Molecular Human Reproduction, Vol.22, No.2 pp. 119–129, 2016 Advanced Access publication on December 11, 2015 doi:10.1093/molehr/gav070

ORIGINAL RESEARCH

GnRH regulates trophoblast invasion via RUNX2-mediated MMP2/9 expression Bo Peng1, Hua Zhu 1, Christian Klausen 1, Liyang Ma2, Yan-ling Wang 2, and Peter C.K. Leung 1,* 1 2

Department of Obstetrics & Gynaecology, Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China

*Correspondence address. Department of Obstetrics & Gynaecology, University of British Columbia, Room C420-4500 Oak Street, Vancouver, BC V6H 3N1, Canada. Tel: +1-604-875-3121; Fax: +1-604-875-2717; E-mail: [email protected]

Submitted on October 29, 2014; resubmitted on November 1, 2015; accepted on November 9, 2015

study hypothesis: We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-releasing hormone (GnRH)-induced human extravillous trophoblast (EVT) cell invasion.

study finding: Our finding indicates that GnRH-induced RUNX2 expression enhances the invasive capacity of EVT cells by modulating the expression of MMP2 and MMP9.

what is known already: GnRH is expressed in first-trimester placenta and exerts pro-invasive effects on EVT cells in vitro. RUNX2 regulates MMP2 and MMP9 expression and is often associated with invasive phenotypes.

study design, samples/materials, methods: First-trimester human placenta (n ¼ 9) was obtained from women undergoing elective termination of pregnancy. The localization of RUNX2, MMP2 and MMP9 in first-trimester human placenta was examined by immunohistochemistry. Primary or immortalized (HTR-8/SVneo) EVT cells were treated alone or in combination with GnRH, GnRH antagonist Antide, MAPK kinase inhibitor PD98095, phosphatidylinositol 3-kinase inhibitor LY294002, MMP2/9 inhibitor or small interfering RNAs (siRNAs) targeting RUNX2, MMP2 and/or MMP9. Protein and mRNA levels were measured by western blot and RT –PCR, respectively. Cell invasiveness was evaluated by transwell Matrigel or collagen I invasion assays.

main results and the role of chance: RUNX2, MMP2 and MMP9 were detected in the cell column regions of human firsttrimester placental villi. GnRH treatment increased RUNX2 mRNA and protein levels in HTR-8/SVneo cells and primary EVTs, and these effects were attenuated by co-treatment with Antide, PD98095 or LY294002. Down-regulation of RUNX2 by siRNA reduced basal and GnRH-induced MMP2/9 expression and cell invasion. Moreover, pharmacological inhibition or siRNA-mediated knockdown of MMP2/9 reduced basal and GnRH-induced cell invasion. limitations, reasons for caution: The lack of an in vivo model is the major limitation of our in vitro study. wider implications of the findings: Our findings provide important insight into the functions of the GnRH - GnRH receptor system in early implantation and placentation.

large scale data: Not applicable. study funding and competing interest(s): This research was supported by Canadian Institutes of Health Research (Grant #143317) to P.C.K.L. The authors have nothing to disclose. Key words: GnRH / trophoblast / RUNX2 / MMP / invasion

Introduction Extracellular matrix (ECM) components undergo dynamic changes in the first trimester of human pregnancy. Elevated expression of collagen types I, III, and V, as well as de novo expression of collagen IV has been

detected in first-trimester human decidua (Kisalus et al., 1987; Aplin et al., 1988). Degrading such a complex ECM requires the presence of multiple matrix metalloproteinases (MMPs), and almost all MMP family members are detected in first-trimester human placenta (Weiss et al., 2007). MMP2 and MMP9 are abundantly expressed in invading

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120 extravillous cytotrophoblast (EVT) cells (Bai et al., 2005), and the expression of these two gelatinases is highly related to trophoblast cell invasiveness (Suman and Gupta, 2012). Indeed, decreased levels of MMP9 have been reported in preeclamptic placenta as compared with normal placenta (Shokry et al., 2009; Omran et al., 2011), which could be related to impaired trophoblast invasion in pre-eclampsia. Additionally, trophoblastic MMP2 and MMP9 expression levels decline during the second and third trimesters, paralleling the time frame of reduced trophoblast invasion in late or term placentas (Shimonovitz et al., 1994). The temporal and spatial expression of MMP2 and MMP9 in EVTs is regulated by multiple autocrine and paracrine factors. Numerous hormones, cytokines and growth factors, such as human chorionic gonadotrophin, epidermal growth factor, and interleukin-10, have been reported to either up- or down-regulate MMP2 and MMP9 in trophoblast cells (Roth and Fisher, 1999; Fluhr et al., 2008; Dilly et al., 2010). In addition, the gonadotrophin-releasing hormone (GnRH)-GnRH receptor (GnRHR) system is present in first-trimester human placenta (Lin et al., 1995; Cheng et al., 2000; Chou et al., 2004), and GnRH has been shown to regulate MMP2 and MMP9 in human EVT cells (Chou et al., 2003). GnRH appears to exert these effects via transactivation of epidermal growth factor receptor (Liu et al., 2009), however the downstream transcription factor(s) involved have not been studied. Runt-related transcription factor 2 (RUNX2) is one of three related transcription factors that control many biological functions in organ development, tissue differentiation and cell fate (Komori, 2002, 2008). RUNX2 was first identified as a regulator of chondrocyte maturation and osteoblast differentiation (Prince et al., 2001). Interestingly, the effects of RUNX2 on chondrocyte and osteoblast differentiation were shown to be coupled with osteoblast cell migration (Fujita et al., 2004). Moreover, RUNX2 has been studied in cancer biology because of its important role in controlling cancer cell migration and invasion (Pratap et al., 2006). In breast cancer cells, RUNX2 can regulate MMP2/9 gene expression, by which it regulates cancer cell invasion (Pratap et al., 2005). To date, the expression of RUNX2 has not been reported in firsttrimester placenta, and the relevance of RUNX2 to trophoblast cell MMP expression and invasiveness is unknown. In this study, we examined the expression of RUNX2 in first-trimester human placenta and trophoblastic cells. We also studied the role of RUNX2 in regulating GnRH-induced MMP2/9 expression as well as trophoblastic cell invasion.

Materials and Methods Reagents and antibodies Native human GnRH I, GnRH II, and the GnRH receptor antagonist, Antide, were purchased from Bachem (Belmont, CA). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 were obtained from Sigma (St. Louis, MO). The MMP2/9 inhibitor I ((2R)-2-[(4-Biphenylylsulfonyl)amino]-3-phenylpropionic Acid) was purchased from Millipore (Billerica, MA). Growth factor reduced Matrigel and acid-extracted rat tail collagen I were purchased from BD Biosciences (Franklin lakes, NJ). Rabbit polyclonal antibodies against mouse RUNX2 (M-70, sc-10758; also reactive with human RUNX2) and human actin (C-11, sc-1615-R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against human MMP2 (clone VB3) was obtained from Thermo Scientific

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(Waltham, MA) and rabbit polyclonal anti-MMP9 antibody (ab38898) was purchased from Abcam (Cambridge, MA). Mouse monoclonal antibodies against human cytokeratin 7 (clone OV-TL-12/30) and HLA-G (clone 4H84) were purchased from Millipore Chemicon (Billerica, MA) and Exbio (Vestec, Czech Republic), respectively. Rabbit polyclonal total AKT (9272), rabbit polyclonal phospho-AKT (Ser473; 9271), rabbit polyclonal total ERK1/2 (9102) and mouse monoclonal phospho-ERK1/2 (Thr202/ Tyr204; clone E10) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Normal rabbit control IgG (sc-2027) and mouse IgG1 kappa isotype control (clone MOPC-21) were purchased from Santa Cruz Biotechnology and Sigma, respectively.

Tissues and immunohistochemistry This study was approved by the Research Ethics Board of the University of British Columbia and all patients provided informed written consent. A total of six first-trimester human placentas (6 – 12 weeks) were obtained from women undergoing elective termination of pregnancy. Samples were fixed in 4% formaldehyde and embedded in paraffin for sectioning. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and processed for wet heat-induced antigen retrieval in a steamer for 20 min with a modified citrate buffer (pH 6.1; Dako, Burlington, ON). Sections were incubated in 3% H2O2 in phosphate-buffered saline (PBS) for 30 min at room temperature to quench endogenous peroxidase, and then blocked with serum-free protein block (Dako) for 1 h at room temperature. Sections were incubated with antibodies against RUNX2 (4 mg/ml), MMP2 (4 mg/ml) or MMP9 (4 mg/ml) overnight at 48C. Immunoreactivity was detected using the universal Dako-labeled streptavidin biotin horseradish peroxidase (HRP) system (Universal LSAB+ Kit/HRP) and 3,3′ -diaminobenzidine chromogen solution (Dako). Slides were counterstained with Harris hematoxylin (Sigma, St. Louis, MO), dehydrated through graded ethanol to xylene, mounted in a xylene-based mounting medium, and observed under a light microscope (Leica, Wetzlar, Germany).

Cell isolation and culture The method for isolation of EVT cells from first-trimester human placenta has been previously described (Chou et al., 2003). Briefly, placental tissues (6– 12 weeks, n ¼ 3) were washed three times in sterile Dulbecco’s PBS (Thermo Scientific, Waltham, MA) and chorionic villi tips were separated from stem villi and minced into fine particles before being transferred to a Falcon tube. Floating chorionic membrane debris was removed and the remaining placental villi fragments were plated in tissue culture flasks in Dulbecco’s Minimum Essential Medium (DMEM; Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). After 1 – 3 days of culture, non-adherent tissues were removed and the villous explants were cultured for another 1 – 2 weeks prior to passaging of the outgrown EVT cells with 0.25% trypsin-EDTA (Life Technologies). The purity of primary EVT cell cultures was assessed by immunocytochemical staining for cytokeratin 7 and HLA-G as demonstrated previously (Peng et al., 2015a), and was further validated by immunoblot of HLA-G in primary EVT cell lysates (Supplementary Fig. S1). Only cultures with more than 99% cytokeratin 7 and HLA-G positive EVTs were used for experiments. The HTR-8/SVneo immortalized EVT cell line was a kind gift from Dr. P.K. Lala (Western University, London, ON). BeWo, JAR and JEG-3 human choriocarcinoma cell lines were purchased from American Type Culture Collection (Manassas, VA). HTR-8/SVneo, JAR and JEG-3 cells were cultured in DMEM supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). BeWo cells were maintained in a 1:1 mixture of DMEM and Ham’s F-12K supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). All cells were maintained at 378C in a humidified atmosphere with 5% CO2.

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GnRH regulates MMP2/9 via RUNX2 HTR-8/SVneo cells (2 × 105) were seeded in 60 mm tissue culture dishes and switched to serum-free medium the next day. Cells were incubated with a fixed concentration (100 nM) of GnRH I or GnRH II for different amounts of time (0, 12, 24, 48 or 72 h). Alternatively, cells were incubated for 48 h with varying concentrations of GnRH I or GnRH II (0, 1, 10 or 100 nM). The GnRH receptor antagonist Antide was added at different concentrations (0, 1, 10 or 100 nM) 1 h prior to treatment with GnRHs (100 nM) for 48 h. For AKT and ERK phosphorylation studies, cells were incubated with GnRH I or GnRH II (100 nM) for 15 or 30 min. Primary EVT cells (2 × 105) were treated with GnRH I or GnRH II for 24 h. Culture media containing GnRH was refreshed every 12 h.

Small interfering RNA transfection ON-TARGETplus nontargeting control pool and ON-TARGETplus SMARTpool siRNAs (Thermo Scientific, Waltham, MA) targeting human RUNX2, MMP2 and MMP9 (25 nM each) were transfected into HTR-8/ SVneo cells using Lipofectaminew RNAiMAX Reagent (Life Technologies). The concentration of siRNA used for primary EVT cells was optimized to 50 nM. HTR-8/SVneo cells were transfected 24 h prior to treatment with GnRHs (100 nM).

Reverse transcription, semi-quantitative PCR and quantitative real-time PCR RNA was extracted using TRIzol reagent (Life Technologies) and 1 mg of total RNA was reverse transcribed into first-strand cDNA with a mix of oligo-dT and random primers using the Quantitect Reverse Transcription Kit with integrated genomic DNA removal (Qiagen, Mississauga, ON). Reverse transcription semi-quantitative PCR (RT – PCR) was performed using an Eppendorf Master Cycler and each 15 ml reaction contained 1× HotStarTaq Master Mix, 50 ng cDNA and 300 nM of each specific primer. The primers used were: RUNX2, 5′ -TCTGGCCTTCCACTCTCTCAGT-3′ (forward) and 5′ -GACTGGCGGGGTGTAAGTAA-3′ (reverse); and GAPDH, 5′ -ATGTTCGTCATGGGTGTGAACCA-3′ (forward) and 5′ -TGGCAGGTTTTTC TAGACGGCAG-3′ (reverse). Amplifications were performed as follows: 5 min at 958C, 27 – 30 amplification cycles (1 min at 958C, 1.5 min at 558C and 1 min at 728C) and final extension for 15 min at 728C. PCR products were analyzed by 1% agarose gel electrophoresis, stained with ethidium bromide, quantified by densitometry (GeneTools software; Syngene, Frederick, MD) and normalized to GAPDH. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed on an Applied Biosystems 7300 Real-Time PCR System equipped with 96-well optical reaction plates. Each 20 ml reaction contained 1×SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 25 ng cDNA and 300 nM of each specific primer. The amplification parameters were 508C for 2 min, 958C for 10 min and 40 cycles of 958C for 15 s and 608C for 1 min. The sequences of primers used in RT-qPCR were as follows: RUNX2, 5′ -AGCCCTCGGAGAGGTACCA-3′ (forward) and 5′ -TCATCGTTACCCGCCATGA-3′ (reverse); MMP2, 5′ -CGTCTGTCCCAGGATGACATC-3′ (forward) and 5′ -ATGTCAGGAGAGGCCCCA TA-3′ (reverse); MMP9, 5′ -CGCCAGTCCACCCTTGTG-3′ (forward) and 5′ -CAGCTGCCTGTC GGTGAGA-3′ (reverse); and GAPDH, 5′ -ATGGAAATCCCATCACCATCTT-3′ (forward) and 5′ -CGCCCCACTTGATTTTGG-3′ (reverse). The specificity of each assay was validated by dissociation curve analysis and agarose gel electrophoresis of PCR products. Assay performance was validated by evaluating amplification efficiencies by means of calibration curves, and ensuring that the plot of log input amount versus DCq has a

slope , |0.1|. Three to four separate experiments were performed on different cultures and each sample was assayed in duplicate. A mean value was used for the determination of mRNA levels by the comparative Cq method with GAPDH as the reference gene and using the formula 2 – DDCq.

Western blot analysis Cells were washed twice with cold PBS prior to being lysed with Cell Extraction Buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate; Life Technologies) supplemented with protease inhibitor cocktail (Sigma, P8340) and 1 mM phenylmethanesulfonyl fluoride (Sigma, P7626). Supernatants were collected following centrifugation at 15 000g for 15 min and protein concentrations were quantified using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts (30 mg) of protein were subjected to 8% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were immunoblotted with primary antibodies against RUNX2 (0.4 mg/ml), MMP2 (0.4 mg/ml), MMP9 (1 mg/ml), total AKT (1:1000), phospho-AKT (1:1000), total ERK1/2 (1:1000), phospho-ERK1/2 (1:1000) or actin (0.2 mg/ml) overnight at 48C. Following incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), immunoreactive bands were detected using enhanced chemiluminescence substrate (ECL, Thermo Scientific). RUNX2, MMP2 and MMP9 data were quantified by densitometry (GeneTools software) and normalized to actin. Alternatively, total AKT or ERK1/2 levels were used to normalize phospho-AKT or phospho-ERK1/2 levels, respectively.

Transwell Matrigel or rat tail collagen I invasion assays Cell culture inserts (12-well, pore size 8 mm; BD Biosciences) pre-coated with either growth factor-reduced Matrigel (0.5 mg/ml) or acid-extracted rat tail collagen I (0.1 mg/ml) were seeded with 1.5 × 104 cells suspended in 250 ml DMEM with 0.1% FBS. DMEM containing 10% FBS (1 ml) was added to the lower chamber and served as a chemotactic agent. Inserts were cultured for 24 h after which non-invading cells were wiped from the upper side of the membrane and cells on the lower side were fixed and stained using the hematoxylin quick stain system (Sigma Aldrich, St. Louis, MO). Mean values from at least three experiments, each counting five microscopic fields in triplicate inserts, were used to calculate fold changes in cell number.

Statistical analysis Results were analyzed by Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Results are presented as the mean + SEM of at least three independent experiments and means considered significantly different from each other are indicated by different letters (P , 0.05).

Results RUNX2, MMP2, and MMP9 are co-expressed in cell columns of first-trimester human placenta We first sought to detect RUNX2, MMP2 and MMP9 at sites of invasion in first-trimester human placenta. Abundant RUNX2 immunoreactivity was detected in the nucleus of both cell column EVT and cytotrophoblast cell populations (Fig. 1A, a and b). MMP2 immunoreactivity was high in the distal end of the cell column approaching the invasive

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Figure 1 Expression of RUNX2, MMP2 and MMP9 in first-trimester human placenta and trophoblastic cells. (A) Representative images showing the immunolocalization of RUNX2 (a and b), MMP2 (c and d) and MMP9 (e and f) in first-trimester human placenta (n ¼ 6). A lack of staining was observed in adjacent control sections incubated with rabbit control (Ctrl) IgG (g) or mouse Ctrl IgG1k (h). The cell column region with EVT cells (Column EVT) in Ctrl IgG is indicated with dash lines (g). 100× scale bar ¼ 200 mM, 400× scale bar ¼ 50 mM. (B) Semi-quantitative RT– PCR and western blot analysis were used to examine the expression of RUNX2 in primary extravillous cytotrophoblast (EVT) cells, immortalized EVT cells (HTR-8/SVneo) and choriocarcinoma cell lines (JAR, JEG-3 and BeWo). GAPDH and actin were used to normalize RT– PCR and western blot results, respectively.

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front, but not in the villous cytotrophoblast (Fig. 1A, c and d). MMP9 immunoreactivity was evident in the cell column and was universally observed in the cytotrophoblast cell population (Fig. 1A, e and f). No immunoreactivity was observed after incubation of placenta sections with rabbit Ctrl IgG (Fig. 1A, g) and mouse Ctrl IgG1k (Fig. 1A, h).

RUNX2 expression is associated with cell invasiveness in trophoblastic cells Having demonstrated the localization of RUNX2 in first-trimester placenta, we next used RT–PCR and western blot analysis to examine the mRNA and protein levels of RUNX2 in primary and immortalized EVTs as well as choriocarcinoma cell lines. Highly invasive primary and immortalized (HTR-8/SVneo) EVT cells displayed higher RUNX2 mRNA and protein levels compared with less invasive choriocarcinoma cells (JAR, JEG-3 and BeWo; Fig. 1B).

GnRH stimulates RUNX2 expression in EVT cells Next, we used RT-qPCR and western blot analysis to investigate the effects of GnRH on RUNX2 expression in HTR-8/SVneo and primary EVT cells. HTR-8/SVneo cells were treated with GnRH I or GnRH II at different concentrations (1, 10 and 100 nM), and only the 100 nM concentration was able to significantly increase RUNX2 mRNA and protein levels at 24 and 48 h, respectively (Fig. 2A and B). Treatment of HTR-8/ SVneo cells with 100 nM GnRH I or GnRH II significantly elevated RUNX2 mRNA levels at 24, 48 and 72 h compared with time-matched controls (Fig. 2C) without altering the mRNA levels of a housekeeping gene, GAPDH (Supplementary Fig. S2). RUNX2 protein levels were increased in HTR-8/SVneo cells between 24 and 72 h following treatment with GnRH (Fig. 2D). Consistent with these findings, treatment of primary EVT cells for 24 h with 100 nM GnRH I or GnRH II significantly increased RUNX2 mRNA and protein levels (Fig. 2E and F). The GnRH receptor antagonist Antide was used to confirm the role of the GnRH receptor in mediating GnRH-induced RUNX2 expression.

Figure 2 GnRH induces RUNX2 expression in primary and immortalized extravillous cytotrophoblast (EVT) cells. (A and B) HTR-8/SVneo immortalized EVT cells were treated with vehicle control (Ctrl) or different concentrations (1, 10 or 100 nM) of GnRH and RUNX2 mRNA (A, 24 h) and protein (B, 48 h) levels were examined by RT-qPCR and western blot, respectively. (C and D) HTR-8/SVneo cells were treated with 100 nM of GnRH I or GnRH II for 0, 12, 24, 48 or 72 h, after which RUNX2 mRNA (C) and protein (D) levels were examined by RT-qPCR and western blot, respectively. (E and F) Primary EVT cells were treated with 100 nM GnRH I or GnRH II for 24 h and RUNX2 mRNA (E) and protein (F) levels were examined by RT-qPCR and western blot, respectively. GAPDH and actin were used to normalize RT– PCR and western blot results, respectively. Results are presented as the mean + SEM of three independent experiments, and values without a common letter are significantly different (n ¼ 3, P , 0.05, ANOVA followed by Tukey’s test).

124 Different concentrations of Antide (1, 10 or 100 nM) were added prior to treatment with a fixed concentration of GnRH (100 nM). Pretreatment of HTR-8/SVneo cells with 100 nM Antide abolished GnRH Iand GnRH II-induced RUNX2 mRNA (Fig. 3A) and protein (Fig. 3B) levels.

AKT and ERK1/2 signaling mediates GnRH-induced RUNX2 expression

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phosphorylated AKT and ERK1/2 at both time points (Fig. 4A). Co-treatment with the PI3K inhibitor LY294002 or the MEK inhibitor PD98059 was used to investigate the involvement of AKT and ERK1/2 signaling in GnRH-induced RUNX2 expression. Pretreatment of HTR-8/SVneo cells for 1 h with either LY294002 or PD98059 attenuated both GnRH I- and GnRH II-induced RUNX2 protein production (Fig. 4B). Interestingly, basal RUNX2 protein levels were also significantly decreased by treatment with either LY294002 or PD98059 alone.

To examine the intracellular signaling mediating the effects of GnRH, we incubated HTR-8/SVneo cells with 100 nM GnRH for 15 or 30 min. Treatment with GnRH I or GnRH II increased the levels of

Figure 4 PI3K/AKT and MEK/ERK1/2 signaling mediates

Figure 3 The GnRHR antagonist Antide attenuates GnRH-induced RUNX2 expression. RUNX2 mRNA (A) and protein (B) levels were examined in HTR-8/SVneo cells following treatment for 48 h with or without 100 nM GnRH I or GnRH II in the absence or presence of different concentrations of Antide (1, 10 or 100 nM). GAPDH and actin were used to normalize RT– PCR and western blot results, respectively. Results are presented as the mean + SEM of three independent experiments, and values without a common letter are significantly different (n ¼ 3, P , 0.05, ANOVA followed by Tukey’s test).

GnRH-induced RUNX2 expression. (A) Western blot analysis was used to examine AKT and ERK1/2 phosphorylation in HTR-8/SVneo cells following treatment with 100 nM GnRH I or GnRH II for 15 or 30 min. Total AKT or ERK1/2 levels were used to normalize phospho-AKT (pAKT) or phospho-ERK1/2 (pERK1/2) levels, respectively. Quantified results from three independent experiments are presented numerically as the mean fold change, and values without a common letter are significantly different (n ¼ 3, P , 0.05, ANOVA followed by Tukey’s test). (B) Alternatively, western blot analysis was used to measure RUNX2 protein levels (normalized to actin) in HTR-8/ SVneo cells following treatment for 48 h with 100 nM GnRH I or GnRH II in the absence or presence of a PI3K inhibitor (10 mM LY294002) or a MEK inhibitor (10 mM PD98059). Results are presented as the mean + SEM of three independent experiments, and values without a common letter are significantly different (n ¼ 3, P , 0.05, ANOVA followed by Tukey’s test).

GnRH regulates MMP2/9 via RUNX2

RUNX2 regulates MMP2 and MMP9 expression and cell invasion in EVT cells RUNX2 has been shown to regulate MMP2 and MMP9 expression in prostate cancer cells (Akech et al., 2010). Thus, siRNA-mediated downregulation of endogenous RUNX2 was performed to investigate its effects on MMP2 and MMP9 expression in HTR-8/SVneo and primary EVT cells. Knockdown of RUNX2 significantly attenuated the mRNA and protein levels of RUNX2 in HTR-8/SVneo cells (Fig. 5A), without altering the mRNA levels of other runt related transcription family members RUNX1 and RUNX3 (Supplementary Fig. S3), and was associated with significant reductions in the mRNA levels of both MMP2 and MMP9 (Fig. 5B). Likewise, depletion of RUNX2 in HTR-8/SVneo and primary EVT cells suppressed pro-MMP2 levels as well as pro- and active forms of MMP9 (Fig. 5C and D). Importantly, knockdown of endogenous RUNX2 attenuated transwell Matrigel invasion of HTR-8/ SVneo and primary EVT cells (Fig. 5E and F), without altering the overall viability of these cells (Supplementary Fig. S4). To confirm the involvement of MMP2 and MMP9 in HTR-8/SVneo cell invasion, we performed transient knockdown of MMP2 or MMP9 prior to assaying transwell Matrigel or collagen I invasion, and RT-qPCR and western blot were performed to examine the knockdown efficiency (Supplementary Fig. S5). Significant reductions in invasive cell numbers were observed following depletion of MMP2 or MMP9 in both the Matrigel-coated (Fig. 5G) or rat tail collagen I-coated transwell assays (Fig. 5H).

RUNX2-induced MMP2 and MMP9 expression mediates GnRH-induced HTR-8/SVneo cell invasion Having demonstrated the involvement of RUNX2 in basal MMP2/9 expression and trophoblast invasion, we next sought to test the role of RUNX2 in GnRH-induced cell invasion. Knockdown of RUNX2 in HTR-8/SVneo cells completely abolished the GnRH-induced increases in MMP2 and MMP9 protein levels (Fig. 6A). Importantly, treatment with RUNX2 siRNA also significantly reduced the GnRH-stimulated Matrigel invasion of HTR-8/SVneo cells (Fig. 6B). In addition, siRNA-mediated knockdown and MMP2/9 inhibitor I were used to examine the involvement of MMP2 and MMP9 in GnRH I- and GnRH II-induced HTR-8/SVneo cell invasion. Co-transfection with MMP2 and MMP9 siRNA significantly inhibited GnRH-induced HTR-8/SVneo cell invasion (Fig. 6C). Similarly, co-treatment with MMP2/9 inhibitor I attenuated GnRH-induced HTR-8/SVneo cell invasion (Fig. 6D).

Discussion In addition to its well-known expression and function in osteoblasts (Komori et al., 1997; Tsuji et al., 1998), RUNX2 expression has also been demonstrated in reproductive tissues, such as testis (Jeong et al., 2008) and ovary (Park et al., 2010). Here we demonstrate for the first time that RUNX2 is expressed in the human placenta, particularly in villous cytotrophoblast and EVT cell populations. These findings are in agreement with a report that Runx2 is expressed in mouse placenta (Blyth et al., 2010); however the specific cell types involved and functional significance were not investigated. Nevertheless, the presence of RUNX2

125 in both mouse and human placenta strongly suggests an important role for RUNX2 in placental development and function. Our immunostaining results demonstrate that RUNX2 and MMP2/9 are co-expressed in first-trimester column EVT cells. Interestingly, we have also detected abundant GnRH receptor protein in column EVT cells (Peng et al., 2015a). In human placenta, peak levels of MMP2 and MMP9 are detected at 6–12 weeks gestational age (Staun-Ram et al., 2004), paralleling the elevated expression of GnRH (Siler-Khodr and Khodr, 1978) and GnRH receptor (Lin et al., 1995) in first trimester. Abundant expression of GnRH-GnRH receptor, RUNX2 and MMP2/ 9 in column EVT cells is spatiotemporally correlated with the highly invasive behavior of these cells during the first trimester of human pregnancy. The present results provide the first characterization of the pro-invasive effects of RUNX2 in human EVT cells. Together with previous studies demonstrating their pro-invasive effects in a variety of normal and malignant cells (Masson et al., 2005; Pratap et al., 2006), co-expression of RUNX2, MMP2 and MMP9 in column EVT cells reflects trophoblast differentiation towards an invasive phenotype. Interestingly, positive immunostaining for MMP2 was only observed in the distal region of the cell column, whereas MMP9 immunoreactivity was detected in both villous cytotrophoblasts and column EVTs. These findings are consistent with previous studies (Isaka et al., 2003), and suggest divergent roles for MMP2 and MMP9 in placental biology, particularly with respect to villous cytotrophoblasts. Moreover, RUNX2 appears to be co-expressed with MMP9 but not MMP2 in villous trophoblastic cells, suggesting RUNX2 alone may not be sufficient to induce MMP2 expression in villous cytotrophoblasts. In LNCaP prostate cancer cells, over-expression of RUNX2 was sufficient to induce MMP9 expression, but MMP2 levels remained low in these cells (Akech et al., 2010). Thus, additional transcriptional regulators may cooperate with RUNX2 to control gene expression in both a gene- and cell type-specific manner. For example, interactions between the AP-1 transcription factor c-Fos and RUNX2 are required for transcriptional activation of MMP13 in osteosarcoma cells (Porte et al., 1999). Interestingly, c-Fos has been detected in EVT cells but not in villous cytotrophoblasts (Bamberger et al., 2004; Peng et al., 2015b). Thus, the absence of c-Fos in villous cytotrophoblasts may explain the lack of MMP2 expression in these cells, especially considering the AP-1 binding site in the human MMP2 promoter is critical to its transcriptional activation (Song et al., 2006). Future studies investigating the transcriptional regulation of MMP2 by RUNX2, alone or in combination with other transcriptional regulators (e.g. AP-1), would be of great interest. We have also established that knockdown of RUNX2 reduces the mRNA and protein levels of both MMP2 and MMP9 in EVT cells. Silencing of endogenous RUNX2 has also been reported to down-regulate MMP2 and MMP9 in other cell types, such as prostate and thyroid cancer cells (Pratap et al., 2005; Niu et al., 2012). Multiple RUNX2 binding sites have been identified in the MMP9 promoter region (approximately 200–800 bp upstream of the transcription starting site) and chromatin immunoprecipitation analysis has confirmed that RUNX2 directly associates with the MMP9 promoter (Pratap et al., 2005), suggesting that RUNX2 may regulate MMP9 via direct promoter binding. The expression of RUNX2 in villous cytotrophoblasts suggests that it may also play a role in villous cytotrophoblast behavior. Cell proliferation and differentiation towards either EVTs or syncytiotrophoblast are the major roles of villous cytotrophoblasts. RUNX2 has welldescribed roles in osteogenic cell differentiation (Komori et al., 1997;

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Figure 5 Knockdown of RUNX2 reduces EVT cell MMP2 and MMP9 expression and cell invasion. (A– F) HTR-8/SVneo or primary EVT cells were transfected for 24 or 48 h with transfection reagent (Vehicle Ctrl), control siRNA (Ctrl siRNA) or RUNX2 siRNA (siRUNX2). RT-qPCR with GAPDH as the reference gene was used to measure the mRNA levels of RUNX2 (A, left panel) MMP2 (B, upper panel) and MMP9 (B, lower panel) in transfected HTR-8/SVneo cells. Western blot analysis was used to measure RUNX2 (A, right panel), pro-MMP2 and pro-/active-MMP9 protein levels (normalized to actin and numerically presented as the mean fold change) in HTR-8/SVneo (C) or primary EVT (D) cells following transfection for 48 h. HTR-8/SVneo (E) or primary EVT (F) cells were transfected with siRNA for 48 h and cell invasiveness was examined by Matrigel-coated transwell assay. (G and H) HTR-8/ SVneo cells were transfected for 48 h with transfection reagent, control siRNA, MMP2 siRNA (siMMP2) or MMP9 siRNA (siMMP9), and cell invasiveness was examined by Matrigel-coated (G) or collagen I-coated (H) transwell assays. Results are presented as the mean + SEM of three independent experiments, and values without a common letter are significantly different (n ¼ 3, P , 0.05, Student’s t-test (A, C, D and F) or ANOVA followed by Tukey’s test (B, E, G and H)).

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Figure 6 Loss of function of RUNX2 or MMP2/9 attenuates GnRH-induced HTR-8/SVneo cell invasion. Cells were transfected for 24 h with control siRNA (Ctrl siRNA), RUNX2 siRNA (siRUNX2) or combined MMP2 and MMP9 siRNAs (siMMP2 siMMP9). (A) Transfected cells were treated for an additional 48 h with 100 nM GnRH I or GnRH II, and western blot analysis was used to measure pro-MMP2 and pro-/active-MMP9 protein levels (normalized to actin and numerically presented as the mean fold change). (B and C) Alternatively, transfected cells were treated for an additional 24 h with 100 nM GnRH prior to examining cell invasiveness by Matrigel-coated transwell assay. (D) Cells were treated for 24 h with 100 nM GnRH after which the MMP2/9 inhibitor was added for 1 h prior to seeding the cells in Matrigel-coated transwell inserts. Results are presented as the mean + SEM of three independent experiments, and values without a common letter are significantly different (n ¼ 3, P , 0.05, ANOVA followed by Tukey’s test).

Prince et al., 2001), and has also been reported to modulate cell proliferation and cell cycle in both osteoblasts (Pratap et al., 2003) and human osteosarcoma cells (Lucero et al., 2013). Knockdown of

RUNX2 in HTR-8/SVneo cells did not affect total cell number as assessed by Trypan blue cell counting assay (Supplementary Fig. S4). However, whether or not RUNX2 can regulate cell differentiation or

128 proliferation/apoptosis in villous cytotrophoblasts requires further investigation. The present study is the first to demonstrate the induction of RUNX2 by GnRH in any system. The up-regulation of RUNX2 by GnRH involves MEK/ERK and PI3K/AKT signaling, both of which are known to be activated by GnRH. Previous studies have demonstrated that MEK/ERK and PI3K/AKT signaling are essential for the induction of RUNX2 expression by growth factors, mechanical sensation or oxidative stress (Byon et al., 2008; Shi et al., 2011; Niger et al., 2012). However, there is also evidence for the differential involvement of ERK and AKT signaling in IGF-induced RUNX2 expression in endothelial cells (Qiao et al., 2004). Interestingly, RUNX2 expression has been demonstrated in pituitary cells (Breen et al., 2010), though whether or not it is regulated by GnRH in the pituitary has yet to be investigated. In summary, our studies describe, for the first time, the expression and localization of RUNX2 in first-trimester human placenta, as well as its functional impact on trophoblast invasion. In addition, we show that RUNX2 mediates GnRH-induced trophoblast invasion by modulating the expression of MMP2 and MMP9.

Supplementary data Supplementary data are available at http://molehr.oxfordjournals.org/.

Authors’ roles B.P., C.K., Y.L.W. and P.C.K.L. designed this study. B.P., H.Z. and L.Y.M. carried out the experiments. B.P. analyzed the data. B.P. and C.K. prepared the manuscript. All authors criticized and reviewed the manuscript.

Funding This research was supported by Canadian Institutes of Health Research (Grant #143317) to P.C.K.L.

Conflict of interest None declared.

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9 expression.

We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-rel...
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