BIRC5 Expression Is Regulated in Uterine Epithelium during the Estrous Cycle

Abstract

Baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), also known as survivin, is a member of the inhibitor of apoptosis (IAP) family of proteins and regulates the size of tissues through cell division control. The uterus is the most dynamically sized organ among tissues during the estrous cycle. Although Birc5 is expressed in some terminally differentiated cells, the regulation of its expression in the uterus remains unknown. We investigated the regulation of Birc5 expression in the mouse uterus. RT-PCR analysis showed that Birc5 was expressed in various tissues, including the uterus; the expression level of Birc5 was significantly higher at the diestrus stage. Immunohistochemistry and Western blotting analysis revealed that Birc5 was more active in luminal and glandular epithelium than in endometrial stroma. In ovariectomized mice, Birc5 expression in the uterus was gradually increased by estrogen treatment; however, progesterone injection decreased its expression. Estrogen-induced Birc5 expression was blocked by treatment with estrogen receptor antagonist, ICI 182, 780 and progesterone-reduced Birc5 expression was inhibited by the progesterone receptor antagonist RU486. These results suggest that Birc5 expression is dynamically regulated by a combination of estrogen and progesterone via their receptor-mediated signaling.

1. Introduction

The uterus is a female reproductive organ that plays an important role in several pregnancy processes such fertilization, implantation, and embryonic development [1]. The endometrium of the uterus is periodically regulated according to the estrous cycle. In humans, the menstrual cycle has an interval of ≈28–30 days. It corresponds to the estrous cycle of 4–5 days in the rodents (reviewed in [2]). The estrous cycle consists of four stages viz. diestrus, proestrus, estrus, and metestrus. The precise regulation of the estrous cycle within ≈4–5 days is very important for a successful pregnancy. The uterine endometrium is dynamically regulated by the ovarian sex hormones estrogen and progesterone. The endometrium undergoes proliferation, differentiation, and shedding (humans and primates) or apoptosis (rodent) in response to hormones during the cycle [2].

Baculoviral inhibitor of apoptosis repeat-containing 5, (Birc5, also known as survivin) is an inhibitor of apoptosis (IAP). Birc5 was originally identified and characterized in human cancers [3]. Birc5 was highly expressed in various cancers such as lung cancer, ovarian cancer, breast cancer, brain tumor, colon cancer, pancreatic cancer, osteosarcoma, and cervical cancer (reviewed in [4]). It was used as a prognostic or survival marker in cancer patients. Birc5 acts as inhibitor of apoptotic processes by repressing caspase activity via binding to caspase-3 and -7 in cancer cells, leading to the survival of cancer cells during tumorigenesis [5].

Recently, many studies o Bir5 expression in various tissue have been reported [4]. The expression of Birc5 has been detected in various normal tissues such as the liver [6], arterial muscle [7], stomach [8], brain [9], ovary [10], testes [11,12,13], and uterus [1], though at lower levels than in cancers. Birc5 has various functions in cellular processes such as differentiation and proliferation of stem cells and progenitor cells and its deficiency results in embryonic lethality at early stage of embryogenesis, viz. at embryonic day E4.5 [14]. This implies that Birc5 plays a crucial role in cell differentiation and proliferation. In fact, T cell-specific Birc5-knocked-out mice showed that it plays a role in T cell development and differentiation [15,16]. Further, Birc5 is involved in regulating proliferation of hematopoietic stem cells [17] and mesenchymal stromal cells [18]. Moreover, Birc5 expression is regulated by various factors including p53 [19], PTEN [20], SIRT1 [21], HDAC2, and HDAC5 [22].

Almost 20 years ago, Konno and colleagues showed Birc5 expression in normal uterus for the first time [1]; there was no further report in this regard about the uterus until recently. A recent study demonstrated that Birc5 is aberrantly expressed in patients with endometrial hyperplasia [23]. They reported that Birc5 is highly detected in the endometrium of patients with endometrial hyperplasia. Endometrial hyperplasia is caused by abnormally excessive proliferation of endometrial cells in the uterus, which is related to high estrogen level and low progesterone level. However, the regulatory relationship between Birc5 expression and hormones in the normal uterus remains unknown. Therefore, we investigated whether Birc5 is expressed and regulated by hormones in mouse uterus.

2. Materials and Methods

2.1. Animals

All animal studies were performed using 6–7-week-old ICR mice provided by KOATECH (Pyeongtaek, Korea). Mice were housed under controlled temperature and light conditions with lights on for 12 h daily and fed ad libitum. Animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University.

2.2. Estrous Cycle and Uterus Sampling

The stages of estrous cycle were determined by a vaginal smear as described in previous studies [24,25,26]. Approximately 0.2 mL of Dulbecco’s phosphate buffered saline (DPBS) was drawn into the pipette tip. The tip of the pipette was pushed gently into the entrance of the vagina at a depth of 2–5 mm, and the fluid was flushed into the vagina and then backed up into the pipette; this was repeated two to three times. The collected DPBS was dropped onto a glass slide and dried. Cell staining was carried out by hematoxylin and eosin. After incubating the cells in 50%, 75%, and 90% ethanol for 5 min each, they were stained with hematoxylin (Vector Laboratory, USA). The slides were washed in tap water for 3 min and soaked in eosin Y (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. After the eosin treatment, the dyed slides were in turn dipped in 90% ethanol and then 100% ethanol for 5 min. After a short incubation in xylene, the slides were covered with cover slips using Permount mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The staining was observed with a microscope and the stage of the estrous cycles was determined by vaginal smear cytology [27]. Once the estrous cycle was correctly identified, the uterus was isolated. Half of the uterus was processed for RNA and protein extraction and the other half was used for paraffin block.

2.3. Ovariectomy and Hormone Treatments

To examine the effects of hormones on Birc5 expression in the uterus, 7-week-old ICR mice were ovariectomized (OVX). After a stabilization period of 2 weeks, the ovariectomized mice were subcutaneously injected with E₂ (200 ng/mouse, Sigma-Aldrich, St. Louis, MO, USA) and/or P₄ (2 mg/mouse, Sigma-Aldrich, St. Louis, MO, USA). Mice were sacrificed by cervical dislocation and uteri were collected at 0, 3, 6, and 24 h after E₂ or/and P₄ treatment. To investigate whether the expression of Birc5 in uterus is dependent on E₂ or/and P₄, OVX mice were injected with an ER antagonist, ICI 182,780 (500 μg/mouse, Sigma-Aldrich, St. Louis, MO, USA) or PR antagonist, RU486 (1 mg/mouse) 30 min before E₂ or P₄ injection. Sesame oil (100 μL/mouse, Sigma-Aldrich, St. Louis, MO, USA) was used for the control mice.

2.4. RNA Preparation, Reverse Transcription PCR (RT-PCR), and Quantitative Real-Time PCR (qRT-PCR)

The tissues isolated from the mice were stored in RNA later solution (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 ℃. After removing RNA later solution, the tissue was homogenized in Trizol reagent (Invitrogen, Waltham, MA, USA) using a homogenizer and then purified with chloroform (Sigma-Aldrich, St. Louis, MO, USA). Total RNA (1 μg) was reverse transcribed using SensiFast™ cDNA Synthesis Kit (Bioline, London, UK) according to the manufacturer’s protocol. The product was used for reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analyses. RT-PCRs were carried out using C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR conditions and size of gene-specific primers are shown as Supplementary Table S1. The PCR products stained with Loading Star (Dyne Bio, Seoul, Korea) were analyzed by gel electrophoresis on 2% agarose gel using Chemidoc™ XRS+ system (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR (qRT-PCR) was performed by the CFX96 Touch (Bio-Rad, Hercules, CA, USA) using iQ™ SYBR Green Super Mix (Bio-Rad, Hercules, CA, USA). qRT-PCR conditions were as follows: 40 cycles of denaturation at 95 ℃ for 15 s, annealing at 63 ℃ for 15 s, and extension at 72 ℃ for 20 s followed by 10 min at 72 ℃, 10 s at 95 ℃, and 5 s at 65 ℃. To quantify the level of gene expression, the cycle threshold (C_T) value was calculated. The C_T value for each gene was determined in the linear phase of the amplification and normalized to the C_T value of either ribosomal protein L-7 (Rpl7) or Gapdh to obtain the ΔΔC_T. The 2^−ΔΔCT method was used for obtaining the fold change of each gene [28].

2.5. Immunohistochemistry

Uteri collected from mice were fixed for 1 week in 4% paraformaldehyde (PFA) at 4 ℃ in a paraffin block. The embedded uteri were sectioned at 5 µm thickness using a paraffin microtome (Macroteck, Ilsan-si, Korea) and put on adhesion microscope slides (Paul Marienfeld, Lauda-Konigshofen, Germany). Sectioned uteri on the slide were deparaffinized twice in xylene (Duksan, Seoul, Korea) for 7 min and rehydrated by immersing once each in 100% ethanol (DaeJung, Seoul, Korea), 90% ethanol, 75% ethanol, and 50% ethanol for 5 min, followed by washing the slides once for 5 min under tap water. Slides were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxide. Deparaffinized slides were boiled in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), cooled at room temperature (RT) for 1 h, and then washed in tap water for 5 min followed by three washes using phosphate buffered saline containing 0.05% Tween-20 (PBS-T). After removing PBS-T around tissue section using 3M paper, a hydrophobic barrier was drawn with ImmEdge hydrophobic pen (Vector Laboratories, USA) on the periphery of the tissue. Sections were blocked with PBS-T containing 4% bovine serum albumin (BSA) and 5% normal goat serum (Vector Laboratories, USA) for 1 h at RT in a humidified chamber. Blocking sections were incubated with anti-survivin mouse monoclonal IgG (1:500 dilution, NB500-238, Novus Biologicals, Centennial, CO, USA) in PBS-T with 4% BSA at 4 ℃ overnight. Slides were washed with PBS-T three times and subsequently were treated with secondary antibody viz. anti-mouse antibody conjugated with HRP (1:200 dilution, G21040, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT. Normal mouse IgG was used as a negative control. Peroxide signals were developed using DAB substrate kit (SK-4100, Vector Labs, Burlingame, CA, USA) according to the manufacturer’s instruction. After washing, the slides were counter-stained with hematoxylin, washed in tap water, and dehydrated in 50% ethanol, 75% ethanol, 90% ethanol, 100% ethanol for 5 min each followed by two washes in xylene for 7 min each. IHC sections were mounted on cover slips with Permount Mounting Medium (StatLab, Lodi, CA, USA). Images were captured and analyzed by Primo Star (Zeiss, Oberkochen, Germany).

2.6. Western Blotting

Uteri from adult mice were collected and frozen in liquid nitrogen. The samples were added to 600 µL RIPA lysis buffer containing protease inhibition cocktail and grounded using a homogenizer (VWR International, Radnor, PA, USA) with vortexing every 10 min while being placed on ice for 30 min. After the incubation, the samples were centrifuged at 13,000 rpm for 30 min at 4 ℃ and the supernatant containing proteins was collected and stored at –80 ℃. Equal amounts of protein samples (10~20 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10%–12% running gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) for 1 h and 10 min. Membranes were washed in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), blocked in ProNA™ phospho-block solution (TransLab, Dajeon-si, Korea) for 1 h at RT, and incubated with anti-survivin mouse monoclonal IgG (1:1000 dilution, NB500-238, Novus, Centennial, CO, USA) overnight at 4 ℃. After washing three times in TBS-T, the blots were incubated with HRP-conjugated goat anti-mouse antibody (1:5000 dilution, sc2005, Santa Cruz Biotechnology Inc., Dalls, TX, USA) in ProNA™ phospho-block solution for 1 h at RT. The membranes were washed three times using TBS-T, and subsequently, the blots were developed using ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL USA). To check whether the same amount of protein was loaded for each sample, anti-GAPDH antibody (1:2000 dilution, 14C10, Cell signaling technology, Danvers, MA, USA) was used as a loading control. The relative expression of the protein bands was quantified by Image Lab program.

2.7. Statistical Analysis

The experimental data were reported as mean ± standard error of mean (±SEM). The results were analyzed by one-way ANOVA for statistical evaluation. A p-value of less than 0.05 was considered as statistically significant.

3. Results

3.1. Expression of Birc5 in the Mouse Uterus

In order to confirm the expression of Birc5 mRNA, we performed RT-PCR analysis using total RNAs, which were extracted from various tissues in the mouse (Figure 1a,b). As shown in Figure 1, Birc5 expression was barely detected in the rest of the tissues including the kidney, liver, heart, brain, and lung (Figure 1a). These results are almost consistent with those from the previous studies indicating that Birc5 expression is detected in normal tissues such as the liver [6], stomach [8], brain [9], ovary [10], and testes [11,12,13]. However, Birc5 transcripts were detected in high quantities in several tissues including the small intestine, stomach, spleen, ovary, and uterus in the female mice (Figure 1b). After confirming the expression of Birc5 mRNA in the mouse uterus, we examined the expression pattern of Birc5 during the estrous cycle. Vaginal smear assay was used to distinguish each stage of the estrous cycle viz. diestrus, proestrus, estrus, and metestrus (Figure 1c). The results of RT-PCR and qRT-PCR showed a cyclic change in Birc5 expression during the estrous cycle (Figure 1d,e). Birc5 expression level was the highest in diestrus stage and then reduced in the rest of the cycle including in the proestrus, estrus, and metestrus stages (Figure 1e).

3.2. Localization and Expression Level of BIRC5 Protein in Mouse Uterus during the Estrous Cycle

To investigate the localization and expression of BIRC protein in the mouse uterus, immunohistochemistry (IHC) was performed using mouse monoclonal BIRC5 antibody in the uterus. IHC results showed that BIRC5 was present in all layers of the endometrium (viz. luminal epithelium (LE), glandular epithelium (GE), stroma) (Figure 2a). Interestingly, strong staining was observed in the luminal epithelium and glandular epithelium compared to that in the endometrial stroma. Western blotting showed a relatively similar BIRC5 expression level during the estrous cycle (Figure 2b,c). Results showed that BIRC5 expression was slightly higher in the diestrus stage than that in metestrus (Figure 2c).

3.3. Regulation of Birc5 Expression by Estrogen Treatment

Previous results have indicated that Birc5 transcript and its protein were dynamically regulated during the estrous cycle of the uterus. Cyclic modulation of the normal uterine endometrium is regulated by two ovarian steroid hormones, namely, estrogen and progesterone. Therefore, we first examined the effect of estrogen (E₂) on the expression of Birc5 using ovariectomized (OVX) mice. The OVX mice were treated with E₂ and their uteri were obtained at 0, 3, 6, 12, and 24 h after E₂ treatment. RT-PCR analysis indicated that the expression of Birc5 mRNA gradually increased with time after E₂ administration (Figure 3a). Early growth response 1 (Egr1) and Lactoferrin (Ltf) expressions were used as referral genes marking estrogen early and late response genes, respectively (Figure 3b). The gradual induction of Birc5 expression by estrogen treatment was confirmed by semi-quantitative RT-PCR. The relative increase in Birc5 expression peaked at 24 h after E₂ treatment (Figure 3b). Next, we examined the status of BIRC5 protein in uterine endometrium of OVX mice by immunostaining with BIRC5 antibody. IHC results showed that BIRC5 protein was weakly detected without E₂ treatment (0 h) and was heavily stained for in all layers of the uterus over time (Figure 3c). Unlike the RT-PCR results, the signal for BIRC5 was the strongest at 6 h post E₂ injection. This suggests that E₂ promotes increase in Birc5 expression in the uterus during the estrous cycle.

3.4. Regulation of Birc5 Expression by Progesterone Treatment

Next, we analyzed whether progesterone (P₄) affects the expression of Birc5 in the uterus. As in the previous studies, we used ovariectomized (OVX) female mice. The OVX mice were injected with P₄ (2 mg/mouse), and the uterus samples were collected at 0, 3, 6, and 24 h. Interestingly, P₄ treatment in OVX mice showed no significant effect on Birc5 expression unlike estrogen treatment. (Figure 4a,b). Amphiregulin (Areg) and Homeobox A10 (Hoxa10) were used to confirm that the reaction with progesterone occurred properly (Figure 4a). qRT-PCR analysis showed that the expression level was lowest at 6 h after P₄ treatment, but it was not a significant difference (Figure 4b).

IHC analysis also showed that BIRC5 protein was detected as basal level in the uterine endometrium of OVX mice without P₄ treatment. However, its signal in the uterine endometrium of OVX mice was rapidly reduced after the administration of P₄ (Figure 4c). This indicates that P₄ regulates the time difference in Birc5 expression in the uterus during the estrous cycle. In fact, P₄ like E₂, is secreted alternately from the ovary during the estrous cycle and acts jointly or solely on endometrial changes.

3.5. Hormone-Dependent Regulation of Birc5 Expression via Hormone Receptors in the Uterus

The expression of Birc5 rapidly responded to hormones E₂ and P₄ in the uterine endometrium (Figure 3 and Figure 4). This rapid regulation of target gene expression by hormones occurs via their receptors. Therefore, we investigated whether Birc5 expression is regulated via the estrogen receptor (ER) and progesterone receptor (PR). First, an ER antagonist, ICI 182,780 (ICI), was used to determine whether ER was involved in the regulation of Birc5 expression. ICI was injected in OVX mice for 30 min before E₂ administration. Birc5 expression was examined in the absence or the presence of E₂ administration (24 h). As shown in Figure 5a, the relative expression of Birc5 mRNA was dramatically increased by E₂ treatment. However, its induction was prevented with ICI pretreatment in OVX mice (Figure 5b).

Next, a PR antagonist, RU486, was used to examine whether PR was involved in the downregulation of Birc5 expression. In addition, we looked at the action of PR on ER-dependent increase in Birc5 expression in the uterus. As shown in Figure 5c,d, the increase in Birc5 expression by E₂ treatment (6 h) was significantly reduced by co-treatment with E₂ and P₄ (Figure 5d). This effect of P₄ on the estrogen-dependent increase was reversed by the treatment with PR antagonist, RU486 (Figure 5d). BIRC5 protein was detected at higher level in E₂-treated uterine endometrium compared with the sesame oil-treated group. The effective induction by E₂ was reduced by progesterone, whereas this effect was again reversed while using P₄ antagonist, RU486, in the presence of estrogen in the uterine endometrium (Figure 5e). These results indicate that Birc5 expression in the uterus is precisely and dynamically regulated by E₂ and P₄.

4. Discussion

In this report, we presented the regulation of Birc5 expression in the uterus. We found that Birc5 is periodically expressed in the uterus during the estrous cycle (Figure 1 and Figure 2). Moreover, we showed that its cyclic expression is related to two ovarian hormones, namely, estrogen and progesterone (Figure 3 and Figure 4). Interestingly, we demonstrated that estrogen positively regulates Birc5 expression through its receptor in the uterine epithelium (Figure 5). This needs further study to prove the involvement in Birc5 expression. This implies that Birc5 plays a role in the uterine dynamics during the estrous cycle in normal status.

As a member of the IAP family, Birc5 is expressed in various cells including hematopoietic cells, immune cells and vascular endothelial cells, and mesenchymal stromal cells [4,17,18]. Additionally, Birc5 plays a role in regulating cell proliferation and survival. One of its functions is to inhibit apoptosis by acting on caspases in the cytoplasm and mitochondria. Further, Birc5 is also located in the nucleus and regulates cell cycle factors [29,30].

The uterus, which is composed of endometrium and myometrium, is a part of the female reproductive system. The uterine endometrium consists of a single columnal epithelium and a connective tissue stromal layer. The uterine layers are cyclically changed with periodic proliferation, differentiation, and apoptosis. This cycle is called the estrous cycle in the rodents. The remodeling of the uterus is controlled by female sex steroid hormones such as estrogen (E₂) and progesterone (P₄). Therefore, we investigated the relationship between Birc5 expression and uterine endometrium.

Nabilsi et al. have reported the relationship between BIRC5 and sex hormones in the human uterus [31]. They analyzed the expression of BIRC5 in proliferative and secretory phases of normal menstrual cycle and found that BIRC5 expression was higher in the proliferative phase than in the secretory phase. They further observed that BIRC5 expression was increased in the human endometrium after estrogen treatment, which then decreased upon progestin treatment [31]. It was the first evidence showing that BIRC5 expression is regulated by female sex hormones. However, it remained unclear whether the hormones acted alone or together.

In fact, although progesterone and estrogen secretion stages are different, they are organically associated rather than acting separately. Skinner et al. reported that progesterone is critical for the full effect of estrogen in ewes [32]. Therefore, we used the OVX model to show their individual effect on BIRC5 expression in the uterus. Each treatment on OVX mice showed a similar pattern as that observed in the study by Nabilsi and colleague. Birc5 transcripts were increased with E₂ administration into OVX mice and this increase was significantly reduced by pretreatment with ER antagonist ICI 182,780. This suggests that Birc5 is regulated in the uterine epithelium via estrogen receptor-associated hormonal signaling. It has been shown that Birc5 is regulated by various signaling pathways such as PI3K/Akt, mTOR, ERK, and NFκB pathways [33,34]. Interestingly, the increased expression of Birc5 was decreased by P₄ administration. This suggests that Birc5 is regulated by both E₂ and P₄, during the dynamic uterine cycle.

In conclusion, the present study demonstrated that Birc5 may be periodically regulated by estrogen and progesterone in the endometrium of the uterus during the estrous cycle. This finding provides insights into understanding the regulatory mechanism underlying the endometrial dynamics.