular wall. In this study, we developed competitive RT-PCR methods for measuring the absolute and relative mRNA levels of both ER{alpha} and ERß, by which we are able to quantify both ER{alpha} and ERß mRNAs in cultured UAECs. Of interest, the mRNA level of ER{alpha} is significantly higher than that of ERß in cultured UAECs. This result suggests that ER{alpha} may play a more important role than ERß in response to estrogen treatment in this vascular endothelial cell type. Now we are testing an idea that ER{alpha} and ERß possess different functional activities in regulating estrogen responses in UAECs by using a selective ER{alpha} and ERß gene silencing approach with specific ER{alpha} or ERß small interfering RNA. Regardless, a recent physiological study showing that selective ER{alpha} ligands are more potent than ERß ligands for increasing uterine blood flow supports this idea [54]. Nonetheless, our present study is the first to quantify the absolute mRNA levels of both ER{alpha} and ERß in any vascular endothelial cells. Establishment of these assays provides us with opportunities to further study the expression profiles of both ER{alpha} and ERß in UA during the estrous cycle and pregnancy. Because the expression levels of ER{alpha} and ERß may vary under different physiological conditions and clinical settings [55–57], studies of such will provide additional, important information for advancing our knowledge of estrogen actions on UAE.

Following estrogen administration, uterine blood flow starts to rise within 30–45 min and increases up to 10-fold within 90–120 min [8, 58]. This rapid vasodilatory effect of estrogen in the uterus has been attributed to both acute activation of eNOS in UAECs, possibly via membrane ER-mediated nongenomic pathways [19] and increased eNOS protein expression [15–17]. We reported previously E2-BSA-fluorescein isothiocyanate binding to the plasma membrane of UAECs [19]. However, because UAECs also expresses other E2 binding proteins, such as the sex hormone-binding globulin (W.X. Liao and D.B. Chen, unpublished data), the binding of E2-BSA to UAECs does not directly support the presence of classical ER at the plasma membrane of UAECs. In this study we report that, in addition to its nuclear localization, ER{alpha} colocalizes with caveolin-1 at the plasma membrane of a UAEC. This result is consistent with previous observations that ER{alpha} is colocalized with caveolin-1 on the plasma membrane in bovine aortic endothelial cells [59] and ovine fetal pulmonary artery endothelial cells [60, 61]. In keeping with recent reports that ER{alpha}, caveolin-1, and eNOS form a functional regulatory complex within the plasma membranous caveolae of endothelial cells to regulate NO production [33], our data suggest that such a mechanism may also be implicated in the regulation of NO production in UAECs. In other endothelial cell systems, others have shown lately that ERß is also localized on the plasma membrane [33]. In the present study, our attempt to detect ERß by immunocytochemical analysis with the same antibody used for Western immunoblot analysis of sheep ERß was unsuccessful. The colocalization of ERß and caveolin-1 is definitely of critical interest to further delineate the role of ERß in UAEC response to estrogen. Additional experiments will be performed to this end once an appropriate ERß antibody is identified.

In this study, UASM cells were also found to be immunostained by both ER{alpha} and ERß antibodies. These data demonstrate the presence of these receptors in UASM cells as well. Furthermore, by using similar immunoblotting and RT-PCR analyses as applied in the present study, we also confirmed the presence of both ER{alpha} and ERß mRNAs and proteins in UASM in vivo (Fig. 1) and cultured UASM cells (a cell culture model recently developed in our laboratory; D.B. Chen, unpublished data). The expression of ER{alpha} and ERß in UASM demonstrates that estrogen is also capable of directly acting on the smooth muscle of UA. Because previous studies have shown that an endothelium/ NO-dependent mechanism accounts for only ~65%–70% of estrogen-induced uterine vasodilatation [13], direct actions of estrogen on the smooth muscle may provide alternative explanations for the endothelium-independent mechanism of estrogen-induced uterine vasodilatation.

In summary, we provide in this study direct evidence for the presence of both ER{alpha} and ERß mRNAs and proteins in the UA and UAE in vivo and in cultured UAECs in vitro, as well as their relative mRNA expression levels in UAECs. In addition, ER{alpha} was found located at both plasma membranes and nuclei, which is expected to mediate both nongenomic and genomic responses to estrogen administration. The identification of a variant ERß in UA that may have a dominant negative effect on both ER{alpha} and ERß signaling pathways add to the complexity of the molecular mechanism or mechanisms underlying estrogen-induced uterine blood flow. The information provided in this study suggesting that UAECs are direct targets of estrogen demonstrates for the first time that estrogen can directly act on this reproductive vascular bed. The maintenance of ER{alpha} and ERß expression in UAECs in culture suggests that the UAEC culture model is a valuable tool for dissecting the cellular and molecular mechanisms of estrogen-induced uterine vasodilatation.


   ACKNOWLEDGMENTS
 
We thank Drs. Cecilia Y. Cheung and Robert A. Brace for their assistance with the animals.


   FOOTNOTES
 
1 Supported in part by National Institutes of Health (NIH) RO1 grants HL70562 and HL74947, by an Academic Senate grant from the University of California San Diego to D.B.C., and by NIH RO1 grants HD33255 and HL49210 to R.R.M. Back

2 Correspondence: Dong-bao Chen, Division of Maternal-Fetal Medicine (MC0802), Department of Reproductive Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0802. FAX 619 543 2919; dochen{at}ucsd.edu Back

Received: 2 September 2004.

First decision: 13 October 2004.

Accepted: 8 November 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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