2024 Volume 71 Issue 2 Pages 199-206
Endometriosis, a common gynecological disorder characterized by the growth of endometrial gland and stroma outside the uterus, causes several symptoms such as dysmenorrhea, hypermenorrhea, and chronic abdominal pain. 17β estradiol (E2) stimulates the growth of endometriotic lesions. Although estetrol (E4), produced by human fetal liver, is also a natural estrogen, it may have the opposite effects on endometriotic cells. We investigated different effects of E4 and E2 on the invasion and migration of immortalized human endometrial stromal cells (HESCs) and evaluated whether E4 affects the expression of Wiskott-Aldrich syndrome protein (WASP) family member 1 (WASF-1). We measured the invasion of HESCs by a Matrigel chamber assay. Cell migration was measured by wound healing assay and cell tracking analysis. The expression of WASF-1 was confirmed by independent real-time PCR analysis. Transfection of cells with siRNAs was carried out to knock down the expression of WASF-1 in HESCs. E4 significantly inhibited E2-induced invasion and migration of HESCs. WASF-1 was found to be a potential mediator based on metastasis PCR array. WASF-1 was upregulated by E2 and downregulated by E4. Knockdown of WASF-1 inhibited migration. Our results suggest that E4 may inhibit E2-induced growth of endometriotic lesions. Downregulation of WASF-1 is involved in the inhibitory effects of E4 on migration. The use of E4 combined with progestins as combined oral contraceptives may cause endometriotic lesions to regress in women with endometriosis.
ENDOMETRIOSIS, a gynecological chronic disease, is characterized by the growth of endometrial tissue outside the uterine cavity, has characteristic symptoms including dysmenorrhea, hypermenorrhea, and chronic abdominal pain, and is often accompanied by infertility. Endometriosis is diagnosed in up to 10% of premenopausal women and 20–50% of infertile women. Endometriosis is classified into 3 categories, ovarian endometriomas, superficial peritoneal endometriosis, and deep infiltrating endometriosis, based on the location. In addition, endometriosis is frequently present in the myometrium. Endometriosis is more common in the ovaries, fossa ovarica, uterosacral ligaments, posterior cul-de-sac, rectum and sigmoid, and less frequently in the pericardium, pleura and brain.
Circulating estrogen reaches endometriotic tissues, but it is also produced locally in endometriotic lesions. Local estrogen accumulation has been considered to play an important role in the development and progression of endometriotic lesions by binding and activating estrogen receptors [1]. Estrogen synthesis is upregulated in endometriotic tissue because of high expression of two enzymes, aromatase and 17β-hydroxysteroid dehydrogenase-1, and a reduction in 17β-hydroxysteroid dehydrogenase type 2, which converts E2 to the less potent estrone [2-4].
Estetrol (E4) is produced by the human fetal liver, reaches maternal circulation through the placenta and can be detected in amniotic fluid and maternal urine [5-7]. E4 is synthesized during pregnancy from E2 and estriol by two fetal liver enzymes, 15 and 16 α-hydroxylase, through hydroxylation. Plasma E4 levels increase during pregnancy and show a peak at term, with fetal levels approximately 10–20 times higher than those in the mother. After delivery, plasma levels of E4 rapidly decrease and become undetectable [8-10]. Although E4 is a natural human estrogen, there are several different effects from other estrogens. E4 may have agonistic or antagonistic activity on estrogen receptors. E4 has estrogen agonistic effects on bone, the brain, the vagina, and the endometrium, while it acts as an estrogen antagonist by preventing dimethylbenz(a)anthracen-induced breast tumor initiation and the growth of existing tumors in the presence of E2 [11]. Compared with E2, E4 possesses two additional hydroxyl groups in the five-membered ring of steroids. Since one of the two hydroxyl groups forms an additional bond to the estrogen receptor, the binding affinity of E4 is higher than that of E2 [12]. However, the effect of E4 on downstream signaling may be much weaker than that of E2. Therefore, E4 appears to be an ideal antagonist of the estrogen receptor based on the structural analysis. In fact, it inhibits the migration and invasion of breast cancer cells [13]. Thus, E4 can be classified as a natural estrogen with selective action in tissues [14].
E2 has been established to promote the invasion and migration of eutopic endometrial stromal cells in women with endometriosis [15], but E4 may have different effects on these cells.
We previously established a cellular endometriosis model using immortalized human endometrial stromal cells (HESCs). Employing this model we reported recently that the NF-κB inhibitor DHMEQ inhibited MLCK-dependent migration and invasion of HESCs [16]. Tissue and cell movement are involved in the etiology of endometriosis, therefore, cell migration/invasion were examined in our E4 study. In the present study, we investigated the effects of E4 and E2 on the invasion and migration of HESCs. In addition, we evaluated whether E4 affects the expression of Wiskott-Aldrich syndrome protein (WASP) family member 1 (WASF-1), a member of the WASP family, which is associated with cellular migration [17].
E4 and E2 were purchased from Sigma. HESCs were purchased from Applied Biological Materials Inc. (Vancouver, Canada). These cells express IGFBP-1, PRL, TF, and PAI-1 as markers for decidualization endpoints (Supplier’s manual). The cells were grown in Prigrow IV medium (Abm, Richmond, Canada) supplemented with 10% charcoal-stripped fetal bovine serum (Gibco, Grand Island, NY), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY) in a humidified atmosphere with 5% CO2 at 37°C.
Cell viability assayHESCs (5 × 103) were seeded in 96-well microplates and incubated for 24 hrs. E4 or E2 were then added to each well and the cells incubated for another 24 hrs. MTT solution (10 μL; Cayman Chemical Company) was added to each well, followed by incubation for 2 hrs in the incubator. Subsequently, 100 μL of DMSO was added to each well to replace the culture supernatant and dissolve purple formazan crystals. Then, the absorbance of the samples was measured at 570 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Matrigel chamber invasion assayHESCs (4 × 104) were suspended in 500 μL of serum-free medium containing chemicals or DMSO and seeded into the upper chambers coated with BD Matrigel Basement Membrane Matrix (Corning Inc., Corning, NY). The lower chambers were filled with 750 μL of medium containing 10% FBS and incubated for 24 hrs at 37°C in an incubator. After incubation, non-invading cells were removed by wiping with a cotton swab from the upper surface of the membrane. Invading cells on the lower surface of the membrane were stained with Diff-Quick (Sysmex, Kobe, Japan) and counted.
Wound healing assayCells (1 × 105) were seeded in 24-well plates until confluent. Confluent cells in 24-well plates were uniformly scratched across the center of each well with a 200-μL pipette tip. The wells were then rinsed twice with serum-free media to remove floating cells and growth media, and then the cells were cultured in serum-free media for 8 hrs. The wound areas at the initial and end time endpoints were recorded as the movement of the cells into the scratched area. Each determination was performed in triplicate in three independent experiments.
Cell tracking analysisApproximately 500 cells were seeded in 96-well ImageLock plates (Essen BioScience, Ann Arbor, MI) precoated with collagen type I (Sigma, Saint Louis, MO). Images were taken every 15 minutes continuously for 24 hrs using IncuCyte ZOOM (Essen BioScience, Ann Arbor, MI). The trajectories of motile cells were tracked manually using ImageJ (NIH, Bethesda, MD) along with a plugin for manual tracking (Fabrice Cordelieres, Institute Curie, Orsay, France).
Metastasis PCR arrayTotal RNA was extracted from HESCs using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was carried out with an RT2 First Strand Kit (Qiagen, Germantown, MD). cDNA was added to the qPCR Master Mix and the aliquot mixture across the Human Tumor Metastasis PCR Array (Qiagen, Germantown, MD). Data analysis was carried out by the comparative Ct method.
RNA isolation and quantitative RT-PCR analysisTotal RNA was extracted from cultured cells using TRIzol reagent (Life Technologies, Carlsbad, CA). Reverse transcription was carried out at 37°C for 120 min with a High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). The prepared cDNA was used for PCR amplification with Quick Taq® HS DyeMix (Toyobo, Tokyo, Japan). The primer, number of PCR cycles, and annealing temperature were as follows: WASF-1, 5'-AGCACTGCCTAGAGGCATTA-3' (forward) and 5'-CCACACGTTCTTGCAATGAG-3' (reverse), 32 cycles, 58°C; β-actin, 5'-CTTCTACA ATGAGCTGCGTG-3' (forward) and 5'-TCATGAGGTA GTCAGTCAGG-3' (reverse), 21 cycles, 58°C. PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and visualized with a UV illuminator.
Knockdown of WASF-1 by siRNAsiWASF-1 (sc-36831) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX). Transfection of cells with siRNAs was carried out using Lipofectamine RNAiMax transfection reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The efficiency of transfection was determined by mRNA expression.
Statistical analysisAll experiments were performed independently at least three times. Data are expressed as the mean ± SEM. Differences between groups were examined for significance with ANOVA and/or Student’s t-test where appropriate.
We first studied the effect of E2 and E4 on the proliferation of HESCs. Neither E2 nor E4 influenced the proliferation of HESCs (data not shown). The HESCs have been treated with virus to activate the growth activity. Then, it is likely that the growth property of this cell line is saturated before the addition of E2.
Next, we studied the effects of E2 and E4 on the invasion of HESCs. E4 is often used at 10–9 M (0.3 ng/mL) in cellular experiments. As shown in Fig. 1A, E2 enhanced the invasion of HESCs at 10–9 M (0.3 ng/mL) for 16 hrs and 24 hrs. E4 at 10–10 M (0.03 ng/mL), 10–9 M (0.3 ng/mL), and 10–8 M (3 ng/mL) inhibited invasion in 16 hrs (Fig. 1B). E4 inhibited invasion even in HESC growth medium-starved cells without E2 for 16 hrs (Fig. 1C).
Activation and inhibition of HESC invasion by E2 and E4, respectively. A. Increase in cellular invasion by E2, time course. The cells were incubated for 8 hrs, 16 hrs and 24 hrs. B. Inhibition of invasion by E4. The cells were incubated for 16 hrs. C. Inhibition of invasion by E4 in serum-starved HESCs. The cells were starved for 16 hrs without HESC growth medium, and then, E4 was added for another 16 hrs. Growth medium was added only in the lower chamber to induce invasion. Invasion was measured by Matrigel chamber assay.
*, p < 0.05; **, p < 0.01; ***, p < 0.001
E2, 17β estradiol; E4, estetrol; HESCs, immortalized human endometriosis stromal cells
We used a wound healing assay and employed cell tracking analysis to measure the migration of HESCs. In the wound healing assay, E2 at 10–9 M (0.3 ng/mL) enhanced the migration in 8 hrs, but the addition of E4 at 10–9 M (0.3 ng/mL) significantly inhibited E2-induced increases in migration (Fig. 2A). In the cell tracking analysis, E2 enhanced mobility at each time point until 24 hrs, and these movements were inhibited by the addition of E4 (Fig. 2B). Thus, the results of both techniques showed that E2 significantly increased migration, but the addition of E4 significantly inhibited E2-induced increases in migration.
Inhibition of migration by E4 in HESCs. A. Activation by E2 and inhibition by E4 of cellular migration. The cells were incubated for 8 hrs, and migration was measured by wound healing assay. B. Activation and inhibition of cellular migration by E2 and E4, respectively. Each cell movement is shown in color. Mean distance after the movement (bar graph, n = 8). Images were taken every 15 minutes continuously for 24 hrs.
*, p < 0.05; **, p < 0.01
E2, 17β estradiol; E4, estetrol; HESCs, immortalized human endometriosis stromal cells
Next, we looked for the mediator in the inhibition of invasion and migration by E4. E2 was added to the cells for 24 hrs with or without E4. PCR array analysis is a comprehensive gene expression analysis, and we employed a metastasis PCR array in which metastasis-related genes are collected. Comparatively, there were changes in expression of small number of genes. Above all, the expression of WASF-1 decreased with the addition of E4 (Table 1).
Cell Mobility Array analysis with HESCs.
| Gene | E2 + E4/E2 | Description | |
|---|---|---|---|
| BCAR1 | 1.90 | Breast cancer anti-estrogen resistance 1 | cell adhesion, migration, invasion, apoptosis, hypoxia and mechanical forces |
| DIAPH1 | 2.02 | Diaphanous homolog 1 (Drosophila) | |
| PTPN1 | 1.95 | SH3 and PX domains 2A | |
| TLN1 | 2.02 | Talin 1 | affecting adhesion, trans-endothelial migration and the invasion stages. linking integrins to the actin cytoskeleton either directly or indirectly by interacting with vinculin and α-actinin |
| WASF1 | 0.43 | WAS protein family, member 1 | regulating the actin cytoskeleton required for membrane ruffling |
Metastasis PCR array analysis of HESCs treated with E2 and E4. The cells were added by 10–9 M estradiol with or without 10–9 M E4 for 24 hrs. Value >1 shows an increase of expression, while value <1 shows a decrease of expression.
E2, 17β estradiol; E4, estetrol; HESCs, immortalized human endometriosis stromal cells.
E2 significantly enhanced the expression of WASF-1 (Fig. 3A). Downregulation of WASF-1 by E4 was confirmed by independent real-time PCR analysis in a concentration-dependent manner (Fig. 3B). WASF-1 expression was decreased by siRNA (Fig. 3C). Moreover, knockdown of WASF-1 by siRNA significantly inhibited cellular migration in HESCs either with or without E2 (Fig. 3D). The addition of E4 further inhibited migration in WASF-1 knockdown cells (Fig. 3D).
Inhibition of cellular migration by siRNA knockdown of WASF-1. A. Activation of WASF-1 expression by E2 in PCR analysis. The cells were incubated for 24 hrs. B. Inhibition of WASF-1 expression by E4 in an independent PCR analysis. The cells were incubated for 24 hrs. C. Knockdown of WASF-1 by siRNA. The amount of WASF-1 is shown by PCR analysis. D. Inhibition of migration by knockdown of WASF-1. The cells were incubated for 8 hrs.
*, p < 0.05; **, p < 0.01; ***, p < 0.001
E4, estetrol; WASF-1, Wiskott-Aldrich syndrome protein family member 1
In this study, the cell invasion assay demonstrated that E2 increased the invasion of HESCs. We also evaluated cell migration by using 2 techniques: wound healing assay and cell tracking analysis. Similar to the cell invasion assay, both techniques showed that cell migration was increased by the addition of E2. E2 is essential for endometrial tissue attachment to the peritoneum, lesion survival, production of inflammatory substances, such as metalloproteinases, cytokines, prostaglandins and growth factors, and angiogenesis. Local E2 levels are increased in women with endometriosis because of upregulation of the aromatase gene and 17β-hydroxysteroid dehydrogenase-1 and downregulation of 17-hydroxysteroid dehydrogenase type 2 [2-4]. Elevated E2 levels may result in enhanced E2 binding and activation of estrogen receptors (ERs) in endometriotic tissues.
In contrast to E2, the addition of E4 inhibited the invasion of HESCs with and without E2. E4 inhibited E2-induced invasion of HESCs in a concentration-dependent manner. Similar to the cell invasion assay, E4 inhibited E2-induced migration of HESCs in both techniques. These inhibitory effects of E4 on the invasion and migration of HESCs may counteract the E2-induced progression of endometriotic tissues in women with endometriosis.
For the mechanistic study, we employed PCR array analysis. We selected WASF-1 from the results of array analysis for the mediator, because it is downregulated by E4 and is reported to be related to the membrane raffling which is related to the cellular movement. WASF-1 is highly expressed in the brain, testes, ovary and endometrium (NIH National Library of Medicine, www.ncbi.nlm.nih.gov/gene/8936). WASF-1 plays a critical role downstream of Rac, a Rho-family small GTPase, in regulating the actin cytoskeleton required for membrane ruffling [17, 18]. WASF-1 has been shown to associate with an actin nucleation core Arp2/3 complex while enhancing actin polymerization in vitro. Wiskott-Aldrich syndrome is a disease of the immune system that is likely due to defects in the regulation of the actin cytoskeleton. It was reported that E2 administration to rat cortical neurons leads to the phosphorylation of WASF-1 on serine residues 310, 397, and 441 and to WASF-1 redistribution toward the cell membrane at sites of dendritic spine formation [19]. The phosphorylation was mediated by the small GTPase Rac1. In the present study, E4 decreased the expression of WASF-1 in a dose-dependent manner. Since membrane ruffling is essential for cellular migration, our results indicate that downregulation of WASF-1 by E4 is one of the mechanisms of the inhibitory effects of E4 on the migration of HESCs. It is possible that E4 blocks E2-induced Rac1 action to regulate WASF-1 in HESCs. Next, we evaluated the effects of E4 on the migration of WASF-1 knockdown HESCs, and the results showed that E4 further inhibited migration. These results suggest that other pathways are likely to be involved in the mechanism of the inhibitory effects of E4 on migration.
ERs such as nuclear receptor family members like ERα and β and membrane estrogen receptors such as G protein-coupled estrogen receptor 1 (GPER) are the principal mediators of estrogen functions. Regarding ERα binding, E4 and E2 have similar binding characteristics and act as agonists for nuclear ER [12]. Although E4 activates the nuclear ER, it has antagonist effects on the membrane ER that are different from those of other estrogens [12, 20, 21]. E2 enhances the migration and invasiveness of human T47D breast carcinoma cells, while E4 reduces the stimulatory effects of E2 on breast cancer cell cytoskeletal rearrangement, migration, and invasion [13]. Therefore, the inhibitory effects of E4 on the migration, and invasion of breast cancer cells may be due to its antagonistic effects on membrane ER. Based on this evidence, the antagonistic effects of E4 on GPER may be associated with the inhibitory effects of E4 on the E2-induced invasion and migration of HESCs in the present study.
Combined oral contraceptives (COCs) contain an estrogen and a progestin component, and the most commonly used estrogen is ethinylestradiol (EE). COCs are commonly used in women with endometriosis to improve dysmenorrhea and reduce the monthly menstrual blood volume. A systematic review and meta-analysis demonstrated that the risk of endometriosis was lower in current COC users [22]. Progestins may prevent the implantation and growth of regurgitated endometrium and inhibit the expression of matrix metalloproteinases and angiogenesis [23]. Moreover, progestins have several anti-inflammatory effects that may reduce the inflammatory state generated by the metabolic activity of the ectopic endometrium and the consequent immune response [23]. Although estrogens are used to regulate menstrual bleeding and to reduce follicle development, concurrent use of EE may counteract the beneficial effects of progestin on endometriotic cells. The inhibitory effects of E4 on the invasion and migration of HESCs in the present study revealed that the use of E4 instead of EE combined with progestins may further suppress the growth of endometriosis.
The results of the present study showed that E4 inhibits E2-induced invasion and migration of HESCs. In addition, E4 also inhibits the expression of WASF-1, and this downregulation of WASF-1 may be partially involved in the inhibitory effects of E4 on the migration of HESCs. The precise mechanisms by which E4 inhibits the invasion and migration of endometriotic cells are unclear. Further studies are needed to clarify whether E4 inhibits the growth of endometriotic lesions in women with endometriosis.
This work was financially supported in part by JSPS Kakenhi (grant no. 22H03062) and the AMED grant (grant no. JP18fk0310118JSPS).
This study was conducted without any commercial supportive grant. K. U. belongs to a fund-donated laboratory. It is supported financially by Shenzhen Wanhe Pharmaceutical Company (Shenzhen, China), Meiji Seika Pharma (Tokyo, Japan), Fukuyu Medical Corporation (Nisshin, Japan), and Brunaise Co., Ltd. (Nagoya, Japan). Other authors declare no conflict of interest. All authors contributed significantly, read the final version of the manuscript, and approved this submission.
