Molecular Human Reproduction, Vol. 10, No. 2, pp. 115-122, 2004
© European Society of Human Reproduction and Embryology 2004
Augmented endothelial nitric oxide synthase (eNOS) protein expression in human pregnant myometrium: possible involvement of eNOS promoter activation by estrogen via both estrogen receptor (ER)
and ERß
1Department of Gynecology and Obstetrics and 2Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
3 To whom correspondence should be addressed. e-mail: fetus{at}kuhp.kyoto-u.ac.jp
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The aim of the present study was to investigate the possible contribution of estrogen to pregnancy-associated modulation of nitric oxide production in the human myometrium during pregnancy. Both endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) proteins were clearly expressed in the non-pregnant myometrium and were elevated in the first trimester of pregnancy. Oral contraceptive pills augmented eNOS, but not iNOS, protein expression in the non-pregnant human myometrium. In cultured human myometrial cells, estrogen receptor (ER)
and ERß expression was extremely low. Therefore, we used either ER or ERß expression vector to investigate the effect of 17ß-estradiol treatment on eNOS promoter activity using eNOS promoter/luciferase vector in cultured human myometrial cells. 17ß-estradiol treatment significantly augmented eNOS promoter activity in cells co-transfected with either ER or ERß, and this augmentation was dose-dependently suppressed by ICI 182780, an estrogen antagonist. These data suggest the possibility that both ER and ERß are involved in the estrogen-associated regulation of eNOS gene expression in the human myometrium. Key words: Key words: endothelial nitric oxide synthase/estrogen/estrogen receptor/myometrium/pregnancy
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During pregnancy, uterine smooth muscle in a state of so-called uterine quiescence relaxes and consequently distends dramatically to accommodate the growing fetus. The mechanisms by which uterine quiescence is maintained are not fully understood and have been postulated to be related to the pregnancy-induced alterations of various bioactive substances including prostaglandins, nitric oxide and others (Cunningham et al., 2001). We previously reported that maternal plasma nitric oxide metabolite concentrations were elevated in pregnancy and declined prior to the onset of labour at term, suggesting possible involvement of nitric oxide in the maintenance of myometrial quiescence and in the onset of labour (Nanno et al., 1998). Subsequently we reported that the activity of soluble guanylate cyclase, a target enzyme of nitric oxide (Garbers et al., 1994), in the human myometrium is decreased prior to the onset of labour at term, suggesting that a possible down-regulation of the myometrial response to endogenous nitric oxide may also be a factor involved in the onset of labour (Telfer et al., 2001). Indeed, some investigators have hypothesized that nitric oxide contributes to the establishment of uterine quiescence as a relaxant of the myometrium in humans and other animals (Natuzzi et al., 1993; Sladek et al., 1993; Yallampalli et al., 1993; Bansal et al., 1997; Riemer et al., 1997). In this regard, nitric oxide donors were used as tocolytic agents promoting uterine relaxation (Black et al., 1996). However, other investigators disagree with these hypothetical roles of nitric oxide in humans (Jones et al., 1997; Bartlett et al., 1999).
Nitric oxide can be synthesized from L-arginine via two constitutive calcium-sensitive isoforms of nitric oxide synthase (NOS), i.e. endothelial NOS (eNOS) and neuronal NOS (nNOS), or an inducible isoform (iNOS) which is calcium-independent (Knowles et al., 1994). Nitric oxide production was reported to be increased in uterine tissues of pregnant rats and rabbits, and to be decreased at term (Sladek et al., 1993; Yallampalli et al., 1993; Riemer et al., 1997). By contrast, in human myometrium, the expression of NOS isoforms as well as the possibility of pregnancy-associated changes in their expression remains controversial (Bansal et al., 1997; Thomson et al., 1997; Bartlett et al., 1999; Norman et al., 1999).
Sex steroids modulate nitric oxide production in the gravid uterus of rats (Yallampalli et al., 2000), sheep (Figueroa et al., 1995; Zhang et al., 1999) and rabbits (Batra et al., 1998) and the non-gravid uterus of humans (Khorram et al., 1999; Weeks et al., 1999; Zervou et al., 1999). In vascular endothelium (MacRitchie et al., 1997; Vagnoni et al., 1998; Tan et al., 1999; Yang et al., 2000) and myocardium (Nuedling et al., 1999), eNOS protein and/or mRNA expression has been reported to be elevated by estrogen stimulation. However, to our knowledge, in vitro studies have yet to clarify whether estrogen directly contributes to the NOS expression in human myometrial cells from pregnant women. To date, no culture model of human myometrium suitable for this purpose has been available because of decreases of eNOS as well as estrogen receptor expression during the process of multiple passages of human myometrial cells, which initially retain some characteristics of pregnant myometrium.
There are two estrogen receptors, ER and ERß (Enmark et al., 1999; Gustafsson et al., 1999; Mendelsohn et al., 1999; Gray et al., 2001), both of which are reported to be expressed in the human myometrium (Wu et al., 2000; Gargett et al., 2002). However, their tissue distribution in the human uterus is controversial. ERß expression was found to be much higher than that of ER in the pregnant human myometrium by Wu et al. (2000). On the other hand, Gargett et al. (2002) demonstrated that human myometrial cells expressed ER and myometrial microvascular endothelial cells expressed ERß.
The best-characterized effect of ER is the modulation of gene transcription by direct interaction of the estrogen receptor DNA-binding domain with estrogen-response elements in the target gene promoter region (Enmark et al., 1999; Mendelsohn et al., 1999; Gray et al., 2001). The eNOS promoter region contains at least six half-palindromic sequences, which form effective estrogen-response elements (Miyahara et al., 1994). Since the ERß DNA-binding domain has 96% homology with that of ER (Mendelsohn et al., 1999), it is hypothesized that ERß and ER affect identical estrogen-response elements in the target gene promoter region (Enmark et al., 1999). However, biological roles of ERß are reported to be partly different from those of ER (Gustafsson et al., 1999; Gray et al., 2001). Moreover, a recent study revealed that the cell-specific availability of nuclear co-regulator proteins is more important than the structure of estrogen-response elements in the target gene promoter region (Enmark et al., 1999; Mendelsohn et al., 1999; Routledge et al., 2000; Gray et al., 2001). Accordingly, for the study of the human myometrial response to estrogen stimulation, it is important to utilize myometrial cells prepared from pregnant women as an in vitro model which retain characteristics of pregnant myometrium.
To evaluate the possible contribution of estrogen to the pregnancy-associated modulation of nitric oxide production in human myometrial tissues, we investigated eNOS and iNOS protein expression in the intrauterine tissues and/or myometrial tissues from first and third trimester pregnant women as well as non-pregnant women with or without oral contraceptive medication. We further investigated the effect of estrogen on eNOS promoter activities in cultured human myometrial cells using transient transfection of ER or ERß expression vectors.
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Reagents
All reagents were of analytical grade and purchased from Sigma Chemical Co. (USA), unless otherwise indicated.
Materials
Uterine corpus myometrial tissues were obtained from pre-menopausal non-pregnant (34–40 years old; n = 10) women who had received total hysterectomy for uterine myoma. Five non-pregnant women were at proliferative follicular phase without any medication. Five additional non-pregnant women were treated with a daily oral contraceptive pill containing 0.05 mg of ethinylestradiol and 0.5 mg of norgestrel for 3–5 weeks until the day of operation to alleviate prolonged genital bleeding. Uterine corpus myometrial tissues were also obtained from pregnant women in the first trimester (9–14 weeks of gestation; n = 6) at total hysterectomy due to uterine cervical cancer (n = 2), ovarian cancer (n = 1) and uterine myoma (n = 3) and from those in the third trimester (37–38 weeks of gestation; n = 6) complicated with uterine cervical cancer (n = 5) and uterine myoma (n = 1). The amnion, chorion laeve and decidua vera tissues were obtained at elective Caesarean delivery of two pregnant women (both 37 weeks of gestation). All samples were obtained with written informed consent. Tissues were immediately frozen in liquid nitrogen in blocks for protein and mRNA extraction and/or in OCT compound (Sakura Finetek Inc., USA) for immunostaining and kept at –80°C. Part of the myometrial tissues from three pregnant women in the first trimester (10, 12 and 13 weeks of gestation) were also utilized for establishment of the cultured human myometrial cells. The experimental protocol was approved by the ethics committee of Kyoto University Graduate School of Medicine.
RT–PCR analysis of ER, ERß and eNOS mRNA expression
Total RNA was extracted as previously described (Masuzaki et al., 1997). After reverse transcription of 5 µg of total RNA using oligo (dT) primer (Promega, USA) and SuperscriptTM II (GibcoBRL, USA), the resulting single-stranded cDNA was subjected to PCR. The forward and reverse primers used were: ER forward, 5'-TGTGCAATGACTATGCTTCA-3'; ER reverse, 5'-GCTCTT CCTCCTGTTTTTA-3' (Green et al., 1986); ERß forward, 5'-GTC CATCGCCAGTTATCACATC-3'; ERß reverse, 5'-GCCTTACATCCT TCACACGA-3' (Enmark et al., 1997); eNOS forward, 5'-GCACAG GAAATGTTCACCTAC-3' and eNOS reverse, 5'-CACGATGGTGAC TTTGGCTAG-3' (Miyahara et al., 1994). Forward and reverse primers for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding regions were purchased from Clontech Laboratories, Inc. (USA). The expected final PCR products from human ER, ERß and eNOS cDNA were 150, 242 and 645 bases respectively. The PCR programs used were: 27 cycles of 30 s at 94°C, 30 s at 54°C, 30 s at 72°C for ER; 32 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C for ERß; and 32 cycles of 30 s at 94 °C, 60 s at 55°C, 60 s at 72°C for eNOS. Final products were extended to full length by incubation for 6 min at 72°C.
Western blot analysis of ER, ERß, eNOS and iNOS
Protein extraction, sodium dodecyl sulphate–polycrylamide gel electrophoresis and immunoblotting were carried out as previously described (Yoshida et al., 2002). Positive control tissues used were human umbilical vein endothelial cells (HUVEC) for the eNOS-positive control and cultured human promyelocytic leukaemia cells (HL-60) after 24 h of stimulation with 10 nmol/l 12-O-tetradecanoylphorbol-13-acetate and 10 ng/ml interleukin-1 for the iNOS-positive control. The amount of protein loaded was 60 µg/lane for myometrial protein, 20 µg/lane for HUVEC protein, 20 µg/lane for stimulated HL-60-cell protein, 20 µg/lane (with and without ER transfection) and 200 µg/lane (concentrated by final 10% trichloroacetic acid after ERß transfection) for cultured human myometrial cell protein, and 6 µg of recombinant ER and ERß (The Protein Company, USA) according to the manufacturer’s recommendation. The antibodies used were a monoclonal antibody raised against human ER (1:1000 dilution; Transduction Laboratories, USA), a rabbit polyclonal antibody raised against human ERß (1:500 dilution; Santa Cruz Biotechnology, Inc., USA), a monoclonal antibody raised against human eNOS (1:250 dilution; Transduction Laboratories) and a monoclonal antibody raised against mouse iNOS, having cross-reactivity for human iNOS (1:1000 dilution; Transduction Laboratories). The expression of both eNOS and iNOS protein was expressed as arbitrary units (AU) based on quantitative densitometric analysis of the blots. In cases of analysis of >10 specimens, several blots were accessed by comparison with three identical non-pregnant myometrial tissues used as internal positive controls in each blot.
Immunohistochemistry of eNOS
After 1 h of incubation at room temperature with the same anti-human eNOS antibody used for Western blot analysis (1:100 dilution), staining was detected using an avidin–biotin–peroxidase (ABC) method kit (Elite ABC; Vector Laboratories, USA) with 3,3'-diaminobenzidine, as previously described (Yoshida et al., 2001). Human placenta was used as a positive control (data not shown).
Preparation of cultured human uterine myometrial cells
Myometrial tissues were obtained from three first trimester (10, 12 and 13 weeks of gestation) pregnant women as described above. The tissues were minced, placed into tubes containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, USA) and 0.2% collagenase (Wako, Japan) and incubated for 4 h at 37°C with continuous mixing, then filtered through a 40 µm cell strainer, diluted in an equal volume of DMEM, and centrifuged at 200 g for 10 min. The cells thus obtained were placed in 10–20 10 cm collagen-coated culture plates (3x105 cells for each plate). When the cells in each plate reached sub-confluency, they were frozen and stored in liquid nitrogen (as first passage cells) until used. Incubation was carried out in a humidified 5% CO2 95% air atmosphere at 37°C. Third passage cells were used in the experiments as cultured human myometrial cells, and showed 99 and 80% positive immunostaining for vimentin and -smooth muscle actin respectively, and <1% positive staining for cytokeratin.
Reporter gene analysis of eNOS with co-transfection of ER or ERß
eNOS promoter (–1600/+26)/luciferase vector (pGV-B2; Toyo Inc. MFG Co. Ltd, Japan) was prepared as previously reported (Nakayama et al., 1999). It has six half-palindromic sequences of the estrogen-response element, which form an effective estrogen-response element (Miyahara et al., 1994). Short eNOS promoter (–265/+26)/luciferase vector (pGV-B2) has no estrogen-response element and was used as a negative control. Human ER and ERß pSG5-SV40 vector were kindly supplied by Professor Pierre Chambon (CNRS, INSERM, University Louis Pasteur) (Kumar et al., 1987; Tora et al., 1989). The steroid hormones in the fetal calf serum (FCS) used were reduced by five rounds of charcoal treatment for a total of 48 h incubation at 4°C. The 17ß-estradiol concentration in medium supplemented with 10% charcoal-stripped FCS was <200 fg/ml, which was much lower than that without charcoal treatment, 1.6 pg/ml. Charcoal-treated FCS (final 10%) was added to phenol red free DMEM throughout the procedures of transfection and the subsquent experiments, because the transfection rate of the vectors as well as the survival rate of the cells was decreased when 1 or 5% FCS were used in a pilot study (data not shown). We transfected 0.5 µg/ml eNOS promoter/luciferase vector, 5 ng/ml pRL-SV40/Renilla (sea pansy) luciferase vector (Toyo) as well as 0.5 µg/ml ER or ERß pSG5-SV40 vector into confluent cultured human myometrial cells at the third passage in 6-well plates for 3 h using Lipofectamine PlusTM Reagent (Invitrogen Co., USA) according to the manufacturer’s recommendations, as previously described (Korita et al., 2002). After pre-incubation for 12 h, the medium was replaced with fresh DMEM supplemented with 10% FCS plus 0.4–400 ng/ml 17ß-estradiol and/or 0.2–20 µmol/l ICI 182780, a common antagonist of ER and ERß. After 24 h of incubation, the eNOS promoter activity was measured as the eNOS promoter luciferase activity/pRL-SV40 Renilla luciferase activity using a picagene dual sea-pansy assay system (Toyo) following the manufacturer’s instructions and expressed as arbitrary units of activity (AUA).
Statistical analysis
The statistical significance was assessed by Mann–Whitney U test for comparison of two groups and analysis of variance (ANOVA) followed by Fisher’s protected least significant difference test for comparison of more than three groups. P < 0.05 was regarded as significant.
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eNOS and iNOS expression in the intrauterine tissues of pregnant women
Western blot analysis of eNOS revealed clear 145 kDa bands in HUVEC and human pregnant myometrial tissues and only faint bands in chorion laeve (Figure 1A). The eNOS protein expression in the amnion and decidua vera tissues was below the limit of Western blot detection (Figure 1A). Western blot analysis of iNOS showed 135 and 150 kDa bands in stimulated HL-60 cells, as a positive control, and in human pregnant myometrial tissues respectively (Figure 1B), which was consistent with previous reports (Forstermann et al., 1991). Moreover, the 150 kDa iNOS band in the myometrial tissues was greatly reduced after adsorption of the iNOS antibody by mouse macrophage extract (data not shown). The iNOS protein expression was below the limit of Western blot detection in the amnion, chorion laeve and decidua vera tissues (Figure 1B). Thus, both eNOS (Figure 1A) and iNOS (Figure 1B) protein expression were observed, mainly in the myometrial tissues.
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eNOS and iNOS protein expression in the non-pregnant and pregnant myometrium
The level of eNOS protein expression in the myometrial tissues of pregnant women in the first trimester was 2.2 ± 0.4 arbitrary units (AU; n = 6), which was significantly higher than that of non-pregnant women, 1.4 ± 0.3 AU (P < 0.05; n = 6; Figure 1C). The level of eNOS protein expression in the myometrial tissues of pregnant women in the third trimester was 1.3 ± 0.1 AU (n = 6), which was significantly lower than that in the first trimester and was similar to the level in non-pregnant women (Figure 1C). The levels of iNOS protein expression in myometrial tissues in the first and third trimesters of pregnancy were 1.6 ± 0.5 AU (n = 6) and 1.6 ± 0.6 AU (n = 6) respectively, both of which were significantly higher than that in non-pregnant women, 0.4 ± 0.2 AU (n = 6) (P < 0.05 for both; Figure 1D).
The level of eNOS protein expression in the myometrial tissues of non-pregnant women with ethinylestradiol and norgestrel treatment was 2.1 ± 0.4 AU (n = 5), which was significantly higher than that in women without oral contraceptive pills, 1.1 ± 0.3 AU (P < 0.05; n = 5; Figure 1E). In contrast, iNOS protein expression levels in the myometrial tissues of non-pregnant women with and without ethinylestradiol and norgestrel treatment were similar (Figure 1F).
Immunohistochemistry of eNOS revealed positive staining in vascular endothelial cells, vascular smooth muscle cells and myometrial stromal cells in the first trimester pregnant myometrial tissues (Figure 2A). A negative control using normal mouse IgG (Dako Co., USA) showed greatly reduced staining (Figure 2B).
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ER
, ERß and eNOS expression in cultured human myometrial cellsER
and ERß protein expression in cultured human myometrial cells after ER and ERß transfection was confirmed by Western blot analysis (Figure 3A, C). Immunoblotting of the ERß protein after transfection showed only a weak band even after concentration of the protein extract, suggesting rather low ERß protein expression (Figure 3C). However, the transfection rate of both ER and ERß expression vectors estimated by the co-transfection of GFP expression vector was 
10% (K.Kakui, S.Yura, H.Itoh, N.Sagawa, unpublished findings). The mRNA and protein expression of ER (Figure 3A, B), ERß (Figure 3C, D) and eNOS (Figure 1A) was detected in the human myometrial tissues, from which the cultured human myometrial cells were prepared. Weak ER (Figure 3B) and very faint ERß (Figure 3D) mRNA expression was detected in the cultured human myometrial cells, although the protein expression of both ER and ERß was below the limit of Western blot detection (Figure 3A, C). The eNOS mRNA expression in the cultured human myometrial cells was below the limit of RT–PCR detection (data not shown).
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The effect of estrogen on the eNOS promoter activity in cultured human myometrial cells
The eNOS promoter activity was up-regulated by co-transfection of ER
(Figure 4A) or ERß (Figure 5A) even without 17ß-estradiol treatment, in agreement with the original reports about these ER expression vectors (Tora et al., 1989). In cells co-transfected with ER, the eNOS promoter activity with 4, 40 and 400 nmol/l 17ß-estradiol treatment was 11.4 ± 0.4, 14.0 ± 1.4 (P < 0.05) and 14.2 ± 0.9 (P < 0.05) AUA, which was significantly higher than that without 17ß-estradiol treatment (9.5 ± 0.7 AUA) (n = 3 for all, Figure 4A). With ER co-transfection, dose-dependent elevation of the eNOS promoter activity was observed by treatment with 4–40 nmol/l 17ß-estradiol. Such augmentation of eNOS promoter activity by 17ß-estradiol treatment was not noted when ER expression vector and short eNOS promoter vector lacking estrogen-response element were used (Figure 4B). In the presence of 40 nmol/l 17ß-estradiol, which approximately corresponds to the maternal plasma 17ß-estradiol concentration in the first–second trimester, the eNOS promoter activity was significantly and dose-dependently suppressed to 7.9 ± 1.7 and 6.1 ± 0.7 AUA by co-treatment with 0.2 and 2 µmol/l ICI 182780, a pure antagonist of both ER and ERß, as compared to the activity without ICI 182780 treatment, 10.2 ± 1.0 AUA (n = 3 and P < 0.05 for both, Figure 4C). Treatment with 2 µmol/l ICI 182780 completely blocked the augmentation of eNOS promoter activity by 40 nmol/l 17ß-estradiol treatment (Figure 4C).
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In cells co-transfected with ERß, the eNOS promoter activity with 0.4, 4, 40 and 400 nmol/l 17ß-estradiol treatment was 5.0 ± 0.2, 6.6 ± 0.3, 6.7 ± 0.6 and 6.8 ± 0.3 AUA respectively, which was significantly higher than the activity without the treatment, 3.8 ± 0.2 AUA (n = 3 and P < 0.05 for all, Figure 5A). Dose-dependent elevation of eNOS promoter activity in cells co-transfected with ERß was observed by treatment with 0.4–4 nmol/l 17ß-estradiol. Such enhancement of eNOS promoter activity by 17ß-estradiol treatment was not noted when ERß expression vector and short eNOS promoter vector lacking an estrogen-response element were used (Figure 5B). In the presence of 40 nmol/l 17ß-estradiol, the eNOS promoter activity in cells co-treated with 0.2, 2 and 20 µmol/l ICI 182780 was 5.2 ± 1.1, 4.5 ± 0.1 and 4.0 ± 0.1 AUA respectively, which was significantly and dose-dependently lower than the activity without ICI 182780 treatment, 6.7 ± 0.5 AUA (n = 3 for all, Figure 5C). Treatment with 20 µmol/l ICI 182780 completely blocked the augmentation of eNOS promoter activity by 40 nmol/l 17ß-estradiol (Figure 5C).
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Discussion |
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In the present study, eNOS protein expression was more prominent in myometrial tissues than in amnion, chorion laeve, and decidua vera tissues from third trimester pregnant women. Similarly, iNOS protein was also more abundant in myometrial tissues than in amnion, chorion laeve, and decidua vera tissues from third trimester pregnant women. On the other hand, our pilot study suggested that nNOS protein and mRNA expression in intrauterine tissues was below the limit of detection (K.Kakui, H.Itoh, N.Sagawa, unpublished findings). These distribution patterns of NOS isoforms in intrauterine tissues during human pregnancy are not completely consistent with those previously reported (Bansal et al., 1997; Thomson et al., 1997; Bartlett et al., 1999; Norman et al., 1999). Based on the results of the present study, we suggest that eNOS protein as well as iNOS protein is most abundantly expressed in the myometrium amongst intrauterine tissues of pregnant women.
In the present study, we also measured eNOS and iNOS protein expression in the myometrial tissues of non-pregnant and pregnant women in the first and third trimesters of pregnancy. The protein expression of eNOS was elevated in the first trimester of gestation and declined to a level similar to that in non-pregnant women in the third trimester of gestation before the onset of labour. Since eNOS up-regulation by estrogen was reported in ovine (Zhang et al., 1999) and rat (Yallampalli et al., 2000) myometrial tissues, we speculate that the elevation observed in the present study may be an estrogen effect. On the other hand, iNOS protein expression was increased in both the first and third trimesters of gestation compared to that in non-pregnant women. Since it was difficult to obtain myometrial tissues after medication with only estrogen derivatives, we examined eNOS and iNOS protein expression in the myometrial tissues obtained at hysterectomy of non-pregnant women treated with daily oral contraceptive pills containing both estrogen and progestogen derivatives. Interestingly, oral contraceptive pill treatment significantly elevated the eNOS protein expression, but not iNOS protein expression, in the myometrial tissues of non-pregnant women, suggesting that estrogen and/or progestogen may up-regulate eNOS protein expression in the myometrium.
Recently, Aggelidou et al. (2002) reported that corticotrophin-releasing hormone contributes to augmentation of eNOS expression in the human pregnant myometrial cells. However, we focused in this study on the effect of estrogen on eNOS expression in the myometrium in view of previous reports of estrogen-induced eNOS expression in the vascular endothelium (MacRitchie et al., 1997; Vagnoni et al., 1998; Tan et al., 1999; Yang et al., 2000) in addition to the observation in the present study of oral contraceptive pill-induced up-regulation of eNOS protein expression. Moreover, eNOS immunostaining was detected widely in myometrial stromal cells, vascular endothelial cells, and vascular smooth muscle cells. Therefore cultured human myometrial cells were prepared and used in in vitro studies. In these cultured human myometrial cells, only weak ER expression was retained concomitant with almost complete disappearance of ERß expression, in contrast with their stronger expression in the pregnant myometrial tissues which were used to establish the cultured human myometrial cells. When we measured eNOS promoter activity with 0.4–40 nmol/l 17ß-estradiol treatment in the cultured human myometrial cells without co-transfection of ER nor ERß, the response was equivocal (data not shown). Moreover, the ER and ERß expression vectors (kind donations from Professor Pierre Chambon) used in this study have been widely used in co-transfection studies with various kinds of promoter vectors with estrogen-response elements to investigate promoter activities (Routledge et al., 2000; Zhu et al., 2001; Yamashita et al., 2003). Therefore, we used the cultured human myometrial cells after co-transfection of ER or ERß.
Treatment with 4–40 nmol/l 17ß-estradiol dose-dependently augmented the eNOS promoter activity in the cells with ER co-transfection. Similarly, treatment with 0.4–4 nmol/l 17ß-estradiol dose-dependently augmented the eNOS promoter activity in the cells with ERß transfection. Short eNOS promoter vector lacking estrogen-response element did not show such up-regulation by 17ß-estradiol treatment, suggesting possible direct effects of transfected ER or ERß on estrogen-response elements of eNOS promoter. The elevation of eNOS promoter activity by 40 nmol/l 17ß-estradiol treatment in the transfected cells was dose-dependently suppressed by treatment with ICI 182780, a common antagonist of ER and ERß, in both ER and ERß studies, with complete suppression at high concentrations of ICI 182780. These in vitro findings demonstrated that both ER and ERß augment eNOS promoter activity in cultured human myometrial cells. These data suggest the possibility that estrogen might augment eNOS expression in the myometrial tissues at the transcriptional level via both ER and ERß during pregnancy. However, a short eNOS promoter vector, used as a negative control, also lacks numerous other transcriptional initiation sites by the deletion procedure, such as activator protein-2 (AP-2) site, T cell factor-1 (TCF-1) site, nuclear factor kappa B (NF
B) site and so on (Miyahara et al., 1994). Since AP-2 site (Guccione et al., 2002), TCF-1 site (El-Tanani et al., 2001) and NFB site (Deshpande et al., 1997) were reported to be potentially involved in the cell signal tranasduction associated with estrogen treatment, the present data are not direct proof that the estrogen receptors interact directly with the several half palindromic sequences of estrogen-response elements on the human eNOS promoter region. Therefore, more detailed mutational analysis of eNOS promoter region in transcription and in vitro protein binding studies will have to be performed to clarify this point.
Since the cultured human myometrial cells used in this study had lost not only the ER but also eNOS expression, we cannot rule out the possibility that various intracellular co-factors and/or nuclear co-regulator proteins might also have been lost in the culture procedure. Moreover, recent studies have revealed that heterodimer formation between ER and ERß also plays an important role in the regulation of transcription (Gustafsson et al., 1999; Gray et al., 2001), which cannot be accessed by this culture model. In the present study, the eNOS promoter activity was up-regulated by either ER or ERß co-transfection even without 17ß-estradiol treatment. We reduced the 17ß-estradiol concentrations in the FCS to <200 fg/ml by intensive charcoal treatment. Furthermore such activation of transfected ER without 17ß-estradiol treatment was also reported in the original publication on these ER expression vectors (Tora et al., 1989). At present we cannot fully explain the mechanism of such activation but suggest that growth factors in the FCS might activate ER and/or ERß, because growth factor-associated ER activation was previously reported in other cells, especially in the case of low serum estrogen concentrations (Karas et al., 1998; Mendelsohn et al., 1999). Furthermore, it is possible that even very low concentrations of 17ß-estradiol, <200 fg/ml, might be able to affect ER activation, augmenting the basal eNOS promoter activity. Although the induction of eNOS promoter activities by treatment with 17ß-estradiol was significant, the magnitude of augmentation was not so prominent. At present, we have no clear explanation for such a low response of eNOS transcription in this culture model. However, no significant elevation of the eNOS promoter activities was observed when short eNOS promoter lacking estrogen-response element was used. These data strongly suggest that such induction of eNOS promoter activities by estradiol, even small augmentation, was caused directly by ER or ERß. In any case, the cultured human myometrial cells used in this in vitro culture model had lost some physiological characteristics of the in vivo response of human myometrium to estrogen exposure. Nevertheless, we recently demonstrated that the cultured human myometrial cells at third passage used in the present study retained the feature of pregnancy-induced augmentation of prostacyclin production (Korita et al., 2002). Moreover, the presence of cell-type-specific nuclear co-regulator proteins was recently postulated to be a major contributor to a variety of cell-type-specific responses to estrogen stimulation in various kinds of tissue (Enmark et al., 1999; Gustafsson et al., 1999; Mendelsohn et al., 1999; Gray et al., 2001), indicating the potential importance of utilizing the cells from human pregnant myometrium as an in vitro experimental model. Based on these facts, we suggest that our culture model is useful, despite the potential limitations of the interpretation of the results, for investigating human myometrium-specific reactions to estrogen during pregnancy.
A stimulatory effect of estrogen on eNOS expression in the myometrium was demonstrated in sheep (Figueroa et al., 1995; Zhang et al., 1999) and rats (Yallampalli et al., 2000), while an inhibitory effect on NOS activity was reported in rabbits (Batra et al., 1998). As for human studies, Khorram et al. (1999) reported that eNOS expression in the myometrium was highest in the late secretory phase, while Telfer et al. (1997) demonstrated no change during the menstrual cycle. Weeks et al. (1999) reported that GnRH treatment had no effect on the eNOS expression in the myometrium of non-pregnant women, while Zervou et al. (1999) found that eNOS expression in the myometrial tissues of non-pregnant women with menorrhagia was elevated by treatment with progesterone and 17ß-estradiol. Bartlett et al. (1999) demonstrated that eNOS is unlikely to play any role in the regulation of myometrial tonus during pregnancy. Thus, the reported effects of estrogen and/or progesterone on eNOS expression in myometrial tissues remain controversial. To our knowledge, the present study is the first to provide direct in vitro evidence of a possible involvement of estrogen in the pregnancy-associated elevation of eNOS expression in human myometrial cells in a receptor-mediated manner. Taking all the findings together, it may be suggested that during early pregnancy estrogen might elevate eNOS expression in the myometrial tissues via ER and/or ERß, and that this elevation of eNOS may play some roles in the maintenance of the pregnancy, such as maintaining uterine quiescence (Cunningham et al., 2001), increasing uterine blood flow (Magness et al., 1997) and other effects. Such estrogen-associated modulation of myometrial eNOS expression cannot explain the decrease in eNOS protein expression in the myometrial tissues in the third trimester of pregnancy. The mechanism of down-regulation of eNOS expression before labour onset will be addressed in future investigations.
In summary, the present study revealed that high levels of eNOS and iNOS protein expression were observed in human myometrial tissues as compared with other intrauterine tissues from pregnant women. The eNOS protein expression was elevated both in pregnant women during the first trimester of gestation and in non-pregnant women after oral contraceptive pill medication. In vitro studies revealed that both ER and ERß contribute to the 17ß-estradiol-induced augmentation of eNOS promoter activity in cultured human myometrial cells. These findings suggested a possible involvement of both ER and ERß in the regulation of eNOS expression in the human pregnant myometrium, although further intensive studies will be necessary to verify this possibility.
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The authors acknowledge Ms Akiko Kishimoto and Ms Akiko Abe for secretarial and technical assistance. We thank Professor Ronald R.Magness, PhD, Department of Obstetrics and Gynecology Perinatal Research Laboratories and Department of Animal Sciences, University of Wisconsin–Madison, for thoughtful comments regarding preparation of the manuscript. This work was supported in part by Grants-In-Aid for the Scientific Research from the Ministry of Education, Science, and Culture, Japan (No. 13470352, 13557139, 13671707, 14704042, 14657419), a grant from the Ministry of Health and Welfare, and grants from the Smoking Research Foundation and the Kanzawa Medical Research Foundation.
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Submitted on August 1, 2003; resubmitted on October 24, 2003; accepted on November 13, 2003.
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