MDL-71782 | MMPs signal

Putrescine Stimulates the mTOR Signaling Pathway and Protein Synthesis in Porcine Trophectoderm Cells1

Xiangfeng Kong,3,4,5 Xiaoqiu Wang,3,4 Yulong Yin,5 Xilong Li,4 Haijun Gao,4 Fuller W. Bazer,4 and Guoyao Wu2,4,6
4Department of Animal Science, Texas A&M University, College Station, Texas
5Hunan Provincial Engineering Research Center of Healthy Livestock and Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan, China 6State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China

1This work was supported by United States Department of Agriculture National Institute of Food and Agriculture/Agriculture and Food Research Initiative competitive grant 2008-35203-19120 and 2011- 67015-20028, National Natural Science Foundation of China grants 31270044 and 31272450, and the Chinese Academy of Sciences/the State Administration of Foreign Experts Affairs International Partnership Program for Creative Research Teams.
2Correspondence: Guoyao Wu. E-mail: [email protected]
3These authors contributed equally to this study.
Received: 8 September 2013.
First decision: 7 October 2013.
Accepted: 8 September 2014.
© 2014 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363

ABSTRACT

Insufficient placental growth is a major factor contributing to intrauterine growth retardation in mammals. There is growing evidence that putrescine produced from arginine (Arg) and proline via ornithine decarboxylase is a key regulator of angiogenesis, embryogenesis, as well as placental and fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that putres- cine stimulates protein synthesis by activating the mechanistic target of rapamycin (mTOR) signaling pathway in porcine trophectoderm cell line 2 cells. The cells were cultured for 2 to 4 days in customized Arg-free Dulbecco modified Eagle Ham medium containing 0, 10, 25, or 50 lM putrescine or 100 lM Arg. Cell proliferation, protein synthesis, and degradation, as well as the abundance of total and phosphorylated mTOR, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. Our results indicate that putrescine promotes cell proliferation and protein synthesis in a dose- and time-dependent manner, which was inhibited by difluoro-methylornithine (an inhibitor of ornithine decarboxylase). Moreover, supplementation of culture medium with putrescine increased the abundance of phosphorylated mTOR and its downstream targets, 4EBP1 and p70 S6K1 proteins. Collectively, these findings reveal a novel and important role for putrescine in regulating the mTOR signaling pathway in porcine placental cells. We suggest that dietary supplementation with or intravenous administration of putres- cine may provide a new and effective strategy to improve survival and growth of embryos/fetuses in mammals. mTOR, placental cells, protein synthesis, putrescine

INTRODUCTION

Intrauterine growth retardation (IUGR), defined as impaired growth and development of the mammalian conceptus (embryo/fetus and its associated placental membranes) or its organs during pregnancy, is commonly observed in mammals, including humans and swine [1]. Studies with the swine model have revealed that 25% of newborn piglets suffer from IUGR [2], which reduces neonatal survival and has permanent stunting effects on the postnatal growth and efficiency of food utilization in offspring [1]. Moreover, IUGR negatively affects whole-body composition, organ function, and health [3, 4]. Since IUGR is positively associated with a high rate of neonatal mortality [1, 2], a better understanding of the underlying mechanisms is necessary to develop strategies to ameliorate the negative effects of IUGR on the health of the fetus and offspring.
Growth and development of embryos/fetuses are influenced by many factors, such as genetic or epigenetic alterations, maternal maturity, and placental environment [2, 5, 6], all of which are associated with the size and functional capacity of the placenta, uteroplacental transfer of nutrients and oxygen from mother to fetus, nutrient availability, fetal endocrine milieu, and metabolic pathways [7–9]. The placenta is the key organ for nutrient uptake, waste elimination, and gas exchange between conceptus and maternal blood [9], required for fetal growth and development [2, 10]. It is generally accepted that impairment of placental growth contributes to IUGR in mammals [11, 12]. In this scenario, a better understanding of regulatory factors is an essential prerequisite to alleviating negative effects of IUGR on the reproductive efficiency in mammals. Trophoblast cells that form the outer layer of the blastocyst provide nutrients to the embryo and then form the chorion of the mature placenta. This layer of trophoblasts is collectively referred to as ‘‘the trophectoderm.’’ After gastru- lation, the trophectoderm, which derives from ectoderm and then, along with mesoderm from the embryo, forms the functional chorion of the conceptus [13].
Polyamines, including putrescine, spermine, and spermi- dine, are produced from amino acids (e.g., ornithine, arginine [Arg], and proline) in mammalian cells. Polyamines regulate expression of key genes involved in cell proliferation, angiogenesis, embryogenesis, and protein synthesis, as well as placental and fetal growth [14–16]. At present, it is not known whether putrescine activates the mechanistic target of rapamycin (mTOR) cell signaling pathway and enhances protein synthesis in placental cells. These potential roles of putrescine were evaluated in the present study using the porcine trophectoderm cell line 2 (pTr2), our well-established cell line model [17]. To achieve this goal, pTr2 cells were treated with putrescine, followed by measurements of total and phosphorylated (active form) levels of mTOR and its two downstream targets, ribosomal protein S6 kinase 1 (p70S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4EBP1) [18], as well as intracellular protein turnover.

MATERIALS AND METHODS

Chemicals
The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): antibiotic solution (penicillin/streptomycin [P/S]; catalog no. A5955); Dulbecco modified Eagle Ham/F12 medium (DMEM/F-12; catalog no. D2906); fetal bovine serum (FBS; charcoal striped; catalog no. F6765); insulin solution (from bovine pancreas, 10 mg/ml; catalog no. I6634, CAS no. 11070- 73-8); L-Arg hydrochloride (catalog no. A6969, CAS no. 1119-34-2); L-phenylalanine (catalog no. P2126, CAS no. 63-91-2), putrescine (catalog no. D13208); and the irreversible inhibitor of ornithine decarboxylase (ODC) difluoromethylornithine (DFMO; catalog no. D193). Customized Arg-free DMEM containing physiological concentrations of other amino acids in plasma and 5 mM D-glucose (formula 08-5009EF) and 2.5% trypsin solution (catalog no. 25095-019) were obtained from Gibco (Carlsbad, CA). Phosphatase inhibitor cocktail set II (catalog no. 524625) was procured from Calbiochem (San Diego, CA), BCA protein assay kit (catalog no. 23228) from Pierce Protein Research (Rockford, IL), and ethylenediaminetetraacetic acid (EDTA; disodium salt; catalog no. 12839521-23) from Boehringer Mannheim (Indianapolis, IN). L-[ring-2,4-3H] phenylalanine was purchased from American Radiolabeled Chemicals (St. Louis, MO). Before use, this isotope was purified on ion-exchange chromatography (AG 1-X8 resin, acetate form, 200–400 mesh) as we described previously [19]. Other chemicals used in this study were reported previously [20].

Cell Culture and Preparation
The pTr2 cells, which were previously established from elongated porcine blastocysts and characterized [17, 21], were used in this study. The cells (passages 30–37) were grown in 75-cm2 flasks containing 15 ml DMEM/F-12 with 5% FBS, 1% P/S, and 0.05% insulin. The medium was changed every other day. When cells reached 70%–100% confluence, they were collected using 0.125% trypsin solution (1.25 ml of 2.5% trypsin in 23.75 ml of 0.02% EDTA). After counting the number of cells, they were diluted to 4.0 3 104 cells/ml in DMEM/F-12 containing 5% FBS, 1% P/S, and 5 mg/L insulin, and used in the following experiments.

Determination of Effects of Putrescine on pTr2 Cell Growth
Cell growth was determined as described previously [22]. Briefly, cells were seeded at 10 000 cells/cm2 (0.5 ml per well) in 24-well culture dishes, and maintained overnight (;16 h) in the complete DMEM/F-12 medium containing 5% FBS, 1% P/S, and 5 mg/L insulin. The cells were maintained for 6 h in the customized DMEM medium supplemented with 5% FBS, 1% P/S, and 0.05% insulin. The concentrations of amino acids in the DMEM were based on their physiological levels in the plasma of gestating swine [23].
To determine the effects of putrescine on cell growth, pTr2 cells were seeded on Day 0 in 0.5 ml customized DMEM containing 5% FBS, 1% P/S, 5 mg/L insulin, and 100 lM Arg in the presence of 0, 10, 25, or 50 lM putrescine. In some experiments, culture medium contained 0, 25, 50, 250, 500, or 1000 lM putrescine. The medium was changed every other day. Eight replicates were trypsinized on Day 4, and cell numbers were counted using a hemacytometer [24]. In some experiments, cells were cultured in DMEM containing 5% FBS, 1% P/S, 5 mg/L insulin, and 350 lM Arg, in the presence of different concentrations of DFMO (a specific inhibitor of ODC1), and cell numbers were counted at the end of experiments. Eight independent experiments were conducted on the basis of statistical power calculation [25]. Using the published enzymatic assay involving [1-14C] ornithine [16], we found that 5 mM DFMO inhibited ODC activity in pTr2 cells by 96.1% 6 1.4% (mean 6 SEM; n ¼ 6).

Determination of Protein Turnover and Polyamines in pTr2 Cells
To determine protein synthesis, pTr2 cells were cultured as described for the cell growth assay. The medium was removed at the end of a 4-day culture period, and the cells were washed once with 2 ml DMEM. Then, cells were cultured for another 3 h in 2 ml customized DMEM containing 1 mM L phenylalanine, 0.1 lCi L-[ring-2,4-3H] phenylalanine, 100 lM Arg, and 0, 10, or 25 lM putrescine. Thereafter, the medium was collected and cells were rapidly washed three times with 2 ml ice-cold calcium and magnesium-free phosphate-buffered saline (PBS; pH 7.4). Protein in the medium was precipitated with 2 ml of 10% trichloroacetic acid (TCA) for 3H counting [26]. The cells attached to the bottom of each well were scraped after addition of 2 ml of 10% TCA to the well. After the whole-cell extract was centrifuged at 3000 3 g for 5 min at 48C, the supernatant fluid was used for analysis of polyamines [27] and the pellet was washed three times with 5 ml of 2% TCA and then dried in the air at 378C. An aliquot (0.5 ml) of 1 M of NaOH was added to each cell pellet and the solution incubated at 378C for several hours until the pellet was completely dissolved. Part of the solution (0.4 ml) was transferred to a 20-ml scintillation vial, followed by addition of 19 ml of Hionic Fluor Scintillation cocktail (PerkinElmer) for 3H counting after standing overnight at room temperature to calculate rates of protein synthesis [28]. Concentrations of polyamines in pTr2 cells were calculated on the basis of the cell volume (1.14 6 0.07 ll/106 cells [mean 6 SEM]; n 6), which was determined using 3H O and [14C] inulin (an extracellular marker), as we have described previously [29].
To determine rates of protein degradation, cells were treated as described for the protein synthesis assay, except that the culture medium was removed at the end of a 3-day culture period. Porcine trophectoderm cell line 2 cells were then cultured in customized DMEM containing L-[ring-2, 4-3H] phenylalanine, 100 lM Arg, and various concentrations of putrescine. After a 24-h culture to label cellular proteins, the cells were washed with fresh DMEM containing 1 mM L-phenylalanine to deplete intracellular free [3H] phenylalanine [26]. Then, 2 ml of customized DMEM containing 5% FBS, 1% P/S, 5 mg/L insulin, 100 lM Arg, and 0, 10, or 25 lM putrescine was added to each well. At the end of a 3-h culture period, the medium was collected and the cells were washed rapidly with PBS. The cells were scraped from the wells after addition of 2 ml 10% TCA. After the whole-cell extract was centrifuged at 3000 3 g for 5 min at 48C, the supernatant fluid was removed and the cell pellet washed three times with 5 ml 2% TCA and dried in the air at 378C.
For determining the amount of [3H] phenylalanine released from prelabeled proteins into culture medium, the medium was centrifuged at 3000 3 g at 48C for 2 min to remove dead cells. An aliquot (1 ml) of the supernatant fluid was transferred to a 15-ml tube, followed by addition of 2 ml 10% TCA. The solution was centrifuged at 3000 3 g for 5 min at 48C. All the supernatant fluid was transferred to a 20-ml scintillation vial, followed by addition of 15 ml of Hionic Fluor Scintillation cocktail for measurement of 3H radioactivity. The pellet was washed three times with 2% TCA and then dissolved in 0.5 ml of 1 M NaOH for 3H counting after standing overnight at room temperature. The percentage of protein-bound [3H] phenylalanine released into the culture medium was calculated to indicate protein degradation in pTr2 cells (namely, [3H] phenylalanine in medium/[3H] phenylalanine in cell protein 3 100).

Western Blot Analysis of mTOR Pathway Proteins
The pTr2 cells were cultured for 4 days in the presence of 100 lM Arg and 0, 10, or 25 lM putrescine, as described above. Thereafter, the medium was removed and cells were rinsed rapidly three times with ice-cold PBS. Cells were then lysed for 30 min at 48C in 0.5 ml of a buffer consisting of 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 1 mM ethylene glycol tetra-acetic acid, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonylfluoride, 50 mM NaF, 30 mM Na4P2O7, and 1% protease inhibitor cocktail. The cell lysates were centrifuged (16 000 3 g for 15 min at 48C) [30]. Protein concentration in the supernatant fluid was determined using the bicinchoninic acid assay (Pierce) with bovine serum albumin as the standard. All samples were adjusted to an equal protein concentration and then diluted with 23 loading buffer (0.63 ml of 0.5 M Tris-HCl [pH 6.8], 0.42 ml 75% glycerol, 0.125 g SDS, 0.25 ml b-mercaptoethanol, 0.2 ml 0.05% solution of bromphenol blue, and 1 ml water) to a final volume of 2.5 ml and heated in boiling water for 5 min. After the solution was cooled on ice, it was used for Western blot analysis of soluble denatured proteins.
Denatured proteins were separated using SDS-PAGE (4%–12% gradient gel) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) overnight at 12 V using the Bio-Rad Transblot. Membranes were blocked in 5% fat-free milk in Tris-Tween buffered saline (TTBS; 20 mM Tris/150 mM NaCl [pH 7.5], and 0.1% Tween-20) for 3 h, and then incubated with primary antibodies at 48C overnight with gentle rocking [31]. All antibodies used in these experiments were purchased from Cell Signaling Technology and are listed in Table 1. After washing three times with TTBS, the membranes were incubated at room temperature for 3 h with secondary antibodies. For Western blot analysis of b-actin, the same blots used for mTOR, p70 S6K1, and 4EBP1 analysis were washed with Stripping Buffer (Pierce). The primary antibody and secondary antibodies (horseradish peroxidase-linked anti-rabbit IgG) were used at dilutions of 1:1000 and 1:3000, respectively. Finally, the membranes were
TABLE 1. Antibodies and dilution factors used for Western blot analyses.*
Antibody Catalog no. Dilution
Rabbit monoclonal anti-mTOR (7C10) 2983 1:1000
Rabbit polyclonal anti-phospho-mTOR (Ser2481) 2974 1:1000
Rabbit polyclonal anti-p70S6 kinase 9202 1:1000
Mouse monoclonal anti-phospho-p70S6 kinase (Thr389) (1A5) 9206 1:1000
Rabbit polyclonal anti-4EBP1 9452 1:1000
Rabbit polyclonal anti-phospho-4EBP1 (Thr70) 9455 1:1000
Rabbit monoclonal anti-b-actin 4970 1:1000
Horseradish peroxidase-linked anti-rabbit IgG 7074 1:3000
*See the Materials and Methods for the sources of the antibodies.
washed with TTBS, followed by development using Supersignal West Dura Extended Duration Substrate according to the manufacturer’s instructions (Pierce). The images were detected on Fujifilm LAS-3000 (Fujifilm, Tokyo, Japan). Multiple exposures of each Western blot were performed to ensure linearity of chemiluminescence signals. The Western blots were quantified by measuring the intensity of correctly sized bands using a ChemiDoc EQ system and Quantity One software (Bio-Rad).

Cell Proliferation in the Presence of Rapamycin
To determine effects of putrescine on cell growth via mTORC1 cell signaling pathway, the pTr2 cells were seeded at 10 000 cells/cm2 (0.4 ml/well) in 24-well culture dishes, maintained overnight (;16 h) in the complete DMEM/F-12 medium containing 10% FBS, 1% P/S, and 5 mg/L insulin, and then switched to serum- and insulin-free customized DMEM medium. The concentrations of amino acids in the DMEM were based on their physiological levels in plasma of gestating swine [23]. After starvation for 24 h, cells (n 4 wells/treatment) were subcultured in 0.4 ml fresh DMEM containing 5% FBS, 1% P/S, and 5 mg/L insulin at 0 min as a blank control, and then treated with putrescine at indicated doses (0, 10, 25, or 50 lM) in the presence of 100 lM Arg with or without 50 nM rapamycin (an mTOR inhibitor). The medium was changed every 2 days, and treated cells were maintained for 2, 4, or 6 days. Cell numbers were determined as described previously [22]. Briefly, DMEM was removed from cells by vacuum aspiration, and cells were fixed in 50% ethanol for 30 min, followed by vacuum aspiration of the fixative. Fixed cells were stained with Janus Green B in PBS (0.2% w/v) for 3 min at room temperature. The stain was immediately removed using a vacuum aspirator, and the whole plate was sequentially dipped into water and destained by gentle shaking. The remaining water was removed by shaking, after which stained cell were immediately lysed in 0.5 N HCl and absorbance readings were taken at 595 nm using a microplate reader. Cell numbers were calculated from absorbance readings using the following formula: cell number (absorbance 0.00462)/ 0.00006926. The entire experiment was repeated independently three times with different batches of pTr2 cells between passages 7 and 12.
FIG. 1. Putrescine promotes pTr2 cell growth. Culture of pTr2 cells in DMEM supplemented with 0, 10, or 25 lM putrescine resulted in a dose- dependent increase (P , 0.05) in cell numbers on Day 4 of culture. Values are means 6 SEM; n 8. Means sharing different letters (a–c) differ significantly (P , 0.05).

Protein Turnover in pTr2 Cells in the Presence of Rapamycin
To determine protein synthesis in the presence of rapamycin, pTr2 cells were cultured as described for the cell proliferation assay. The medium was removed at the end of a 4-day culture period, and the cells were washed once with 2 ml customized DMEM. Then, 2 ml of fresh DMEM containing 1 mM L- phenylalanine, 0.1 lCi L-[ring-2,4-3H] phenylalanine, Arg (0 or 100 lM), putrescine (0 or 25 lM), or rapamycin (0 or 50 nM) were added to cells in each well, and cultured for another 3 h. Thereafter, rates of protein synthesis in pTr2 cells were determined as described previously [31].
To determine protein degradation in the presence of rapamycin, cells were treated as described for the protein synthesis assay, except that the culture medium was removed at the end of a 3-day culture period. Porcine trophectoderm cell line 2 cells were then cultured in customized DMEM containing L-[ring-2, 4-3H] phenylalanine, and combinations of Arg (0 or 100 lM), putrescine (0 or 25 lM) or rapamycin (0 or 50 nM). After a 24-h culture to label cellular proteins, the cells were washed with fresh DMEM containing 1 mM L-phenylalanine to deplete intracellular free [3H] phenylalanine [26]. Then, 2 ml of DMEM containing 5% FBS, 1% P/S, and 5 mg/L insulin, and combinations of Arg (0 or 100 lM), putrescine (0 or 25 lM), or rapamycin (0 or 50 nM), was added to each well. At the end of a 3-h culture period, the medium was collected and the cells were washed rapidly with PBS. Thereafter, rates of protein degradation in pTr2 cells were determined as described previously [31].

Statistical Analysis
Data are expressed as means 6 SEM. Results were analyzed by one-way ANOVA and the Student-Newman-Keuls multiple comparison test or by regression analysis using SPSS 13.0 (SPSS Inc., Chicago, IL). Probability values of P ≤ 0.05 indicated statistical significance.

RESULTS

Effects of Putrescine and DFMO on pTr2 Growth
During the 4-day culture, increasing concentrations of putrescine in culture medium from 0 to 25 lM dose- dependently increased (P , 0.05) the number of pTr2 cells, while 50 lM putrescine decreased (P , 0.05) the number of pTr2 cells (Fig. 1). Cell numbers on Day 4 of culture did not differ (P . 0.05) between 0 and 50 lM putrescine (Fig. 1). The effects of 10 and 25 lM putrescine on stimulating cell growth were also observed on Days 2 and 6 (Fig. 2). Increasing extracellular concentrations of DFMO from 0 to 5 mM dose- dependently decreased (P , 0.05) the number of pTr2 cells on Day 4 of culture (Fig. 3).
To determine whether high concentrations of putrescine may inhibit the growth of pTr2 cells, they were cultured for 2 and 4 days in the presence of 0, 25, 50, 250, 500, or 1000 lM. On Day 2 of culture, the number of cells decreased progressively (P , 0.05) as extracellular concentrations of putrescine increased from 50 to 1000 lM (see Table 3). On Day 4 of culture, the number of cells decreased (P , 0.05) progressively as extracellular concentrations of putrescine increased from 25 to 1000 lM (see Table 3).
FIG. 2. Time course of pTr2 cell growth at various concentrations of putrescine. The pTr2 cells were cultured in DMEM supplemented with 0, 10, or 25 lM putrescine, and cell numbers were determined on Days 0, 2, 4, and 6. Values are means 6 SEM; n 8. Within a putrescine concentration, means sharing different letters (a–d) differ significantly (P , 0.05) among the four time points. *Cell growth differed (P , 0.05) between the 0 and 25 lM putrescine groups at the same time point; †cell growth differed (P , 0.05) between the 0 and 10 lM putrescine groups at the same time point.

Effects of Putrescine on Intracellular Protein Turnover and Polyamine Concentration in pTr2 Cells
Increasing extracellular concentrations of putrescine from 0 to 25 lM dose-dependently increased (P , 0.05) protein synthesis in pTr2 cells, but did not affect (P . 0.05) protein degradation in the pTr2 cells (Table 2). Intracellular concen- trations of putrescine increased (P , 0.05) from 1.22 to 3.14 mM as extracellular concentrations of putrescine increased from 0 to 25 lM (Table 2). Intracellular concentrations of spermidine and spermine were also augmented (P , 0.05) by the addition of putrescine to the culture medium (Table 3).

Effects of Putrescine on the mTOR Cell Signaling Pathway Proteins in pTr2 Cells
The abundance of total and phosphorylated mTOR, p70 S6K1, and 4EBP1 was readily detected in pTr2 cells. Addition
FIG. 3. DFMO (an inhibitor of ODC1) decreased proliferation of pTr2 cells. Culture of pTr2 cells in DMEM supplemented with 1 to 5 mM DFMO resulted in a dose-dependent decrease (P , 0.05) in cell numbers on Day 4 of culture, as analyzed by regression analysis. Values are means 6 SEM; n 8. Based on one-way ANOVA and the Student-Newman-Keuls multiple comparison test, means sharing different letters (a–b) differ significantly (P , 0.05). of 25 lM putrescine to the culture medium increased (P , 0.05) the abundance of both total and phosphorylated mTOR (Fig. 4), as well as phosphorylated p70S6K1 (Fig. 5B). The abundance of total and phosphorylated mTOR (Fig. 4) and p70S6K1 (Fig. 5) did not differ (P . 0.05) between the 0 and
10 lM putrescine treatments. Increasing concentrations of putrescine in culture medium from 0 to 25 lM dose- dependently increased (P , 0.05) the abundance of both total and phosphorylated 4EBP1 (Fig. 6).

Cell Proliferation and Protein Turnover in the Presence of Rapamycin
To further understand the mechanisms for the roles of putrescine in regulating the proliferation of, and protein turnover in, pTr2 cells, these parameters were determined in the cells cultured in the presence or absence of rapamycin. Addition of 100 lM Arg to culture medium enhanced (P , 0.05) pTr2 cell growth, compared with the absence of Arg. In comparison with the absence of both putrescine and Arg, addition of 10–50 lM putrescine increased (P , 0.05) the rate of cell proliferation in the absence of rapamycin during 48, 96, and 114 h of culture (Fig. 7). Notably, the effect of putrescine or Arg was blocked (P , 0.05) by addition of 50 nM rapamycin to the culture medium (Fig. 7). Similarly, the stimulatory effect of putrescine or Arg on the rate of protein synthesis in pTr2 cells was abolished (P , 0.05) by the presence of 50 nM rapamycin in the culture medium (Fig. 8A). Compared with the absence of rapamycin, the presence of 50 nM rapamycin increased (P , 0.05) the rate of protein degradation in pTr2 cells by ;100% (Fig. 8B). Putrescine and Arg did not affect the rate of intracellular protein degradation in pTr2 cells cultured in the presence of rapamycin (Fig. 8B).

DISCUSSION

Polyamines are key regulators of angiogenesis, early mammalian embryogenesis, growth of the trophectoderm and placenta, and embryonic development through modulating gene expression, signal transduction, ion channel function, and
TABLE 2. Effects of putrescine on protein turnover and polyamine concentrations in pTr2 cells.
Putrescine concentration in culture medium*
Variable 0 lM 10 lM 25 lM
Intracellular protein turnover
Protein synthesis (nmol Phe/mg
protein/3 h) 41.2 6 1.0c 45.9 6 1.2b 51.6 6 1.5a Protein degradation
(%/3 h) 8.03 6 0.49 7.74 6 0.52 7.65 6 0.46
Intracellular concentrations of polyamines
Putrescine (mM) 1.22 6 0.07c 2.06 6 0.13b 3.14 6 0.19a
Spermidine (mM) 5.28 6 0.28c 7.61 6 0.40b 10.9 6 0.47a
Spermine (mM) 6.47 6 0.35c 9.34 6 0.44b 14.2 6 0.58a
*Values are means 6 SEM; n 8.
a–cWithin a row, means sharing different superscript letters differ significantly (P , 0.05).
DNA and protein synthesis, as well as cell proliferation and differentiation [2, 32, 33]. Physiological levels of polyamines are also scavengers of reactive oxygen species, thereby protecting DNA, proteins, and lipids from oxidative damage [34]. Due to the multiple functional roles of putrescine in physiological and pathophysiological processes, research related to putrescine has received increasing attention during the last two decades [15]. To our knowledge, the present study provides the first biochemical, cellular, and molecular evidence for an important role of putrescine in activating the mTOR cell signaling pathway to stimulate protein synthesis and growth of porcine trophectoderm cells, and by inference, the porcine placenta.
Animal cells can concentrate certain substances, including polyamines, through endogenous synthesis and uptake [14, 15]. For example, between Days 30 and 140 of gestation, intracellular concentrations of total polyamines (putrescine, spermidine plus spermine) in ovine placentomes are 0.66–3.5 mM, despite very low concentrations of polyamines in maternal and fetal plasma (2–5 lM) [35]. Furthermore, concentrations of total polyamines in porcine placentae are 0.25–0.6 mM, although concentrations of polyamines in maternal and fetal plasma are only 4–6 lM [16]. Results of the present study indicate that intracellular concentrations of putrescine, spermidine, and spermine in pTr2 cells increased from 1.22 to 3.14 mM, from 5.28 to 10.9 mM, and from 6.47 to 14.2mM, respectively, as concentrations of putrescine in the culture medium increased from 0 to 25 lM (Table 2). These findings provide an experimental basis for the use of putrescine to effectively enhance intracellular concentrations of poly- amines in placental cells.
We reported that supplementation of culture medium with Arg promotes growth of porcine placental cells primarily via a nitric oxide-independent pathway [31]. This observation is
FIG. 4. Amounts of total (A) and phosphorylated (B) mTOR proteins in pTr2 cells. The pTr2 cells cultured for 96 h in DMEM supplemented with 25 lM putrescine had a greater (P , 0.05) abundance of both total and phosphorylated mTOR proteins, as compared with the 0 and 10 lM putrescine groups. Data are expressed as means 6 SEM; n 8. Means sharing different letters (a and b) differ significantly (P , 0.05).
confirmed in the current work (Fig. 7). Results of the present study indicate that putrescine is another factor responsible for stimulating proliferation of placental cells. Arg is a common substrate for the formation of ornithine and proline via arginase in maternal tissues, but this enzyme is virtually absent from the porcine placenta [11]. Through the systemic blood circulation, arterial ornithine is utilized for polyamine production by the placenta and uterine tissue via ODC1 [36]. Interestingly, arterial proline is the major amino acid for the synthesis of polyamines in the porcine placenta via proline oxidase and ODC1 [16]. The lack of Arg degradation via arginase in the porcine placenta maximizes placental transfer of Arg from maternal to fetal blood [1]. This finding aids in understanding the role of the unusually high abundance (4–5 mM) of Arg in
TABLE 3. Effects of high extracellular concentrations of putrescine on pTr2 cell growth.
Concentrations of putrescine in culture medium*
Culture time 0 lM 25 lM 50 lM 250 lM 500 lM 1000 lM
Cell numbers (104/well) 48 h
4.46 6 0.11c
6.24 6 0.14a
6.05 6 0.15a
5.23 6 0.12b
4.57 6 0.09c
4.12 6 0.08d
96 h 7.92 6 0.16b 10.8 6 0.20a 8.01 6 0.18b 7.44 6 0.15c 6.85 6 0.12d 6.03 6 0.10e
*Values are means 6 SEM; n 8.
a–eWithin the time point, means sharing different letters differ significantly (P , 0.05).
FIG. 5. Amounts of total (A) and phosphorylated (B) p70S6K1 proteins in pTr2 cells. The pTr2 cells cultured for 96 h in DMEM supplemented with either 0 or 25 lM putrescine had an increased (P , 0.05) abundance of phosphorylated p70S6K1 protein. Data are expressed as means 6 SEM; n ¼ 8. Means sharing different letters (a and b) differ significantly (P , 0.05).
FIG. 6. Amounts of total (A) and phosphorylated (B) 4EBP1 proteins in
pTr2 cells. The culture of pTr2 cells for 96 h in DMEM supplemented with 0, 10, or 25 lM putrescine resulted in a dose-dependent increase (P , 0.05) in the abundance of both total and phosphorylated 4EBP1 proteins. Data are expressed as means 6 SEM; n 8. Means sharing different letters (a–c) differ significantly (P , 0.05). porcine allantoic fluid during early gestation [37] when the greatest rates of polyamine synthesis occurs in the porcine placenta [16]. An important role for putrescine in pTr2 cell growth is further substantiated by our finding that increasing extracellular concentrations of DFMO from 0 to 5 mM dose- dependently decreased the number of pTr2 cells (Fig. 3). Thus, through the generation of putrescine as an intermediate in cell metabolism, amino acids are essential for successful pregnancy outcome in mammals. Interestingly, 5 mM DFMO inhibited ODC activity in the extracts of pTr2 cells by 96%, but only modestly reduced cell growth on Day 4 of culture (Fig. 3). It is possible that putrescine may be produced from Arg and other amino acids via a yet-unknown pathway or from agmatine (present in the culture medium) via agmatinase in these cells. Determining cell numbers is the best available method to assess cell growth [22]. Using this technique, we found that, on Day 4 of culture, putrescine at 10 and 25 lM increased pTr-2 cell numbers by 12% and 23%, respectively, whereas 50 lM putrescine did not affect cell growth, as compared with the control group (Fig. 1). Further analysis of our results revealed that 50 lM putrescine increased pTr2 cell numbers on Day 2 of culture in comparison with the 0-lM putrescine group (Fig. 7). Thus, 50 lM putrescine could initially stimulate cell proliferation, but inhibit this biochemical event in long-term culture. Consequently, when extracellular concentrations of putrescine increased from 25 to 50 lM, cell numbers decreased 26.1% on Day 4 of culture (Fig. 1). Of note, on both Days 2 and 4, increasing extracellular concentrations of putrescine from 50 to 1000 lM reduced pTr2 cell growth in a dose- dependent manner (Table 3). Mechanisms for high concentra- tions of polyamines to inhibit cell growth are likely complex. Emerging evidence shows that the deregulation of polyamine metabolism may induce apoptosis [38]. Moreover, spermidine and spermine at elevated levels have also been reported to be cytotoxic metabolites [39]. This may explain, in part, why excessive amounts of Arg in the maternal diet actually reduce placental growth in gestating swine [40]. Based on our study, an optimal extracellular concentration of putrescine to stimulate pTr2 cell proliferation appears to be 25 lM. Accordingly, we conducted all subsequent experiments with 0–25 lM putrescine in culture medium.
Cell growth depends on the balance between protein synthesis and degradation [32]. To quantify intracellular protein degradation, we measured the release of protein-bound [3H] phenylalanine into culture medium under the conditions (e.g., the presence of 1 mM unlabeled phenylalanine) that minimize the incorporation of [3H] phenylalanine into protein [26]. Rates of proteolysis in pTr2 cells were not affected by 10 or 25 lM putrescine in culture medium (Table 2). In contrast, as compared with the control, the addition of 10 or 25 lM putrescine to culture medium dose-dependently increased rates of protein synthesis in pTr2 cells (Table 2). In response to 10– 25 lM putrescine in the culture medium, an increase in protein synthesis and no change in protein degradation resulted in protein accretion and growth of pTr2 cells. Interestingly,
FIG. 7. Putrescine enhanced pTr2 cell proliferation via mTOR activation at different time points (2, 4, and 6 days). The pTr2 cells were treated with putrescine at indicated doses (0, 10, 25, or 50 lM) in the presence of 100 lM Arg with or without 50 nM rapamycin (an mTOR inhibitor). Cell numbers were determined on Days 2 (A), 4 (B), and 6 (C). Values are means 6 SEM; n 4. Different superscript letters (a–d) denote significant effects of treatment (P , 0.05). A black bar represents cell numbers at Day 2, 4, or 6 in response to different treatments, whereas a patterned bar represents cell numbers at the beginning of cell culture as a reference. concentrations of putrescine in porcine placentae are positively correlated with placental weight during early pregnancy [16]. TOR is a serine-threonine kinase that was originally identified as a TOR in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes [41]. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in the control of
FIG. 8. Effects of putrescine on protein synthesis (A) and protein degradation (B) via mTOR activation. The pTr2 cells were cultured for 96 h in DMEM containing 5% FBS, 1% P/S, and 5 mg/L insulin at 0 min as a blank control, and then treated with putrescine at indicated doses (0, 10, 25, or 50 lM) in the presence of 100 lM Arg with or without 50 nM rapamycin (an mTOR inhibitor). At the end of a 96-h culture period, cells were used for the measurement of protein synthesis. Release of 3H-phenylalanine from prelabeled proteins into culture medium was determined to indicate protein degradation in the cells. Values are means 6 SEM; n ¼ 8. Different superscript letters (a–c) denote significant effects of treatment (P , 0.05). cell growth [42–44]. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, also resulted in reduced cell size and proliferation arrest in embryonic stem cells [45]. Thus, in mammals, mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells [45] through stimulating protein synthesis by phosphor- ylating downstream substrates, including p70S6K1 and 4EBP1 [46, 47]. Consistent with this notion, we found that, as compared with its absence from the culture medium, 25 lM putrescine increased amounts of both total and phosphorylated mTOR proteins in pTr2 cells by 1.3- and 2.5-fold, respectively (Fig. 5), and the amount of phosphorylated p70S6K1 protein by 1.2-fold (Fig. 6). Furthermore, putrescine dose-dependently enhanced the abundance of both total and phosphorylated 4EBP1 proteins in pTr2 cells (Fig. 6). These findings are consistent with the role of putrescine and Arg in augmenting protein synthesis in pTr2 cells (Table 2) and conceptus development [48–52]. In the presence of 50 nM rapamycin (an inhibitor of mTOR), 10–25 lM putrescine or 100 lM Arg could not enhance pTr2 cell growth (Fig. 7) or protein synthesis (Fig. 8A), further supporting an important role of mTOR in mediating effects of putrescine and Arg in the cells.
One of the most critical effects of polyamines on cell growth is the availability of spermidine for the post-translational modification of eIF-5A [53]. Because hypusine-containing eIF- 5A is required for cell proliferation, depletion of cellular polyamines suppresses cell growth by reducing cellular levels of the modified eIF-5A. Excessive putrescine accumulation in DH23A/b cells induces apoptosis and suppresses the formation of hypusine-containing eIF-5A [54]. Treatment of DH23A/b cells with diaminoheptane also reduces the formation of the modified eIF-5A and induces apoptosis [54]. Further research is needed to elucidate the underlying mechanism.
Results of the present study may have important implica- tions for the use of putrescine to improve placental and fetal growth in mammals. Compelling evidence shows that placental size is positively correlated with birth weight and, therefore, neonatal survival [55, 56]. For example, in gestating swine, a 16% increase in placental growth is associated with increases in the number of live-born piglets by 2.1 per litter and litter birth-weight by 11% [57]. In lambs born of multiple-pregnancy ewes, a 1% increase in placental weight per fetus is associated with 0.92% and 2.4% increases in birth weight and neonatal survival, respectively [55, 58]. In human IUGR fetuses, a 10% increase in placental weight may result in an approximately 5% gain in fetal growth [59, 60], which would contribute to substantial decreases in stillbirth and neonatal mortality [61, 62]. Thus, while putrescine at 10 and 25 lM increased placental cell growth by only 12% and 23% (Figs. 1 and 2), respectively, such a moderate increase in placental size could be translated into tremendous benefits in animal production and human medicine.
In summary, supplementation with putrescine promotes proliferation of pTr2 cells, which is mediated by increased protein synthesis via the activation of the mTOR signaling pathway. Besides serving as a common intermediate of Arg and proline metabolism, physiological levels of putrescine play a key role in regulating placental growth and develop- ment. We suggest that dietary supplementation with or intravenous administration of putrescine offers a potentially novel and effective strategy to improve survival and growth of embryos/fetuses in mammals, including humans and pigs. Our results not only provide a new biochemical mechanism for an important role of putrescine in enhancing protein synthesis in placental cells, but will also guide the development of a novel strategy in using putrescine to prevent and treat IUGR in mammals (including humans and swine).

ACKNOWLEDGMENT
We thank Bie Tan, Junjun Wang, and Zhenlong Wu for helpful discussion.

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