Human chorionic gonadotropin stimulates trophoblast invasion through extracellularly regulated kinase and AKT signaling.

Placental tissues of early pregnancy (n = 17, between 7 and 10 wk) and late (n = 3 between 36 and 40 wk) were obtained from legal abortions and cesarean sections of uncomplicated pregnancies, respectively. Use of tissues was approved by the local ethical committee and required informed consent of patients. Cytotrophoblasts of term (n = 3) and first-trimester (n = 3) placentae were isolated by enzymatic dispersion, Percoll (5–70%) density gradient centrifugation, and immunopurification as described ( 15 , 29 , 30 ). Cell preparations were cultivated on gelatin-coated (1%) dishes and were routinely checked at the time of isolation (24 h) using immunofluorescence with cytokeratin 7 (clone OV-TL 12/30, 8.3 μ g/ml; Dako, Glostrup, Denmark) and vimentin antibodies (clone Vim 3B4, 1.2 μ g/ml; Dako) to detect trophoblasts (>99%) and stromal cells (<1%), respectively. First-trimester villous fibroblast (n = 3) were isolated from gradients of trypsinized placental material (between 25 and 35% Percoll) and passaged two times in DMEM supplemented with 10% fetal calf serum (FCS). Villous fibroblasts were characterized by vimentin immunocytochemistry (100%), and a contamination with trophoblasts was excluded by cytokeratin 7 staining. Decidual fibroblasts (n = 3) were isolated from decidual tissue attached to first-trimester placentae using enzymatic digestion with 2 mg/ml collagenase I (484 IU/ml; Life Technologies, Inc., Paisley, UK) and 0.5 mg/ml DNase I (Sigma, St. Louis, MO) as described ( 31 ). Isolated cells were seeded in DMEM/F-12 supplemented with 10% FCS (Biochrom AG, Berlin, Germany). Fibroblasts were characterized after first passage by immunofluorescence and were positive for vimentin (100%) and pan-keratin (1%) but negative for CD45 and CD56. Small pieces (2 × 2 mm) of villous tissue of different first-trimester placentae (n = 14) were dissected under the microscope and cultivated on collagen I-coated dishes in serum-free medium (DMEM/Ham’s F12 with 50 μ g/ml gentamicin) as described elsewhere ( 32 , 33 ). For analyses of LH/CG receptor expression, pure extravillous trophoblasts, which had migrated from anchoring sites, were mechanically separated from villous material after 72 h and RNA/protein were extracted as previously mentioned ( 34 , 35 ). Approximately 50 dissected villi per placenta were attached on plastics coated with a thin layer of collagen I using coverslips. After formation of EVTs (72 h), villi were mechanically removed from the dishes using tweezers as mentioned ( 34 , 35 ). The residual migratory EVT (100% cytokeratin 7 positive) were incubated with serum-free medium the absence or presence of hormone. For analyses of ERK and AKT phosphorylation, pure EVT of each three different placentae were incubated with 50 IU/ml CG for 10 and 15 min. Pure, urinary CG (C-0434; Sigma) used in this study was previously shown to lack contaminating EGF ( 27 ). Subsequently each EVT pool was lysed to obtain cellular protein extracts. For detection of secreted amounts of matrix metalloproteinase (MMP)-2 and MMP-9, two different pure EVT pools were treated with 5 and 50 IU/ml CG for 48 h. After concentration (see below) supernatants were snap frozen and stored at −80 C for further analyses. Cytotrophoblastic SGHPL-5 cells exhibiting features of invasive trophoblasts cells, such as human leukocyte antigen-G and cytokeratin 7 expression, were cultivated in a 1:1 mixture of DMEM/Ham’s F-12 supplemented with 10% FCS (Gibco BRL Life Technologies, Paisley, UK) as described ( 36 , 37 ). To analyze effects of CG on trophoblast migration, serum-free explant cultures were seeded for 4 h on collagen I, allowing for anchorage, and then stimulated with 5 IU or 50 IU/ml CG. Eight anchoring villi were analyzed per condition and experiments were repeated with three different placentae. After 48 h explant cultures were digitally photographed. For quantification the area of outgrowth was measured using the imaging software CellP̂ (Olympus, Hamburg, Germany). To analyze migration and invasion of SGHPL-5, cells were seeded on either uncoated transwells or growth factor-reduced Matrigel invasion chambers (BD Biosciences, Bedford, MA; 50,000 cells/chamber). After attachment cells were starved for 6 h and subsequently incubated with either 5 IU or 50 IU/ml CG. After 24 h noninvaded cells on the upper side of the inserts were removed by a cotton swap. Cells on the lower surface were fixed in ice-cold methanol and stained with Papanicolaou’s solution 1a Harris’ hematoxylin solution (Merck, Darmstadt, Germany). Filters were excised and mounted in Aquatex (Merck). For evaluation cells were counted using Olympus cell imaging software. In blocking studies organ cultures or SGHPL-5 cells were preincubated for 1 h with 10 μ m UO126 [MAPK kinase (MEK) inhibitor] or 10 μ m LY294002 [phosphatidylinositide 3-kinase (PI3K) inhibitor] before supplementation of CG. For detection of proliferation in first-trimester villous explant cultures, 5-bromo-2′-deoxyuridine (BrdU) labeling and detection kit I (Roche, Mannheim, Germany) was used as previously mentioned ( 34 , 35 ). Briefly, serum-free floating villous explants were treated with 10 μ m BrdU and incubated for 24 h in the absence or presence of CG. Subsequently tissues were snap frozen in liquid nitrogen, and eight serial sections per explant were cut and fixed in ethanol-Fixans (15 m m glycine per 70% ethanol, 20 min, −20 C). BrdU was detected with primary monoclonal anti-BrdU and secondary antimouse Ig fluorescein antibodies. Sections were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) and cytokeratin 7 and digitally photographed. The ratio (percentage) of BrdU/4′,6′-diamino-2-phenylindole labeling was counted in cytotrophoblasts of cell columns using Olympus CellP̂ imaging software. Seventeen untreated explants (36207 nuclei), 18 explants stimulated with 5 IU/ml CG (39803 nuclei), and 17 explants incubated with 50 IU/ml CG (36446 nuclei) of three different placentae were analyzed. For evaluation of SGHPL-5 cell proliferation, serum-free cultures were seeded on plastics (1000 cells/cm 2 ) and stimulated with 5 IU or 50 IU/ml CG for 24, 48, and 72 h. Cumulative cell numbers were counted using a multichannel electronic cell counter (CASY-I; Schärfe Systems, Reutlingen, Germany). Tissues of total placenta and explant cultures were frozen in liquid nitrogen and homogenized with a microdismembrator (Braun; Biotech International, Melsungen, Germany). Total RNA of primary cultures, cell lines, and tissues was isolated using Tri-reagent (Molecular Research Center Inc., Cincinnati, OH) as previously mentioned ( 35 ). Quality and quantity of the extracted RNA was analyzed with the Bioanalyzer 2100 (Agilent, Palo Alto, CA). Reverse transcription and PCR were done as previously mentioned ( 38 ). Cycle numbers were optimized within the linear range of individual PCRs. Oligonucleotide primers, annealing temperatures, product sizes, and cycle numbers were as follows: LH/CG-R receptor-s, 5′-ACCTTGAAGTTGTCCACAT TGC-3′, LH/CG receptor-a, 5′-ACCGAATTG AACAGTGCA TCTC-3′ (57 C, 508 bp, 40 cycles), β -actin-s, 5′-GACAGCAGTCGG TTGGAG C-3′, β -actin-a, 5′-CAGGTAAGCCCT GGCTGC-3′ (55 C, 398 bp, 22 cycles). In all experiments, a possible DNA contamination was checked by negative control RT-PCR in which reverse transcriptase was omitted in the reverse transcription step. The PCR products were analyzed on 1.5% agarose gels containing ethidium bromide and photographed under UV radiation. PCR fragments were sequence verified on a 16-capillary sequencer by using the nonradioactive ABI PRISM terminator cycle sequencing ready reaction kit as specified by the supplier (Applied Biosystems, Foster City, CA). Western blot analyses were performed using standard protocols as recently done ( 35 , 38 ). For MMP analyses, 1 ml of supernatant of either SGHPL-5 cells or pure EVT pools, which had been stimulated for 48 h with CG, was concentrated 25-fold (to ~200 μ g/ml) using Ultrafree-MC filter tubes (Millipore, Billerica, MA). Equal amounts of protein lysate (10 μ g) were separated on 10% sodium dodecyl sulfate/polyacrylamide gels and transferred onto polyvinyl difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK). After blocking filters were incubated overnight (4 C) with polyclonal rabbit antibodies against human LH/CG receptor [1:5000 (39)], p44/42 MAPK (1:1000, Cell Signaling, Beverly, MA), phospho(Thr202/ Tyr204)-p44/42 MAPK (1:1000; Cell Signaling), AKT (1:1000; Cell Signaling), phospho-AKT (Ser473, 1:1000; Cell Signaling, Danvers, MA), MMP-2 (1:1000; Cell Signaling), or MMP-9 (1:1000; Cell Signaling). After 1 h of treatment (room temperature) with secondary antibodies (antimouse or antirabbit Ig horseradish peroxidase linked; Amersham; 1:50,000) signals were developed by using ECL Plus Western blotting detection system (GE Healthcare). To analyze protein loading, filters were stripped as previously mentioned ( 35 ) and incubated with actin antibodies (mouse monoclonal, 1:5000; Sigma). PageRuler prestained protein ladder (Fermentas, St. Leon-Rot, Germany) was used as a molecular size marker. Gelatinase (MMP-2, MMP-9) secretion was evaluated in 50-fold concentrated supernatants of SGHPL-5 cells or pooled villous explant cultures (eight explants per stimulation, three different placentae) using substrate gel zymography as recently mentioned ( 34 ). Briefly, conditioned medium containing 500 ng of protein was mixed with the same volume of 2× sample buffer [0.125 m Tris-HCl (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate, 0.005% bromophenol blue], incubated for 10 min at room temperature, and loaded onto nonreducing 10% polyacrylamide gels containing 0.1% gelatin (Sigma). Proteins were separated by electrophoresis at a constant voltage of 125 V/gel for 90 min as described by the manufacturer. Gels were washed with Novex renaturing buffer (Invitrogen, Carlsbad, CA) with gentle agitation for 30 min and incubated overnight at 37 C with Novex developing buffer. Gels were stained with 0.5% Coomassie brilliant blue R-250 and destained overnight in 10% acetic acid per 30% methanol. Statistical analyses were performed with Student’s t test or ANOVA using SPSS 14 (SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant.

To test the suitability of cell systems for analyses of CG-dependent effects, LH/CG receptor expression was investigated in different trophoblast cultures ( Fig. 1 ). Semiquantitative RT-PCR analyses suggested receptor expression in placental tissues, primary villous and extravillous trophoblasts, and villous and decidual fibroblasts as well as trophoblast cell lines ( Fig. 1A ). Accordingly, in Western blot analyses, specific signals at 90 and 75 kDa, corresponding to the mature and precursor forms of the LG/CG receptor ( 40 ), were detected in the different cultures systems ( Fig. 1B ). Subsequently the influence of CG on trophoblast cell migration and invasion was tested in a dose-dependent manner ( Fig. 2 ). Compared with controls (100%), 50 IU/ml of the hormone increased migration and invasion of trophoblastic SGHPL-5 cells to 192 and 247%, respectively ( Fig. 2A ). EGF (5 ng/ml), a well-known stimulator of trophoblast motility ( 27 , 41 , 42 ), increased migration and invasion to 415 and 444% each. Similarly, 5 and 50 IU/ml of CG stimulated trophoblast outgrowth and migration in first-trimester villous explant cultures seeded on collagen I ( Fig. 2B ). Quantification revealed that the area of outgrowth increased to 271, 301, and 480% in the presence of 5 IU/ml CG, 50 IU/ml CG, and 5 ng/ml EGF, respectively ( Fig. 2C ). To analyze whether CG may influence SGHPL-5 cell proliferation, cumulative cell numbers were determined in the absence or presence of CG ( Fig. 3A ). The hormone did not significantly change proliferation at 24, 48, or 72 h of incubation. Similarly, CG-dependent proliferation was investigated in villous explant cultures using BrdU labeling ( Fig. 3B ). No significant changes in numbers of BrdU-labeled nuclei of cell columns could be observed at 24 h of stimulation using 5 or 50 IU/ml CG. To gain insights into the molecular mechanisms of CG-induced trophoblast migration and invasion, activation of critical signal transduction pathways was investigated in SGHPL-5 cells ( Fig. 4 ). Western blot analyses revealed that CG dose-dependently induced phosphorylation of ERK with peak levels at 15 min of treatment ( Fig. 4A ). Densitometrical scanning of Western blots (n = 3) revealed that phosphorylation of both ERK-1 and -2 increased to 260% ± 49 sd at 15 min of incubation with 50 IU/ml CG. (controls set at 100%). Similarly, strongest CG-induced phosphorylation of AKT could be observed at 15 min of stimulation with either 5 or 50 IU/ml of hormone ( Fig. 4B ). At the latter condition, signals of phospho-AKT increased to 228% ± 13 sd . To study whether CG had similar effects on invasive primary trophoblasts, activation of ERK and AKT was studied in pure EVTs isolated from villous explant cultures ( Fig. 5 ). Western blot analyses suggested that stimulation with 50 IU/ml CG increased phosphorylation of both ERK ( Fig. 5A ) and AKT ( Fig. 5B ). Western blot analyses were performed to study the effects of either ERK or AKT inhibition in SGHPL-5 cells. Treatment of cultures with the specific MEK inhibitor UO126 reduced basal as well as CG-stimulated ERK phosphorylation ( Fig. 6A ). Similarly, basal and CG-induced AKT phosphorylation was diminished in the presence of the PI3K inhibitor LY294002 ( Fig 6B ). To evaluate whether CG-induced activation of ERK and AKT might play a role in CG-mediated trophoblast invasion, Matrigel transwell assays in the presence of the inhibitors were performed ( Fig. 6C ). Compared with controls, basal migration was not significantly affected upon preincubation with LY294002 or UO126. However, compared with CG-stimulated motility at either 5 or 50 IU/ml, invasion was significantly reduced in the presence of either UO126 or LY294002. Similar to their effects on SGHPL-5 cells, pretreatment of villous organ cultures with UO126 or LY294002 significantly diminished the area of CG-stimulated outgrowth ( Fig. 6D ). Gelatin zymography revealed that CG stimulated accumulation of the pro-form (72 kDa) of MMP-2 in SGHPL-5 cell supernatants ( Fig. 7A ). Pro-MMP-9 was hardly detectable in the concentrated supernatants, and its expression was only slightly induced in the presence of the hormone. Similarly, CG induced secretion of 72 and 62 kDa (active) MMP-2 in first-trimester villous explant culture ( Fig. 7B ), whereas pro-MMP-9 was less stimulated. CG-stimulated secretion of the 72-kDa pro-MMP-2 was also noticed in Western blot analyses of SGHPL-5 supernatants ( Fig. 8A ). Production of active MMP-2 could be observed in the presence of 50 IU/ml CG. However, this was not consistently observed in every experiment and required long exposure of films. Densitometrical scanning (n = 3) revealed that compared with controls (100%), CG increased pro-MMP-2 to 166% ± 36 sd and 266% ± 45 sd at 5 IU and 50 IU/ml CG, respectively. Low levels of pro-MMP-9 were only significantly stimulated at 50 IU/ml (164 ± 8%). Accordingly, CG treatment of two different pools of pure EVT promoted expression of soluble, pro- and active MMP-2 ( Fig. 8B ). However, MMP-9 was undetectable in these highly concentrated supernatants (not shown). Incubation of SGHPL-5 cells with either UO126 or LY294002 reduced basal as well as CG-stimulated MMP-2 and MMP-9 expression ( Fig. 8C ). Compared with controls (100%), MMP-2 stimulated by 50 IU/ml CG (255% ± 50 sd ) decreased to 51% ± 25 sd and 35 ± 17 sd in the presence of UO126 and LY294002, respectively. Similarly, MMP-9 expression at 50 IU/ml CG (170% ± 28 sd ) decreased to 29% ± 14 sd and 18% ± 11 sd upon incubation with UO126 and LY294002, respectively.

Expression of the LH/CG receptor in diverse trophoblast and fibroblast subtypes of the fetal-maternal interface supports the assumption that CG has a pivotal role in implantation, decidualization, and placental differentiation. Here we confirm previous results demonstrating LH/CG receptor expression in villous and invasive trophoblasts as well as in fibroblasts of the placental bed ( 20 , 40 , 43 ). Whereas the multiple functions of CG on diverse target cells of reproductive tissues are still being elucidated, we attempted to shed light on its role in trophoblast invasion. Besides using SGHPL-5 trophoblast cells, retaining features of EVTs ( 36 , 37 ), first-trimester villous explant cultures and pure EVT were used to analyze CG-dependent effects on proliferation, motility, signaling, and MMP expression. CG-dependent Matrigel invasion assays of SGHPL-5 cells as well as evaluation of trophoblast migration in villous explants cultures suggest that the hormone indeed promotes trophoblast motility. Therefore, data obtained in primary trophoblasts and nonimmortalized SGHPL-5 cells are in accordance with previous results demonstrating that CG increased invasion/migration of immortalized and tumorigenic trophoblasts ( 23 , 24 ). With respect to proliferation, CG may negatively affect growth ( 44 ) but was also shown to provoke a mitogenic response in various reproductive cell types ( 45 – 47 ). However, in SGHPL-5 cells, CG did not affect proliferation up to 72 h of stimulation, suggesting that the increase in invasion cannot be explained by an elevated mitogenic response. Similarly, CG did not significantly alter BrdU labeling in cell column trophoblasts of first-trimester explant cultures, indicating that CG-dependent outgrowth could be mainly due to elevated cell motility. Hence, abundant levels of the hormone during early pregnancy may predominantly stimulate trophoblast invasion but not affect placental growth. Different forms of CG are present in urinary preparations such as intact, cleaved, nicked, and glycosylated proteins ( 48 ). Whether these various hormones may have distinct effects on proliferation or invasion still has to be determined. Along those lines, it was recently suggested that supernatants of primary EVT may promote invasion through its high proportion of hyperglycosylated CG ( 49 ). Interestingly, CG of villous trophoblast supernatants was inefficient in promoting motility, compared with the hormone of EVT supernatants ( 49 ). This may suggest that the motility-promoting effects of the urinary hormone used in our study could be mainly due to its portion of hyperglycosylated CG. Signaling pathways involved in CG-induced trophoblast invasion remain largely elusive. In HTR-8/SVneo cells, CG-stimulated trophoblast migration may involve the IGF-II system. The hormone was suggested to increase the externalization rate of IGF-II/mannose 6-phosphate receptor and hence bioavailability of IGF-II ( 24 ). Here we demonstrate that activation of ERK and AKT signaling are crucially involved in CG-dependent trophoblast motility. Indeed, Raf/MEK/ERK and PI3K/AKT signaling are thought to play key roles in the invasive differentiation process because abundant growth factors of the fetal-maternal interface, such as EGF or IGF-II, stimulate trophoblast migration through activation of both pathways ( 28 ). CG activated ERK and AKT in not only SGHPL-5 cells but also pure, nonproliferating EVT, suggesting a direct role of the signaling pathways in the regulation of invasive trophoblasts. Although ERK and/or AKT signaling are known to mediate a mitogenic response upon activation of G protein-coupled receptors ( 50 ), we failed to detect significant CG-dependent changes in proliferation. However, inhibition of the kinases reduced CG-stimulated SGHPL-5 cell invasion as well as trophoblast motility in explant cultures. Interestingly, inhibition also affected basal migration in villous explant cultures but not SGHPL-5 cells. This could suggest that the organ cultures/EVT produce higher levels of ERK/AKT-activating growth factors acting in a paracrine or autocrine manner. CG-induced signaling through these pathways may have multiple effects on migratory trophoblasts such as changes in the cytoskeleton, expression of invasion-promoting growth factors, or secretion of matrix-digesting proteinases. Among the latter, expression of the gelatinases, MMP-2 and MMP-9, has been closely linked to trophoblast invasiveness because various promigratory growth factors were shown to activate the particular enzymes ( 51 ). MMP-2 is thought to be the predominant enzyme in early placentae, but its expression decreases after 11 wk gestation. In contrast, MMP-9 is undetectable at 6 wk of pregnancy but gradually increases after wk 7. Accordingly, blocking of MMP-9 did not affect trophoblast invasion before the ninth week of pregnancy ( 52 ). Compared with MMP-2, gelatin zymography and Western blot analyses detected only low levels of the proform of MMP-9 in concentrated supernatants of SGHPL-5 cells or first-trimester villous explant cultures. This could be due to the fact that the majority of first-trimester placentae were usually obtained between 7 and 9 wk gestation. Interestingly, MMP-9 was absent from two different pure EVT pools of 8 and 10 wk of pregnancy despite its weak expression in explant cultures. This might be explained by the fact that during early pregnancy MMP-2 is predominantly expressed in EVT, whereas MMP-9 is mainly secreted from villous trophoblasts ( 53 ), the latter being present in the whole organ cultures. Cell models used in this study mainly mimic interstitial trophoblast invasion of early pregnancy. In these cultures low levels of pro-MMP-9 were only marginally stimulated by CG, compared with MMP-2, suggesting that MMP-9 may play only an ancillary role in early, hormone-dependent trophoblast invasion. However, it is possible that the effects of CG on MMP-9 secretion become more important when expression of the protease increases at later stages of pregnancy. Interestingly, CG induced not only total MMP-2 expression but also production of its active 62-kDa form in the different trophoblast model systems. Hence, it is possible that the hormone also activates MMP-2-processing enzymes such as MT1- or MT2-MMP, which had been detected in first-trimester placentae ( 54 , 55 ). Both AKT and ERK signaling are involved in CG-dependent regulation of MMP expression because inhibition of either kinase reduced hormone-stimulated MMP-2 or MMP-9 production. Thus, CG acts similar to other promigratory growth factors such as EGF increasing MMP expression through activation of both ERK and AKT signaling ( 56 ). Besides direct effects of the hormone on protease expression, CG may also indirectly affect trophoblast invasiveness by stimulating other inducers of gelatinases such as vascular EGF (vEGF) ( 57 ). However, CG could also play a more central role in trophoblast invasion. Growth factors such as leptin may promote MMP expression partly through stimulation of CG secretion ( 58 ). In conclusion, the data suggest that CG increases trophoblast invasion and migration through activation of ERK and AKT signaling. CG-stimulated production of active MMP-2, particularly in nonproliferating EVT, supports the assumption that the gelatinase might be crucially involved in hormone-dependent trophoblast invasion. Further studies are needed to gain more insights into the diverse effects of the pregnancy hormone on invasive trophoblasts.