Activation of G Protein-coupled Receptor 30 Promotes Proliferation of Goat Mammary Epithelial Cells via MEK/Erk&PI3K/Akt Signaling Pathway

were harvested with 0.25% (w/v) trypsin and resuspended in fresh medium. Cell numbers were determined using a hemocytometer, and proliferation was expressed as population doubling time (T D ). T D = t × log 2 / (log N t - log N 0 ), where t is the culturing time; N 0 is the initial cell numbers; N t is the cell numbers after culturing time.


Background
Goat is an important dairy animal. The synthetic capacity of milk from the mammary gland relies largely on the quantity and e ciency of functional mammary epithelium [1]. During a pregnancy/lactation cycle, the mammary epithelial cells proliferate rapidly to form ducts and secretory alveoli through hormonal regulation, resulting in remodeling of the glandular tissue architecture into a milk-secretory organ [2,3].Estrogen (mainly 17β-estradiol) is a key regulator of mammary gland development, especially for its essential in the epithelial cell proliferation and ductal morphogenesis of the mammary gland [4,5].
As a membrane estrogen receptor, G protein-coupled receptor 30 (GPR30) is widely expressed in reproductive tissues, such as mammary glands [5], ovary [6], bone [7], uterus [8], and others [9,10]. After binding with estrogen, GPR30 mediates the rapid non-genomic actions of estrogen by stimulating the intracellular second messenger signals [11]. In normal human breast epithelial cells, estrogenic GPR30 signaling promoted cell proliferation via the MEK/Erk pathway, but GPR30 knockdown caused a reverse [12]. Similarly, this phenomenon was also found in endometrial carcinoma cells [13] and Sertoli TM4 cells [9]. On the other hand, estrogen-stimulated and GPR30-mediated promotion of uterine epithelial cell growth was regulated via PI3K/Akt signaling, but GPR30 antagonist G15 inhibited the effect induced by estrogen [14,15]. Moreover, depletion of GPR30 abolished estrogen-induced PI3K/Akt signaling activation and led to the suppression of endometrial cancer cell proliferation [8]. These ndings indicate that GPR30-mediated non-genomic signaling could play a signi cant role in cell proliferation.
Normal cell-cycle progression is a crucial event for every multicellular organism, due to it deciding body size and shape, tissue renewal and senescence, and is also important for reproduction [16], which is closely related to cell proliferation. In bovine mammary epithelial cells, an abundance of S and G2 phases [17,18] and up-regulated expression of cyclin D1 mRNA [17] were accompanied in the promotion of cell proliferation. In addition, G1 promoted the proliferation of GPR30-positive human thyroid cancer cells and upregulated the expression of cyclin A and D1 [19]. Interestingly, G1 repressed the survival ability via cell arrest in the G2 phase of prostate cancer cells [20], which was veri ed in the mouse melanoma cells [21]. Parallelly, the expression of cyclin A2, cyclin B1, cdc25c, and cdc2 was down-regulated after G1 treatment [20], suggesting that estrogen/GPR30 signaling played a vital role in the initiation and progression of cell proliferation. Although estrogen/GPR30 signaling has been demonstrated involving in cell proliferation regulation of breast cancer cells [12,19], endometrial carcinoma cells [13], prostate cancer cells [20], ovarian cancer cells [6], and others [9,10], the relationship between estrogen/GPR30 signaling and proliferation of goat mammary epithelial cells has not been reported. And the molecular mechanisms underlying the proliferative effect of estrogen via GPR30 on GMECs remain unclear.
In this study, we explored whether estrogen/GPR30 signaling affected the proliferation of GMECs. The results presented here may provide a new perspective on the effect of estrogen/GPR30 signaling on the regulation of goat mammary gland development.

Methods
Goat mammary epithelial cell isolation and culture Goat mammary gland tissues were surgically isolated from a 2-year-old lactating Guanzhong dairy goat at 36 d of lactation as described previously [22], which were reared in Shaanxi Province (P. R. China). The mammary parenchyma was obtained under sterile conditions, placed in sterilized PBS with penicillin, streptomycin, and amphotericin B at 100 U/mL, 100 µg/mL, and 25 ng/mL (Sigma, St. Louis, MO, USA), and immediately transported to the laboratory. Tissue pieces were nely minced and put into a 24-well culture plates, one piece for each hole, and cultured in Dulbecco's Modi ed Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; BasalMedia, Shanghai, China) containing 10% KnockOut Serum Replacement (SR; Gibco, Waltham, MA, USA) and 10 ng/mL epidermal growth factor (EGF; Sigma). The medium was replaced every 3 d, until cells had spread across the bottom of the plate. All cells were cultured in a 37°C incubator with 5% CO 2 .

Cell lines and cell culture
The HEK-293T cells was purchased from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China), and were cultured in Dulbecco's modi ed Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) prior to further treatment. All cells were cultured in a 37°C incubator with 5% CO 2 .
All sequences are as described in Table 1. The method of constructing short hairpin RNA lentivirus vectors was performed as previous description [23]. After detecting by ampicillin (Solarbio, Beijing, China), drugresistant colonies were delivered to be analyzed by Sangon Biotech (Shanghai) Co., Ltd. For knockdown of the target GPR30, synthesized vectors and packaging vectors were transfected into HEK-293T cells using TurboFect Transfection Reagent (Thermo Fisher Scienti c, Waltham, MA, USA) to form lentivirus. GMECs were incubated with lentivirus of shGPR30 and shRNA for 18 h before removing. After transfection, cells were allowed to grow, and then gained for protein extraction. Western blot analysis was used to evaluate the knock-down e ciency of GPR30. The GMECs were seeded in 24-well culture plates (1 × 10 4 cells/well) and allowed to grow for 6 d. After this, cells were washed 3 times with PBS for 5 min each time, and xed for 30 min using 4% paraformaldehyde (Solarbio) at room temperature. All xed cells were washed 3 times with PBS and incubated with a blocking buffer (1% FBS in PBS) for 2 h at room temperature. Subsequently, cells were incubated with primary antibody against cytokeratin 18 (CK18; ab668, abcam, Cambridge, MA, USA) and GPR30 (ab39742, abcam) overnight at 4°C. The nuclei were stained with Hoechst 33342 (Sigma) in dark for 5 min at room temperature. Images were captured using a uorescence microscope (Olympus, Tokyo, Japan).

Cell cycle analysis
Flow cytometry analysis was used to measure cell cycle distribution. GMECs were cultured in 6-well culture plates at a density of 1. Cell counting assay Cell proliferation was tested by cell counting. Brie y, 1 × 10 4 cells/well were seeded in 24-well plates for 24 h, and then cultured with estrogen (Sigma), G1 (Cayman Chemical Co), and estrogen + G15 (Cayman Chemical Co) for 5 d. Next, cells were harvested with 0.25% (w/v) trypsin and resuspended in fresh medium. Cell numbers were determined using a hemocytometer, and proliferation was expressed as population doubling time (T D ). T D = t × log 2 / (log N t -log N 0 ), where t is the culturing time; N 0 is the initial cell numbers; N t is the cell numbers after culturing time.

Cell viability assay
Cell viability was evaluated using the Cell Counting Kit-8 kit (BOSTER, Wuhan, China). In detail, 6 × 10 3 cells/well were seeded in 96-well plates for 24 h and then cultured with estrogen (Sigma), G1 (Cayman Chemical Co), and estrogen + G15 (Cayman Chemical Co) for 5 d. Next, CCK-8 solution (10 µL/well) was added and incubated for 2 h, and the absorbance value was measured at 450 nm.
Bromodeoxyuridine labeling and immuno uorescence assay Cell proliferation was detected using the BrdU assay. Brie y, GMECs were seeded in 24-well culture plates at

Statistical analysis
The data were presented as average values ± standard error of the means from 3 independent experiments of cell counting assay, cell viability assay, BrdU assay, and Western blot analysis. Statistical analyses were performed with SPSS (version 20.0; SPSS Inc., Chicago, IL, USA), which were checked using Tukey's test.

Estrogen promotes GMECs proliferation via GPR30
The isolated cells expressed cytokeratin 18 and β-casein tested by immuno uorescence and Western blot respectively ( Fig. 1a and b), de ning their mammary alveolar epithelium origination. Furthermore, the expression of GPR30 was identi ed by immuno uorescence and Western blot. As the results showed, GMECs continuously express GPR30 during the experiments (P3-P9) ( Fig. 1c and d). Since estrogen is the ligand of GPR30, GMECs were treated with different concentrations of estrogen. After treatment with 0.1 µM and 1 µM estrogen, population doubling time was obviously decreased compared with control (0 µM) (Fig.   1e). Meanwhile, the CCK-8 assay and BrdU assay showed the similar results that 0.1 µM and 1 µM estrogen evidently promoted the survival and mitosis of GMECs compared to control (0 µM) (Fig. 1f, g, and h). These results suggested that estrogen promoted the proliferation of GMECs. As the speci c agonist of GPR30, G1 was used to treat GMECs. Similar to estrogen, G1 group presented a decrease of population doubling time (Fig. 1e) as well as an improvement of cell survival (Fig. 1f) and mitosis of GMECs ( Fig. 1g and h) compared with control group (0 µM), and there was no signi cance between 0.1 µM and 1 µM estrogen group. G15, the speci c antagonist of GPR30, was added to determine whether the effect of estrogen could be inhibited. As predictably, in the presence of G15, estrogen-induced GMECs proliferation was obviously repressed compared with estrogen alone (Fig. 1e, f, g, and h). These results indicated that estrogen promoted the proliferation of GMECs via GPR30.

RNAi-mediated silence of GPR30 suppresses estrogen-induced GMECs proliferation
To further explore the role of GPR30 activation induced by estrogen in GMECs proliferation, GPR30 expression was silenced by RNAi. As showed in Fig. 2a, shGPR30-2 treatment had the greatest downregulated effect on GPR30 expression (Fig. 2a). Therefore, shGPR30-2 was chosen to use in followed experiments. When GPR30 was knocked down, in contrast to negative control, population doubling time was markedly increased in spite of the presence of estrogen and G1 (Fig. 2b). Similarly, cell viability was visibly inhibited as measured by the CCK-8 assay (Fig. 2c), and the proportion of BrdU-positive cells was also clearly downregulated ( Fig. 2d and e) after GPR30 knockdown. These data revealed that RNAi-mediated silence of GPR30 suppressed estrogen-induced and G1-driven promotion of cell proliferation, suggesting a proliferative role of GPR30 activation in GMECs.

GPR30 activation and inactivation altered cell cycle distribution
Because the proliferation of GMECs was obviously promoted in response to estrogen exposure, we then explored whether the cell cycle was also altered following estrogen treatment.  . 3a). In particular, GPR30 knockdown altered cell cycle distribution, accumulating in the G2/M phase compared to negative control in spite of the presence of estrogen and G1 (10.93 ± 0.62% vs. 8.12 ± 0.15%; 9.87 ± 0.57% vs.7.75 ± 0.43%) (Fig. 3b).
To further elucidate the underlying mechanisms, the expression of cell cycle checkpoint regulators was examined. Estrogen treatment improved the level of cyclin D1, cyclin B1, CDK1, and p-CDK1 contrasted with control. Similar to estrogen, G1 treatment presented a positive effect on the expression of cyclin D1, cyclin B1, CDK1, and p-CDK1. Nevertheless, G15 treatment cause a reverse, suppressing estrogen-induced expression of cyclin D1, cyclin B1, CDK1, and p-CDK1 (Fig. 3c). When GPR30 was knocked down, the levels of cyclin D1, cyclin B1, CDK1, and p-CDK1 were downregulated than negative control (Fig. 3d). These results demonstrated that GPR30 activation driven by estrogen upregulated S phase proportion, and GPR30 inactivation induced cell cycle arrest in the G2/M phase. serves as a loading control. Data are shown as mean ± SEM, n = 3. Statistical signi cance was determined using Tukey's test. *, p < 0.05; **, p < 0.01; ns, not signi cant.

MEK/Erk and PI3K/Akt signaling pathway are involved in GMECs proliferation
For in-depth investigation of the molecular regulation mechanisms of GPR30 activation induced by estrogen involved in cell proliferation, small molecular inhibitors were applied. Western blot results revealed that estrogen treatment obviously promoted the phosphorylation of Erk1/2 and Akt compared with control. After G1 treatment, an abundance of phosphorylated Erk1/2 and Akt were detected by Western blot. Conversely, G15 treatment visibly inhibited the estrogen-induced phosphorylation of Erk1/2 and Akt (Fig. 4a). When GPR30 was silenced, in comparison to negative control, a decrease in p-Erk1/2 and p-Akt was determined (Fig. 4b). Additionally, with the treatment of MEK inhibitor U0126, the rise in protein levels of Cyclin D1, Cyclin B1, CDK1, and p-CDK1 induced by estrogen and G1 was abolished. Similarly, LY294002, the PI3K inhibitor, markedly decreased the expression of cyclin D1, cyclin B1, CDK1, and p-CDK1 driven by estrogen and G1 (Fig. 4c).
To investigate the relationship between MEK/Erk&PI3K/Akt signaling pathway and GMECs proliferation further, we detected the growth of GMECs after treatment with U0126 and LY294002. In the presence of estrogen and G1, U0126 treatment upregulated the population doubling time (Fig. 5a). In addition, the CCK-8 assay discovered that U0126 treatment obviously impaired the viability of GMECs elicited by estrogen and G1, respectively (Fig. 5b). BrdU assay further con rmed the result that U0126 treatment inhibited the estrogen-induced and G1-driven promotion of mitosis ( Fig. 5c and d). In parallel, LY294002 treatment evidently suppressed the growth of GMECs originated from estrogen and G1 (Fig. 5). These data suggested that GPR30 activation induced by estrogen promoted GMECs proliferation via the MEK/Erk and PI3K/Akt signaling pathway.

Discussion
In this study, we discovered that estrogen promoted GPR30-mediated proliferation of mammary epithelial cells by altering cell cycle distribution via MEK/Erk&PI3K/Akt signaling pathway, providing a new perspective on the effect of estrogen/GPR30 signaling on the regulatory action of goat mammary gland development.
Estrogen is a key regulator of mammary gland development, especially for its essential role in the proliferation of epithelial cells and ductal morphogenesis of mammary gland [4,5]. As a novel estrogen membrane receptor, GPR30 plays an important role in the regulation of estrogen mediated signaling pathways [11]. In mammary epithelial cells, GPR30 agonist G1 promoted cell growth, nevertheless, GPR30 antagonist G36 partly abolished G1-mediated cell proliferation [12]. In addition, estrogen signi cantly enhanced the proliferation of cervical adenocarcinoma cells [24] and endometrial carcinoma cells [8,25] by stimulating GPR30. Intriguingly, GPR30 activation by G1 inhibited the growth of mouse-derived neural stem/progenitor cells, what's more, GPR30 siRNA reversed the inhibitory effect of G1 on cell proliferation [26]. Here, this study found that estrogen/GPR30 signaling was involved in GMECs proliferation. Activation of GPR30 induced by estrogen could promote GMECs proliferation, as predictably, G15 suppressed estrogenstimulated and GPR30-mediated GMECs proliferation (Fig. 1). Importantly, RNAi-mediated silence of GPR30 inhibited estrogen-induced and G1-driven promotion of cell proliferation (Fig. 2), suggesting a proliferative role of GPR30 activation in GMECs.
Generally, cell proliferation is closely associated with cell cycle [27,28]. In breast cancer cells, estrogen promoted cell progression by signi cant accumulation in the S phase and a high expression of cyclin D1 [29]. In addition, a high proportion in the S and G2/M phases was also observed in the promotion of mouse osteoblast cell growth [30,31], and up-regulated expression of cyclin B, cyclin D, and cyclin E at the transcription and translation levels were accompanied in the positive proliferation of porcine granulosa cells [32]. Particularly, GPR30 activation by G1 inhibited prostate cancer cell growth and downregulated the expression of cyclin A2, cyclin B1, cdc25c, and cdc2, resulting in the cell cycle arrest in the G2 phase [20]. In this study, GPR30 stimulation caused obvious differences in the cell cycle distribution and checkpoint regulator expression. GPR30 activation driven by estrogen upregulated S phase proportion, and GPR30 inactivation induced cell cycle arrest in the G2/M phase via down-regulation of cyclin D1, cyclin B1, CDK1, and p-CDK1 expression (Fig. 3).

Conclusions
In summary, this work determines that estrogen acting on GPR30 contributes to the proliferation of mammary epithelial cells by affecting cell cycle progression via MEK/Erk&PI3K/Akt signaling pathway, which is helpful to improve milk yield. This work may provide a new insight into the effect of estrogen/GPR30 signaling on the regulation of goat mammary gland development.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.