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. 2016 Aug 12;22(8):890–899. doi: 10.1093/molehr/gaw032

Branched-chain amino acids regulate insulin-like growth factor-binding protein 1 (IGFBP1) production by decidua and influence trophoblast migration through IGFBP1

Kei Tanaka 1,*, Keiji Sakai 1, Miho Matsushima 1, Yukiko Matsuzawa 1, Tomoko Izawa 1, Takashi Nagashima 1, Seishi Furukawa 1, Yoichi Kobayashi 1, Mitsutoshi Iwashita 1
PMCID: PMC4986415  PMID: 27193429

Abstract

STUDY QUESTION

Do branched-chain amino acids (BCAAs) influence the migration of human extravillous trophoblast (EVT) cells through changes in insulin-like growth factor-binding protein 1 (IGFBP1) production in decidual cells?

STUDY FINDING

Decidua-derived IGFBP1 had a stimulating effect on migration of EVT.

WHAT IS KNOWN ALREADY

IGFBP1 is abundantly secreted from human decidual cells and influences trophoblast migration in human placenta of early pregnancy. In hepatic cells, the expression of IGFBP1 is influenced by nutritional status and BCAAs regulate IGFBP1 production.

STUDY DESIGN, SAMPLES/MATERIALS, METHODS

This is a laboratory-based study using human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy (n = 50) and grown as primary cultures. Production of IGFBP1 from decidual cells was examined by enzyme-linked immunosorbent assay and immunoblotting after incubation with or without BCAAs. EVT migration was evaluated using the media conditioned by decidual cells. The effect of conditioned media on phosphorylation of focal adhesion kinase (FAK) in EVT was also analyzed by immunoblotting. The same experiments were repeated in the presence of RGD peptide, which inhibits IGFBP1 binding to α5β1 integrin. An EVT migration assay and the immunoblotting of phosphorylated FAK were also conducted with exogenous IGFBP1. The effect of the conditioned media on cytotrophoblast cell number was also assessed using WST-1 in a cell proliferation assay.

MAIN RESULTS AND THE ROLE OF CHANCE

Deprivation of BCAAs on decidual cells significantly suppressed IGFBP1 secretion (P < 0.05, versus BCAA+). Exogenous IGFBP1-stimulated EVT migration (P < 0.05) and phosphorylation of FAK (P < 0.05), and the RGD peptide inhibited these effects. EVT migration and phosphorylation of FAK were stimulated by the conditioned media, presumably by IGFBP1 in the media. RGD treatment abrogated the stimulating effects of conditioned media. The conditioned media deprived of BCAAs had suppressive effects on EVT migration (P < 0.05, versus BCAA+) and phosphorylation of FAK (P < 0.05, versus BCAA+). The conditioned media did not affect number of cytotrophoblast cells.

LIMITATIONS, REASONS FOR CAUTION

The conclusions are based on in vitro experiments with human decidual cells and trophoblast cells isolated from placental tissue of early pregnancy, and we were unable to ascertain whether these mechanisms actually operate in vivo. We investigated the effect of decidua-derived IGFBP1 on EVT migration, however, we cannot completely rule out the possibility that endogenous IGF could also influence cell migration.

WIDER IMPLICATIONS OF FINDINGS

Interruption of the BCAA supply to uterine decidual cells in early pregnancy may suppress EVT migration through reduced IGFBP1 secretion, which may be one of the pathophysiological conditions responsible for pre-eclampsia.

LARGE SCALE DATA

None.

STUDY FUNDING/ AND COMPETING INTEREST(S)

All funds were obtained through Kyorin University School of Medicine. The authors have no conflict of interest to declare.

Keywords: decidua, insulin-like growth factor-binding protein 1, branched-chain amino acids, extravillous trophoblast, cell migration

Introduction

Migration of extravillous trophoblast (EVT) cells into decidualizing endometrium is one of the fundamental process of human implantation and placentation (Fisher and Damsky, 1993). Appropriate EVT invasion is a critical event for normal placental growth and development, and shallow invasion of EVT has been implicated in pre-eclampsia and fetal growth restriction (FGR) (Kaufmann et al., 2003). EVT migration is regulated by various factors in the surrounding microenvironment, including growth factors, growth factor-binding proteins, extracellular matrix components and many adhesion molecules, which act in both an autocrine and paracrine manner (Bichof et al., 2000; Chakraborty et al., 2002).

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP) is one of the family of IGFBPs that can bind to IGF with high affinity and modulate IGF actions on target cells (Jones and Clemmons, 1995). There is also evidence to suggest that IGFBP1 is able to exert IGF-independent actions by binding to integrins (Jones et al., 1993; Gockerman et al., 1995). IGFBP1 is abundantly expressed in decidualized endometrial stromal cells and is believed to play important roles at the maternal–fetal interface during embryonic implantation (Chakraborty et al., 2002; Tseng and Mazella, 2002; Hills et al., 2004). A previous study has revealed that EVT migration is stimulated in a paracrine manner by decidua-derived IGFBP1 (Gleeson et al., 2001). However, little is known about the regulatory mechanisms of IGFBP1 secretion in decidual cells, therefore elucidation of this mechanism(s) may lead to the treatment and prevention of pre-eclampsia and FGR.

The branched-chain amino acids (BCAAs), namely leucine, isoleucine and valine, are among the nine essential amino acids for human, and are not produced by human body. BCAAs are essential for protein synthesis in humans and animals. The expression of IGFBP1 is dynamically influenced by nutritional status and it has been demonstrated that deprivation of BCAAs, especially leucine, induces IGFBP1 expression in hepatic cells (Straus et al., 1993; Jousse et al., 1998; Takenaka et al., 2000). However, it is not known whether BCAA deprivation treatment also affects IGFBP1 production in decidual cells.

The objectives of the current study were to examine possible changes in IGFBP1 secretion from decidua cells induced by BCAA deprivation and to investigate the physiological effects on migration of EVT.

Materials and Methods

Materials

Tissue culture media, penicillin, streptomycin and amphotericin B were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Amino acid-free Medium 199 (without HEPES) was synthesized by Cell Science & Technology Institution, Inc. (Miyagi, Japan). Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad (Hercules, CA, USA). Several antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), including anti-prolactin (PRL), anti-focal adhesion kinase (FAK) and anti-phosphotyrosine (PY99), anti-vimentin and anti-cytokeratin 7. Anti-IGFBP1 antibody was purchased from Millipore (Temecula, CA, USA). Fibronectin and BCAAs (l-Leucine, l-Isoleucine and l-Valine) were purchased from Wako-Junyaku-Kogyo (Osaka, Japan). Human recombinant IGFBP-1 was purchased from Prospec Technogene (East Brunswick, NJ, USA). Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide was purchased from AnaSpec Inc. (Fremont, CA, USA). All other chemicals were purchased from Sigma-Aldrich Co.

Ethical approval and acquisition of human samples

Placental tissue at between 6 and 10 weeks of gestation was surgically obtained during legal and elective termination of pregnancy from 50 women. All patients provided written informed consent for collection and investigational use of placental tissues. This study protocol was approved by the ethics committee of Kyorin University, School of Medicine, Tokyo, Japan (IRB No.338).

Primary culture of decidual cells and cytotrophoblast cells

Decidual cells and cytotrophoblast cells were obtained using a previously described method (Braveman et al., 1984; Iwashita et al., 1992; Sakai et al., 1997; Kabir-Salmani et al., 2005). Placental tissues were washed with cold phosphate-buffered saline (PBS, pH7.4) and decidua and villous tissues were isolated. The blood vessels or clots and membranes were removed manually. The decidual tissue and villous tissue were separated, sliced into small pieces and each were incubated with the enzymes 0.1% type-I collagenase, 0.1% hyaluronidase and 0.1% DNase-I in PBS for 1 h at 37°C to disperse the cells. The cell suspension was filtered through nylon mesh with pore size of 105 μm (Yazawa-Kagaku-Kogyo, Tochigi, Japan) to remove undigested tissue debris. Cells were collected from the filter by rinsing and centrifugation at 800g for 5 min at room temperature. The viability of dispersed cells, evaluated by trypan blue exclusion, was more than 90%. Then cells were suspended on 6-well cell culture plates in Medium 199 supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, 2.5 μg/ml amphotericin B and 10% fetal calf serum (FCS) (Sigma-Aldrich Co.) in 5% CO2 and 95% air at 37C. Media was replaced every 48 h and the cultures were continued for 5–7 days until they reached confluence. Homogeneity of the cultures was verified by immunocytochemical localization of vimentin and cytokeratin 7.

The culture media containing BCAAs (BCAA+) was formulated by adding l-Leucine (to 4.5 × 10−5 M), l-isoleucine (1.5 × 10−5 M) and l-Valine (1.5 × 10−5 M) (equivalent to Medium 199) and 1% non-essential amino acid solution (Sigma-Aldrich Co.) to amino acid-free Medium 199. The media deprived of BCAAs (BCAA−) was prepared by adding only 1% non-essential amino acid solution.

Once the decidual cells reached confluence, the cells were washed twice with serum-free medium (amino acid-free) and incubated either with serum-free BCAA+ or BCAA− media for 18 h in 5% CO2 and 95% air at 37°C. After incubation, the conditioned media were collected, centrifuged at 15 000g for 10 min at 4°C and stored at −20°C for further studies.

For further analysis, the decidual cells were washed using ice-cold PBS three times and solubilized with lysis buffer (50 mM Tris–HCl [pH7.5], 150 mM NaCl, 1% Nonidet P-40, 2 mM EGTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride [AEBSF], 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin). The insoluble materials were removed by centrifugation at 15 000g for 10 min at 4°C and the supernatants were used for further experiments. The decidual cells were never passaged and the cells were prepared from different placental samples for each experiment.

Primary culture of EVT

EVT were obtained from the placental tissues between 6 and 10 weeks of gestation using a previously described method (Yagel et al., 1989; Kabir-Salmani et al., 2003). Briefly, the tissue was washed with cold PBS prior to dissection. The tissue was cut into small pieces and any blood vessels or clots, membranes and decidual tissue were manually removed. Villous tissue fragments were cultured in Medium 199 supplemented with antibiotics and 10% FCS on 6-well cell culture plates precoated with 20 μg fibronectin diluted in 1 ml PBS. Tissues were allowed to attach to the bottom of the plate for 2 h before adding 200 μl of the same medium. Medium 199 (1 ml) supplemented with antibiotics and 10% FCS was added after 24 h, and culture was continued for an additional 1–2 weeks. The medium was changed every 48 h until the outgrowing cells were confluent, in 5% CO2 and 95% air at 37°C. Cells were passaged using trypsin. EVT between passages two and four were used for all experiments. The cells were identified as EVT by immunohistochemical staining using anti-cytokeratin 7 and 8/18, anti-α5β1 and αvβ3 integrins, anti-vimentin, CD9 and factor VIII, as described previously (Kabir-Salmani et al., 2003).

Protein assay

A protein assay was performed based on the method of Bradford using the Bio-Rad Protein Assay (Bio-Rad). According to the manufacturer's instruction, 100 μl of the decidual cell lysate was added to 5.0 ml of the reagent and incubated at room temperature for 5 min. The absorbance at 595 nm was measured with Gene Quant pro RNA/DNA calculator (Amersham pharmacia biotech, Cambridge, UK).

IGFBP-1 measurement

IGFBP-1 levels in the conditioned media from decidual cells were assayed by enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems, Inc. (Minneapolis, USA) according to the manufacturer's instruction. The sensitivity of this assay was 6.4 pg/ml. Intra-assay coefficients of variation were 4.1–5.6% and inter-assay coefficients of variation were 6.7–9.3%.

Immunoblotting of IGFBP1 and PRL

Each lot of medium (20 μl) conditioned by decidual cells was added to 4 × Laemmli buffer, which contained 200 mM dithiothreitol, and boiled for 5 min at 95°C. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 15% polyacrylamide gels and transferred to PVDF membranes by electrophoresis. After blocking with Tris-buffered saline (10 mM Tris and 140 mM NaCl, pH7.4) containing 0.1% Tween-20 and 3% bovine serum albumin for 2 h at room temperature, blots were exposed to a primary antibody against IGFBP1 (1:5000) or PRL (1:1000) overnight at 4°C. The blots were then incubated with secondary horse-radish peroxidase-conjugated antibodies for 20 min at room temperature and visualized using enhanced chemiluminescence (Immobilon Western; Millipore), following the manufacturer's instructions. The images obtained were scanned using EZ capture II (ATTO Corporation, Tokyo, Japan). Densitometric analyses of the images were performed using Image J (version 1.41, NIH, Bethesda, MD, USA).

Cell migration assay

EVT were plated on 6-well plates in Medium 199 containing 10% FCS and grown to confluence. Wounding was performed using a single razor blade to scrape cells on culture plates, leaving a denuded area and sharp visible demarcation line at the wound edge, as described in a previous report (Kabir-Salmani et al., 2004). The wounded monolayers were rinsed twice with serum-free medium (amino acid-free) and were inspected immediately after wounding. The sections of the wounds to be used for quantifying migration were selected according to the criteria described previously (Irving and Lala, 1995; Jones et al., 1996). The cells were then incubated for 48 h with the media conditioned by decidual cells. At the end of the incubation, more than 95% of cells were viable as assessed by the Trypan blue dye exclusion. Cells were rinsed in PBS, fixed and stained using the Diff-Quick kit (International Reagents Co., Kobe, Japan) and examined using phase-contrast microscopy. The migrated EVT cells were counted in 1.0-mm-long sections, allowing for a 20-μm space from the demarcation line in order to minimize the possible physical effects of cell movement resulting from cell proliferation. A calibrated eyepiece grid was used to determine the mean number of migrated cells. The results were calculated by averaging a mean of four sections per test substance for each experiment. To detect the influence of BCAAs on EVT cells migration, the same experiments were performed using the serum-free culture medium, either with or without BCAAs, that does not contain IGFBP1. In another set of experiments, GRGDSP (10−5 M), that inhibits IGFBP1 from binding to integrins, was added to each medium. To confirm the effect of IGFBP-1 on EVT migration, the same experiments were conducted in the presence of human recombinant IGFBP-1 (50 ng/ml), either with or without GRGDSP (10−5 M), in the serum-free Medium 199.

Immunoprecipitation and immunoblotting of phosphorylated FAK

EVT cells were grown to confluent on 6-well plates, rinsed twice with serum-free Medium 199 (amino acid-free) and incubated for 24 h with same medium. The cells were washed twice and incubated for 30 min with the medium conditioned by decidual cells. Cells were washed with ice-cold PBS three times and solubilized with lysis buffer. The insoluble materials were removed by centrifugation at 15 000g for 10 min at 4°C and the supernatants were incubated overnight at 4°C with 2 μg/ml anti-FAK antibody. The immunocomplexes were incubated with protein A-Sepharose (Sigma, St. Louis, USA) at 4°C for 2 h. The immunoprecipitates were washed three times with lysis buffer. The bound proteins were eluted in 20 μl of Laemmli sample buffer, heated for 5 min at 95°C and then separated by SDS–PAGE on 7.5% polyacrylamide gels. After transfer to PVDF membrane, the membrane was probed with PY99 (1:1000) and visualized by ECL. To detect the influence of BCAAs on phosphorylation of FAK, the same experiments were performed using the serum-free culture medium (with or without BCAAs) that does not contain IGFBP1. In another set of experiments, GRGDSP (10−5 M), that inhibits IGFBP-1 from binding to integrins, was added. To confirm the influence of IGFBP-1 on phosphorylation of FAK, the same experiments were conducted in the presence of IGFBP-1 (50 ng/ml), with or without GRGDSP (10−5 M), in the serum-free Medium 199.

Cell number assay

A cell number assay, using mitochondria-dependent reduction of 4-(3-[4-lodophenyl]-2-[4-nitropheny]-2H-5-tetrazolio-)-1,3-benzene disulfonate (WST-1; Roche, Masel, Switzerland), was performed according to the manufacturer's instructions. Briefly, the cytotrophoblast cells were seeded in flat-bottomed 96-well plates at 1 × 104 cells/well and cultured for 48 h in Medium 199 with 10% FCS in 5% CO2 and 95% air at 37°C. The cells were rinsed twice with serum-free medium (amino acid-free) and treated for 24 h with the media conditioned by decidual cells. Following incubation, cell proliferation reagent WST-1 was added at a 1/10 volume directly to each well and incubated for an additional 4 h. The absorption was measured with a multi-plate reader (iMark; Bio-Rad, Tokyo, Japan) set to wavelength 430 nm. To detect the influence of BCAAs on cytotrophoblast cell proliferation, the same experiments were performed using the serum-free culture medium, with or without BCAAs, that does not contain IGFBP1. The cytrophoblast cells passaged once were used for the experiments and cells from different placental samples were prepared for each experiment to assess reproducibility (n = 3).

Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Statistics ver19, IBM®, Japan). The Mann–Whitney U-test was used to compare differences between the control and test groups. A value of P < 0.05 was considered statistically significant. The experiments were repeated at least three times in each group to assess reproducibility. All values were expressed as mean ± SD.

Results

The results shown in the paper were derived from the samples of 25 women. The other 25 samples either failed in primary culture or were used for preliminary experiments.

Effects of BCAA deprivation on protein synthesis of decidual cells

To determine the effects of BCAAs (in the presence of NEAA) on protein synthesis of decidual cells, a Bradford protein assay was carried out. There was no significant difference in total protein concentration in cell lysates between decidual cells incubated with and without BCAAs (722.8 ± 55.7 µg/ml versus 725.0 ± 58.1 µg/ml, respectively).

Effects of BCAA deprivation on IGFBP1 and PRL secretion from decidual cells

The average concentration of IGFBP-1 in the conditioned media with BCAAs was 51.9 (±12.7) ng/ml, while that in the conditioned media deprived of BCAAs was 28.1 (±2.5) ng/ml (P < 0.05) (Fig. 1A). On the basis of these results, the concentration of exogenous IGFBP-1 used in further experiments was determined as 50 ng/ml.

Figure 1.

Figure 1

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Effects of BCAAs deprivation on IGFBP1 and PRL secretion from human decidual cells. Decidual cells were grown to confluence and incubated with serum-free medium with (+) and without (−) BCAAs for 18 h. The concentrations of IGFBP1 in the conditioned media were measured by ELISA kits (A). Each value is the mean ± SD from eight separate sets of samples (n = 8). The conditioned media were analyzed by SDS–PAGE followed by immunoblotting using anti-IGFBP1 (B) and anti-PRL antibodies (C) (upper panels). The cell lysates were analyzed by SDS–PAGE and the results of immunoblotting by anti-β-actin antibody were used as internal controls (lower panels). Relative indexes of scanning densitometry values represent the mean ± SD from three separate experiments (n = 3). *P < 0.05 NEAA, non-essential amino acids.

The immunoblot analysis showed BCAAs deprivation significantly reduced IGFBP1 level in the conditioned medium of decidual cells (Fig. 1B, P < 0.05). On the other hand, BCAAs deprivation did not influence PRL secretion from decidual cells (Fig. 1C).

EVT migration assay

The incubation of EVT with exogenous IGFBP-1 (50 ng/ml) significantly enhanced EVT cells migration (Fig. 2A, Bar 1, 2). To investigate the association of integrins with IGFBP1-stimulated migration of EVT cells, the cells were incubated in media containing GRGDSP peptide (10−5 M) that inhibits IGFBP1 from binding to integrins. GRGDSP peptide significantly decreased the number of migrated cells (Fig. 2A, Bar 3). Exposure to GRDGDSP peptide itself did not affect EVT cells migration (Fig. 2A, Bar 4).

Figure 2.

Figure 2

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Effects of IGFBP1 and conditioned media from human decidual cells on EVT cells migration. Migration of EVT following wounding of the cells and incubating with one of the following for 48 h: (A) the serum-free Medium 199 and 50 ng/ml IGFBP1 alone or in the presence of GRGDSP (10–5 M) and (B) the media conditioned by decidual cells for 18 h and the serum-free culture medium with or without BCAAs (Panel 1–4). The same experiments were repeated by adding GRGDSP (10–5 M) to each group (Panel 5–8). Scale bar = 50 μm. A calibrated eyepiece grid was used to determine the mean number of migrated EVT cells/1.0 mm section of wound. Statistical evaluation was calculated by averaging a mean of four sections per test substance for each experiment. The experiments were repeated three times in each group (n = 3). The results are shown as the mean ± SD. The solid bars (▪) represent the groups using the conditioned medium and the blank bars (□) represent those using the culture medium. *P < 0.05. GRGDSP, Gly-Arg-Gly-Asp-Ser-Pro hexapeptide.

To determine whether IGFBP1 secreted from decidual cells can affect EVT cells migration, the migration assay was conducted using condition media. As shown in Fig. 2B, the incubation with the conditioned media from decidual cells enhanced EVT cells migration. When incubated with the conditioned medium deprived of BCAAs (Fig. 2B, Panel 1), EVT cells migration was significantly suppressed, compared with the conditioned medium containing BCAAs (Fig. 2B, Panel 2). BCAAs deprivation itself showed no significant effects on EVT cells migration in the culture media (Fig. 2B, Panel 3, 4). Co-exposure to GRGDSP peptide and media decreased numbers of migrated cells in the conditioned media both with and without BCAAs and no significant difference was detected between them (Fig. 2B, Panel 5, 6). Exposure to GRGDSP peptide itself did not affect the migration of EVT cells in the culture media (Fig. 2B, Panel 7, 8).

Profiles of FAK phosphorylation in EVT cells

We investigated whether IGFBP1 activates the integrin signaling pathway in EVT cells. The results of anti-phosphotyrosine immunoblotting showed incubation with exogenous IGFBP-1 (50 ng/ml) enhanced phosphorylation of FAK in EVT cells (Fig. 3A). Addition of GRGDSP peptide (10−5 M) suppressed IGFBP-1 enhancement of FAK phosphorylation, and GRGDSP peptide itself did not affect phosphorylation status of FAK (Fig. 3A).

Figure 3.

Figure 3

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Effects of IGFBP1 and conditioned media from human decidual cells on phosphorylation of FAK in EVT. EVT were grown to confluence and incubated with one of the following for 30 min after 24 h starvation: (A) serum-free Medium 199 and 50 ng/ml IGFBP1 alone or in the presence of GRGDSP (10–5 M) and (B) media conditioned by decidual cells for 18 h and serum-free culture medium with or without BCAAs. The cells were lysed and immunoprecipitated with anti-FAK antibody. The proteins were analyzed by SDS–PAGE followed by immunoblotting using anti-phosphotyrosine antibodies (PY99) (upper panels) and anti-FAK antibodies (lower panels). The same experiments were repeated with adding GRGDSP (10–5 M) to each group (C). Relative indexes of scanning densitometry values of PY99 and FAK represent the mean ± SD from three separate experiments (n = 3). The solid bars (▪) represent the groups using the conditioned medium and the blank bars (□) represent those using the culture medium. *P < 0.05.

The incubation with the conditioned media from decidual cells also stimulated phosphorylation of FAK in EVT cells (Fig. 3B). When using the conditioned medium from BCAAs-deprived decidua, phosphorylation was significantly suppressed compared with the cells in the medium containing BCAAs (Fig. 3B, Lane 1, 2). Deprivation of BCAAs itself in the culture media did not alter phosphorylation status of FAK in EVT cells (Fig. 3B, Lane 3, 4). Incubation with GRGDSP peptide eliminated difference in phosphorylation between EVT cells in the conditioned media with and without BCAAs (Fig. 3C, Lane 1, 2).

Numbers of cytotrophoblast cells

We investigated whether changes in IGFBP1 secretion from decidual cells induced by BCAA deprivation affect the number of cytotrophoblast cells. The result of the assays on the cells before incubation (control) are represented by the shaded bar. In an assay which determines cell number, the absorbance values for samples incubated with the conditioned media were not increased compared with those without conditioned media (Fig. 4). There was also no difference in the absorbance when using the culture media that does not contain IGFBP1, represented by open bars.

Figure 4.

Figure 4

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Effects of conditioned media from human decidua cells on number of cytotrophoblasts. Cytotrophoblast cells were seeded in 96-well culture plates at 1 × 104 cells/well and cultured for 48 h in 10% FCS media. The cells were treated with one of the following for 24 h: media conditioned by decidual cells (▪) without BCAAs (BCAA−) or with BCAAs (BCAA+), and serum-free culture medium (□) with or without BCAAs. Following incubation, 1/10 of the well volume of WST-1 was added and incubation was continued for an additional 4 h. The absorption was measured with a multi-plate reader at 430 nm. Ten wells per group in the culture plate were evaluated for each experiment. The results represent the mean ± SD of three separate experiments (n = 3). The shaded bar represents the absorbance measured before the incubation as control (cont).

Discussion

In the current study, we examined possible changes in IGFBP1 production in decidual cells induced by BCAA deprivation and investigated its physiological effects on EVT and cytotrophoblast cells.

By subjecting decidual cells to BCAA deprivation, we demonstrated suppression in IGFBP1 secretion from decidual cells both by ELISA and immunoblotting. BCAA deprivation treatment did not affect total protein synthesis in decidual cells. We also investigated secretion of PRL, another representative protein derived from decidual cells, and showed BCAA deprivation did not alter PRL production from decidual cells. These results suggest that IGFBP1 secretion from decidual cells is selectively regulated by the presence of BCAAs.

Next, we examined the physiological significance of alteration of IGFBP1 secretion from BCAA-deprived decidual cells on cell–cell communication between fetal trophoblast cells and maternal decidual cells at the maternal–fetal interface.

First, to confirm the effects of IGFBP1 on EVT migration, a wounding and migration assay was. EVT migration was evidently stimulated by exogenous IGFBP1 (50 ng/ml). IGFBP1 and IGFBP2 contain an Arg-Gly-Asp (RGD) domain capable of binding to the RGD recognition sites of certain integrins (Brewer et al., 1988; Bourner et al., 1992). In vitro studies have demonstrated that IGFBP1, but not IGFBP2, via its RGD domain, can bind to α5β1 integrin, leading to an IGF-independent effect on a variety of cells including EVT (Jones et al., 1993; Gockerman et al., 1995). To examine whether the effects of IGFBP1 on EVT migration is caused via α5β1 integrin, GRGDSP peptide treatment was performed and this completely abrogated the migration-stimulating effect of IGFBP1, which indicates GRGDSP peptide can occupy the RGD binding domain on α5β1 integrin and inhibit the IGFBP-1 effect.

To assess the effects of IGFBP1 secreted from decidual cells on EVT migration, a migration assay was performed with the conditioned media. EVT migration was stimulated by incubation with the media conditioned by decidual cells. In addition, when incubated with the media conditioned by BCAA-deprived decidual cells, EVT migration was significantly suppressed. BCAA deprivation treatment itself did not influence cell migration in the culture media that does not contain IGFBP1. Incubation with GRGDSP inhibited the migration-stimulating effect of the conditioned media irrespective of the presence of BCAAs. IGFBP1 was the representative protein with a RGD domain among the soluble factors in the conditioned media; therefore, the inhibitory effect of GRDGSP may be attributed to IGFBP1 in the conditioned media. These results demonstrated that decidua-derived IGFBP1-stimulated EVT migration via α5β1 integrin, and that IGFBP1 secretion from decidual cells decreased and EVT migration was suppressed when BCAAs were absent from the media.

Integrins have short cytoplasmic domains without endogenous catalytic activity. To function in signal transduction for cell migration, these domains must interact with other catalytic molecules for internalization of signals. Occupation of integrins with ligands can increase phosphorylation of a FAK tyrosine kinase, known as FAK (Guan and Shalloway, 1992; Kornberg et al., 1992). To confirm biological effects of decidua-derived IGFBP1 on EVT through α5β1 integrin, FAK phosphorylation was investigated using immunoprecipitation and immunoblotting.

We confirmed that exogenous IGFBP1 enhanced phosphorylation of FAK in EVT and that GRGDSP peptide treatment had an inhibitory effect against IGFBP1.

A decrease in IGFBP1 secretion from BCAAs-deprived decidual cells led to significantly lower levels of phosphorylation of FAK in EVT. In addition, GRGDSP peptide treatment eliminated differences in phosphorylation of FAK caused by IGFBP1 in the conditioned media. These results were consistent with the finding that decidua-derived IGFBP-1 stimulated EVT migration by binding to α5β1 integrin, as shown in migration assay experiment. The change in secreted IGFBP1 level from decidual cells by deprivation of BCAAs affected intracellular signaling through α5β1 integrin in EVT.

These results indicate that decidua-derived IGFBP1 stimulates EVT cell migration by binding to α5β1 integrin, independent of IGF, in the maternal–fetal interface. Therefore, a decreased secretion of IGFBP1 by BCAA deprivation of decidual cells resulted in suppression of EVT migration.

Insulin is another well-known counter-regulator of IGFBP1 production in decidual cells. The previous studies revealed dose-dependent inhibition of IGFBP1 secretion from decidual cells when incubated with insulin (Giudice et al., 1991; Lathi et al., 2004). Moreover, maternal hyper-insulinemic states, such as gestational diabetes and obesity, is known increase risk of pre-eclampsia (Solomon and Seely, 2001; Miyakoshi et al., 2004). Maternal hyper-insulinemia may cause suppression of IGFBP1 secretion from decidual cells in the maternal–fetal interface, leading to suppressed EVT migration and finally placental dysfunction. This is compatible with the results of the present study, that the decreased IGFBP1 production caused suppression of EVT migration.

Deprivation of essential amino acids, including BCAAs, stimulates IGFBP1 expression in hepatocytes and hepatoma cell line (Straus et al., 1993). We believe this conflicting effect of BCAAs deprivation treatment on IGFBP1 production can be attributed to the tissue-specific functions of IGFBP1. Liver is a major organ responsible for systemic metabolism, and IGFs stimulate amino acid and glucose uptake and general protein synthesis of hepatic cells (Jones and Clemmons, 1995). When deprived of metabolic resources, such as BCAAs, IGFBP1 expression is stimulated to inhibit IGF effects in order to spare the limited energy resources for survival rather than growth. As for hepatic cells, the biological significance of IGFBP1 is to bind IGFs and control IGFs metabolic effects (Fowler et al., 2000). On the other hand, as shown in the present study, decidua-derived IGFBP-1 acted directly on EVT via α5β1 integrin and stimulated migration IGF-independently in the maternal–fetal interface. Under conditions of maternal malnutrition and fasting, pregnancy itself carries a significant risk for maternal survival (Mircea et al., 2007). The inhibitory effects on placental development by reducing IGFBP1 secretion under conditions of poor BCAA supply are a reasonable response for maternal decidual cells.

There have been some reports suggesting inhibitory effects of IGFBP1 on EVT invasion (Irwin and Giudice, 1998; Irwin et al., 1999). On the other hand, the previous in vitro study (Gleeson et al., 2001) showed the stimulating effects of IGFBP1 by binding α5β1 integrin, which is consistent with our data. The idea of an invasion-blocking effect of IGFBP-1 was based on the results of enhanced EVT invasion in an in vitro decidualized cell layer in the presence of insulin (5 μg/ml), which inhibits IGFBP1 expression, and a reversal of the effects with a high dose (1 μg/ml) of IGFBP1 (Irwin and Giudice, 1998). In that study, other effects of insulin on decidual cells or trophoblast cells were not ruled out. Moreover, the significance of the relatively high concentration of IGFBP1 used in the experiments should be carefully examined, considering that its concentration in maternal serum during normal pregnancy ranges between 0.08 and 0.36 μg/ml (Anim-Nyame et al., 2000). The concentration of IGFBP-1 in the conditioned media measured by ELISA was 25.0–68.7 ng/ml; therefore, we believe the conditioned medium from decidual cells in primary culture used in our study was a more physiologically appropriate in vitro model of the maternal–fetal interface of early pregnancy. Moreover, the stimulating effects of IGFBP1 on EVT migration were confirmed by adding exogenous IGFBP1 at 50 ng/ml in our study, which was consistent with the results of the previous report showing the migration-stimulating effects of IGFBP1 at 1 nmol/l, i.e. 30 ng/ml (Gleeson et al., 2001). In addition, the recent study has demonstrated that IGFBP1 expression in decidua was significantly decreased in pre-eclamptic placenta (Gratton et al., 2002). This finding is consistent with the idea that IGFBP1 stimulates EVT migration and decreased IGFBP1 expression in decidua may contribute to the abnormal trophoblast invasion in pre-eclampsia.

Cytotrophoblasts either differentiate as well as fuse to form syncytiotrophoblasts or differentiate into EVT to form stratified cell columns. Therefore, in addition to shallow invasion of EVT, insufficient cytotrophoblast growth at the villous cell column may be also associated with poor placental development, leading to pre-eclampsia. We counted cytotrophoblasts incubated in the conditioned media of decidual cells and showed that BCAA deprivation treatment on decidual cells did not affect cytotrophoblasts number. As shown in the culture media, BCAAs deprivation treatment itself did not influence cell proliferation either. These results demonstrated decidua-derived IGFBP1 itself may not have a proliferative effect on cytotrophoblasts at the villous cell column. It may be attributed to a selective integrin profile of the villous trophoblast cells as the cytotrophoblasts do not have α5β1 integrin but gain it during differentiation into EVT (Damsky et al., 1992; Vicovac et al., 1995).

In conclusion, decidua-derived IGFBP1 has a stimulating effect on EVT migration by binding to α5β1 integrin, independent of IGF, in the maternal–fetal interface. When deprived of BCAAs, secretion of IGFBP1 from decidual cells is selectively decreased and therefore EVT migration is suppressed.

Not only chronic maternal malnutrition, but also the conditions which may impair vascular perfusion to uterine endometrium, including maternal thrombophilia and heavy smoking, could interrupt the BCAAs supply to decidual cells in early pregnancy. As we have shown, BCAA deprivation could reduce IGFBP1 secretion from decidual cells, leading to suppressed EVT cells migration. Maternal poor nutrition status is well known as a risk factor for restricted fetal growth, and maternal thrombotic disease, such as anti-phospholipid antibody syndrome, is highly associated with pre-eclampsia. Therefore, loss of the BCAA supply to decidual cells may be one of the pathophysiological conditions responsible for pre-eclampsia. Moreover, this implication brings us to the idea of BCAA supplementation in early pregnancy to possibly prevent development of pre-eclampsia. Although further study is surely needed, the idea of pre-eclampsia prevention by BCAA supplementation is attractive from the point of safety, since we could utilize food and dietary supplements instead of chemical drugs.

The precise regulatory mechanism of BCAA stimulation of IGFBP1 production by decidual cells should be investigated in further studies. It has been reported that the forkhead transcription factor forkhead box O1A (FOXO1) plays an important role in progesterone-dependent decidualization of the uterine endometrium. FOXO1 is also indispensable for the induction of the decidual marker genes, including PRL and IGFBP1 (Takano et al., 2007; Kajihara et al., 2013). Takenaka et al. reported that the amino acid-responsive element, which is involved in the induction of IGFBP1 gene in response to amino acid deprivation, includes the insulin-responsive element (IRE) and glucocorticoid responsive element of IGFBP1(Takenaka et al., 2000). Since FOXO1 is known to bind to the IRE of IGFBP1 and regulate IGFBP1 gene expression in hepatic cells (Hall et al., 2000), FOXO1 might be also a key regulator of IGFBP1 expression in decidual cells in response to BCAAs, which should be investigated in further experiments.

Authors' roles

K.T. and K.S. designed the study. K.T. executed the experiments, analyzed the data and drafted the manuscript. K.T., K.S. and M.I. revised the manuscript. M.M., Y.M. and T.I. helped to collect the tissue and perform the experiments. T.N., S.F. and K.Y assisted the analysis of data.

Funding

All funds were obtained through Kyorin University School of Medicine. No specific grants were sought or obtained for this research.

Conflict of interest

There are no conflicts of interest, sources of financial support, corporate involvement, patent holdings for any author to be disclosed.

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