TOB1 modulates the decidualization of human endometrial stromal cells via the Notch pathway.

Background Decidualization is critical for embryo implantation and the success of pregnancy; however, the mechanisms underlying this process remain largely unknown.

In the present study, RNA sequencing was used to detect the expression levels of transducer of ERBB2/1(TOB1) in endometrial samples derived from proliferative and secretory phases. A decidualization model was induced using the combination of estrogen (E2) and progestin (P4) in human endometrial stromal cells (HESCs). The cell counting kit-8 assay was used to detect the viability of HESCs. Related proteins were detected by qPCR and western blot.

The results indicated that TOB1 expression was upregulated in the secretory endometrial samples compared with the corresponding expression observed in the proliferative samples. The expression levels of TOB1 and Notch1 were markedly increased in E2P4-treated HESCs compared with those in the control cells. Treatment with E2P4 strongly suppressed the proliferation of HESCs and induced a G1-phase cell cycle arrest. These effects were abolished by knockdown of TOB1 or treatment with of the cells with the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester.

Therefore, these findings highlighted an important role for TOB1/Notch signaling in E2P4-induced decidualization in HESCs, which may provide novel targets for improving the endometrial receptivity. Supplementary Information The online version contains supplementary material available at 10.1007/s10815-021-02277-z.

Human endometrial stromal cells, Decidualization, Transducer of ERBB2/1, Notch1

Despite the significant advances in assisted reproduction techniques, a low embryo implantation rate remains the major rate-limiting step in in vitro fertilization success [1]. A viable embryo and an appropriately decidualized endometrium are two indispensable conditions required for successful implantation [2, 3]. Decidualization is a differentiation process of endometrial stromal cells into secretory cells during the menstrual cycle. It is primarily determined by sex steroids and involves various changes in cellular morphology and function [4, 5]. Increasing evidence suggests that an unreceptive endometrium has a higher impact on reproductive disorders than the embryonic quality [5, 6]. Therefore, understanding the molecular mechanism of endometrial decidualization is essential to increase the possibility of pregnancy. Transducer of ERBB2.1 (TOB1) is a member of the TOB/B cell translocation gene (BTG) family and is distributed extensively throughout the cytoplasm and nucleus of germ cells [7]. TOB1 and transducer of ERBB2 2 (TOB2) include the longest C-terminal region among the TOB/BTG protein family. These proteins are involved in protein-protein interactions and play a vital role in transcription, mRNA turnover, and cell proliferation [8, 9]. TOB1 can maintain cells in a quiescent state via downregulation of cyclin-dependent kinase expression, which leads to an antiproliferative effect [10]. Chen et al. [11] reported that TOB1 inhibited the proliferation of mouse embryonic stem cells via the degradation of Id3 mRNA. TOB genes also play an important role in germ cell differentiation and embryonic development [7]. However, the mechanism underlying the role of TOB1 in endometrial decidualization remains largely unknown. Notch is a cell-surface receptor that transduces short-range signals by interacting with transmembrane ligands and regulates cell proliferation, differentiation, and cell death [12]. Notch signaling is critical for endometrium decidualization in both human and mouse cells [13, 14]. It has been reported that inhibition of Notch signaling in the endometrium of women with endometriosis can impair decidualization [14]. Suppression of Notch signaling reduces deciduoma in a murine artificial decidualization model, confirming the important role of the Notch pathway in successful decidualization [15]. The estrogen receptor (ER) and the progesterone receptor (PR) play a pivotal role in decidual differentiation. Lack of these receptors impairs implantation and decidualization [16]. The estradiol (E2)/progestin (P4)-induced decidualization model is commonly used in decidual studies [17, 18]. In the present study, we sought to investigate the function and mechanism of TOB1 in E2P4-induced decidualization with human endometrial stromal cells (HESCs).

Antibodies and reagents Anti-TOB1 (AF9216, 1:1000) antibody was purchased from Affinity; anti-prolactin (ab188229, 1:1000), anti-insulin-like growth factor–binding protein 1 (ab228741, 1:1000), anti-Notch1 (ab52627, 1:1000), anti-cyclin D1 (ab16663, 1:1000), anti-cyclin E1 (ab208696, 1:1000), anti-CDK2 (ab32147, 1:1000) antibodies, and anti-GAPDH (ab181602, 1:1000) were purchased from Abcam. Anti-mouse or anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Alexa Fluor488-conjugated secondary antibody was from Cell Signaling Technology. N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) was acquired from Sigma-Aldrich. Tissue collection Endometrial specimens from different phases of the endometrial cycle were obtained from 20 female patients with normal body mass index (aged 29–42; weight, 50–60 kg; proliferative phase, n = 10; secretory phase, n = 10) who underwent surgical operation (June 2018—June 2019) at the Third Affiliated Hospital of Guangzhou Medical University, with the permission of the ethical committee of the Third Affiliated Hospital of Guangzhou Medical University (No. 2018PGY260K), and informed written consent was obtained from all patients. Cellular structure of proliferative and secretory endometria in the menstrual cycle was distinguished by pathological sections. Cell culture The telomerase-immortalized HESCs were obtained from the American Type Culture Collection (ATCC), and the cells were cultured with DMEM/F12 (Thermo Fisher Scientific) supplemented with 2% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. For in vitro decidualization of HESCs, HESCs were divided into two groups (the control and E2P4 groups) with 3 wells per group. At 80~90% confluence, HESCs were trypsinized and collected by centrifugation at 1000 rpm for 5 min. 2.5 × 105 HESCs were placed in 24-well plates and cultured overnight. HESCs were treated with 10 nM E2 (Sigma-Aldrich) and 1 μM P4 (Sigma-Aldrich) or teal oil (control) for 3 or 6 days. The medium with stimuli was changed every 3 days. RNA sequencing Total RNA was isolated from the total tissues of human endometrial samples (~50 mg) using the TRIzol® reagent. The quality of RNA was analyzed on an RNA NanoPhotometer spectrophotometer (IMPLEN, Munich, Germany). RNA (2 μg) was used to construct cDNA library using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, Inc.). Library quality and quantity were checked on 2100 Bioanalyzer, and the cDNA library was sequenced on an Illumina HiSeq 2500 platform. Approximate 5.0 G reads were obtained for each sample. FPKM were used to evaluate the gene expression. DESeq package was applied to analyzed differentially expressed genes. Genes with an adjusted P-value lower than 0.05 were considered to be differentially expressed. RT-qPCR Total RNA from endometrial specimens (the proliferative and secretory endometria) and cells treated without (control) or with E2 and P4 (E2P4) for 6 days was extracted using the TRIzol® reagent. Subsequently, the RNA (3 μg) was reverse transcribed to cDNA using a Transcriptor cDNA Synthesis kit (Roche Diagnostics). RT-qPCR was performed using SYBR Green (Soyetin Biotech) under the following conditions: Initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. GAPDH was used as a normalization control, and the analysis for each gene was performed according to the 2−ΔΔCq method [18]. Immunohistochemical analysis Human endometrial specimens from patients were fixed with buffered formalin at 4 °C and embedded in paraffin. The tissue samples were cut into at 10-μm sections, which were dewaxed and dehydrated in gradient ethanol. The slides were incubated in a microwave in citrate buffer (20 min) and subsequently immersed in 3% H2O2 at room temperature (20 min). Following washing with PBS, TOB1 antibody (1:200) was incubated with the sections at 4 °C overnight prior to application of the biotinylated secondary antibodies (37 °C, 30 min). The Vectastain ABC kit (Vector Laboratories) was used for visualizing specific antigens, and hematoxylin was selected for counterstaining of the sections. TOB1 expression was observed and recorded with an Olympus BX-51 microscope (Olympus Corporation), and the intensity of TOB1 staining was quantified by ImageJ software. Immunofluorescence analysis The cells were seeded on glass coverslips, incubated overnight, fixed with 4% paraformaldehyde for 30 min, and blocked using 2% BSA (BD, USA) for 30 min at room temperature. Subsequently, they were incubated with TOB1 antibody (1:200) overnight at 4 °C. Following rinsing with PBS, Alexa Fluor555–conjugated secondary antibody (1:400) was applied to HESCs for 1 h at room temperature in the dark. DAPI was used for nuclear staining. The immunofluorescence images of the cells were recorded with a confocal microscope (LSM710; Carl Zeiss AG), and the fluorescence intensity was quantified by ImageJ software. Western blotting The cells were lysed and scraped by the lysis buffer (Beyotime Institute of Biotechnology). Following centrifugation for 15 min (12,000×g), the supernatant was collected. The BCA protein assay kit was used for quantification. Equivalent amounts of protein (20 μg) were resolved by 10% SDS-PAGE and transferred on 0.45-μm PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight with anti-TOB1, anti-PRL, anti-IGFBP1, anti-Notch1, anti-cyclin D1, anti-cyclin E1, and anti-CDK2 antibodies (1:1,000) at 4 °C. The secondary antibodies were incubated with the membrane for 1 h at room temperature. The bands were detected with the Chemiluminescent HRP Substrate lit and semi-quantified by the ImageQuant LAS 4000TM (GE Healthcare Dharmacon, Inc.) and ImageJ software (National Institutes of Health). An anti-GAPDH antibody was used as a loading control (1:10,000). CCK-8 assay HESCs were seeded in 96-well plates at a density of 5000 cells/well and treated with E2P4 (10 nM E2 and 1 μM P4) alone or together with TOB1-targeted small interfering (si)RNA (50 ng). Cell viability was determined with a CCK-8 kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer’s instructions. In the CCK-8 assay, WST-8 produces a water-soluble formazan dye (orange) by dehydrogenases in cells, the amount of which is directly proportional to the number of living cells. Cell cycle analysis The cells were collected by centifugation, washed with PBS, and fixed with ice-cold 70% ethanol at 4 °C overnight. The fixed cells were washed twice in cold PBS, resuspended in a volume of 100 μl containing 10 mg/ml RNase (Nanjing KeyGen Biotech Co., Ltd.) and 25 mg/ml propidium iodide (KeyGen Biotech Co., Ltd.), and incubated for 30 min at 37 °C in the dark prior. Then, cell cycle distribution was measured by flow cytometry analysis (BD Biosciences). siRNA interference HESCs were grown to 30–50% confluence and transfected with siRNA negative control (5′-UUCUCCGAACGUGUCACGUTT-3′) and TOB1-targeted siRNA (5′-GCUGUAAGCCCUACCUUCATT-3′) (Shanghai GenePharma Co., Ltd.) for 6 h using Lipofectamine® 3000. The cells were cultured in medium with 10% FBS for 48 h, and the siRNA interference efficiency was evaluated using western blotting. Statistical analysis All data were expressed as mean ± SD from three independent experiments. Statistical analyses were performed with SPSS 19.0 (SPSS, Inc.). The GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for histogram drawings and scatter plots. The independent samples t-test was used to evaluate the statistical significance between the two groups. The one-way ANOVA was used for comparisons among different groups followed by a Tukey post hoc test. A P < 0.05 was considered to indicate a statistically significant difference.

TOB1 expression is upregulated in secretory endometrial specimens To identify differentially expressed genes in human endometrial specimens, RNA sequencing was performed. TOB1 is one of most significantly upregulated gene in the secretory samples compared with the proliferative samples (Fig. 1A). Consistently, higher expression levels of TOB1 were observed in the secretory specimens compared with those noted in the proliferative samples as demonstrated by RT-qPCR(Fig. 1B), immunohistochemical analysis (Fig. 1C), and western blotting (Fig. 1D). Interestingly, Notch1, but not Notch3, was also significantly increased in the secretory specimens compared to the proliferative samples (Fig. 1D). Taken together, these results demonstrated an increased expression of TOB1 in secretory endometrial samples. Establishment of the E2P4-induced decidualization model E2P4 was applied for inducing the decidualization model in HESCs. RT-qPCR analysis indicated that the mRNA levels of IGFBP-1 and PRL, two key markers of decidualization [5], were strongly induced following stimulation of HESCs with E2P4 (Fig. 2A). Moreover, the protein levels of IGFBP1 and PRL were markedly increased in E2P4-treated HESCs, as determined by western blot analysis (Fig. 2B). Therefore, the E2P4-induced decidualization model was established successfully. It is interesting to note that the expression levels of TOB1 and Notch1 were markedly increased in E2P4-treated HESCs compared to those of the control cells (Fig. 2C and D), indicating an involvement of TOB1 and Notch1 in decidualization. E2P4 inhibits the proliferation of HESCs and induces G1-phase arrest To determine the effects of E2P4 on HESC proliferation, the CCK-8 assay was performed. The results indicated that the viability of E2P4-treated HESCs was significantly reduced compared to that of the control cells (Fig. 3A). Flow cytometry analysis revealed that E2P4 exposure had no significant effect on apoptosis of HESCs (Supplementary Fig. S1). E2P4 treatment greatly increased the population of G1-phase cells (Fig. 3B), indicating G1-phase cell cycle arrest. The mRNA and protein levels of the G1-phase regulators, cyclin D1, cyclin E1, and CDK2, were significantly decreased in HESCs treated with E2P4, which was consistent with the results of the cell cycle analysis (Fig. 3C and D). Effects of TOB1 knockdown on E2P4-induced decidualization Based on the increased levels of TOB1 expression in the secretory samples, it was suggested that this protein may contribute to the decidualization of HESCs. To address this hypothesis, siRNA-mediated knockdown of TOB1 was performed. Three different si-TOB1 sequences were applied to silence TOB1 expression in HESCs. As shown in Fig. 4A, TOB1 expression was efficiently reduced by si-TOB1-3, and this sequence was selected for further experiments. The CCK-8 assay indicated that knockdown of TOB1 expression partially restored cell viability inhibited by E2P4 (Fig. 4B). The increased population of HESCs accumulating at the G1 phase was induced by E2P4 and was reversed following TOB1 knockdown (Fig. 4C). Moreover, the increase noted in the mRNA and protein levels of cyclin D1, cyclin E1, and CDK2 by E2P4 was attenuated by TOB1 depletion (Fig. 4D and E). Taken together, these results suggested that TOB1 was involved in E2P4-induced inhibition of HESC proliferation and G1 cell cycle arrest. TOB1 modulates decidualization via Notch signaling Notch1 expression levels were upregulated following E2P4 incubation (Fig. 2D), indicating that TOB1 may regulate the decidualization process via Notch signaling. Knockdown of TOB1 decreased the protein levels of Notch1, which were increased following treatment with E2P4 (Fig. 5A), suggesting that TOB1 was an upstream regulator of Notch1. Moreover, treatment with the Notch inhibitor DAPT partially reversed the effects caused by E2P4 (Fig. 5B). The increased cell number at the G1 phase in E2P4-treated HESCs was partially reversed following DAPT administration (Fig. 5C). In addition, treatment of the cells with DAPT partially abolished E2P4-mediated suppressive effects on cyclin D1, cyclin E1, and CDK2 expression (Fig. 5D and E). These data suggested that the TOB1/Notch cascade was involved in the progression of decidualization.

The improvement of decidualization is critical for successful implantation, and it is crucial to fully understand the mechanisms involved in this process. In the present study, TOB1 expression levels were increased in the secretory endometrial samples compared with the corresponding expression noted in the proliferative samples. Increased TOB1 expression was observed in the E2P4-induced decidualization model in HESCs. Further studies demonstrated that TOB1 exerted an antiproliferative effect on HESCs by regulating the Notch pathway. Therefore, the findings revealed a novel mechanism of TOB1/Notch modulating decidualization. Decidualization is a process driven by the rise in progesterone levels. The increase in local cAMP production indicates the transformation of endometrial stromal fibroblasts into specialized secretory decidual cells, which is necessary for embryo implantation and pregnancy establishment [4]. Lim et al. [19] reported that knockout of the decidualization-associated proteins HOXA, ER, and PRB in mice caused failure in implantation, infertility, and intrauterine lethality. Decidualization can be established in vitro as the endometrium is relatively accessible and stromal cells can be readily isolated and cultured. The protocols include the treatment of cells with a combination of E2 and P4 [18, 20, 21]. E2 induces epithelial proliferation to build endometrial thickness during the proliferative phase, and, subsequently, P4 inhibits E2-induced proliferation and promotes decidualization during the secretory phase of the menstrual cycle [22]. The expression levels of genes involved in the decidualization of HESCs may vary. To assess the differentiation status of HESCs in culture, PRL and IGFBP-1 were used as markers [5]. In the present study, the combination of 10 nM E2 and 1 μM P4 was used to induce the decidualization model as reported previously [18]. E2P4 treatment upregulated the expression levels of PRL and IGFBP-1 in HESCs, suggesting that the decidualization model was successfully established in vitro. Multiple studies have suggested an important role of the BTG/Tob family proteins as cell cycle regulators and tumor suppressors [10, 23, 24]. It has been demonstrated that the interaction between TOB1 and cyclin D1 and its nuclear translocation are involved in the gefitinib-induced anti-proliferative cell cycle arrest [25]. TOB1 has been reported to play a crucial role in germ cell differentiation and embryonic development [11, 26]. This protein is expressed in endometriotic cells and treatment of endometriotic stromal cells with IL-1β reduces its levels [26]. An additional study has reported that TOB1 inhibits the proliferation of mouse embryonic stem cells via the degradation of Id3 mRNA [11]. In the present study, the expression levels of TOB1 protein and mRNA were increased following E2P4 treatment. Moreover, knockdown of TOB1 reversed the effects of E2P4 on the inhibition of cell proliferation and cell cycle arrest. The Notch proteins play important roles in cell survival [12]. Among them, Notch1 is suggested to be involved in endometrial decidualization and embryo implantation [14]. It has been reported that poFUT1 can promote endometrial decidualization by enhancing the O-fucosylation of Notch1 [27]. Blockade of Notch signaling by DAPT downregulates Sox2 transcription and retards embryo hatching [28]. It has been reported that the expression levels of G1/S cyclins and cyclin D2 are induced in decidualizing stroma [29]. In the absence of Notch1 signaling, stromal cell proliferation is inhibited by downregulating the expression levels of cyclin D2 and cyclin K6 [30]. In the present study, E2P4 treatment–induced cell cycle arrest at the G1 phase and reduced the expression levels of cyclin D1, cyclin E1, and CDK2, while knockdown of TOB1 or treatment with DAPT reversed the antiproliferative effects mediated by E2P4. These results indicated that the TOB1/Notch1 cascade contributed to E2P4-induced decidualization in HESCs. In conclusion, the data demonstrated an important role of TOB1/Notch1 signaling in the regulation of E2P4-induced decidualization. TOB1 or Notch signaling may act as promising targets for enhancing the success of embryo implantation and pregnancy establishment. Supplementary information Availability of data and materials All datasets generated and analyzed during the present study are available from the corresponding author on reasonable request. Author contribution YJ designed and performed the experiments. XY analyzed the data and wrote the manuscript. BL and ML carried out the experiments. YS and JF performed part of the experimental research. HZ, MO and XH participated in the experimental design. YJ provided financial support and supervised the manuscript. All authors read and approved the final manuscript. Funding The present study was supported by the National Natural Science Foundation of China (grant no. 81601349). Declarations Ethics approval and consent to participate The research was approved by the ethical committee of The Third Affiliated Hospital of Guangzhou Medical University (No. 2018PGY260K). Consent for publication All patients consent for publication. Competing interests The authors declare no competing interests. Footnotes Yaling Jiang and Xinhua Yuan should be considered co-first authors. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Yaling Jiang and Xinhua Yuan contributed equally to this work.

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