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. 2019 Jan 26;13(4):503–521. doi: 10.1007/s12079-019-00505-x

HGF regulate HTR-8/SVneo trophoblastic cells migration/invasion under hypoxic conditions through increased HIF-1α expression via MAPK and PI3K pathways

Piyush Chaudhary 1,2, Gosipatala Sunil Babu 2, Ranbir Chander Sobti 3, Satish Kumar Gupta 1,✉
PMCID: PMC6946778  PMID: 30684191

Abstract

Hepatocyte growth factor (HGF) is reported to be down-regulated in pregnancy complications like intrauterine growth retardation and preeclampsia, which are associated with abnormal trophoblast migration/invasion. In this study, role of HGF and associated signaling pathways has been investigated in HTR-8/SVneo trophoblastic cells migration/invasion under normoxia (20% O2) and hypoxia (2% O2). HTR-8/SVneo cells exposed to hypoxia showed increase in migration and invasion as compared to cells incubated under normoxic conditions. The migration/invasion under both normoxic and hypoxic conditions was further enhanced after treatment with HGF. Subsequent to treatment with HGF, a significant increase in expression of MMP2 & MMP3 under normoxia and MMP1 & MMP9 under hypoxia was observed. Treatment of HTR-8/SVneo cells with HGF under hypoxia also led to decrease in TIMP1. Treatment of the cells with HGF led to activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. Inhibition of MAPK by U0126 and PI3K by LY294002 led to concomitant decrease in the HGF-mediated migration/invasion of HTR-8/SVneo cells. HGF treatment under hypoxia also led to a significant increase in hypoxia inducible factor (HIF-1α) expression. Additionally, inhibition of HIF-1α by siRNA led to decrease in HGF-mediated migration of HTR-8/SVneo cells under hypoxic conditions. Inhibition of HGF activated MAPK and PI3K signaling led to reduction in HIF-1α expression under hypoxia. In conclusion, HGF facilitates HTR-8/SVneo cell migration/invasion by activation of MAPK/PI3K signaling pathways and increased expression of MMPs. HIF-1α has a role in HGF-mediated increase in migration under hypoxic conditions.

Electronic supplementary material

The online version of this article (10.1007/s12079-019-00505-x) contains supplementary material, which is available to authorized users.

Keywords: HGF, HIF-1α, Invasion, Migration, MAPK, MMPs, PI3K, TIMPs

Introduction

During first trimester of pregnancy, trophoectoderm of the blastocyst give rise to highly proliferative and undifferentiated cells known as cytotrophoblast cells (CTBs). Cytotrophoblasts differentiate to form villous cytotrophoblast that fuse to form multinucleated syncytiotrophoblast cells (STBs), which cover floating villi and extravillous trophoblast (EVTs) that proliferates to form cell column of the anchoring villi. The EVTs further differentiate into interstitial EVTs which invade the maternal decidua and reach up to the inner third of myometrium and endovascular EVTs, which replace the maternal endothelial cells and remodel the spiral arteries (Aplin 1991). The partial pressure of oxygen during this period (8-10th week of pregnancy) in intervillous space is ~20 mmHg (approximately 2–3% O2) (Rooth et al. 1961). This concentration of O2 is essential for EVT differentiation and vascular remodelling of spiral arteries (Genbacev et al. 1996; Zhou et al. 1998). During this early hypoxic phase, EVT cells plug the lumen of spiral arteries to prevent maternal blood flow in the developing placenta and thereby maintain a low O2 environment (Genbacev et al. 1997). This physiological hypoxia is essential to protect the immature embryonic tissues from oxidative stress (Burton et al. 2003). At the end of first trimester, on completion of embryogenesis, EVT plugs dissolve, subsequent to which the oxygenated blood flow into intervillous space increases and oxygen partial pressure becomes >50 mmHg (Rodesch et al. 1992; Burton et al. 1999). However, insufficient migration and invasion of EVT’s into spiral arteries and hence failure to plug maternal arteries can lead to early onset of increased maternal blood flow, which may damage villous architecture, resulting in oxidative stress (Jauniaux et al. 2003; Fisher 2015) and pregnancy disorders like intrauterine growth restriction (IUGR) and preeclampsia (Poston and Raijmakers 2004). Thus, it is important to investigate the regulatory mechanisms associated with trophoblast migration/invasion under hypoxic conditions.

Secretion of matrix metalloproteinases (MMPs), which break down extracellular matrix (ECM), increases during migration and invasion of EVT cells (Cockle et al. 2007; Zhu et al. 2012). Although, some MMPs like MMP1, MMP2 and MMP3 are primarily expressed in trophoblast cells, the production of others like MMP9 are induced by external factors like cytokines, growth factors, oxygen and pathological conditions (Staun-Ram et al. 2004; Weiss et al. 2007). For example, MMP2 expression dominates during 6-8th weeks of pregnancy, while expression of MMP9 increases from 10 - 12th weeks onwards till the end of pregnancy (Staun-Ram et al. 2004). The activities of MMPs are regulated by tissue inhibitors of matrix metalloproteinases (TIMPs) (Visse and Nagase 2003), which inhibit their activity. It is the ratio of MMPs/TIMPs which regulate the integrity of extracellular matrix and thus controls migration and invasion of the trophoblast cells in pre-term placenta (Luo et al. 2011).

In addition to oxygen tension, trophoblast migration/invasion is also regulated by several growth factors, cytokines, hormones as well as extracellular matrix proteins present at the maternal-fetal interface. Hepatocyte growth factor (HGF), a pleotropic growth factor secreted by syncytiotrophoblast, extravillous trophoblast, mesenchymal and endothelial cells, plays an important role in placental growth and development (Wolf et al. 1991). Low levels of placental HGF production is reported in preeclampsia and IUGR patients (Somerset et al. 1998). HGF binds to C-met, a transmembrane glycoprotein with tyrosine kinase activity, mainly expressed on trophoblast cells (Somerset et al. 1998). During normal physiological conditions, HGF induced trophoblast cells migration involves activation of mitogen activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways and up-regulation of MMP2 under the transcription regulation of HLX gene (Cartwright et al. 2002; Liu et al. 2012). However, the significance of HGF and associated signaling pathways in trophoblast migration under hypoxic condition has not been fully elucidated.

It is well known that placenta responds to hypoxia through stabilization and increase in expression of oxygen sensitive transcription factors such as hypoxia-inducible factors (HIFs) (Semenza and Wang 1992). HIFs are heterodimer and comprised of the oxygen sensitive α subunit (HIF-1α, HIF-2α & HIF-3α) and constitutively active β subunit (HIF-1β, also known as aryl hydrocarbon receptor nuclear translocator, ARNT) (Semenza 2003). In hypoxic conditions, HIF-1α stabilizes in the cytoplasm and is directed towards the nucleus where it dimerizes with HIF-1β and activates target genes involved in cell proliferation, invasion and apoptosis (Semenza 2003). The expression of HIF-1α increases during early gestation (5-8th weeks) in low oxygen concentration and gradually drops around 10-12th weeks of pregnancy with an increase in the placental oxygen level (Caniggia et al. 2000). Although, HIF-1α is constitutively expressed throughout pregnancy (Pringle et al. 2010), its abnormal expression has been reported in preeclamptic women (Caniggia and Winter 2002). HIF-1α plays an important role during trophoblastic cells migration and invasion by regulating expression of certain genes like uPAR (Meade et al. 2007) and TGF-β3 (Nishi et al. 2004). In general, expression of HIF-1α is primarily regulated by changes in oxygen concentration; however, during pregnancy different cytokines and hormones also regulate HIF-1α under different physiological conditions (Pringle et al. 2010). Previous studies have shown that HGF stabilizes and stimulates the expression of HIF-1α via NF-kB signaling in various carcinomas cell lines (Tacchini et al. 2004). Similarly, under hypoxic condition HGF regulates expression of HIF-1α via PI3K and JNK signaling pathways in Hep G2 cells (Tacchini et al. 2001). However, we still do not know how HIF-1α is regulated under hypoxic condition during trophoblast migration in presence of HGF.

In the present study, we have used trophoblast derived HTR-8/SVneo cell line as a model system, to study the trophoblast migration/invasion in response to HGF treatment under hypoxic conditions (2% O2, 93% N2, 5% CO2). This cell line has been extensively employed to study molecular aspect of placental genes expression and trophoblast migration in normoxic (20% O2) as well as under hypoxic conditions (Ha et al. 2015; Tamaru et al. 2015; Liu et al. 2016). The expression of various MMPs and TIMPs responsible for trophoblast migration/invasion in the presence and absence of HGF was also studied. We have also studied the significance of activated MAPK and PI3K signaling pathways during HGF-mediated migration/invasion of HTR-8/SVneo cells under normoxic/hypoxic conditions using respective inhibitors. Moreover, silencing of HIF-1α and its effect on HGF-mediated HTR-8/SVneo cell migration under hypoxia was also investigated.

Materials and methods

Cell line and culture conditions

HTR-8/SVneo (a human immortalized EVT cell line, kindly provided by Prof. P. K. Lala, Queen’s University, Kingston, ON, Canada) was maintained in Dulbecco’s modified Eagle medium (DMEM) and Ham’s Nutrient Mixture F12 medium (HAM’s F12, Sigma-Aldrich Inc., Missouri, USA) in 1:1 ratio supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco®, NY, USA) and antibiotic-antimycotic cocktail pencillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (0.25 μg/mL; MP Biomedicals, Illkrich, France) at 37 °C in a humidified chamber under normoxic conditions containing 20% O2 and 5% CO2. For various experiments under hypoxic conditions, cells were incubated at 37 °C in humidified chamber containing 93% N2, 2% O2 and 5% CO2 (Esco Technologies Inc., Singapore).

Scratch wound migration assay

To measure cell migration, in vitro scratch wound assay was performed. HTR-8/SVneo cells (0.2 × 106 cells/well) cultured in 6-well culture plate were grown to form monolayer in humidified chamber at 37 °C and 5% CO2 supplemented under normoxic conditions (20% O2). To inhibit cell proliferation, monolayer of HTR-8/SVneo cells was treated with 5 μM mitomycin-C (Sigma-Aldrich Inc.) for 2 h. Subsequently, cells were scratched with 200 μL pipette tips to create wounds in horizontal as well as in vertical directions parallel to the diameter of the culture plate. Wells were washed with plain medium to remove detached cells and fresh medium containing 1% FBS and recombinant human HGF (Gibco®) was added to wells. Cells were imaged from different regions at 0 h using phase contrast microscope (Nikon Instruments Inc., Melville, NY, USA) and incubated in hypoxic chamber (2% O2 and 5% CO2) for 24 h. To measure the area of wound closure, images were taken from the same area and cell bordering the wounds were traced using ImageJ software (https://rsb.info.nih.gov/ij/). The percentage migration was calculated from the equation {(Wi-Wz)/Wi} × 100; where Wi is the area of wound at t = 0 h and Wz is the area of wound closure after 24 h.

Matrigel matrix invasion assay

The in vitro invasive potential of HTR-8/SVneo cells under normoxic and hypoxic conditions in presence and absence of HGF was also assessed using Matrigel matrix-based invasion assay (Malik et al. 2017). The Matrigel matrix was prepared by adding 50 μl of matrigel matrix (1 μg/mL, BD Biosciences, California, USA) in the transwell insert (Greiner Bio-One, Kremsmünster, Austria) of 8 μm filter pore size kept in a 24-well cell culture plate and incubated overnight at 37 °C under normoxic conditions to form a semisolid gel-like matrix. Culture medium (300 μl, 1:1 DMEM + HAM’s F12 supplemented with 1% FBS) was added to the lower chamber of all the wells with the transwell insert. Following which, cells (0.1 × 106 cells/transwell) suspended in reduced serum (1%) medium were seeded in the upper chamber over the Matrigel matrix in a total volume of 150 μl. HGF (50 ng/mL) was added in the upper and lower chambers. Cells were allowed to invade for 24 h either under normoxic or hypoxic conditions at 37 °C. Non-invading cells along with the Matrigel remaining in the upper chamber of the transwell inserts were carefully removed using moist cotton swab. Transwell inserts were washed with 50 mM phosphate buffered saline (PBS), pH 7.4 and cells were fixed for 10 min in chilled methanol. Fixed cells were washed with PBS and stained with 0.2 μM Hoechst 3342 (Thermo Fisher Scientific, Massachusetts, USA) nuclear dye for 10 min. After PBS wash, the membranes were cut and placed on a slide and cells counted on the whole membrane under oil immersion in a fluorescent phase contrast microscope (Nikon Instruments Inc.). The number of cells of untreated control was taken as one and fold change was calculated by dividing number of cells on membrane of treated transwell insert by the number of cells on untreated control transwell insert membrane.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Relative expression of mRNA transcripts encoded by target genes either under normoxic or hypoxic conditions was quantified by RT-qPCR. HTR-8/SVneo cells were serum starved for 6 h in normoxic conditions before treatment with HGF (50 ng/mL) and incubated either under normoxic or hypoxic conditions for 24 h at 37 °C. Total RNA was extracted using Ribo-Zol™ (AMERSCO®, Pennsylvania, USA) according to the manufacturer’s protocol. RNA purity and quantity was analysed by NanoDrop 3300 spectrophotometer (Thermo Scientific, NanoDrop Products, Wilmington, USA). Isolated RNA (2 μg) was reverse transcribed into cDNA using oligo (dT) 18 primer, random hexamer primer, dNTP mix, RiboLock RNase Inhibitor and Maxima reverse transcriptase enzyme (Fermentas International Inc., Burlington, Canada) according to the manufacturer’s instructions. Subsequently, RT-qPCR was performed in duplicates in 20 μL reaction mixture containing Maxima™ SYBR green master mix (Fermentas International Inc.), synthesized cDNA (diluted 3 times) and gene specific primers (Supplementary Table S1) in Stratagene Mx3005P (Agilent Technologies Inc., Santa Clara, CA, USA). The cycle profile for target gene amplification: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, primer specific annealing temperature (Supplementary Table S1) for 60 s. Finally, a melting curve analysis was carried out at a temperature range of 60 to 95 °C for 20 min. A single peak in the melting curve analysis confirmed gene-specific amplification. The fold change in expression of genes was calculated from relative Ct values using ∆∆Ct (Ct; cycle threshold) method after normalized with TOP1 (DNA topoisomerase type I), which acted as loading control in the same sample.

Western blotting

Preparation of whole cell lysate

HTR-8/SVneo cells (0.2 × 106/well) were seeded in 6-well culture plate in DMEM + HAM’s F12 medium and grown overnight under normoxic conditions at 37 °C. Next day, cells were serum starved for 6 h under normoxic conditions and then treated with HGF (50 ng/mL) for 24 h either under normoxic or hypoxic conditions at 37 °C. Thereafter, cells were lysed in cell lysis buffer (20 mM Tris-HCl, 10% glycerol, 0.2 mM EDTA, 0.137 M NaCl, 1% NP-40) supplemented with complete protease and phosphatase inhibitor cocktail (Roche Diagnostic, Mannheim, Germany). This was followed by three rapid freeze-thaw cycles to ensure complete cell lysis. Cell lysates were centrifuged at 12,000 x g for 10 min at 4 °C, supernatants collected and stored at −70 °C till used.

Preparation of nuclear and cytoplasmic fractions

For HIF-1α expression, cells were harvested in ice-cold PBS containing 1 mM EDTA. The cell pellet was suspended in cytoplasmic extraction buffer (1 M HEPES-KOH pH -7.9, 3 M KCl, 0.5 M EDTA, 10% NP-40) and lysed by vortexing for 3 min followed by incubation on ice for 1 min (3 cycles). Cell suspension was immediately centrifuged for 5 min at 10,000 x g at 4 °C, the supernatant thus obtained represented cytoplasmic extract. The residual pellet was dissolved in the nuclear extraction buffer (1 M Tris pH 7.5, 3 M KCl, 0.5 M EDTA) followed by rapid freeze-thaw (three times) in liquid nitrogen. Nuclear fraction was collected by centrifugation at 10,000 x g for 5 min. The protein content in whole cell lysate, nuclear & cytoplasmic fractions was quantitated by bicinchoninic acid colorimetric assay (BCA) using bovine serum albumin (BSA) as standard (Thermo Fisher Scientific, Rockford, USA).

Procedure

Proteins in cell lysate/cytoplasmic & nuclear fractions (40 μg/lane) were resolved by 0.1% SDS-10% polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane (0.45 μm) by wet transfer method. After transfer of proteins, membrane was blocked in 5% BSA in Tris Buffer Saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH -7.4) for 1 h at room temperature. Further, blots were incubated overnight at 4 °C with an optimized dilution (1:1000) of antibodies against MMP1, MMP2, MMP3, HIF-1α, tata binding protein (TBP, Cell Signaling Technology®, Massachusetts, USA), MMP9 (Abcam Technologies, Cambridge, USA), TIMP1, TIMP3 (CloudClone Corp., Texas, USA), and 1:5000 dilution of antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Abgenex, Bhubaneswar, India) in TBST (TBS + 0.1% Tween 20) containing 5% BSA. After subsequent washings with TBST, membrane was incubated with horseradish peroxidase (HRP) conjugated either anti-rabbit antibody (1:2500) or anti-mouse antibody (1:5000) (Thermo Scientific Inc.) for 1 h at room temperature in TBST containing 5% BSA. Blots were washed thrice with TBST and developed using Immobilon chemiluminescent substrate (Millipore Corp. MA, USA). Pictures of the chemiluminescent blots were taken by FluorChem E system (ProteinSimple, SJ, California, USA). The densitometry analysis of bands on Western blots was performed by ImageJ software.

MAPK and PI3K signaling pathways

To study activation of MAPK and PI3K pathways, HTR-8/SVneo cells (0.2 × 106/well) were seeded in 6-well culture plates and allowed to adhere overnight at 37 °C in humidified atmosphere of 5% CO2 under normoxic conditions (20% O2). Cells were serum starved for 6 h under same conditions before treatment with HGF (50 ng/mL) either under normoxic or hypoxic conditions for 10, 30 and 60 min. After each time point, cells were harvested in cell lysis buffer to prepare whole cell lysate. The cell lysates were processed for Western blot using primary antibodies at a dilution of 1:1000 against p44/42 MAPK, phospho-p44/42 MAPK, Akt, phospho-Akt (Thr308) (Cell Signaling Technology®) as described above.

To inhibit MAPK and PI3K phosphorylation, serum starved HTR-8/SVneo cells were pre-treated with MAPK inhibitor, U0126 (10 μM) and PI3K inhibitor, LY294002 (10 μM) (Cell Signaling Technology®) for 2 h as per manufacturer’s instructions before treatment with HGF. Cells were further processed for either Scratch wound migration assay or Matrigel matrix invasion assay in presence or absence of HGF (50 ng/mL) under either normoxic or hypoxic conditions as described above. Inhibition of ERK½ and Akt phosphorylation was confirmed by Western blotting.

Indirect immunofluorescence

HTR-8/SVneo cells (0.3 × 105/well) were seeded on coverslips in 24-well plate and incubated overnight under normoxic conditions at 37 °C. Next day, after serum starvation for 6 h in normoxic conditions, cells were incubated in presence of HGF (50 ng/mL) for 24 h under hypoxic conditions. Cells were fixed in chilled methanol for 10 min at 4 °C. Subsequently, coverslips were washed with PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl and pH -7.4) and blocked in 3% BSA in PBS for 30 min. Cells were incubated with primary antibody against HIF-1α diluted (1:100) in PBS containing 1% BSA for overnight at 4 °C. Cells were washed thrice with PBS, followed by incubation with anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen Corporation, Oregon, USA) diluted (1:500) in PBS and supplemented with 1% BSA for 45 min at room temperature. After extensive washings, coverslips containing fixed cells were mounted on a glass slide using ProLong® Diamond Antifade containing DAPI (Invitrogen Corporation). Cells were examined under fluorescent phase contrast microscope (Nikon Instrument Inc.) and images were captured and processed using Image-Pro Plus software (Media cybernetics, USA).

Zymography

The activity of secreted MMP1 was evaluated by collagen (HIMEDIA®, Mumbai, India) (0.1%), MMP2 and MMP9 by gelatin (Sigma Aldrich Inc.) (0.1%) and MMP3 by casein (Sigma Aldrich Inc.) (0.1%) gel zymography. HTR-8/SVneo cells (0.1 × 106/well) were seeded in 6-well culture plate and incubated overnight under normoxic conditions at 37 °C. After serum starvation for 24 h in same culture condition, cells were treated with HGF (50 ng/mL) for 24 h in plain medium under either normoxic or hypoxic conditions. Culture medium was collected, concentrated and was resolved in 8% polyacrylamide gels containing 0.1% collagen/gelatin/casein. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 1 h to remove SDS and then incubated in buffer containing 50 mM Tris-HCl (pH -7.5), 150 mM NaCl, 0.5 mM ZnCl2 and 10 mM CaCl2 for MMP2 and MMP9 and in 50 mM Tris-HCl (pH -7.5), 150 mM NaCl, 10 mM CaCl2 and 0.05% Brij 35 (Sigma Aldrich Inc.) for MMP1 and MMP3 at 37 °C for 36 h to allow proteolysis of the collagen/ gelatin/casein substrate. After incubation, gels were stained in 0.5% Coomassie brilliant blue R-250 in 40% methanol and 10% acetic acid for 3 h followed by washing with destaining solution (40% methanol and 10% acetic acid in water). Proteolytic activities appeared as clear band on gel against dark background. Intensity was measured by ImageJ software.

Gene silencing by siRNA

HTR-8/SVneo cells (0.2 X 106/well) were seeded in 6-well culture plate and cultured overnight under normoxic conditions at 37 °C. At 70% confluencey, cells were transfected with control siRNA, siRNA for HIF-1α (SantaCruz Biotechnology Inc., Texas, USA) using lipofectamine™ RNAiMAX transfection reagent (LifeTechnologies, NY, USA) and Opti-MEM® medium (Gibco®). HIF-1α and control siRNA (optimized concentration of 20 pmol) were mixed with Opti-MEM® medium to make a total volume of 150 μL. In separate tube, 6 μL of lipofectamine was mixed with 144 μL Opti-MEM® medium and incubated for 5 min at room temperature. Both solutions were mixed and incubated for 20 min at room temperature. Cells in culture plates were washed with Opti-MEM® and fresh medium was added into each well. After incubation, mixed solutions were added drop by drop in respective wells and incubated under normoxic conditions at 37 °C. After 48 h of transfection, control siRNA and HIF-1α silenced cells were processed for Scratch wound migration assay and Western blotting as described above under hypoxic conditions subsequent to treatment with HGF.

Results

HGF increases HTR-8/SVneo cells migration/invasion under normoxic as well as hypoxic conditions through up-regulation of MMPs

Hypoxia plays a crucial role during the trophoblast cells migration and invasion through secretion of various cytokines and growth factors. In order to study the effect of HGF on the migration of HTR-8/SVneo cells, in vitro Scratch wound migration assay was performed. Using this assay, our group recently showed that treatment of HTR-8/SVneo cells with HGF (50 ng/mL) under normoxic (20% O2) conditions led to a significant increase in their migration (Chaudhary et al. 2018). In the present study, HTR-8/SVneo cells were incubated under hypoxic conditions (2% O2, 5% CO2) at 37 °C in the presence and absence of optimized concentration of HGF (50 ng/mL) for 24 h. As a control, the migration assay was also performed in HTR-8/SVneo cells exposed to normoxic conditions without any treatment with HGF for 24 h at 37 °C. We observed ~1.4 and ~1.7-fold increase in HTR-8/SVneo cells migration in untreated and HGF treated cells incubated under hypoxic conditions, which was statistically significant, as compared to untreated cells under normoxia (Fig. 1). Interestingly, a significant (p = 0.0008) increase in migration under hypoxia was also observed after treatment of the cells with HGF as compared to untreated cells (Fig. 1). To rule out that increase in migration of HTR-8/SVneo cells observed in untreated and HGF treated cells under hypoxic conditions was not due to increase in the proliferation, HTR-8/SVneo cells were pre-treated with mitomycin-C, prior to use in scratch wound assay as described in Materials and Methods. In addition to migration, treatment of HTR-8/SVneo cells with HGF (50 ng/mL) also led to a significant increase in their invasion (~5 and ~ 6 fold) under both normoxic as well as hypoxic conditions (Fig. 2). Further, a significant (p = 0.006) increase in the invasion under hypoxia as compared to normoxia was also observed in absence of treatment with HGF (Fig. 2).

Fig. 1.

Fig. 1

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Effect of HGF treatment on HTR-8/SVneo cells migration under hypoxic condition: HTR-8/SVneo cells (0.2 × 106/well) were grown overnight in 6-well culture plate under normoxia (20% O2) in a humidified chamber at 37 °C with 5% CO2 to form a monolayer. Cells were pre-treated with mitomycin-C (5 μm) for 2 h to inhibit cell proliferation. Subsequently, cells growing as monolayer were scratched in horizontal as well as vertical directions. Detached cells were removed by washing with plain medium, subsequently treated with and without HGF (50 ng/mL) for 24 h under hypoxia (2% O2). Another set of cells were cultured for 24 h under normoxic conditions without any treatment with HGF. The fold change in migration was calculated based on the area of wound closure after 24 h. The results are shown as mean ± s.e.m of three independent experiments. Representative images at 0 and 24 h with and without HGF (50 ng/mL) treatment are appended alongside. Scale bar represents 20 μm. *p ≤ 0.05 was considered statistically significant as compared to untreated cells under normoxia

Fig. 2.

Fig. 2

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Effect of HGF treatment on HTR-8/SVneo cells invasion under normoxic and hypoxic conditions: To measure invasion by Matrigel invasion, HTR-8/SVneo cells (0.1 × 106/well) were seeded in upper chamber of transwell either in presence or absence of HGF (50 ng/mL) for 24 either under normoxic or hypoxic conditions at 37 °C as described in Materials and Methods. Each bar graph shows relative fold change in invasion of HTR-8/SVneo cells in HGF treated cells under normoxic conditions and untreated & HGF treated cells under hypoxic conditions as compared to the untreated control cells cultured under normoxia. Values are expressed as mean ± s.e.m of three independent experiments. *p ≤ 0.05 was considered statistically significant as compared to untreated cells under normoxia

To determine which class of MMPs are involved in HGF-mediated HTR-8/SVneo cells migration/invasion under both normoxic as well as hypoxic conditions; the expression of MMP1, 2, 3 and 9 both at the transcript as well as at the protein levels were checked. Under normoxic conditions, treatment of HTR-8/SVneo cells with HGF for 24 h led to a significant increase in the expression of MMP2, MMP3, and MMP9 at the transcript level (Table 1). However, at protein level, only an increase in MMP2 and MMP3 was observed by Western blot in HTR-8/SVneo cells treated with HGF (Fig. 3b, c). Moreover, under similar culture conditions, significant increase in enzymatic activity of MMP2 by gelatin zymography in HGF treated cells as compared to untreated cells was also observed (Fig. 4a). However, no significant MMP3 activity was observed in both HGF treated as well as control cells by casein zymography (data not shown). Under hypoxic conditions, treatment of the cells with HGF led to a significant increase in the MMP1 and MMP9 at the transcript level (Table 1), which was also confirmed at the protein level (Fig. 3a, d). Further, increased MMP9 enzymatic activity in the culture supernatant of HGF treated cells as compared to untreated cells incubated under hypoxic conditions was also observed by gelatin zymography (Fig. 4b). However, under similar experimental conditions MMP1 in the culture supernatant harvested from untreated and HGF treated cells did not show any enzymatic activity in collagen zymography (data not shown). Interestingly, a significant increase in the transcript levels of MMP1, MMP2 and MMP9 was also observed under hypoxic conditions as compared to normoxic conditions without any treatment with HGF (Table 1). However, at protein level, only a significant increase in MMP1 was observed under hypoxic versus normoxic conditions in absence of treatment with HGF (Fig. 3a).

Table 1.

Relative transcript levels of MMPs, TIMPs and MMPs/TIMP1 ratio in HTR-8/SVneo cells with and without HGF treatment under normoxic/hypoxic conditions by RT-qPCR

MMPs & TIMPs Relative ΔCt of the transcript levels with respect to untreated cells under normoxia (Mean ± s.e.m)
Normoxia Normoxia + HGF Hypoxia Hypoxia + HGF Normoxia vs Normoxia+ HGF (p value) Hypoxia vs Hypoxia + HGF (p value) Normoxia vs Hypoxia (p value)
MMP1 1.0 ± 0.01 1.27 ± 0.11 2.50 ± 0.17c 6.83 ± 0.2b 0.06 0.001b 0.001c
MMP2 1.0 ± 0.01 2.87 ± 0.12a 2.50 ± 0.21c 3.03 ± 0.12 0.001a 0.11 0.002c
MMP3 1.0 ± 0.01 6.73 ± 0.46a 1.43 ± 0.18 2.16 ± 0.14 0.002a 0.08 0.07
MMP9 1.0 ± 0.01 1.34 ± 0.04a 1.96 ± 0.08c 4.60 ± 0.26b 0.001a 0.0006b 0.003c
TIMP1 1.0 ± 0.01 0.69 ± 0.04d 2.12 ± 0.19 1.03 ± 0.08e 0.002d 0.006e 0.004
TIMP2 1.0 ± 0.01 1.51 ± 0.17 0.8 ± 0.08 1.54 ± 0.49 0.03 0.21 0.08
TIMP3 1.0 ± 0.01 1.98 ± 0.44 2.65 ± 0.47 0.44 ± 0.15e 0.09 0.01e 0.02
TIMP4 1.0 ± 0.01 1.45 ± 0.20 0.89 ± 0.34 0.70 ± 0.24 0.08 0.67 0.77
MMP1/TIMP1 1.0 ± 0.01 1.85 ± 0.15f 1.19 ± 0.10 6.71 ± 0.65g 0.004f 0.001g 0.13
MMP2/TIMP1 1.0 ± 0.01 4.18 ± 0.26f 1.20 ± 0.20 2.95 ± 0.14g 0.0002f 0.002g 0.32
MMP3/TIMP1 1.0 ± 0.01 9.87 ± 1.03f 0.68 ± 0.10 2.10 ± 0.07g 0.001f 0.004g 0.04
MMP9/TIMP1 1.0 ± 0.01 1.97 ± 0.19f 0.93 ± 0.08 4.54 ± 0.59g 0.007f 0.003g 0.47
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aSignificant increase in MMPs on treatment with HGF under normoxia

bSignificant increase in MMPs on treatment with HGF under hypoxia

cSignificant increase in MMPs in hypoxia as compared to normoxia

dSignificant decrease in TIMPs on treatment with HGF under normoxia

eSignificant decrease in TIMPs on treatment with HGF under hypoxia

fSignificant increase in MMPs/TIMP1 ratio under normoxia after HGF treatment

gSignificant increase in MMPs/TIMP1 ratio under hypoxia after HGF treatment

Fig. 3.

Fig. 3

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Expression profile of MMP1, MMP2, MMP3 and MMP9 in HTR-8/SVneo cells treated with HGF under normoxic and hypoxic conditions: HTR-8/SVneo cells (0.2 × 106/well) were grown overnight in 6-well culture plate under normoxia (20% O2) in presence of 5% CO2 at 37 °C. Next day, cells were serum starved for 6 h, followed by treatment with HGF (50 ng/mL) for 24 h under normoxic as well as hypoxic conditions. Subsequently, culture supernatant was collected and cell lysates were prepared to determine expression of MMP1, MMP2, MMP3 and MMP9 at protein level by Western blotting as described in Materials and Methods. Panels a, b, c, d show the densitometric analysis and representative Western blots of respective MMPs in cell lysate. GAPDH was used as a loading control. The values are expressed as mean ± s.e.m of band intensity of three independent experiments

Fig. 4.

Fig. 4

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Expression profile of secreted MMPs by zymography. HTR-8/SVneo cells (0.2 × 106/well) were grown overnight in 6-well culture plate under normoxia (20% O2) at 37 °C. Next day, cells were serum starved for 24 h, followed by treatment with HGF (50 ng/mL) for 24 h either under normoxic or hypoxic conditions. Culture supernatant were harvested and concentrated. Concentrated supernatant was analysed by gelatin Zymography as described in Materials and Methods. Panel a shows the densitometric analysis of MMP2 under normoxic conditions and Panel b densitometric analysis of MMP9 under hypoxic conditions. Representative Zymogram are appended below. The values are expressed as mean ± s.e.m of three independent experiments

Expression of TIMP1, TIMP2, TIMP3 and TIMP4 at the transcript level by RT-qPCR was also investigated. TIMP1 was down-regulated in HTR-8/SVneo cells treated with HGF under normoxic conditions, whereas under hypoxic conditions, TIMP1 and TIMP3 were down-regulated in HGF treated HTR-8/SVneo cells as compared to the untreated controls under similar experimental conditions (Table 1). However, Western blot analyses revealed a significant decrease only in TIMP1 in HTR-8/SVneo cells treated with HGF as compared to untreated cells incubated under hypoxic conditions (Fig. 5a). No significant changes in protein expression of TIMP3 was observed between untreated and HGF treated HTR-8/SVneo cells in normoxic as well as hypoxic conditions (Fig. 5a). Since only TIMP1 was decreased in HGF treated cells as compared to their respective untreated counterparts, we calculated the ratio between MMPs versus TIMP1 at mRNA as well as at protein level. Interestingly, determination of MMPs and TIMP1 ratios revealed a significant increase at the transcript levels in MMP1/TIMP1, MMP2/TIMP1, MMP3/TIMP1 and MMP9/TIMP1 ratio under both normoxic and hypoxic conditions subsequent to treatment with HGF as compared to respective untreated controls (Table 1). Moreover, at protein level significant increase in MMP2/TIMP1 and MMP3/TIMP1 ratio was observed in HGF treated cells under normoxia as compared to untreated control cells (Fig. 6a), while significant increase in MMP1/TIMP1 and MMP9/TIMP1 ratio was observed in HGF treated HTR-8/SVneo cells as compared to untreated cells under hypoxic conditions (Fig. 6b).

Fig. 5.

Fig. 5

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Expression profile of TIMP1 and TIMP3 in HTR-8/SVneo cells treated with HGF under normoxic and hypoxic conditions: HTR-8/SVneo cells (0.2 × 106/well) were grown overnight in 6-well culture plate under normoxic conditions. Next day, cells were serum starved for 6 h, followed by treatment with HGF (50 ng/mL) for 24 h under normoxic as well as hypoxic conditions. Subsequently, cell lysates were prepared to determine expression of TIMP1 and TIMP3 at protein level by Western blotting as described in Materials and Methods. Panels a and b shows densitometric analysis and representative Western blots of TIMP1 and TIMP3 proteins in cell lysate. GAPDH was used as a loading control. Values are expressed as mean ± s.e.m of band intensity of three independent experiments

Fig. 6.

Fig. 6

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MMP/TIMP ratio in HGF-mediated increase in HTR-8/SVneo cells migration/invasion. MMP/TIMP ratio was calculated from the densitometric analysis of Western blots of MMPs and TIMPs in untreated cells and subsequently treated with HGF cells under normoxic and hypoxic conditions. Panel a show the bar graph of MMP2/TIMP1 and MMP3/TIMP1 ratio under normoxic conditions whereas Panel b show MMP1/TIMP1 and MMP9/TIMP1 ratio under hypoxic conditions in HGF treated cells and value of HGF untreated cells were taken as 1. Value are expressed as ratio of mean ± s.e.m of three independent experiments

HGF activates ERK½ and PI3K signaling pathways that are involved in trophoblast migration/invasion

We have previously shown relevance of MAPK and PKA signaling pathways during migration of HTR-8/SVneo cells subsequent to treatment with HGF under normoxia (Chaudhary et al. 2018). To investigate, which signaling pathways were activated during HGF-mediated HTR-8/SVneo cells migration/invasion under hypoxic conditions, HTR-8/SVneo cells were serum starved and subsequently treated with and without HGF for 10, 30 and 60 min under hypoxic conditions. After each time point, cells were lysed and processed for Western blotting to analyse ERK½ and Akt phosphorylation. Cells exposed to hypoxia in absence of HGF treatment showed significant increase in pERK2 at 10 min and both pERK1 and pERK2 at 30 and 60 min (Fig. 7a). Cells treated with HGF under hypoxic conditions led to a significant increase in pERK1 and pERK2 at all the time points studied. Time kinetics analysis for pERK½ revealed a maximal increase at 10 min (pERK1 ~4.8 fold; pERK2 ~3.0 fold) after HGF treatment followed by gradual decrease till 60 min (Fig. 7b). However, under hypoxia in absence of HGF, no significant changes in the pAkt (Thr308) were observed, whereas treatment with HGF led to a rapid increase in pAkt (Thr308) level. Time kinetics analysis revealed a sharp increase in pAkt (Thr308) expression level (~25 fold) within 10 min of HGF treatment as compared to untreated cells at 0 min. However, pAkt (Thr308) levels decreases from 10 min onwards, but still significantly higher as compared to untreated control (Fig. 8b). Treatment of HTR-8/SVneo cells with HGF under normoxia also led to a significant increase in pAkt (Thr308) at 10 min (Fig. 8c).

Fig. 7.

Fig. 7

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Activation of MAPK pathway in HGF treated HTR-8/SVneo cells under hypoxia. HTR-8/SVneo cells (0.2 × 106/well) were cultured in 6-well plate for 24 h under normoxic conditions. Subsequently, cells were serum starved for 6 h, followed by treatment with and without HGF (50 ng/mL) for varying time periods (10, 30 and 60 min) under hypoxic conditions. After treatment with HGF, cell lysate was prepared and processed to determine the activation of ERK½ by Western blotting as described in Materials and Methods. Panel a represents the densitometric plot showing the relative increase in pERK½ in untreated cells kept under hypoxic conditions with respect to total ERK½. Panel b represents the densitometric plot showing the relative increase in pERK½ in HGF treated cells with respect to total ERK½. GAPDH was included as loading control for each set of experiments. The data is expressed as fold change with respect to cells at 0 min; values are shown as mean ± s.e.m of three independent experiments. Representative blots of p-ERK½, ERK and GAPDH are shown alongside panels a & b. *p ≤ 0.05 was considered statistically significant as compared to untreated cells at 0 min

Fig. 8.

Fig. 8

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Activation of PI3K pathway in HGF treated HTR-8/SVneo cells under normoxic and hypoxic conditions. HTR-8/SVneo cells (0.2 × 106/well) were cultured in 6-well plate for 24 h under normoxic conditions. Subsequently, cells were serum starved for 6 h, followed by treatment with and without HGF (50 ng/mL) for varying time periods (10, 30 and 60 min) under hypoxic and normoxic conditions. After treatment with HGF, cell lysate was prepared and processed to determine the activation of pAkt by Western blotting as described in Materials and Methods. Panel a represents the densitometric plot showing the relative increase in pAkt (Thr 308) in untreated cells kept under hypoxic conditions with respect to 0 min as compared to total Akt. Panel b represents the densitometric plot showing the relative increase in pAkt (Thr 308) in HGF treated cells with respect to 0 min as compared to total Akt. Panel c represents the densitrometric plot showing the relative increase in pAkt (Thr308) in HGF treated cells with respect to untreated control at 0 min as compared to total Akt under normoxic conditions. GAPDH was included as loading control for each set of experiments. The data is expressed as fold change with respect to cells at 0 min; values are shown as mean ± s.e.m of three independent experiments. Representative blots of pAkt, Akt and GAPDH are shown alongside in panels a, b and c. *p ≤ 0.05 was considered statistically significant as compared to untreated cells at 0 min

To test whether ERK½ and PI3Ksignaling are involved in HGF-mediated HTR-8/SVneo cell migration under hypoxic conditions, Scratch wound healing migration assay was performed in cells pre-treated with pharmacological inhibitors for MAPK (U0126) and PI3K (LY294002) signaling and subsequently treated with HGF for 24 h under hypoxic conditions as described in Materials and Methods. Inhibition of MAPK signaling pathway led to concomitant decrease (p = 0.002) in migration of HTR-8/SVneo cells treated with HGF as compared to HGF treated control cells (Fig. 9). Moreover, inhibition of PI3K signaling pathways also led to significant (p = 0.004) decrease in the migration of HTR-8/Svneo cells treated with HGF as compared to cells that were not pre-treated with LY294002 but subsequently treated with HGF (Fig. 9). In addition to migration, effect of pharmacological inhibitors for MAPK and PI3K was also studied on invasion of HTR-8/SVneo cells under both normoxic and hypoxic conditions. The results are summarized in Fig. 10. Inhibition of both MAPK by U0126 and PI3K by LY294002 led to significant decrease in invasion of these cells treated with HGF as compared to cells that were not pre-treated with MAPK and PI3K inhibitors but subsequently treated with HGF under both normoxic as well as hypoxic conditions (Fig. 10a, b). These findings revealed that both MAPK and PI3K signaling play a crucial role during HGF-mediated HTR-8/SVneo cells migration/invasion both under normoxic and hypoxic conditions.

Fig. 9.

Fig. 9

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Effect of inhibition of MAPK and PI3K signaling pathways on HGF-mediated HTR-8/SVneo cells migration under hypoxic conditions. HTR-8/SVneo cells (0.2 × 106/well) were cultured in 6-well culture plate at 37 °C under normoxic conditions to form a monolayer. After formation of monolayer, cells were serum starved for 6 h followed by treatment with MAPK inhibitor U0126 (10 μm) or PI3K inhibitor LY294002 (10 μM) for 2 h. Subsequently, cells were processed for wound healing assay under hypoxic condition in the presence or absence of HGF with appropriate controls as described in Materials and Methods. The bar graph show fold change in migration under different experimental conditions as compared to untreated control. Data is shown as mean ± s.e.m of three independent experiments. Representative images at 0 h and 24 h are also shown in the panel. Scale bar represents 20 μm

Fig. 10.

Fig. 10

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Effect of inhibition of MAPK and PI3K signaling pathways on HGF-mediated HTR-8/SVneo cells invasion under normoxic and hypoxic conditions. Following U0126 and LY294002 pre-treatment of HTR-8/SVneo cells, invasion assay was performed either in presence or absence of HGF (50 ng/mL) under normoxic and hypoxic conditions as described in Materials and Methods. Panel a shows relative fold change in invasion of varying treatment groups (HGF treated, U0126 pre-treated and U0126 pre-treated cells treated with HGF, LY294002 pre-treated and LY294002 pre-treated cells treated with HGF) as compared to the untreated HTR-8/SVneo cells under normoxic conditions. Panel b shows relative fold change in invasion of varying treatment groups (HGF treated, U0126 pre-treated and U0126 pre-treated cells treated with HGF, LY294002 pre-treated and LY294002 pre-treated cells treated with HGF) as compared to the untreated HTR-8/SVneo cells under hypoxic conditions. Values are expressed as mean ± s.e.m. of three independent experiments performed in duplicates

HIF-1α may be involved in HGF-mediated increase in HTR-8/SVneo cell migration under hypoxia

In response to hypoxia, HIF-1α protein stabilizes inside the cytoplasm and translocate towards nucleus, where it activates genes involved in cell migration and invasion. To determine, whether HGF has any regulatory influence on HIF-1α, HTR-8/SVneo cells were incubated under hypoxia for 24 h in the presence and absence of HGF. Significant increase (p = 0.003) in transcript level of HIF-1α was observed in HGF treated HTR-8/SVneo cells as compared to untreated cells (Fig. 11a). Similarly, significant increase (p = 0.02) in HIF-1α expression at protein level was also observed in nuclear fraction of HGF treated cells (Fig. 11b). To further confirm the nuclear translocation of HIF-1α protein, immunofluorescence studies were carried out in presence and absence of HGF under hypoxia as described in Materials and Methods. HGF treated HTR-8/SVneo cells showed higher HIF-1α protein localization (green dots) inside the nucleus (blue) as compared to untreated counterpart (Fig. 11c).

Fig. 11.

Fig. 11

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Level of HIF-1α in HGF treated HTR-8/SVneo cells under hypoxia. HTR-8/SVneo cells (0.2 × 106/well) were cultured in 6-well plate for overnight at 37 °C under normoxic condition followed by serum starvation for 6 h. Subsequently, cells were treated with HGF (50 ng/mL) for 24 h at 37 °C under hypoxic condition. After treatment, cells were processed to prepare cell lysate and transcript levels of HIF-1α was determined by RT-qPCR. In addition, cells were processed to prepare nuclear fraction to study HIF-1α expression profile by Western blotting. Further, HIF-1α localization in nucleus was confirmed by immunofloresence. Panel a show transcript profile of HIF-1α, the bars represent relative expression after normalization with TOP1 as an internal loading control, and data expressed as mean ± s.e.m of three different experiments performed in duplicates. Panel b show protein profile of HIF-1α, the bar represents relative expression after normalize with TBP1 as an internal loading control and data expressed as mean ± s.e.m of three different experiment. Representative blots are appended alongside the graph. Panel c HTR-8/SVneo cells were cultured on coverslip and treated with HGF (50 ng/mL) for 24 h in hypoxic conditions followed by fixation in chilled methanol. The HIF-1α (green) was detected inside nucleus stained with DAPI as described in Materials and Methods. Scale bar represent 10 μm

As HGF induces HTR-8/SVneo cells migration and also lead to stabilised increased in nuclear expression of HIF-1α, it might be possible that HGF may regulate HTR-8/SVneo cells migration through the increased expression and stabilization of HIF-1α. To delineate the relevance of HIF-1α during migration of HTR-8/SVneo cells under hypoxia, HIF-1α was knock-down by siRNA and its silencing under hypoxic conditions was confirmed by Western blotting (Fig. 12a). HGF treated HIF-1α silenced HTR-8/SVneo cells show significantly reduced (p = 0.002) migration under hypoxic conditions as compared to control siRNA transfected cells incubated with HGF for 24 h (Fig. 12b). However, no significant change was observed in basal migration of HTR-8/SVneo cells transfected with HIF-1α siRNA as compared to control siRNA transfected cells without HGF treatment (Fig. 12b).

Fig. 12.

Fig. 12

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Effect of HIF-1α silencing on HGF-mediated HTR-8/SVneo cells migration under hypoxic conditions. HTR-8/SVneo cells were transfected with control and HIF-1α siRNA. After 48 h of transfection, cells were used to study migration by Scratch wound assay as described in Materials and Methods. Silencing of HIF-1α expression was confirmed by Western Blotting. Panel a shows densitometric analysis of relative intensity of HIF-1α expression in control siRNA and HIF-1α siRNA transfected cells after subsequent treatment with and without HGF. Values are expressed as mean ± s.e.m of three independent experiments. Representative blots are appended alongside. Panel b shows fold change in migration of cells transfected with HIF-1α siRNA as compared to control siRNA, subsequent to treatment with and without HGF (50 ng/mL) for 24 h. Values are expressed as mean ± s.e.m of three independent experiments. Representative images are appended alongside. Scale bar represent 20 μm

HGF regulates HIF-1α expression through activation of MAPK and PI3K signaling pathways

To investigate, if activation of MAPK and PI3K signaling pathways by HGF are also involved in up-regulation of HIF-1α expression, HTR-8/SVneo cells were pre-treated with MAPK inhibitor (U0126) and PI3K inhibitor (LY294002) as described in Materials and Methods. The efficacy of these inhibitors was confirmed by Western blotting. Interestingly, inhibition of MAPK pathway led to significant decrease (p = 0.03) in HIF-1α expression in the nuclear fraction of cells treated with HGF as compared to HGF treated control cells without pre-treatment with inhibitor (Fig. 13). Similarly, significant decrease (p = 0.04) in HIF-1α expression was also observed in nuclear fraction of cells pre-treated with PI3K inhibitor and subsequently treated with HGF (50 ng/mL) as compared to cells not pre-treated with LY294002 but treated with HGF (Fig. 13). On other hand, no significant change in HIF-1α expression was observed in HTR-8/SVneo cells pre-treated with either U0126 or LY294002 as compared to untreated cells under similar experimental conditions (Fig. 13).

Fig. 13.

Fig. 13

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Effect of inhibition of MAPK and PI3K signaling on HGF induced expression of HIF-1α in HTR-8/SVneo cells under hypoxia. HTR-8/SVneo cells (0.2 × 106/well) were cultured in 6-well plate for overnight at 37 °C under normoxic conditions followed by serum starvation for 6 h. Cells were pre-treated with U0126 (10 μm) and LY294002 (10 μm) for 2 h. Subsequently, cells were treated with/without HGF for 24 h under hypoxic conditions. The expression of HIF-1α was assessed by Western blotting as described in Materials and Methods. The bar graph showed the densitometric profiles of HIF-1α in untreated, U0126 and LY294002 pre-treated and further treated in the presence of HGF. Data are shown as mean ± s.e.m of three independent experiments. Representative blots are appended alongside. Inhibition of MAPK and PI3K signaling was confirmed by down regulation of pERK½ and pAkt in the cells treated with U0126 and LY294002 respectively. TBP1 was used as nuclear loading control for normalisation of HIF-1α expression, while GAPDH was used for normalisation of pERK½ and pAkt expression

Discussion

During early phase of pregnancy, physiological hypoxia plays a significant role in different embryonic processes, including placentation, angiogenesis and haematopoiesis (Dunwoodie 2009). Non-physiological hypoxia at utero-placental interface is associated with pregnancy complications like preeclampsia (due to shallow invasion of EVTs) and IUGR (van Patot et al. 2012). Low levels of HGF are also reported in preeclamptic and IUGR patients (Wolf et al. 1991); thus decoding the role of HGF in trophoblast migration and associated signaling pathways under hypoxic conditions might reveal the molecular aspects of the above pregnancy complications. In the present study, significant increase in migration/invasion of HTR-8/SVneo cells exposed to 2% oxygen as compared to cells incubated in presence of 20% oxygen was observed (Figs. 1 and 2), which is in agreement with the previous studies (Wang et al. 2015; Zhu et al. 2017). However, these studies employed different cell line model i.e. JEG3 and different Migration assay system (Transwell assay). The increase in invasion observed under hypoxic condition by other groups was suggested to be due to the up regulation and activation of urokinase receptor expression and matrix metalloproteinases (Graham et al. 1998; Lash et al. 2007). In addition, significant increase in HTR-8/SVneo cell migration and invasion under both normoxic as well as hypoxic conditions in presence of HGF was also observed (Figs. 1 and 2). Significant increase in migration by HGF under normoxic conditions has been previously published by our group (Chaudhary et al. 2018). The increase in migration that we observed in HGF treated HTR-8/SVneo cells might be due to activation of c-Met protein as reported by previous studies in JEG-3 cell line and glioblastoma cell lines under hypoxic conditions (Hayashi et al. 2005; Eckerich et al. 2007). Moreover, under normoxic conditions, c-met dependent HGF-mediated increase in invasion has been reported in trophoblastic cell line ED27 and pancreatic cell line COLO-357 (Kauma et al. 1999; Matsushita et al. 2007). Further, it has been reported that up-regulation of c-Met increase the sensitivity of cells towards HGF, which results in higher amplification of HGF signaling and subsequently cell migration. Thus, hypoxia might be working in synergy with HGF to increase HTR-8/SVneo cell migration/invasion.

MMPs play an important role during migration and invasion of cells. The activity of these proteases is controlled by TIMP, which regulate the proteolytic cleavage of active domain of MMPs in the ECM. In general, trophoblast cells do express wide variety of MMPs, from MMP-1 to MMP-28 except MMP-20 and MMP-25 (Anacker et al. 2011). However, we have investigated the role of four MMPs that is MMP1, MMP2, MMP3 and MMP9 associated with migration/invasion of extravillous trophoblast cells. In this study, significant increase in the expression of MMP1 at transcript and proteins levels in HTR-/SVneo cells treated with HGF as compared to untreated cells under hypoxic condition was observed (Table 1, Fig. 3). MMP1 degrades interstitial collagen and is abundantly expressed in EVTs closer to decidua (Huppertz et al. 1998). MMP1 also plays an important role during cytotrophoblast invasion, as low levels of MMP1 are reported in placental complications like preeclampsia and fetal growth restriction (FGR) (Lian et al. 2010; Deng et al. 2015). Moreover, significant increase in MMP1 expression was also observed under hypoxia as compared to cells incubated under normoxia, which suggest that increased MMP1 may have a role in migration/invasion at basal level under hypoxia. In addition to MMP1, increase in expression of MMP9 both at mRNA and protein levels was also observed (Table 1, Fig. 3) in HGF treated HTR-8/SVneo under hypoxic conditions. However, under normoxia, increase in MMP2 and MMP3 expression was observed in HGF treated HTR-8/SVneo cells as compared to untreated control cells. This is in agreement with previous study, where increase in MMP2 expression under normoxic condition was observed, while no change in MMP9 expression was seen in HTR-8/SVneo cells treated with HGF (Liu et al. 2012). Further, these results concur with the reports that have previously shown that hypoxia favours secretion of MMP9 over MMP2 during trophoblast invasion as depicted in first trimester trophoblast cell lines (Onogi et al. 2011; Kobara et al. 2013). MMP3 also known as stremolysin-1 is widely expressed in placental and trophoblast cultures (Maquoi et al. 1997). Decrease in expression of MMP3 was observed, in EVT cells located near spiral arteries of the placenta of women suffering from preeclampsia (Reister et al. 2006). Besides HGF, IL-1β also stimulate MMP3 expression, which regulate trophoblast cells motility through up regulation of IGF/IFGBP network (Coppock et al. 2004; Husslein et al. 2009).

Imbalances in the MMP:TIMP ratios have been implicated in pregnancy disorders (Fortunato et al. 1999) and may act as biomarker for determining the severity of disease (Ferrer-Agüero et al. 2009). Importantly, higher level of MMP9:TIMP1 and MMP9:TIMP2 ratios was found in women with preterm labor as compared to term (Sundrani et al. 2017). Significant decrease in the expression of only TIMP1 at the protein level in HGF treated HTR-8/SVneo cells under hypoxic conditions was observed (Fig. 5a). A significant increase in the ratio of MMP2:TIMP1 and MMP3:TIMP1 in HGF treated cells under normoxia and MMP9:TIMP1 and MMP1:TIMP1 in HGF treated cells under hypoxia was observed (Fig. 6).

It has been known that HGF regulates trophoblast migration through activation of MAPK and PI3K signaling pathways under normoxic condition (Cartwright et al. 2002). Activation of pERK½ in HTR-8/SVneo cells treated with HGF under normoxia was reported previously by our group (Chaudhary et al. 2018). In the present study, we observed an increase in pAkt (Thr308) in HTR-8/SVneo cells treated with HGF under normoxic conditions (Fig. 8). However, it is not clear which signaling pathways are involved in HGF-mediated increase in HTR-8/SVneo cell migration/invasion under hypoxic conditions. Here in this study, phosphorylation of both ERK½ and Akt proteins has been observed in response to HGF treatment under hypoxic conditions (Figs. 7 and 8). In addition, we have also observed gradual increase in pERK½ levels in untreated cells under hypoxia, which is in agreement with the previous findings done in human microvascular endothelial cells-1 (HMEC-1) (Minet et al. 2000). A previous study has reported a significant increase in the levels of pAkt and p38, but no change in pERK½ levels in human ovarian carcinomas cells Hey-A8 (Xu et al. 2004). However, we have not observed any significant increase in the expression of pAkt (Thr308) in HTR/SVneo cells under hypoxia alone (Fig. 8a). This suggests that hypoxia mediated cell signaling pathways behave differently in cancer and trophoblast cell lines. The activation of ERK1 phosphorylation was observed to be significantly higher than ERK2 in HGF treated HTR-8/SVneo cells, while ERK2 and not ERK1 was up regulated in HGF induced motility of non-small cell lung carcinoma (NSCLC) (Radtke et al. 2013). Thus it may be possible that phosphorylation of ERK1 may be more relevant in regulating HGF-mediated HTR-8/SVneo cell migration, while ERK2 control HGF-mediated increase in migration of cancer cells. The activation of both the ERK½ and PI3K signaling is important in HGF-mediated migration/invasion of HTR-8/SVneo cells, as inhibition of MAPK by U0126 and PI3K pathway by LY294002 led to concomitant decrease in cell migration/invasion as compared to HTR-8/SVneo cells treated with HGF without pre-treatment of pharmacological inhibitor (Figs. 9 and 10).

HIF-1α is known to be expressed in early placenta and its expression decreases as the pregnancy progresses (Caniggia et al. 2000). Although HIF-1α is regulated by hypoxia in general, others factors like cytokines and hormones also regulate its expression under normoxic and hypoxic conditions. In the present study, a significant increase in expression of HIF-1α at protein level in the nucleus of HGF treated HTR-8/SVneo cells incubated under hypoxic conditions was observed as compared to untreated cells in Western blot and immunofluorescence (Fig. 11). This is in agreement with previous studies, where increase in expression of HIF-1α in nuclear extract of HepG2 hepatoma cells treated with HGF under normoxia as well as in hypoxia has been reported (Tacchini et al. 2001; Tacchini et al. 2004). Additionally, ~30% decrease in migration of HIF-1α knockdown cells treated with HGF was observed as compared to control siRNA transfected cells treated with HGF under hypoxic conditions (Fig. 12). This loss in migration of HTR-8/SVneo cells may be due to HGF- mediated decrease in HIF-1α expression by HGF under hypoxia. This conclusion is further supported by the observed decrease in the expression of HIF-1α after inhibition of MAPK and PI3K signaling pathways in HGF treated HTR-8/SVneo (Fig. 13).

Conclusion

To summarize, hypoxia in synergy with HGF increases HTR-8/SVneo cells migration/invasion as compared to cells under normoxia. Further, this increase in migration involve up regulation of MMP2 and MMP3 under normoxia and MMP1 and MMP9 under hypoxia and along with down regulation of TIMP1 (Fig. 14). HGF regulates HTR-8/SVneo cell migration/invasion by activation of MAPK and PI3K signaling pathways and also by up-regulation of HIF-1α expression under hypoxia (Fig. 14). The present findings use a trophoblast cell line i.e. HTR-8/SVneo; hence it is imperative to confirm the main experimental findings in primary EVT cells.

Fig. 14.

Fig. 14

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HGF-mediated migration/invasion of HTR-8/SVneo cells under normoxic and hypoxic conditions. Schematic representation of the role of MMPs and MAPK/PI3K signaling pathways in HTR-8/SVneo cells migration/invasion following treatment with HGF under normoxic and hypoxic conditions. Treatment of HTR-8/SVneo with HGF under normoxia led to activation of ERK½ and Akt concomitant with increased expression of MMP2 and MMP3 which increase cell migration/invasion. Under hypoxia alone, increase in expression of MMP1 and activation of pERK½ were observed, which may be responsible for increase in migration/invasion. Treatment of HTR-8/SVneo cells with HGF under hypoxia led to an increase in expression of MMP1 & MMP9, downregulation of TIMP1 and activation of both ERK½ and Akt signaling pathways which may be responsible for further increase in migration/invasion as compared to cells without treatment with HGF under hypoxia. Activation of ERK½ & Akt under hypoxia after treatment with HGF additionally recruit HIF-1α in the nucleus that facilitate HTR-8/SVneo cells migration under hypoxia

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Acknowledgements

This work was supported by Department of Health Research, Government of India under grant (GIA/28/2014); Department of Biotechnology, Government of India under grant (BT/PR12312/MED/30/1424/2014); J. C. Bose National Fellowship by Science and Engineering Research Board, Department of Science and Technology, Government of India to SKG under grant (SB/S2/JCB-040/2015). SKG would like to acknowledge National Institute of Immunology, New Delhi, India for additional financial support. The funding bodies were not involved in the design of the study, data analysis or writing the manuscript.

Authors’ contributions

PC, SKG and GSB were involved in project conception and experimental design. PC carried out the experiments. PC, SKG, GSB and RCS interpreted the data thus obtained and were involved in writing the manuscript.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

Footnotes

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Contributor Information

Piyush Chaudhary, Email: piyush_own@hotmail.com.

Gosipatala Sunil Babu, Email: sunil_gos@yahoo.com.

Ranbir Chander Sobti, Email: rcsobti@pu.ac.in.

Satish Kumar Gupta, Email: skgupta@nii.ac.in.

References

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