Abstract
Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory, anti-atherogenic, and anti-proliferative effects. The expression of specific adiponectin receptors in the placenta and in the endometrium suggests a role for this cytokine in placental development, but this role has not yet been elucidated. The invasion of trophoblast cells during the first trimester of pregnancy being crucial to placentation process, we have studied adiponectin effects on human trophoblast invasive capacities. We found that adiponectin stimulated human trophoblast cell migration in HTR-8/SVneo cells in a dose-independent manner. In addition, adiponectin also significantly enhanced invasion of HTR-8/SVneo cells and of human extravillous trophoblast from first trimester placenta. These pro-invasive effects of adiponectin in human trophoblasts seem to be mediated in part via increased matrix metalloproteinases (MMP2 and MMP9) activities and via repression of TIMP2 mRNA expression. Our results suggest that adiponectin could be a positive regulator of the early invasion process by modulating the MMP/TIMP balance. Moreover, these results provide an insight into the role of adiponectin in pathological conditions characterized by insufficient or excessive trophoblast invasion.
Introduction
Implantation requires a number of distinct cellular functions including attachment and invasion of the extravillous trophoblasts (EVTs) into the endometrium. Deficient trophoblastic invasion contributes to the poor success rates of IVF and may result in gestational complications including preeclampsia (PE) or intrauterine growth restriction (Norwitz et al. 2001, Anin et al. 2004, Kadyrov et al. 2006). However, excessive invasion of the trophoblast cells occurs in placenta accreta or percreta and choriocarcinoma (Lala et al. 2002). In contrast to tumor invasion, trophoblast invasion during implantation is strictly spatiotemporally regulated (Bischof et al. 2000).
EVT invasion involves proteolytic degradation of decidual and endothelial extracellular matrix (ECM) in the direction of migration followed by EVT adhesion to ECM elements and active cell migration through the degraded matrix (Mareel & Leroy 2003). For these processes, the action of proteases, particularly the matrix metalloproteinases (MMP2 and MMP9), is very important (Staun-Ram et al. 2004, Cohen et al. 2006). These proteases are secreted as latent enzymes, and their activities are further regulated by the local concentration of the major natural tissue inhibitors of metalloproteinases TIMP1 and TIMP2 which bind the MMPs with a 1:1 stoichiometry (Huppertz et al. 1998). Thus, the local balance between MMPs and TIMPs at the invasive site is crucial. Moreover, cell–ECM interactions are mediated by adhesion molecules, such as cadherins and integrins that provide the appropriate adhesion to enable traction and movement of the cells (Burrows et al. 1996). When trophoblasts acquire the invasive phenotype, a downregulation of integrin α6β4 and an upregulation of integrins α5β1 and α1β1 are observed suggesting that switching of integrin expression contributes to this process (Damsky et al. 1994).
Trophoblast invasion depends on a controlled program of intercellular signaling mediated by growth factors, cytokines, and hormones of both fetal and maternal origin (Bischof et al. 2000, Cohen & Bischof 2007, Knofler et al. 2008). Adiponectin, a hormone mainly produced in adipose tissue, has been described as an insulin-sensitizing adipocytokine (Yamauchi et al. 2001). Adiponectin effects are mediated through two specific receptors, AdipoR1 and AdipoR2, which share homology with G protein-coupled receptors (Yamauchi et al. 2003). In the presence of adiponectin, these receptors activate downstream targets such as AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-α, phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK; Kadowaki & Yamauchi 2005, Tilg & Moschen 2006). In addition to its role in energy homeostasis, adiponectin exerts a protective action against gastric, prostate, and breast cancer development (Mantzoros et al. 2004, Bub et al. 2006, Ishikawa et al. 2007). Some evidence suggests that adiponectin could also directly regulate reproductive and placental processes (Mitchell et al. 2005, Campos et al. 2008) as already described for another adipocytokine leptin (Chehab 2000). Furthermore, it was demonstrated that, in human endometrium, adiponectin receptor levels are the highest during the luteal period, which corresponds to the embryo implantation period (Takemura et al. 2006a). More recently, we have shown in our laboratory that adiponectin exerts anti-proliferative effects on trophoblastic cell lines JEG-3 and BeWo and also on human trophoblasts (Benaitreau et al. 2009). McDonald & Wolfe (2009) have shown that adiponectin reduces the endocrine function of human term placenta.
In the present study, we hypothesized that adiponectin might regulate human EVT invasion during early placentation. Owing to the restricted availability of primary human EVTs, extended studies were performed using immortalized EVT cell line, the HTR-8/SVneo cells, followed by more limited studies using primary EVTs isolated from first trimester placenta to validate these findings. We have determined the effects of human recombinant adiponectin on EVTs and HTR-8/SVneo cell migration and invasion, and characterized the cellular signaling pathways involved. Furthermore, we also determined its effects on specific members of the MMP system.
Materials and Methods
Materials
DMEM/F12, RPMI, penicillin, streptomycin, HEPES, leupeptin, aprotinin, 5-aminoimidazole-4-carboxamide 1β-d ribofuranosyl 5′monophosphate (AICAR), 4-(2-aminoethyl)-benzene-sulfonyl fluoride, NaF, Dnase type IV, and BSA were purchased from Sigma Chemical Co. Recombinant human adiponectin and leptin were provided by R&D Systems Europe Ltd (Abingdon, UK), Superscript II Rnase H-RT by Invitrogen Corporation, and RNAguard by Pharmacia Biotechnology. MAPK kinase inhibitor UO126 was obtained from Promega, PI3K inhibitor LY29002 was obtained from Calbiochem (Merck Chemicals), Matrigel was obtained from BD Biosciences (Le Pont-de-Claix, France), and trypsin was obtained from Difco Laboratories (Detroit, MI, USA).
Tissue collection
This study was approved by the local ethical committee (CCPPRB), and informed consent was obtained from each donor before clinical sampling.
Human placental tissues from first trimester (6–8 weeks of pregnancy, n=10) were obtained from healthy pregnant women aged between 18 and 35 years when undergoing legal abortions. Placental tissues were placed in HBSS supplemented with streptomycin (0.1 mg/ml) and penicillin (100 U/ml), washed several times, and aseptically dissected to remove decidual tissues and fetal membranes.
First trimester villous explants culture
Pieces of villous tissue from different first trimester placentae (n=8–10) were dissected and cultured on collagen I-coated 12-well plates. Placental samples were left for 2 h at 37 °C under 5% CO2 and 95% air atmosphere before the addition of DMEM/F12 medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamycin (10 μg/ml).
EVT cell isolation
Human primary EVT cells were prepared from tips of placental terminal villi as previously described (Handschuh et al. 2007). Briefly, terminal villi were incubated in HBSS containing 0.125% trypsin, 4.2 mM MgSO4, 25 mM HEPES, and 50 U/ml Dnase type IV for 30 min at 37 °C without agitation. After tissue sedimentation, the supernatant was filtered through 100 μm nylon screen and centrifuged at 200 g for 10 min. Cells were washed twice and then filtered through 40 μm nylon screen. The cell suspension was layered over a discontinuous Percoll gradient and centrifugated for 25 min at 1000 g. The layer corresponding to 40–45% Percoll containing EVTs was washed twice in HBSS supplemented with 10% FCS. Cells were seeded on Matrigel- (5 mg/ml) coated dishes containing F12/DMEM with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamycin (10 μg/ml), and maintained at 37 °C under 5% CO2 and 95% air atmosphere. To ensure that the cultured cells possessed the invasive phenotype of EVTs, adherent cells were characterized by immunocytochemistry analysis. In total, 90–95% of cells were positive for cytokeratin 7 (Dako, Glostrup, Denmark), and 80–85% of cells were positive for the proto-oncogene c-erbB2 (Ventana Medical System, Illkirch, France; Pavan et al. 2003). Moreover, the expression of EVT marker was confirmed by RT-PCR using specific primers of HLA-G transcript as described by Pavan et al. (2003).
Immortalized EVT cell line (HTR-8/SVneo)
The human EVT cell line HTR-8/SVneo was kindly provided by Dr Nadia Alfaidy (CEA Grenoble France) in agreement with Dr Charles Graham. Cells were used at passage 45. Cells were plated in RPMI medium supplemented with streptomycin (0.1 mg/ml), penicillin (100 U/ml), and 10% FCS, and maintained at 37 °C under 5% CO2 and 95% air atmosphere.
No significant toxic effects of the various factors tested were observed by measuring the lactate dehydrogenase activity released into the culture medium as previously described (Lacasa et al. 1988).
Wound healing assay
Wound healing assays were performed in 35 mm dishes coated with collagen I. HTR-8/SVneo cells were seeded and incubated until confluence. Then, cell layers were wounded with a blade and washed three times with serum-free culture medium. The marking of the blade on the plastic served as the migratory start line. Digital photographs within each wound were taken before, and 24 or 48 h after adiponectin addition in serum-free culture medium. Cells that had migrated and filled the wounded area were quantified using Histolab analysis software (version 5.2.3, Microvision Instrument, Paris, France). Ten percent FCS in the culture medium was used as a positive control.
Invasion assays
Invasion assays were performed in 24-well plates containing Matrigel-coated polycarbonate membrane (8 μm) invasion chamber inserts (Greiner, Frickenhausen, Germany).
Cells were cultured at a density of 5×104 or 1×105 for HTR-8/SVneo cells and human primary EVTs respectively in serum-free medium with or without adiponectin or 10% FCS or 500 μM AICAR (a specific AMPK activator) (Corton et al. 1995). After 48 h, media containing non-invaded cells were removed from the upper wells and conserved at −20 °C until required for zymography. Cells at the lower surface of the inserts were incubated for 1 h at 37 °C with 4 μM calcein AM and detached with trypsin, and their fluorescence was quantified using an Infinite microplate fluorometer (Tecan, Lyon, France).
Zymography
The total gelatinase activities of MMP2 and MMP9 were analyzed by zymography. Aliquots of culture media containing 2 μg protein were resolved under non-reducing conditions in 10% polyacrylamide gels containing 1 mg/ml gelatin (Difco). Gels were washed in 2.5% Triton X-100 for 30 min to remove SDS and incubated overnight at 37 °C in a renaturing buffer (50 mM Tris–HCl, pH 7.5, 5 mM CaCl2, 150 mM NaCl, and 0.02% sodium azide). Gels were stained with Coomassie brilliant blue and destained in methanol/acetic acid (20%/5% v/v). Proteolytic activity was identified as a clear band on a blue background. The images were scanned and quantitative enzyme analysis was carried out using Bio1D Software (Vilbert Lourmat, Marne la Vallée, France).
[3H]-Thymidine incorporation
HTR-8/SVneo cells were plated into 24-well culture dishes at a density of 1.5×104 in DMEM/F12 with 10% FCS. After 24 h, cells were cultured in serum-free medium with various concentrations of human recombinant adiponectin, or 10% FCS used as a positive control of cell proliferation, for the next 24 h. For the last 6 hours, [3H]-thymidine (1 mCi/ml) was added to the culture medium. After washing three times with saline buffer, cells were lysed during 5 min with 1% SDS and treated with 10% trichloroacetic acid for 45 min at 4 °C. Radioactivity was counted after filtration on GF/C filters (Whatman, Clifton, NY, USA).
Quantitative RT-PCR
Total RNA (0.1 μg) was extracted and reverse transcribed as previously described (Machinal-Quelin et al. 2002). Quantitative PCR was performed using a LightCycler 480 instrument from Roche Diagnostics with primer sets indicated in Table 1 and SYBR Green I master mix (Roche Diagnostics). Second derivative maximum method was used to automatically determine the crossing point (Cp) for individual samples. Three reference genes TBP, RP13A, and β2 microglobulin have been tested for their expression stability in HTR-8/SVneo cells, EVT cells, and placental tissue. Analysis using the geNorm software (Vandesompele et al. 2002) revealed that TBP and β2 microglobulin genes were the most stable genes to normalize data in HTR-8/SVneo cells. However, the same analysis indicated that TBP and RP13A were the best genes to normalize data from EVT cells and placental tissue. For each sample and for each cell type, the concentration ratio (target/both reference mRNAs) was calculated using the RelQuant Roche software and expressed in arbitrary units. Calibration curves were log-linear over the quantification range with correlation coefficient (r2)>0.99 and efficiency ranging from 1.8 to 2. The intra-assay variability of duplicate Cp values never exceeded 0.2 cycle, and the inter-assay variability (coefficient of variation value) ranged from 1 to 5% for the five or seven runs of each transcript.
Primer pairs for RT–PCR
| Sequence | PCR product (bp) | |
|---|---|---|
| Primer sets | ||
| AdipoR1 | ||
| Sense | 5′-TTC TTC CTC ATG GCT GTG ATG T-3′ | 71 |
| Antisense | 5′-AAG AAG CGC TCA GGA ATT CG-3′ | |
| AdipoR2 | ||
| Sense | 5′-ATA GGG CAG ATA GGC TGG TTG A-3′ | 76 |
| Antisense | 5′-GGA TCC GGG CAG CAT ACA-3′ | |
| Adiponectin | ||
| Sense | 5′-GTC ATT GTC ATT ATC AGC-3′ | 153 |
| Antisense | 5′-GCT ATG CTC TTC ACC TAT-3′ | |
| TIMP1 | ||
| Sense | 5′-GGG CTT CAC CAA GAC CTA CA-3′ | 71 |
| Antisense | 5′-TGC AGG GGA TGG ATA AAC AG-3′ | |
| TIMP2 | ||
| Sense | 5′-GAA GAG CCT GAA CCA CAG GT-3′ | 85 |
| Antisense | 5′-GGG GGA GGA GAT GTA GCA C-3′ | |
| TBP | ||
| Sense | 5′-TGC ACA GGA GCC AAG AGT GAA-3′ | 132 |
| Antisense | 5′-CAC ATC ACA GCT CCC CAC CA-3′ | |
| β2 microglobulin | ||
| Sense | 5′-TGC TGT CTC CAT GTT TGA TGT ATC T-3′ | 86 |
| Antisense | 5′-TCT CTG CTC CCC ACC TCT AAG T-3′ | |
| RP13A | ||
| Sense | 5′-TTG AGG ACC TCT GTG TAT TTG TCA A-3′ | 125 |
| Antisense | 5′-CCT GGA GGA GAA GAG GAA AGA GA-3′ |
PCR products were separated on a 2% agarose gel in 90 mM Tris-borate and 2 mM EDTA buffer (TBE, pH 8.0), and visualized by staining with ethidium bromide and u.v. transilluminator.
Statistical analysis
All values were expressed as means±s.e.m. of four to eight separate experiments, and statistical analysis was performed using the non-parametric paired Wilcoxon test or Kruskal–Wallis test.
Results
Expression of adiponectin and adiponectin receptors in HTR-8/SVneo cells and human primary EVTs
Before investigating adiponectin effects on HTR-8/SVneo cells and human primary EVTs, we have examined adiponectin and adiponectin receptor expressions in both cell types.
Using quantitative RT-PCR, we demonstrated that AdipoR1 and AdipoR2 mRNAs were expressed in HTR-8/SVneo cells, in human primary EVTs, and also in first trimester human placental tissue (Fig. 1A and B). Adipose tissue was used as a positive control. We have also shown that AdipoR1 mRNA was expressed at a 10- to 20-fold higher level than AdipoR2 mRNA in HTR-8/SVneo cells, in primary EVTs, and in placental tissue (Fig. 1B). Moreover, we have demonstrated that HTR-8/SVneo cells expressed two- to three-fold higher level of AdipoR2 mRNA than primary EVTs (Fig. 1B).
Adiponectin and AdipoR1 and AdipoR2 mRNA expressions. Total RNA was extracted from human HTR-8/SVneo cells, human primary EVTs, and human adipose and placental tissues, and analyzed by RT-PCR with primers as described in Table 1. (A) PCR products were analyzed by agarose gel electrophoresis. This figure shows one representative among four separate experiments. (B) RT-PCR relative quantification of AdipoR1 and AdipoR2 mRNA expressions. Results are the means±s.e.m. of six separate experiments. *P<0.05, **P<0.01, Kruskal–Wallis test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Adiponectin and AdipoR1 and AdipoR2 mRNA expressions. Total RNA was extracted from human HTR-8/SVneo cells, human primary EVTs, and human adipose and placental tissues, and analyzed by RT-PCR with primers as described in Table 1. (A) PCR products were analyzed by agarose gel electrophoresis. This figure shows one representative among four separate experiments. (B) RT-PCR relative quantification of AdipoR1 and AdipoR2 mRNA expressions. Results are the means±s.e.m. of six separate experiments. *P<0.05, **P<0.01, Kruskal–Wallis test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Adiponectin and AdipoR1 and AdipoR2 mRNA expressions. Total RNA was extracted from human HTR-8/SVneo cells, human primary EVTs, and human adipose and placental tissues, and analyzed by RT-PCR with primers as described in Table 1. (A) PCR products were analyzed by agarose gel electrophoresis. This figure shows one representative among four separate experiments. (B) RT-PCR relative quantification of AdipoR1 and AdipoR2 mRNA expressions. Results are the means±s.e.m. of six separate experiments. *P<0.05, **P<0.01, Kruskal–Wallis test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
As shown in Fig. 1A, adiponectin was expressed neither in HTR-8/SVneo cells and human primary EVTs nor in human first trimester placenta. However, adiponectin was expressed in human adipose tissue, which was used as a positive control.
Effects of adiponectin on cell migration and invasion
We have examined the ability of adiponectin to modify the migratory and invasive properties of human EVTs and HTR-8/SVneo cells using wound healing (Fig. 2) and Matrigel transwell invasion assays (Fig. 3) respectively.
Effect of adiponectin on cell migration and proliferation. (A and B) Wound healing assays were performed in HTR-8/SVneo cells as described in Materials and Methods. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS during 24 or 48 h. (A) Bars represent mean values±s.e.m. of seven separate experiments. *P<0.05, Wilcoxon test. (B) Micrographs of wounded, non-fixed cell layers at 0, 24, and 48 h after adiponectin addition as described in Materials and Methods. Representative examples of seven separate experiments are shown. (C) Effect of adiponectin on DNA synthesis in HTR-8/SVneo cells. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS for 24 h in the presence of [3H]-thymidine as described in Materials and Methods. Results are means±s.e.m. of seven to nine experiments and are normalized as percentages of the control value (untreated). *P<0.05; NS, non-significant, adiponectin or 10% FCS versus control, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effect of adiponectin on cell migration and proliferation. (A and B) Wound healing assays were performed in HTR-8/SVneo cells as described in Materials and Methods. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS during 24 or 48 h. (A) Bars represent mean values±s.e.m. of seven separate experiments. *P<0.05, Wilcoxon test. (B) Micrographs of wounded, non-fixed cell layers at 0, 24, and 48 h after adiponectin addition as described in Materials and Methods. Representative examples of seven separate experiments are shown. (C) Effect of adiponectin on DNA synthesis in HTR-8/SVneo cells. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS for 24 h in the presence of [3H]-thymidine as described in Materials and Methods. Results are means±s.e.m. of seven to nine experiments and are normalized as percentages of the control value (untreated). *P<0.05; NS, non-significant, adiponectin or 10% FCS versus control, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effect of adiponectin on cell migration and proliferation. (A and B) Wound healing assays were performed in HTR-8/SVneo cells as described in Materials and Methods. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS during 24 or 48 h. (A) Bars represent mean values±s.e.m. of seven separate experiments. *P<0.05, Wilcoxon test. (B) Micrographs of wounded, non-fixed cell layers at 0, 24, and 48 h after adiponectin addition as described in Materials and Methods. Representative examples of seven separate experiments are shown. (C) Effect of adiponectin on DNA synthesis in HTR-8/SVneo cells. Cells were exposed to human recombinant adiponectin (25 or 250 ng/ml) or 10% FCS for 24 h in the presence of [3H]-thymidine as described in Materials and Methods. Results are means±s.e.m. of seven to nine experiments and are normalized as percentages of the control value (untreated). *P<0.05; NS, non-significant, adiponectin or 10% FCS versus control, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effect of adiponectin on cell invasion in Matrigel invasion chambers. Transwell migration assays of HTR-8/SVneo cells and primary EVTs were performed as described in Materials and Methods. (A) HTR-8/SVneo cells were incubated in the presence or absence of adiponectin (25 or 250 or 500 ng/ml), AICAR (500 μM), or 10% FCS for 48 h. (B) Human EVTs were incubated in the presence or absence of adiponectin (250 ng/ml) or 10% FCS for 48 h. Results are the mean values±s.e.m. of five to seven separate experiments. *P<0.05, **P<0.01; NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effect of adiponectin on cell invasion in Matrigel invasion chambers. Transwell migration assays of HTR-8/SVneo cells and primary EVTs were performed as described in Materials and Methods. (A) HTR-8/SVneo cells were incubated in the presence or absence of adiponectin (25 or 250 or 500 ng/ml), AICAR (500 μM), or 10% FCS for 48 h. (B) Human EVTs were incubated in the presence or absence of adiponectin (250 ng/ml) or 10% FCS for 48 h. Results are the mean values±s.e.m. of five to seven separate experiments. *P<0.05, **P<0.01; NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effect of adiponectin on cell invasion in Matrigel invasion chambers. Transwell migration assays of HTR-8/SVneo cells and primary EVTs were performed as described in Materials and Methods. (A) HTR-8/SVneo cells were incubated in the presence or absence of adiponectin (25 or 250 or 500 ng/ml), AICAR (500 μM), or 10% FCS for 48 h. (B) Human EVTs were incubated in the presence or absence of adiponectin (250 ng/ml) or 10% FCS for 48 h. Results are the mean values±s.e.m. of five to seven separate experiments. *P<0.05, **P<0.01; NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Wound healing assay
HTR-8/SVneo cell migration into the denuded area was increased after 24 h exposure to 25 and 250 ng/ml adiponectin (+32 and +65% respectively) as compared to control situation. After 48 h of treatment, this effect was higher (+100 and +112% for 25 and 250 ng/ml adiponectin respectively). Ten percent FCS was used as a positive control of cell migration (Fig. 2A and B). In parallel, we have measured, by [3H]-thymidine incorporation experiments, adiponectin effect on HTR-8/SVneo cell proliferation. As shown in the Fig. 2C, adiponectin (25 and 250 ng/ml) had no effect on HTR-8/SVneo cell number. However, 10% FCS, used as a positive control, increased (approximately threefold) significantly [3H]-thymidine incorporation.
Matrigel transwell assay
As shown in Fig. 3A, adiponectin increased HTR-8/SVneo invasion towards Matrigel (+75%) compared to control condition, at all concentrations examined (25–500 ng/ml). Under the same experimental conditions, 500 μM AICAR (a specific AMPK activator) did not affect HTR-8/SVneo invasion. To confirm these results, we have also examined the effect of adiponectin on primary EVTs in the transwell assay (Fig. 3B). Adiponectin (250 ng/ml) also significantly induced EVT invasion (+67%). Used as a positive control, 10% FCS induces a +175 and +150% increase in HTR-8/SVneo and EVT invasion respectively.
Effects of adiponectin on MMP2 and MMP9 activities
Since MMP2 and MMP9 are the major metalloproteinases involved in trophoblast invasion, we examined their enzyme activities by gelatin zymography in the conditioned media from first trimester human placental explants and from human primary EVTs. We found that MMP9 and MMP2 activities were increased (approximately twofold) in the presence of both adiponectin concentrations (25 and 250 ng/ml) for 48 h in placental explants compared to control (Fig. 4A and B). Similarly, 250 ng/ml adiponectin significantly increased (∼1.5-fold) MMP activities in primary EVTs (Fig. 4C). Leptin was used as a positive control for MMP activities (Castellucci et al. 2000, Gonzalez et al. 2001).
Effects of adiponectin on MMP activities. Activities of gelatinases in conditioned media from human placental explants (A and B) and human EVTs (C) were measured as described in Materials and Methods. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) or leptin (250 ng/ml) for 48 h. (A and C) Bars represent mean values±s.e.m. of eight to ten separate experiments. *P<0.05, **P<0.005, ***P<0.0005, Wilcoxon test. (B) Representative experiments are shown.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effects of adiponectin on MMP activities. Activities of gelatinases in conditioned media from human placental explants (A and B) and human EVTs (C) were measured as described in Materials and Methods. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) or leptin (250 ng/ml) for 48 h. (A and C) Bars represent mean values±s.e.m. of eight to ten separate experiments. *P<0.05, **P<0.005, ***P<0.0005, Wilcoxon test. (B) Representative experiments are shown.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effects of adiponectin on MMP activities. Activities of gelatinases in conditioned media from human placental explants (A and B) and human EVTs (C) were measured as described in Materials and Methods. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) or leptin (250 ng/ml) for 48 h. (A and C) Bars represent mean values±s.e.m. of eight to ten separate experiments. *P<0.05, **P<0.005, ***P<0.0005, Wilcoxon test. (B) Representative experiments are shown.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effects of adiponectin on TIMP1 and TIMP2 mRNA expressions
We also measured the mRNA expression of MMP inhibitors including TIMP1 and TIMP2 in HTR-8/SVneo cells and human primary EVTs. In both cell types, treatment with adiponectin (25 and/or 250 ng/ml) for 24 h had no effect on TIMP1 mRNA expression compared to control condition (Fig. 5A and B). In contrast, TIMP2 mRNA expression decreased (−25%) in the presence of the same concentrations of adiponectin in HTR-8/SVneo cells and human primary EVTs (Fig. 5A and B).
Effects of adiponectin on TIMP1 and TIMP2 mRNA expressions. Total RNA was extracted from HTR-8/SVneo cells (A) and human primary EVTs (B), and analyzed by RT-PCR with primers as described in Table 1. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) for 24 h. Results are the mean values±s.e.m. of seven separate experiments. *P<0.05, NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effects of adiponectin on TIMP1 and TIMP2 mRNA expressions. Total RNA was extracted from HTR-8/SVneo cells (A) and human primary EVTs (B), and analyzed by RT-PCR with primers as described in Table 1. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) for 24 h. Results are the mean values±s.e.m. of seven separate experiments. *P<0.05, NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Effects of adiponectin on TIMP1 and TIMP2 mRNA expressions. Total RNA was extracted from HTR-8/SVneo cells (A) and human primary EVTs (B), and analyzed by RT-PCR with primers as described in Table 1. Cells were incubated in the presence or absence of adiponectin (25 or 250 ng/ml) for 24 h. Results are the mean values±s.e.m. of seven separate experiments. *P<0.05, NS, non-significant, Wilcoxon test.
Citation: Journal of Endocrinology 207, 1; 10.1677/JOE-10-0170
Discussion
Human pregnancy is associated with extensive growth and remodeling of the uterus and placenta. A successful human pregnancy requires cytotrophoblasts from the fetal portion of the placenta to adopt tumor-like properties. Cytotrophoblasts attach the conceptus to the endometrium by invading the uterus, and they initiate blood flow to the placenta by breaching maternal vessels. So, migration and invasion of cytotrophoblasts into the maternal endometrium are key events in human placentation. However, unlike tumor metastasis, cytotrophoblast invasion is highly spatio-temporally regulated (Bischof et al. 2000). Trophoblast infiltration of maternal decidua and spiral arteries is regulated by a fine balance between the production of multiple MMP1–MMP16 (gelatinases, collagenases, and stromelysins), their physiological activators (e.g. plasmin), and their inhibitors (TIMP1 and TIMP2; Cohen & Bischof 2007). Many factors have been shown to regulate invasive capacities of EVTs. Some cytokines, such as IL6, IL10, IL11, leukemia inhibitory factor, epidermal growth factor, angiotensin II, and leptin, have recently been described to modulate synthesis and release of one or more of these MMPs and TIMPs (Castellucci et al. 2000, Gonzalez et al. 2001, Qiu et al. 2004, Araki-Taguchi et al. 2008, Fitzgerald et al. 2008, Pang et al. 2008). Adiponectin is another cytokine that seemed to play an important role in embryo implantation (Caminos et al. 2005, Takemura et al. 2006b, Benaitreau et al. 2009, McDonald & Wolfe 2009). The presence of adiponectin at the feto-maternal interface originates firstly to human endometrium. Indeed, we and others (Takemura et al. 2006b) observed that adiponectin is expressed (mRNA and protein levels) in human endometrium. This local expression of adiponectin by the endometrium, which expresses mainly AdipoR during the embryo implantation period, is important for adiponectin effects (Takemura et al. 2006b). Secondly, adiponectin at the feto-maternal interface originates from maternal blood. Adiponectin is thus found in this interface very early when remodeling of spiral arteries operates. Finally, adiponectin presence at the feto-maternal barrier is relayed by the production and secretion by fetal tissues (Corbetta et al. 2005).
In this paper, we confirm our previous results demonstrating AdipoR1 and AdipoR2 expression in human first trimester placenta (Benaitreau et al. 2009) and demonstrate, for the first time, their expression in HTR-8/SVneo cells. Once receptors were characterized, we attempted to shed light on the role of adiponectin in trophoblast migration and invasion. Wound healing assays of HTR-8/SVneo cells reveal that adiponectin promotes trophoblast motility in a dose-independent manner. Several studies have also shown no dose–response effect to adiponectin in other cell types (Berner et al. 2004, Dos Santos et al. 2008, Benaitreau et al. 2009). At least one hypothesis could explain such observation. Adiponectin is indeed able to downregulate its own receptors, AdipoR1 and AdipoR2. These downregulations have already been observed in different tissues and cell lines (adipose tissue, prostate cancer cell lines, and MDA-MB 231 mammary cancer cells) (Bauche et al. 2006, Dos Santos et al. 2008) and more particularly in human placenta (Caminos et al. 2005, Mistry et al. 2006). In trophoblastic cell line BeWo, we recently described that adiponectin inhibited AdipoR mRNA expression at the low concentration of 25 ng/ml (Benaitreau et al. 2010). So, it cannot be excluded that the absence of additional adiponectin action on cell migration at concentrations >25 ng/ml is related to the rapid downregulation of AdipoR by adiponectin itself.
Moreover, our results demonstrate that adiponectin does not affect HTR-8/SVneo cell proliferation suggesting that the increase in migration cannot be explained by an elevated mitogenic response. These results are consistent with previous studies performed in prostate cancer (Tang & Lu 2009), in endothelial progenitor (Nakamura et al. 2009), or in chondrosarcoma cells (Chiu et al. 2009), which showed a positive adiponectin effect on cell motility. In these different cell types, adiponectin modulates cell migration by regulating expression of integrins such as α5β1 and α2β1. Further experiments will be needed in order to establish whether adiponectin also modulates integrin expression in human trophoblast cells. In our experiments, adiponectin effects were observed with concentrations (25–500 ng/ml) much lower than those found in normal human circulation or in cord blood during gestation (∼20 μg/ml; Kajantie et al. 2004, Corbetta et al. 2005). These discrepancies can be explained as follows: i) the human recombinant adiponectin used in the present study contains mainly the high molecular weight adiponectin form, which is known to be the active form of adiponectin (Kobayashi et al. 2004); ii) this human recombinant adiponectin was tested in a serum-free culture medium to avoid interactions with plasma proteins; iii) adiponectin could exhibit proliferative and pro-inflammatory effects at subphysiological concentrations in various cell types (Berner et al. 2004, Brakenhielm et al. 2004, Lappas et al. 2005, Bub et al. 2006, Benaitreau et al. 2009).
Since trophoblast migration is generally associated with trophoblast invasion, we studied adiponectin action on invasive capacities of HTR-8/SVneo cells. According to transwell assays, our results clearly demonstrate that adiponectin acts as a pro-invasive factor in trophoblasts. These effects were accompanied by activation of gelatinase activities (MMP2 and MMP9) and repression of TIMP2 with no changes in TIMP1 levels. Similar results were obtained in human EVTs suggesting that MMP/TIMP balance is a part of the mechanism required for adiponectin to induce trophoblast migration. These results are substantiated by two studies that have shown that adiponectin could modulate MMP and/or TIMP expressions in human chondrocytes and macrophages (Kumada et al. 2004).
To gain insights into the molecular mechanisms of adiponectin-induced trophoblast invasion, activation of the major signal transduction pathway, AMPK (Kadowaki & Yamauchi 2005), was investigated in HTR-8/SVneo cells. Transwell analysis revealed that AICAR, a specific AMPK activator, surprisingly did not affect HTR-8/SVneo cell invasion capacities. These results suggest that adiponectin modulates trophoblast invasion via other transduction pathways. We recently demonstrated that in two human trophoblastic cell lines JEG-3 and BeWo, adiponectin anti-proliferative actions seem to be mediated at least in part by the MAPK and PI3K signaling pathways (Benaitreau et al. 2009). Experiments are currently in progress in our laboratory using different selective inhibitors of each pathway in order to understand the signaling pathways implicated in adiponectin effects on human trophoblast invasion.
Impaired placental development due to reduced trophoblast invasion is associated with PE and intra-uterine growth restriction. Altered plasma adiponectin concentrations have been described in women with PE (Takemura et al. 2006a, Nien et al. 2007, Fasshauer et al. 2008, Herse et al. 2009) reinforcing its possible implication on the control of trophoblast invasion.
In conclusion, our data showed that adiponectin stimulated EVT migration and invasion with increased MMP2–MMP9 activities and reduced TIMP2 expression. This study suggests that adiponectin would play a favorable role in the early steps of human implantation.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Acknowledgements
The authors gratefully acknowledge T Fournier, PhD, for his helpful advice in conducting the EVT cell culture assays.
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