Fibroblast growth factor 2 induces proliferation and distribution of G2/M phase of bovine endometrial cells involving activation of PI3K/AKT and MAPK cell signaling and prevention of effects of ER stress
Whasun Lim1,†, Hyocheol Bae2,†, Fuller W. Bazer3 and Gwonhwa Song2
Abstract
Fibroblast growth factor 2 (FGF2) is abundantly expressed in conceptuses and endometria during pregnancy in diverse animal models including domestic animals. However, its intracellular mechanism of action has not been reported for bovine endometrial cells. Therefore, the aim of this study was to identify functional roles of FGF2 in bovine endometrial (BEND) cell line which has served as a good model system for investigating regulation of signal transduction following treatment with interferon-tau (IFNT) in vitro.
Results of present study demonstrated that administration of FGF2 to BEND cells increased their proliferation and regulated the cell cycle through DNA replication by an increase of PCNA and Cyclin D1. FGF2 also increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, and P38 in BEND cells in a dose-dependent manner, and expression of each of those transcription factors was inhibited by their respective pharmacological inhibitor including Wormannin, U0126 and SP600125. In addition, the increase in proliferation of BEND cells and activation of the protein kinases in response to FGF2 was suppressed by BGJ398, a FGFR inhibitor. Furthermore, proliferation of BEND cells was inhibited by tunicamycin, but treatment of BEND cells with FGF2 restored proliferation of BEND cells. Consistent with this result, the stimulated unfolded protein response (UPR) regulatory proteins induced by tunicamycin were down-regulated by FGF2. Results of this study suggest that FGF2 promotes proliferation of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating cell signaling via the PI3K and MAPK pathways and by restoring
Keywords: FGF2, proliferation, uterine epithelial cells, ER stress
Introduction
Establishment of successful pregnancy requires reciprocal interactions between the conceptus and uterine endometrium for enhancing implantation and reducing fetal loss (Spencer and Bazer, 2004; Su and Fazleabas, 2015). In ruminants, including sheep and cattle, interferon-τ (IFNT) derived from the trophoblast acts as a pregnancy signaling molecule during early gestation by silencing expression of estrogen receptor alpha and oxytocin receptor to abrogate the mechanism for pulsatile release of luteolyitic prostaglandin F2α (PGF2α) which prevents luteolysis and ensure continued secretion of progesterone by the corpus luteum (Bazer, 2013). Progesterone then stimulates of morphological changes of endometrial epithelial cells for uterine receptivity, decidualization and placentation through autocrine, paracrine and endocrine signaling (Bazer, 1975; Roberts, 2007). Especially in cattle, the endometrium synthesizes and secretes histotroph that includes IFNT, prostaglandins, growth factors and cytokines for recognition of pregnancy, conceptus elongation, implantation and embryogenesis (Mullen et al., 2012). Unless sufficient histotroph is provided during early gestational period in cattle, embryonic and fetal death losses may be 40 to 50% (Berg et al., 2010; Diskin and Morris, 2008). Therefore, understanding regulatory mechanisms in response of endometrial epithelia to various components of histotroph is critical to reproductive success in cattle. For this reason, bovine endometrial (BEND) cells were established by spontaneously immortalizing endometrial cells from Day 14 of the estrous cycle (Staggs et al., 1998). BEND cells have morphological characteristics of both uterine epithelial cells and stromal cells by expressing cytokeratin and vimentin (Parent et al., 2002; Thatcher et al., 2001). Although these cells are useful for research on prostaglandin biosynthesis by the uterine endometrium that is important for maintenance of the corpus luteum and pregnancy (Godkin et al., 2008; Guzeloglu et al., 2004), further research on regulatory mechanisms of diverse factors affecting endometrial function during pregnancy are required.
Fibroblast growth factors (FGFs) are implicated in the maintenance of pregnancy success by regulating embryogenesis, implantation and placentation through cell survival, migration and differentiation (Cooke et al., 2009; Dorey and Amaya, 2010). One member of the FGF family is FGF2. FGF2, also known as basic FGF, has distinct roles during early embryogenesis in ruminants. FGF2 and its receptor FGFR2 are expressed during development of bovine embryos and a combination of FGF2 and transforming growth factor beta (TGF-β) improves development of bovine embryo (Larson et al., 1992; Lazzari et al., 2002). In addition, endometrial FGF2 stimulates secretion of IFNT by bovine conceptuses (Michael et al., 2006) and increase implantation of mouse blastocysts (Michael et al., 2006; Taniguchi et al., 1998) Moreover, expression of FGF2 mRNA is abundant in bovine endometria during the peri-implantation period of pregnancy (Bai et al., 2015). However, the intracellular signaling mechanisms induced by FGF2 in bovine endometria not well known.
Therefore, the objectives of this study were to: (1) to investigate direct effects of FGF2 on proliferation and cell cycle of bovine endometrial luminal epithelial cells; (2) identify intracellular signaling mechanisms in response to FGF2 that increase proliferation of BEND cells; and (3) to determine regulatory effects of FGF2 on ER stress in BEND cells. Results of present study provide novel insights into the FGF2-induced proliferation and distribution of G2/M phase BEND cells involving activation of PI3K/AKT and MAPK cell signaling and prevention of effects of ER stress.
Materials and Methods
Cell culture
The BEND cell line was purchased from American Type Culture Collection (Manassas, VA, USA) that was derived from the uterine endometrium of a normal female cow on Day 14 of the estrous cycle in 1997 in Laramie, Wyoming, United States. Briefly, monolayer cultures of BEND cells were grown in 1:1 mixture of Ham’s F12 and Eagle’s Minimal Essential medium with Earle’s BSS (D-valine modification) with 1.5 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate supplemented with 0.034 g/L D-valine, 10% heatinactivated fetal bovine serum (FBS) and 10% heat-inactivated horse serum to 80% confluence in 100-mm tissue culture dishes at 37 °C and 5% CO2. For assays, in vitro cultured BEND cells were serum starved for 24 h, and then incubated in the presence of various concentrations of FGF2.
Reagents
Recombinant FGF2 (catalog number: 233-FB/CF) was purchased from R&D Systems (Minneapolis, MN, USA). Tunicamycin (catalog number: T7765) was purchased from Sigma-Aldrich, Inc (St.Louis, MO, USA). The antibodies against phosphorylated AKT (Ser473, catalog number: 4060), ERK1/2 (Thr202/Tyr204, catalog number: 9101), JNK (Thr183/Tyr185, catalog number: 4668), P38 (Thr180/Tyr182, catalog number: 4511), P70S6K (Thr421/Ser424, catalog number: 9204), S6 (Ser235/236, catalog number: 2211), Cyclin D1 (catalog number: 3300) and eIF2α (Ser51, catalog number: 3398) and total AKT (catalog number: 9272), ERK1/2 (catalog number: 4695), JNK (catalog number: 9252), P38 (catalog number: 9212), P70S6K (catalog number: 9202), S6 (catalog number: 2217), Cyclin D1 (catalog number: 2922), eIF2α (catalog number: 5324) and IRE1α (catalog number: 3294) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies 981, catalog number: sc-32577), total PERK (catalog against phosphorylated PERK (Thrnumber: sc-13073), ATF6α (catalog number: sc-166659) and GRP78 (catalog number: sc 13968) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Inhibitors for ERK1/2 (U0126, catalog number: EI282) and JNK (SP600125, catalog number: EI305) were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), and PI3K/AKT inhibitor (Wortmannin, catalog number: 9951) was from Cell Signaling Technology, Inc. The FGFR inhibitor (BGJ398, catalog number: S2183) was purchased from Selleckchem (Houston, TX, USA).
Proliferation assay
Proliferation assays were conducted using a Cell Proliferation ELISA, BrdU kit (Cat No. 11647229001, Roche) according to the manufacturer’s recommendations. Briefly, the BEND cells were seeded in a 96-well plate, and then incubated for 24 h in serum-free 1:1 mixture of Ham’s F12 and Eagle’s Minimal Essential medium. Cells were then treated with FGF2 alone (0, 1, 5, 10, 25, 50, 100, 200 ng), tunicamycin alone (0.25 ng) or with various inhibitors of transcription factors including wortmannin (1 μM), U0126 (20 μM), SP600125 (1 μM) and BGJ398 (20 μM) in a final volume of 100 μL/well. After 48 h of incubation, 10 μM 5-bromo-2′-deoxyuridine (BrdU) was added to the cell culture and the cells were incubated for an additional 2 h at 37°C. After labeling cells with BrdU, the fixed cells were incubated with anti-BrdU-peroxidase (POD) working solution for 90 min. The anti-BrdUPOD binds to BrdU incorporated in newly synthesized cellular DNA and these immune complexes were detected by the reaction to 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate.
The absorbance values of the reaction product were quantified by measuring the absorbance at 370 nm and 492 nm using an ELISA reader.
Cell cycle analysis
Cells were seeded in a 6-well plate, and then incubated for 24 h in serum- free 1:1 mixture of Ham’s F12 and Eagle’s Minimal Essential medium. Cells were then treated with FGF2 in a dose-dependent manner (0, 5, 10, 25 and 50 ng) for 48 h. After treatment, the cells were centrifuged, washed twice with cold 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), and fixed in 70% ethanol at 4 °C for 24 h. The BEND cells were then centrifuged and the supernatant was discarded. Pellets were washed twice with 0.1% BSA in PBS and stained with propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) in the 100 μg/mL RNase A (Sigma-Aldrich) for 30 min in dark. Fluorescence intensity was analyzed using a flow cytometer (BD Biosciences).
Immunofluorescence analysis
The effects of FGF2 on the expression of PCNA and Cyclin D1 were determined by immunofluorescence microscopy. The BEND cells (3×104 cells per 300 μL) were seeded on confocal dishes (catalog number: 100350, SPL Life Science, Republic of Korea) and then incubated for 24 h in serum-free 1:1 mixture of Ham’s F12 and Eagle’s Minimal Essential medium. For detection of proliferating cell nuclear antigen (PCNA) and Cyclin D1 protein, the serum starved cells were treated with FGF2 for 24 h. The cells were fixed using methanol and probed with mouse anti-human monoclonal PCNA and rabbit anti-human polyclonal Cyclin D1 at a final dilution of 1:100. Negative controls for background staining included substitution of the primary antibody with purified non-immune mouse IgG or rabbit IgG. Cells were then incubated with goat anti-mouse IgG Alexa 488 (catalog number: A11017, Invitrogen, Carlsbad, CA, USA) or goat anti-rabbit IgG Alexa 488 (catalog number: A-11008, Invitrogen) at a 1:200 dilution for 1 h at room temperature. Then, the BEND cells were washed using 0.1% BSA in PBS and overlaid with 4′,6-diamidino-2-phenylindole (DAPI). For each primary antibody, images were captured using a LSM710 (Carl Zeiss, Thornwood, NY, USA) confocal microscope that was fitted with a digital microscope AxioCam camera with Zen2009 software. Relative fluorescence intensity was quantified by green/DAPI ratio using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
Western blot analyses
Concentrations of protein in whole-cell extracts were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) with BSA as the standard. Proteins were denatured, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose. Blots were developed using enhanced chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). Immunoreactivewere detected using goat anti-rabbit or mouse polyclonal antibodies against phosphorylated proteins and total proteins at a 1:1000 dilution and 10% SDS/PAGE gel. As a loading control, total proteins were used to normalize results from detection of proteins by western blotting. Multiple exposures of each western blot were performed to ensure linearity of chemiluminescent signals.
RNA isolation
Total cellular RNA was isolated from BEND cells using Trizol reagent (Invitrogen) and purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.
Semiquantitative RT-PCR analysis
The cDNA was synthesized from total cellular RNA (1 μg) using random hexamer (Invitrogen) and oligo (dT) primers and AccuPower RT PreMix (Bioneer, Daejeon, Republic of Korea). The cDNA was diluted (1:10) in sterile water before use in PCR. Specific primers for bovine FGFR1 (forward: 5′- CAG CTG CCA AGA CAG TGA AG -3′; reverse: 5′- ATC TTA CTC CCG TTC ACC TC -3′) amplified a 394-bp product, FGFR2 (forward: 5′- TCG CAT TGG AGG CTA TAA GG -3′; reverse: 5′- TCC GTC ACA TTG AAC AGA GC -3′) amplified a 390-bp product, FGFR3 (forward: 5′- CAC CGA CAA GGA GCT AGA GG 3′; reverse: 5′- CAG GAT GAA GAG GAG GAA GC -3′) amplified a 217-bp product and FGFR4 (forward: 5′- CTT GAA TGG GCA CGT TTA CC -3′; reverse: 5′- ACA CCT TGC AGA GCA GTT CC -3′) amplified a 413-bp product. The bovine GAPDH (housekeeping gene) (forward: 5′- CAC AGT CAA GGC AGA GAA CG -3′; reverse: 5′- CAT AAG TCC CTC CAC GAT GC -3′) designed from sequences in the GenBank database using Primer 3 (ver. 4.0.0) amplified a 352-bp product. All primers were synthesized by Bioneer. The primers, PCR amplification, and verification of their sequences were conducted as described previously (Song et al., 2007; Stewart et al., 2000). PCR amplification was conducted using approximately 120 ng cDNA as follows: 1) 95 °C for 3 min; 2) 95°C for 20 sec, 60°C for 40 sec, and 72°C for 1 min for 35 cycles; and 3) 72°C for 10 min. After PCR, equal amounts of reaction product were analyzed using a 1% agarose gel, and PCR products were visualized using ethidium bromide staining. The amount of DNA present was quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a Gel Doc XR+ system with Image Lab software (Bio-Rad).
Statistical analyses
All quantitative data were subjected to least squares analysis of variance (ANOVA) using the General Linear Model procedures of the Statistical Analysis System (SAS Institute Inc., Cary, NC, USA). Western blot analyses were corrected for differences in sample loading using total protein data as a covariate. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A Pvalue less than or equal to 0.05 was considered significant. Data are presented as leastsquare means (LSMs) with standard errors (SEs).
Results
Effects of dose dependent FGF2 administration on bovine endometrial luminal epithelial cells
To investigate dose-dependent effects of FGF2 (0, 1, 5, 10, 25, 50, 100 and 200 ng) on proliferation of BEND cells, we performed cell proliferation assays. As shown in Figure 1A, FGF2 increased proliferation of BEND cells approximately 119% (P < 0.05), 139% (P < 0.01), 150% (P < 0.001), 149%(P < 0.01) and 149% (P < 0.05) at 10 ng, 25 ng, 50 ng, 100 ng and 200 ng, respectively, compared to non-treated BEND cells (100%). Based on these results, 50 ng FGF2 was used in all future experiments. Then, we confirmed results of the proliferation assays by immunofluorescence analysis to detect expression of proliferating cell nuclear antigen (PCNA) protein which is well-known as a marker for DNA replication. The PCNA proteins were more abundant in nuclei of FGF2-treated BEND cells than non-treated BEND cells (Figure 1B). The intensity of PCNA protein was increased 407% (P < 0.01) in BEND cells by FGF2 (50 ng) compared to non-treated control cells (Figure 1C).
Effects of FGF2 on cell cycle regulation of bovine uterine luminal epithelial cells
Next, we determined effects of FGF2 on phase of the cell cycle for BEND cells as illustrated in Figure 2. We analyzed stage of cell cycle by quantitation of DNA content using a flow cytometry after staining with propidium iodide (PI). The effect of FGF2 to regulate the cell cycle of BEND cells was to decrease cells in the G0/G1 phase and increase cells in the G2/M phase of cell cycle (Figure 2A). In addition, we demonstrated that FGF2 increased the abundance of Cyclin D1 protein which is associated with actively proliferating cells (Figure 2B). Although Cyclin D1 was slightly detectable in nuclei of non-treated BEND cells, there was abundant Cyclin D1 in nuclei of FGF2-treated BEND cells (Figure 2B). The abundance of Cyclin D1 was increased about 240% (P < 0.01) by FGF2 (50 ng) in BEND cells as compared to non-treated control cells (Figure 2C).
FGF2activates PI3K/AKT and MAPK signal transduction in BEND cells
To identify FGF2-mediated signal transduction pathways in BEND cells, we determined phosphorylation of signaling molecules associated with PI3K/AKT and MAPK-mediated proliferation of cells. Phosphorylation of AKT, P70S6K and S6 proteins was increased by FGF2 in a dose dependent manner (0, 5, 10, 25 and 50 ng) in BEND cells at 30 min posttreatment (P < 0.001, P < 0.01 and P < 0.05). For the MAPK pathway, phosphorylated ERK1/2 and JNK proteins increased approximately 1.9- (P < 0.01) and 2.2-fold (P < 0.01) in response to 10 ng FGF2 in BEND cells, and that state of phosphorylation was maintained at doses to 50 ng FGF2 (Figures 3D and 3E). The phosphorylation of P38 in BEND cells increased from 5 to 50 ng of FGF2 (Figure 3F). Furthermore, a downstream signaling molecule of PI3K and MAPK cell signaling, Cyclin D1, was also increased in BEND cells approximately 1.8-fold (P < 0.05) at 50 ng FGF2 compared to 0 and 5 ng FGF2 (Figure 3G).
We next investigated proliferation and activation of protein kinases in BEND cells in response to FGF2 alone or in the presence of pharmacological inhibitors including wortmannin (a PI3K inhibitor, 1 μM), U0126 (an ERK1/2 MAPK inhibitor, 20 μM) and SP600125 (a JNK inhibitor, 20 μM) to confirm FGF2-regulated cell signaling pathways (Figure 4). The induction of phosphorylation of AKT by FGF2 was inhibited by wortmannin (P<0.01) and SP600125 (P<0.05), but increased (P<0.01) by U0126 (Figure 4A).
The phosphorylation of P70S6K and S6 proteins were inhibited significantly by wortmannin, U0126 and SP600125 (Figure 4B and 4C). Moreover, the FGF2-induced phosphorylation of ERK1/2 was completely inhibited by U0126 in BEND cells (Figure 4D), while phosphorylation of JNK was inhibited significantly by U0126 and SP600125, but not wortmannin and U0126 (Figure 4E). The induction of phosphorylation of P38 in response to FGF2 was only inhibited by U0126 (Figure 4F), while FGF2-induced phosphorylation of Cyclin D1 in BEND cells was inhibited significantly by wortmannin, U0126 and SP600125 (Figure 4G). Taken together, these results show that FGF2-induced proliferation of BEND cells is regulated by activation of PI3K and MAPK signal transduction.
Effects of blocking FGF2 receptor on proliferation and PI3K and MAPK pathways in BEND cells
To investigate the role of receptors for FGF2 in BEND cells, we first determined the expression of FGF receptors (FGFR) including FGFR1, FGFR2, FGFR3 and FGFR4 in BEND cells by semi-quantitative RT-PCR (Supplemental figure 1). Among those four genes, only FGFR1 mRNA was strongly detected in BEND cells. Then, we identified effects of blocking FGFR1 on proliferation of BEND cells and phosphorylation of signaling molecules by using BGJ398 (an FGFR inhibitor) (Figure 5). FGF2-increased proliferation of BEND cells by approximately 155% (P < 0.01) was inhibited (P<0.05) by a combination of FGF2 and BGJ398 (Figure 5A). Next, we investigated inhibitory effects of BGF398 on FGF2-mediated cell signaling molecules in BEND cells by western blot analyses (Figure 5B to 5G). FGF2-induced increases in abundances of phosphorylated AKT, P70S6K, S6, ERK1/2, JNK, P38 and Cyclin D1 proteins in BEND cells were all inhibited significantly when by BGJ398 (see Figures 5A-5G). These results show that FGF2-induced cell signaling in BEND cells is dependent on FGFR1.
Restoration of ER stress in BEND cells in response to FGF2
To investigate effects of FGF2 on restoration of endoplasmic reticulum (ER) stress in BEND cells, we determined proliferation and phosphorylation of FGF2-mediated signaling molecules in the presence of tunicamycin that is an inducible factor for ER stress (Figure 6). Tunicamycin decreased proliferation of BEND cells to approximately 40% (P < 0.01) of that for non-treated cells (Figure 6A). However, the combination of FGF2 with tunicamycin significantly increased the proliferation of BEND cells as compared to tunicamycin-treated BEND cells (P < 0.05). Consistent with these results, we analyzed Cyclin D1 phosphorylation under the same condition in BEND cells (Figure 6B). Tunicamycin decreased phosphorylation of Cyclin D1 as compared to non-treated BEND cells (P < 0.001) whereas a combination of tunicamycin and FGF2 indicated restoration of phosphor-Cyclin D1 protein (P < 0.01). Next, we identified ER stress regulatory genes which are mostly unfolded protein response (UPR) genes in BEND cells treated with FGF2 alone, tunicamycin alone or their combination (Figure 6C to 6G). The three major stress sensors, protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6α (ATF6α) increased in BEND cells in response to tunicamycin (0.25 μg) by 2.14- (P < 0.01), 3.76- (P < 0.001) and 1.86-fold (P < 0.01), respectively (Figures 6C to 6E). In addition, downstream proteins or transcription factors of stress sensors, including eukaryotic translation initiator factor 2α (eIF2α) protein and glucose-regulated protein 78 (GRP78) increased in BEND cells in response to tunicamycin by 1.98- (P < 0.01) and 5.34-fold (P < 0.01), respectively (Figures 6F and 6G). However, the abundances of tumicamycin-induced eIF2α and GRP78 were down-regulated by co-incubation of BEND cells with tunicamycin and FGF2. These results indicate that FGF2 increases in proliferation of BEND cells involve its suppression of ER stress in BEND cells.
Discussion
Results of present study revealed stimulatory effects of FGF2 on proliferation of BEND cells that was confirmed by increases in PCNA in nuclei of BEND cells and a greater percent of BEND cells in G2 phase of the cell cycle. The enhanced proliferation of BEND cells in response to FGF2 was regulated via FGFR1, and activation of PI3K/AKT and MAPK signal transduction. Moreover, inhibition of FGF2-induced proliferation of BEND cells was inhibited by tunicamycin-induced ER stressor proteins. These findings support our hypothesis that FGF2 may act on uterine epithelial cells and perhaps stromal cells to increase proliferation and improve uterine functions during pregnancy. In the livestock industry, the high reproductive efficiency of cattle very important because cattle are monotocous and have long periods of gestation (Dobson and Kamonpatana, 1986). For improvements in uterine receptivity, there are various reports of research with growth factors in cattle. The FGF family is closely associated with uterine receptivity and paracrine regulator on porcine uterine endometrium for formation of true epitheliochorial placentation during pregnancy (Ka et al., 2000). Progesterone-induced FGF10 regulates ovine endometrial functions during early gestational by maintaining a uterine environment conducive to pregnancy (Satterfield et al., 2008). In addition, FGF10 is expressed by bovine conceptuses during elongation in a pattern similar to that for IFNT and it is also expressed in bovine endometrium and ovarian follicles (Buratini et al., 2007; Okumu et al., 2014). FGF2 is also essential for successful development of conceptus and uteri in various species. In human placenta, FGF2 acts predominantly on syncytiotrophoblast and cytotrophoblast cells in the first-trimester of pregnancy and promotes uterine receptivity to implantation (Ornitz and Itoh, 2001; Paiva et al., 2011). In primates, FGF2 increases proliferation of uterine luminal epithelial cells and decidual cells during implantation and enhances proliferation of trophectoderm cells (Rider and Psychoyos, 1994; Taniguchi et al., 1998). Moreover, in ruminants, endometrial-derived FGF2 improves ovine placental development and angiogenesis and induces IFNT, the pregnancy recognition signal from bovine conceptus trophectoderm (Bai et al., 2015; Michael et al., 2006; Yang et al., 2011). Although bovine trophectoderm cells and endometrial cells express FGF2, the specific intracellular signaling pathway is unknown. Results of present study indicated that the recombinant FGF2 increased proliferation of BEND cells by stimulating phosphorylation of transcription factors in the PI3K and MAPK signal transduction pathways in BEND cells.
FGF2 signaling induces phosphorylation of tyrosine residues activating protein kinase C (PKC), MAPK and PI3K pathways through ligand-binding dimerization of FGFR (Dorey and Amaya, 2010). Among the reproductive and embryonic tissues, FGF2 stimulates proliferation of avian granulosa cells of ovary by activation of PKC pathways after binding FGFR1 (Lin et al., 2012). In addition, FGF2 activates migration of ovine trophoblast cells through MAPK-dependent signal transduction (Yang et al., 2011).
Moreover, supplementation of FGF2 in vitro increases proliferation and migration of luminal epithelial cells in porcine endometria during early pregnancy through activation of AKT and MAPK-mediated pathways (Lim et al., 2017b). Similar to previous studies, results of the present study showed that FGF2 activates AKT-P70S6K-S6 proteins and MAPK (ERK1/2, JNK and P38) regulatory proteins. In addition, the FGF2-induced phosphorylation of those cell signaling proteins was inhibited in BEND cells by pharmacological inhibitors of FGFR1, PI3K and MAPK. These results indicated that the FGF2-induced proliferation of BEND cells is regulated by activation of PI3K and MAPK through dimerization of FGFR.
In reproduction, ER stress influences gametogenesis, implantation, embryogenesis and uterine functions (Guzel et al., 2017; Michalak and Gye, 2015; Sutton-McDowall et al., 2016). The ER is an organelle in eukaryotic cells involved with protein folding and synthesis, biosynthesis of lipids and maintenance of calcium levels for cellular homeostasis and development (Bravo et al., 2013). In response to extracellular and intracellular stress, ER stress occurs and results in reproductive physiopathology. For instance, immunological changes affected by ER stress with an increase in GRP78 expression in endometrium may lead to a defective window of implantation in infertile women (Galgani et al., 2015). The increased UPR copies cause embryonic death during the pre-implantation period in mice (Hao et al., 2009). In addition, treatment of mice with tunicamycin decreases blastocyst formation from 79% to 4% during early pregnancy that is associated with an increase in abundance of X-box binding protein, a transcription factor in response to ER stress (Basar et al., 2014). And, ER stress disrupts placentation and increases intrauterine growth restriction through regulating post-translational processing of proteins (Yung et al., 2012).
Furthermore, ER stress reduces proliferation of porcine endometrial cells through activation of UPR factors (Lim et al., 2017a). In accordance with previous reports, results of present study indicated that ER stress induced by tunicamycin, reduced proliferation of BEND cells which was reversed by FGF2. In addition, the elevated UPR regulatory proteins in response to tunicamycin were down-regulated by a combination of tunicamycin and FGF2 in BEND cells. Previous studies have shown that FGF2 attenuates ER stress induced by ischaemia regulatory mechanisms of FGF2 in BEND cells (Wang et al., 2015; Wang et al., 2012). In summary, FGF2 stimulates proliferation of bovine uterine endometrial cells by activation of PI3K and MAPK through FGFR1. And, FGF2 suppresses tunicamycin-induced ER stress leading to decreases in proliferation of BEND cells. Collectively, these results provide intracellular signaling mechanism used by FGF2 that may be used to improve uterine receptivity, implantation and development of endometria in cattle, as well as other ruminant and nonruminant livestock species.
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