MMP3 mediates E2-induced bovine endometrial cell proliferation by releasing HB-EGF

Abstract

Cyclic cell proliferation and endometrial remodeling during the estrous cycle are important for maintaining normal endometrial function. However, the regulatory mechanisms underlying this cell proliferation have not yet been elucidated. In this study, the function of matrix metalloproteinase 3 (MMP3) on endometrial cell proliferation was analyzed. Gene expression in bovine endometrial stromal (BES) and epithelial (BEE) cells was analyzed using qPCR. The protein expression of MMP3 and heparin-binding EGF-like growth factor (HB-EGF) was analyzed using casein zymography and western blotting, respectively. Cell proliferation was analyzed using an automated cell counter. The results revealed that MMP3 was highly expressed at the follicular stage compared to that at the luteal and implantation stages. Estrogen (E2) increased the gene expression and release of MMP3 protein in BES in vitro, whereas progesterone (P4) and interferon alpha (IFNα) decreased mRNA and protein expression. E2 also increased the proliferation of BES, but the inhibitors of MMP3 and epidermal growth factor receptor (EGFR) inhibited the proliferation induced by E2. Furthermore, E2 increased the release of HB-EGF from BES, whereas the MMP3 inhibitor suppressed this release. The effect of E2 on BEE cell proliferation was not reported. However, the conditioned medium of BES treated with E2 increased BEE cell proliferation but was inhibited by an EGFR inhibitor. E2 induced MMP3 protein expression and promotes HB-EGF release from BES. These results suggest that MMP3 is involved in endometrial cell proliferation during the follicular stage.

The endometrium is an essential organ for the establishment and maintaining of gestation, and periodic tissue remodeling is required to maintain normal function. Tissue remodeling of the endometrium is driven by various secretory hormones and biological enhancers, such as growth factors and cytokines [1]. Extensive renovation is important for tissue remodeling, which involves disrupting and recasting the extracellular matrix (ECM) [2]. The significance of the changes in the endometrium is assumed to be maintaining endometrial homeostasis and preparation of the reproductive tract to support fertilization and embryonic development. The placentation pattern in cattle is noninvasive, unlike that in humans and rodents, which have decidua in the endometrium [3].

Several proteases are involved in the proteolytic degradation of ECM proteins. The biological behavior of matrix metalloproteinases (MMPs) and their implications in many biological processes have been reported [4]. MMPs belong to a family of zinc-dependent proteases. These endopeptidases are responsible for maintaining equilibrium between ECM protein conservation and decline. MMPs contribute to tissue homeostasis by regulating the release or activation of several biological factors, including chemokines, cytokines, growth factors, and other bioactive molecules [5]. The intricate regulation of MMPs occurs at the gene transcription, post-translational activation, and endogenous inhibition levels. Spatial and transient regulation of MMPs is indispensable during endometrial tissue remodeling. Few studies have reported the mRNA and protein expression of MMPs in the uteri of bovines [6], goats [7], and sheep [8]. Stromelysin-1, also known as matrix metalloproteinase 3 (MMP3), is an important member of the MMP family. It plays a significant role in degrading different types of connective tissue proteins, such as collagens, proteoglycans, elastin, and glycoproteins. MMP3 also plays a role in degrading major components of the basement membrane, such as laminin. The important role of MMP3 also describes activating MMP1, 2, and 9 [9, 10]. Progesterone (P4) suppresses the expression of MMP3 mRNA and protein, which are both confined to endometrial stromal cells, in humans [11,12,13]. The mRNA expression of MMP3 during implantation in the rodent endometrium indicates the participatory role of MMP3 in uterine tissue remodeling [14]. Accumulated evidence implies that species- and stage-specific endometrial MMP3 is differentially expressed and regulated by several factors.

Most studies of MMPs in the bovine endometrium have focused on gelatinases (MMP2 and 9). Although the gene and protein expressions of many MMPs have been reported in the bovine endometrium, reports on MMP3, which belongs to the stromelysin group, is limited. Considering the importance of MMP3 during tissue remodeling in other species, it is necessary to clarify how MMP3 mRNA and protein are expressed and regulated in the bovine endometrium. Thus, the purpose of this study was to elucidate the dynamics of MMP3 expression and its regulatory mechanisms in the bovine endometrium. Moreover, the physiological function of MMP3 in endometrial cell proliferation at the follicular stage was verified using cultured cells.

Materials and Methods

Animals

Endometrial tissues and conceptuses were collected from Japanese Black heifers reared at the Kuju Agricultural Research Center of Kyushu University, and bovine uteri were collected from a local slaughterhouse. In all cases, Japanese Black heifers aged 24–30 months and weighing 700–800 kg were used.

The animals used in this study were treated according to the guidelines for Animal Experiments in the Faculty of Agriculture of Kyushu University (No. A19-297-0) and the laws of the Japanese Government (Law no. 105 with notification no. 6).

Collection of endometrial tissues from hormonal-treated pregnant cows and estrous cycle groups

In this study, the endometrium of five Japanese Black cows, from which elongation-stage embryos were collected using superovulation, was used as the implantation stage. Cows were superovulated using FSH (Antorin R-10; Kyoritsu Seiyaku Co., Tokyo, Japan) 10 days after estrous was detected, following the protocol of Nishino et al. [15]. FSH injections were administered twice a day, that is, in the morning and evening, for 3 days with gradually decreasing doses (5 IU, 3 IU, and 2 IU on days 1, 2, and 3, respectively). In addition, 2 ml of prostaglandin F2α (PGF2α) (Resipron-C; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan) was also injected on the morning of day 3. Then, 48 h after PGF2α injection, animals displaying standing estrus behavior were inseminated (designated as day 0 of pregnancy). The cows were slaughtered on day 18 of pregnancy, representing conceptus elongation. The reproductive tract of each cow was removed and the extraneous tissue was trimmed. The uterus was immersed in 70% (v/v) ethanol and then washed two times with PBS (Dulbecco’s PBS; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 38.5°C. Conceptus samples were obtained from the uterus by flushing with PBS containing 1% (v/v) charcoal-stripped fetal bovine serum (FBS; Hyclone Laboratories, South Logan, UT, USA). Only uterine horns from which an appropriately developed conceptus was recovered were processed further in the implantation group (n = 5). Bovine uteri from the estrous cycle, follicle stage (n = 5), and luteal stage (n = 5) groups were collected from the slaughterhouse. The follicular and luteal stages of the estrous cycle were determined by ovarian morphology, considering the follicular size and the corpus luteum [16]. The uterus was opened longitudinally, and samples were carefully removed from the lamina propria of the caruncular endometrium using scissors and transferred to serum tubes. These samples were then stored at −80°C until further processing.

Isolation and culturing of bovine endometrium cells

Bovine endometrial stromal cells (BES) and epithelial cells (BEE) were separated and purified from the endometrium of bovine uteri collected from a slaughterhouse according to the protocol described by Yamauchi et al. [17]. The endometrial caruncular tissues were surgically dissected with scissors, and the resultant tissues were incubated in culture medium (DMEM/Ham’s F-12; Nacalai Tesque, Inc., Kyoto, Japan) containing 0.1% (w/v) type-I collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 1% (w/w) antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Inc.) for 1 h at 37°C. After incubation, the tissue fragments were centrifuged at 300 × g for 3 min using a tabletop centrifuge (Kubota 2410; Kubota Co., Tokyo, Japan) and washed twice with the culture medium. Then, the tissue fragments were plated in 75 cm² cell culture flasks (TR6002; Nippon Genetics Co., Ltd., Tokyo, Japan) and cultured in a CO₂ incubator (Astec Co., Ltd., Fukuoka, Japan) at 37°C in a humidified atmosphere of 5% CO₂. The primary culture cells were designated as having population doubling levels (PDL) of one, and cells were used at 3–5 PDL for further studies. Different reactivities for trypsin were utilized to separate different cell types: BES was detached from flasks with a lower concentration of trypsin, whereas BEE was detached with a higher level of trypsin. The cultured cells were stored at −80°C until further processing, and the same cells derived from five individual cows were used for all in vitro experiments when necessary. The storage solution for a few weeks of cryopreserving BES and BEE cells at −80°C involves using the culture medium containing 10% dimethyl sulfoxide.

Treatment of BES and BEE

BES was seeded in each well of 48-well cell culture plate at 4 × 10⁴ cells/well, whereas BEE was seeded at 8 × 10⁴ cells/well in 200 µl culture medium (DMEM/Ham’s F-12) containing 10% (v/v) FBS. After the cells reached confluence, the medium was replaced with serum-free medium (DMEM/Ham’s F-12, devoid of phenol red). Cells were treated with 100nM 17β-Estradiol (E2; Sigma-Aldrich Co., Ltd., Tokyo, Japan), 1 µM Progesterone (P4; Sigma-Aldrich Co., Ltd.), and 50 IU/ml human interferon α2 (IFNα; Pestka Biomedical Laboratories, Inc., Piscataway, NJ, USA) for 48 h to analyze the effect of steroid hormones and cytokines. The concentrations of E2 and P4 have been reported previously [15, 21]. Finally, the harvested supernatant was stored at −20°C until further use.

BES and BEE were seeded in each well of 48-well cell culture plate at 5 × 10⁴ cells/well in 200 µl serum-free culture medium (DMEM/Ham’s F-12 devoid of phenol red) containing 4 mg/ml bovine serum albumin (BSA; Nacalai Tesque, Inc.) to analyze the effect of E2 on the growth curve. The cells were then treated with E2 and cultured for 10 days.

The cells were treated with 5 µM MMP3 inhibitor (Enzo Life Science, Inc., Farmingdale, NY, USA), 5 µM epidermal growth factor receptor (EGFR) inhibitor (AG1478) (Enzo Life Science., Inc.), and 100 nM insulin-like growth factor 1 receptor (IGF-1R) inhibitor (AG1024) (Enzo Life Science, Inc.) for 6 days to determine how the inhibitors affected the E2-induced cellular proliferation of BES. In each case, the harvested supernatant was collected and stored at −20°C until further use.

The dose-dependent effects of recombinant heparin-binding EGF-like growth factor (rhHB-EGF; R&D Systems, Inc., Minneaolis, MN, USA) and recombinant MMP3 (rhMMP3; Abcam, Tokyo, Japan) on BES and BEE proliferation were analyzed. In this study, the cells were treated with 0.01, 0.1, 1, 10, and 100 ng/ml rhHB-EGF and 0.1, 1, 10, and 100 ng/ml rhMMP3.

Effect of E2 treated condition media on BEE

BEE was seeded in each well of a 48-well cell culture plate at 5 × 10⁴ cells/well in 200 µl serum-free culture medium (DMEM/Ham’s F-12 devoid of phenol red) containing 4 mg/ml BSA. BEE cells were treated with fresh media along with a combination of 25%, 50%, and 75% (v/v) CM to analyze the effects of BES-conditioned media (CM) of BES. Additionally, cells were treated with 5 µM MMP3 inhibitor and 5 µM EGFR inhibitor (AG1478) for 6 days to analyze the effects of MMP3 and epidermal growth factor (EGF) in CM for BEE proliferation.

Concentrating conditioned medium using heparin-sepharose column beads

A total of 3.7 × 10⁶ BES cells were seeded in 75 cm² cell culture flasks (TR6002; Nippon Genetics Co., Ltd.) and cultured for 6 days in culture media (DMEM/Ham’s F-12 devoid of phenol red) containing 100 nM E2 and 4 mg/ml BSA. The culture medium was changed every 2 days. E2 treated cell culture medium was harvested on day 6 of culture, incubated for 48 h, and centrifuged at 300 × g for 3 min. Finally, the cultured media was filtrated with 2 µm filter to remove the cell particle from the supernatant and stored at −20°C for further use. At the same time the cells remaining in the flask were used for cell lysate preparation. A portion of the detached cell suspension was used to count the cell number for each treatment group. Subsequently, 50 µl of each collected supernatant and lysate were applied to heparin-sepharose columns (Bio Vision, Inc., Milpitas, CA, USA) previously equilibrated with 0.2 M NaCl in 0.01 M Tris-HCl, pH 7.4. The column was washed with 1 × PBS to remove unbound proteins from the column beads. The bound protein was eluted with 50 µl of elution buffer (2 M NaCl in 0.01 M Tris-HCl, pH 7.4). Thereafter, each eluted sample was used to detect the HB-EGF peptide by immunoblotting.

Cell counting

The cells were detached from each well of 48-well plates after 1 min of treatment with PBS containing 0.05% (w/v) Trypsin with 0.53 mM EDTA and 0.5% trypsin with 5.3 mM EDTA for BES and BEE, respectively. Cell counting was performed as described by Yun et al. [18]

RNA extraction, RT-PCR, and RT-qPCR

Total RNA was extracted from endometrial tissues or cells using the RNeasy Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. All the reagents of reverse transcription were purchased from ReverTraAce qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Osaka, Japan). RNA quality was assessed using spectrophotometric UV absorbance at 260/280 nm. Reverse transcription was performed using 6 ng of total RNA in a 10 µl reaction volume, and cDNA was synthesized according to the customized reaction condition according to the protocol of Nishino et al. [15].

RT-PCR was performed in a volume containing 1.0 µl cDNA, 5.0 µl of GoTaq Green Master Mix, 2 × (Promega Corporation, Madison, WI, USA), 0.1 µl of each specific primer, and 3.8 µl of Nuclease-free water (Promega). All primers were selected using the primer design software from the National Center for Biotechnology Information (NCBI). The specific primer sequences and sizes of the resulting fragments for the reference and target genes are shown in Supplementary Table 1. The PCR amplification was performed according to the protocol described by Yun et al. [19]. The relative abundance of the GAPDH transcript was used as an internal control. All primers were validated before use (95–97%) by using the protocol of Taylor et al. [20].

RT-qPCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) utilizing an Mx3000 P qPCR system (Agilent Technologies, Santa Clara, CA, USA) and the protocol of Yun et al. [19]. Relative mRNA abundance was determined using the 2^−ΔΔCTmethod. There was an evaluation of GAPDH and β-actin to evaluate the validity of use as reference genes for qPCR, and there were no significant differences among values. All qPCR experiments were performed using procedures consistent with the Guidelines for Minimum Information for the Publication of Quantitative Real-Time PCR experiments [21]. The specific primers used for the RT-qPCR were the same as those used for the RT-PCR.

Casein zymography

Harvested culture medium (10 µl) was mixed with 5 × SDS sample buffer (125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) Glycerol, and 0.04% (w/v) bromophenol blue) to determine the enzymatic activity of MMP3. SDS-PAGE was performed on 10% (w/v) polyacrylamide gel by adding casein solution (15 mg/ml bovine casein (Sigma-Aldrich Co., Ltd.), 75 mM Tris-HCl (pH 8.8), 0.1 M NaOH) under non-reducing conditions. The gel was pre-run at the current density of 40 mA. Subsequently, the sample was loaded, and the gel was run at 20 mA until the blue line reached the bottom. After running, the gel was washed with a washing buffer (50 mM Tris-HCl (pH 7.5), 5 mM CaCl₂, 1 µM ZnCl₂, 2.5% (v/v) Triton X-100, 0.02% (w/v) NaN₃) to remove SDS and then incubated at 37°C for 18 h in an incubation buffer (50 mM Tris-HCl (pH 7.5), 5 mM CaCl₂, 1 µM ZnCl₂) [22]. Thereafter, the gel was stained with staining buffer (0.1% (w/v) Coomassie brilliant blue R-250, 50% (v/v) methanol, and 20% (v/v) acetic acid), and caseinolytic activity was detected as unstained bands on blue background. Densitometric analysis was performed using ImageJ 1.48 software (ImageJ, NIH, USA).

Western blot analysis

Pieces of follicular stage uterine tissue (0.1 g) were homogenized in 600 µl of buffer containing 20 mM Tris-HCl (pH 7.5) and 2 mM EDTA to detect the HB-EGF protein in tissue. The eluent protein sample was mixed with 5 × electrophoresis Laemmli sample buffer and heated at 99°C for 5 min. SDS-PAGE was performed on 15% (w/v) polyacrylamide gel in reducing condition, and 10 µl of protein sample was applied for each lane. For the tissue sample, 20 µg of proteins for HB-EGF and 5 µg of proteins for β-actin were applied. The proteins were electrotransferred onto Immobilon-PVDF membranes (Millipore, Billerica, MA, USA). After transfer, membranes were incubated for 1 h in blocking buffer (10 mM Tris-HCl (pH 7.5) with 0.15 M NaCl, 1 mM EDTA, 0.05% (v/v) Tween 20, and 5% (w/v) BSA at room temperature). Afterward, membranes were incubated overnight at 4°C on a rotating shaker with mouse monoclonal anti-HB-EGF antibody (1:200 dilution, Santa Cruze Biotechnology, Inc., Dallas, TX, USA, Cat. No. SC-74441) at 1:200 dilution or mouse monoclonal anti-β-actin antibody (1:5000, Abcam, Cat. No. ab8226). After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse-IgG (Cell Signaling Technology, Danvers, MA, USA, Cat. no. 7076) secondary antibody at a 1:2000 dilution in blocking buffer for 1 h at room temperature. After several washes, the membranes were incubated with LumiGlo reagent (Cell Signaling Technology) and exposed to Fuji Medical X-ray film (FUJIFILM Corporation, Tokyo, Japan) to visualize the bound proteins and capture images.

Statistical analysis

The data were presented as means ± SEM from biological replicates (n = 5). A cumulative of three technical replicates for each biological replicate was used for the statistical analyses. One-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test for multiple comparisons between experimental groups was performed using StatView statistical analysis software (version 5; SAS Institute, Cary, NC, USA). Statistical significance was set at P < 0.05.

Results

Expression of MMP3 in bovine endometrial tissue and endometrial cells

The expression of MMP3 in bovine caruncular endometrial tissue during the follicular, luteal, and implantation stages was analyzed using RT-qPCR. mRNA expression was significantly higher in the caruncular tissue at the follicular stage than at the luteal and implantation stages (P < 0.05) (Fig. 1A). In the in vitro analysis, the purity of the cells was confirmed by the same morphological features observed by phase-contrast microscopy, which were characterized as spindle- and cobblestone-shaped morphologies for BES (Fig. 1B) and BEE (Fig. 1C), respectively. RT-PCR results revealed that both cell types expressed MMP3 (Fig. 1D). Expression of MMP3 increased significantly when BES were treated with E2 (P < 0.05). P4 and IFNα significantly reduced expression compared to the control in BES (P < 0.05) (Fig. 1E). In contrast, there were no effects of either of these treatments on MMP3 expression in the BEE (Fig. 1F). BES express MMP3 and its expression is regulated by E2, P4, and IFNα.

Fig. 1.

Expression of MMP3 in bovine endometrium and effects of E2, P4, and IFNα on MMP3 expression in BES and BEE. The expression of MMP3 in endometrial caruncular tissues (n = 5 per group) during the estrus cycle and early pregnancy was analyzed by RT-qPCR (A). Each mRNA expression normalized to GAPDH is shown as means ± SEM relative to follicle phase (= 1.0). Morphological features of BES (B) and BEE (C). The scale bar indicates 50 µm. Expression of MMP3 in BES and BEE was analyzed using RT-PCR (D). Effects of E2, P4, and IFNα on the expression of MMP3 in BES (E) and BEE (F) were also analyzed using RT-qPCR. The mRNA expression was normalized to GAPDH and shown as means ± SEM relative to the control (CT) (= 1.0). Different superscript letters indicate significant differences in each panel (P < 0.05). End, bovine endometrial tissue; NC, negative control without sample; CT, control without treatment; IFNα, human interferon α2.

Release of MMP3 protein in BES and BEE

The conditioned medium was analyzed using casein zymography to analyze the MMP3 protein release from BES and BEE (Fig. 2A). Both the precursor (57 kDa) and mature forms (50 kDa) of MMP3 were strongly detected in E2-treated BES-conditioned medium. In contrast, no MMP3 band was detected in the conditioned medium of BEE due to E2, P4, or IFNα treatment. Figure 2B shows that MMP3 release was significantly higher when BES were treated with E2 compared to the control (P < 0.05). The release of MMP3 was significantly decreased when BES were treated with P4 or IFNα (P < 0.05) (Fig. 2B). E2 upregulated MMP3 release in the BES.

Fig. 2.

Effects of E2, P4, and IFNα on the release of MMP3 in BES and BEE. Cells were cultured in the absence (CT) or presence of E2, P4, or IFNα for 48 h after confluence. The amount of MMP3 in the harvested supernatant was analyzed using casein zymography (A). Zymograms were quantified, and the activity of the precursor and mature forms was summed in BES (B). Each activity is shown as means ± SEM relative to the control (CT) (= 1.0). Different superscript letters indicate significant differences in each panel (P < 0.05). CT, control without treatment; IFNα, human interferon α2.

Effects of E2 on cellular proliferation of BES and BEE

The cells were cultured and counted after 2, 4, 6, 8, and 10 days to investigate the effects of E2 on BES and BEE cell proliferation. From day 2 to 6, the number of proliferating BES was significantly higher in the E2-treated group than in the control group (P < 0.05). They reached a plateau on day 6 of culture. The control group reached a plateau on day 8, and there was no difference compared to the E2-treated group after that (Fig. 3A). However, there was no difference in BEE cell proliferation from days 2 to 10 between the control and E2-treated groups (Fig. 3B). This result suggested that E2 enhances the proliferation of BES.

Fig. 3.

Effects of E2 on the cellular proliferation of BES and BEE. Proliferation of BES (A) and BEE (B) was determined by cell counting. The number of cells is shown as means per well. Error bars show SEM. Asterisks (*) indicate significant differences compared with that of the control group in each panel (P < 0.05). CT, control without treatment.

Effect of inhibitors on E2-induced cellular proliferation of BES

E2 positively affected BES proliferation but not on BEE (Fig. 3). In this study, the effects of other factors were analyzed using inhibitors to clarify this mechanism. The cells were treated with MMP3 and EGFR inhibitors, respectively, to clarify the involvement of MMP3 and EGF family members in E2-induced BES cell proliferation. Casein zymography of the supernatant showed that the EGFR inhibitors had no effects on the release of MMP3, whereas the MMP3 inhibitor itself reduced this release (Fig. 4A). Cell counting on day 6 revealed that the E2-induced cellular proliferation was significantly inhibited by the addition of MMP3 and EGFR inhibitors (Fig. 4B). MMP3 and EGFR inhibitors blocked the effect of estradiol on proliferation. These results indicated the positive involvement of MMP3 and EGF family growth factors in E2-induced BES cellular proliferation. The experiment used an inhibitor of the IGF-1R expressed in BES as a control to examine the effects of other growth factors, inhibitors, and inhibitors themselves. The IGF-IR inhibitor did not affect either the release MMP3 and E2-induced BES proliferation (Figs. 4A and B).

Fig. 4.

Effects of inhibitors for MMP3, EGFR, and IGF-1R on E2-induced cellular proliferation of BES. BES were treated with E2, E2+MMP3 inhibitor, E2+EGFR inhibitor, or E2+IGF-1R inhibitor for 6 days. The amount of MMP3 in the conditioned medium was analyzed using casein zymography (A). Cell proliferation was determined using cell counting (B). The number of cells is shown as means per well. Error bars show SEM. Asterisks (*) indicate significant differences compared with that of the control group in each panel (P < 0.05). PMA, Phorbol myristic acetic acid treated as positive control; CT, control without treatment.

Release of HB-EGF in the conditioned media of BES and BEE

Western blot analysis was performed to examine the expression of the HB-EGF protein in the supernatant of endometrial cells. Initially, bovine endometrial tissues at the follicular stage were analyzed to clarify the state of HB-EGF protein in the endometrium. Western blotting revealed multiple molecular bands of 28, 30, 37, and 45 kDa (Fig. 5A). Figure 5B shows the band of β-actin bands in each lane. Four bands similar to endometrial tissues were detected in the BES cell lysate, but additional bands of 22 kDa and 43 kDa were also detected. In contrast, in the analysis of the conditioned medium concentrated by the heparin-sepharose column, HB-EGF protein was hardly detected in the control group. A band was clearly detected in the conditioned media of the E2 and rhMMP3 treated groups. Bands at 24 and 43 kDa were detected in addition to the four bands detected in the tissue. However, when the cells were treated with an MMP3 inhibitor in the presence of E2, the band in the medium was barely detectable (Fig. 5C). HB-EGF protein was not detected in BEE, cell lysates, or conditioned media, even in the presence of E2 or rhMMP3 (Fig. 5D). E2 increased the release of HB-EGF protein from the supernatant of BES, but the MMP3 inhibitor suppressed this release. This indicated the potential of MMP3 to release HB-EGF from the BES.

Fig. 5.

Western blotting analysis of HB-EGF in bovine endometrium and endometrial cells. Analysis of HB-EGF (A) and β-actin (B) in bovine endometrial tissue at follicular stage. The number of the endometrium shows the different bovine endometrial tissues. Analysis of HB-EGF in BES (C) and BEE (D). Cells were treated with E2, E2+MMP3 inhibitor, or recombinant human MMP3 (rhMMP3) for 24 h. After culture, cell lysates (L) and conditioned media (M) were harvested for all cells. PM, protein marker; ET, endometrial tissue; CT, control without treatment.

Effects of rhHB-EGF and rhMMP3 on the proliferation of BES and BEE

In the case of rhHB-EGF, the number of the cells was significantly high at 0.1 and 1.0 ng/ml (P < 0.05) for BES, whereas the range was 0.1–10 ng/ml (P < 0.05) for BEE. There was no difference at 0.01 and 100 ng/ml both for BES and BEE (Supplementary Fig. 1A). For rhMMP3, the number of cells was significantly higher with 10 and 100 ng/ml for BES (P < 0.05). However, the application of rhMMP3 did not affect BEE cell proliferation at either dose (Supplementary Fig. 1B). HB-EGF has a positive role in endometrial cell proliferation.

mRNA expression of HB-EGF, EGFR, CCND1, and PCNA in BES and BEE

RT-qPCR results showed that the expression of HB-EGF and EGFR was not significantly different between the control and E2-treated groups in BES and BEE (Supplementary Figs. 2A and 2 B). In the BES, the expression of both CCND1 and PCNA was significantly increased in response to E2, rhMMP3, and rhHB-EGF compared with that in the control (P < 0.05) (Supplementary Figs. 2C and 2D). In the case of BEE, there was no difference in CCND1 expression between the treatment groups (Supplementary Fig. 2C). However, the expression of PCNA was significantly higher in the rhHB-EGF group than in the control, E2, and rhMMP3 groups (P < 0.05) (Supplementary Fig. 2D). The results revealed that proliferation marker genes were differentially expressed in BES and BEE owing to the effects of E2, rhHB-EGF, and rhMMP3.

Effects of CM of E2-treated BES on the proliferation of BEE

CM collected from E2-treated BES was applied to BEE to observe its effects on cell proliferation. To determine the ideal ratio of CM for BEE cell culture, different ratios of CM (25%, 50%, and 75%) in fresh medium (FM) were applied on BEE and cultured for 6 days. The result revealed that the number of cells in 25%CM–75%FM and 50%CM–50%FM was significantly higher compared to FM as a control group (P < 0.05). However, there was no significant difference in the cell number between the control and 75%CM–25%FM groups (Fig. 6A).

Fig. 6.

Effects of condition medium (CM) of E2-treated BES on the cell proliferation of BEE. Analysis of the effect of different concentrations of CM on cell proliferation of BEE (A). The effect of inhibitors for MMP3 and EGFR on the proliferation of BEE treated with 50% CM (B). Cell proliferation was determined by cell counting. The number of cells is shown as means per well. Error bars show SEM. Different superscript letters indicate significant differences in each panel (P < 0.05). FM, fresh medium without treatment.

The cells were treated with 50%CM with or without inhibitors of MMP3 and EGFR to demonstrate the involvement of MMP3 and EGF family members in E2-treated CM on BEE cell proliferation. The cell counting result on day 6 revealed that 50%CM without any inhibitor significantly increased the cellular proliferation of BEE (P < 0.05). The cell proliferation of the MMP3 inhibitor-treated group was significantly higher than that of the FM-treated group but significantly lower than that of the control group (P < 0.05). On the other hand, the number of cells treated with EGFR inhibitor significantly decreased compared with without adding the inhibitor in the 50%CM group (P < 0.05) (Fig. 6B). This revealed that CM increased the proliferation of BEE cells, suggesting that BES-released HB-EGF, rather than E2, affected BEE cell proliferation in a paracrine manner.

Discussion

Endometrial remodeling is essential for successful reproduction and tuning of complex hormonal cascades to confirm the structural reorganization and function of mature animals [12]. Continuous cyclic changes in uterine tissue are a unique biological behavior of females. Estradiol and progesterone play significant roles in this cyclic event [23]. In mammals, cyclic cell proliferation is important for maintaining normal endometrial function. E2, a prime hormone in the follicular stage, plays a significant role in cell proliferation in different species. The effects of E2 on cell proliferation have been widely reported in various cell types. A previous study revealed that E2 enhanced the growth and proliferation of human keratinocytes [24]. Additionally, the effects of E2 on mammary cancer cell proliferation have been reported previously [25]. The role of E2 in successful reproduction is well evident, and it has been shown that E2 stimulated uterine cell proliferation in ovariectomized mice [26]. The effects of E2 on endometrial cell proliferation have also been observed in sheep [27]. In connection with onward proliferation, it was found that E2 increased endometrial cell proliferation in bovine as dose-dependent manner [28]. Considering existing reports and accumulated knowledge, it is now well-established that E2 plays a significant role in cellular proliferation. The results of the present study support those of previous studies regarding the effects of E2. Although E2 directly stimulated BES proliferation, it did not directly affect BEE, further suggesting that the growth mechanism differs depending on cell type.

MMPs are prime regulators of cyclic remodeling in the endometrium and are guided by ECM component disruption and reformation. A previous study has shown the influence of MMPs on the proliferation and survival of vascular smooth muscle cells [29]. Limited information is available regarding the relationship between E2 and MMP3, and it has been reported that E2 transiently upregulates MMP3 mRNA and protein expression 4 h post-E2 treatment in the ovariectomized rat endometrium [30]. Coherent estrogen stimulated the mRNA expression of MMP3 in chicken ovarian granulosa cells [31]. In humans, an adjacent report showed that E2 promotes MMP3 protein secretion in cancer cell lines [32]. The results of the present study support the previous findings regarding the effects of E2 on MMP3 mRNA expression and protein release in the BES. To the best of our knowledge, this is the first study on endometrial cells. The action of E2 suggested that MMP3 plays a functional role during the follicular stage.

Accumulated HB-EGF was demonstrated by immunoreactivity in the human uterine decidual stroma [33]. The initial form of HB-EGF is a membrane-fastening growth factor. The structural mingle of HB-EGF has three major components: a heparin-binding domain, an EGF domain, and a cytoplasmic domain. HB-EGF exemption from the membrane is conducted by ectodomain detachment to transform it into a soluble form. An immunohistochemical study demonstrated the presence of membrane-anchored HB-EGF in human fibrosarcoma cells [34]. However, the presence of HB-EGF was first detected in the conditioned medium (CM) of the human histiocytic lymphoma cell line U-937 [35]. The presence of HB-EGF in CM has also been revealed in human liver [36], rat intestinal cells [37], and gastric cells [38]. The western blot results of this study also showed consistent results in E2-treated stroma-derived CM. It has been reported that HB-EGF is released by proteases in the mouse and human endometrium [39,40,41]. Release of anchored HB-EGF from the cell membrane by MMP3 has been reported in human bronchial epithelial cells [42]. HB-EGF is released from the cell membrane in mouse hematopoietic cells [40] and the cecum [43] by MMP3. A particular site in the juxtacrine domain is split by MMP3, which changes the state of HB-EGF to a paracrine or autocrine growth factor [40]. In the present study, E2 induced the release of HB-EGF and was detected in the CM, which is in accordance with the MMP3 functional role. Notably, rhMMP3 treatment increased the release of HB-EGF from CM. This further supports the role of MMP3 in HB-EGF release. The findings of the present study in the BES agreed with those of a previous report on MMP3 and HB-EGF release. In the present study, MMP3 released HB-EGF from BES but not from BEE. It has been suggested that MMP3 promotes the presence of growth factors by releasing them from the cell membrane of the BES.

HB-EGF is a member of the EGF family and a membrane-anchored growth factor with mitogenic effects [44]. The mitogenic activity of HB-EGF is well documented. In the rat pancreas, it is noteworthy that a rapid increase in the number of β-cell is induced by HB-EGF [45]. HB-EGF regulates endometrial stromal decidua and cell proliferation via cyclin D3 in mice [46]. Furthermore, HB-EGF stimulated the proliferation of uterine epithelial cells in primary cultures in rodents [47]. The results of the present study support those of a previous report on the effects of HB-EGF on cellular proliferation. In the present study, HB-EGF was detected in the CM of E2-treated BES, supporting the findings of a previous report. The local synthesis and release of HB-EGF have both autocrine and paracrine roles in cell proliferation. Lessey et al. [48] described a potential paracrine role of HB-EGF in the human endometrium during implantation. In a previous study, CM from human endometrial stromal cells led to a similar stimulation of HB-EGF to uplift integrin on epithelial cells. These results suggest the effects of stromal-derived HB-EGF on epithelial cells. The results of the present study showed similar stimulatory effects of HB-EGF when the CM increased the proliferation of BEE. It has been suggested that stromal-derived HB-EGF participates in BEE cell proliferation. However, BEE did not produce HB-EGF, and the CM of BES increased proliferation, which agrees with the paracrine role of stromal-derived HB-EGF in BEE proliferation. It was further suggested that HB-EGF released from BES regulated BES and BEE proliferation in autocrine and paracrine manners.

The accumulated knowledge of previous findings and the present study suggests a complex mechanism for E2-induced cellular proliferation, which depends on the cell type. An immunohistochemical study revealed that the proliferation marker (Ki-67)-positive cells tended to be higher in stromal and epithelial cells during the follicular stage of the bovine endometrium [49]. The present study demonstrated that MMP3 is necessary for the E2-induced proliferation of BES. The current findings also support the findings in other cell types in which MMP3 involvement was confirmed in the proliferation mechanism. MMP3 is involved in liberating signaling molecule precursors such as HB-EGF from the cell surface and promotes cell proliferation [40]. This study confirmed the involvement of HB-EGF in the E2-induced proliferation of endometrial cells, which were released from the BES by MMP3. The development of uterine tissue is regulated through the close connection and synchronization between epithelial and stromal cells. Expression in the uterine epithelium is led by stromal cells [50].

Finally, the findings of the present study suggest that MMP3 is involved in the proliferation of endometrial cells during the follicular stage of the bovine endometrium. The results are summarized in Supplementary Fig. 3. To the best of our knowledge, this is the first study to demonstrate the complex mechanism underlying bovine endometrial cell proliferation during the follicular stage. These findings provide new insights into the expression mechanisms of MMP3, which are critically regulated in the bovine endometrium.

Conflict of interest

The authors declare that they have no conflict of interest.