Autocrine-Paracrine Regulation of Human Trophoblast Invasiveness byInsulin-like Growth Factor (IGF)-II and IGF-Binding Protein (IGFBP)-1

G. S. Hamilton,* J. J. Lysiak,* V. K. M. Han,*,† and P. K. Lala*,1

*Department of Anatomy and Cell Biology, and †Department of Pediatrics, University of Western Ontario,London, Ontario, Canada N6A 5C1

Trophoblast growth and invasion of the uterus aretightly regulated by locally produced factors. Sinceinsulin-like growth factor (IGF)-II is produced bythe invasive human extravillous trophoblast (EVT)cells and IGF-binding protein (IGFBP)-1 by the de-cidual cells in situ that are in proximity to eachother, we examined the possible influence of thesemolecules on proliferation, migration, and invasive-ness of first-trimester EVT cells in culture. TheseEVT cell functions were respectively measured by3H-TdR uptake, in vitro migration, and invasion as-says. Secretion of invasion-associated enzymes wasassessed by zymography, and IGF-binding moietieson the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cellswas stimulated by addition of serum but unaffectedby a wide range of IGF-I, IGF-II, and IGFBP-1 con-centrations. IGF-II and IGFBP-1 or their combina-tion stimulated EVT cell invasiveness and migra-tion, when assays were conducted in serum-reducedmedia. Affinity cross-linking studies failed to detectthe type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules in-cluding type 2 IGF receptor were identified on theEVT cell surface. IGF-II enhancement of invasionwas unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVTcell invasion, indicating that type 1 IGF receptorwas not responsible for the IGF-II effects. Secretionof gelatinases or plasminogen activators was unal-tered by IGF-II or IGFBP-1. We conclude that tro-phoblast-derived IGF-II and decidua-derived IG-FBP-1 provide autocrine/paracrine enhancement oftrophoblast invasiveness largely by stimulating mi-gration, an essential step in invasion. © 1998 Academic

Press

Key Words: IGF-II; IGFBP-1; trophoblast invasive-ness; trophoblast migration.

INTRODUCTION

In a number of mammalian species including hu-mans, the blastocyst must cross the uterine epitheliumand invade the underlying endometrial stroma, glands,and blood vessels in order to establish proper fetoma-ternal exchange of molecules. This process requiresthat specific embryonic trophoblast cells exhibit aninvasive phenotype. During further development of theplacenta in the human, cytotrophoblast stem cells inthe chorionic villi give rise to a subset of highly prolif-erative, migratory, and invasive population whichbreak out of the villi and invade the decidua and theuteroplacental arteries. These “extravillous tropho-blast” (EVT) cells exhibit a selective phenotype in situ,including some proximo-distal changes in the expres-sion pattern of phenotypic markers [1]. The optimalextent of trophoblast invasion of the uterus and itstransient nature are believed to result from tightlyregulated paracrine and autocrine systems involvingthe trophoblast and cells of the maternal decidua intowhich the trophoblast invade [2, 3]. In fact, a variety ofgrowth factors produced at the fetomaternal interfacehave already been shown to influence trophoblast pro-liferation, migration, and invasion. For example, trans-forming growth factor (TGF)b reduces proliferation [4],migration [5], and invasiveness [6–8] of EVT cellspropagated in vitro, whereas epidermal growth factorreceptor (EGF-R) ligands (EGF, TGFa, and amphi-regulin) enhance proliferation of these cells withoutsignificantly affecting their invasiveness [9–11].

Immunoreactive insulin-like growth factor (IGF)-IIhas been identified in a variety of cells of the humanplacenta and decidua throughout gestation [12]. IGF-IImRNA has been localized in both the villous and theextravillous cytotrophoblast [13, 14]. However, onlythe extravillous trophoblast cells are found to retainthe mRNA expression throughout gestation [14]. This67 amino acid, single chain polypeptide growth factorshares homology with IGF-I and human insulin and isbelieved to play important roles in embryonic and fetaldevelopment [15]. IGF-II mediates many of its effectsthrough binding to the type 1 IGF receptor which pos-

1 To whom correspondence and reprint requests should be ad-dressed at Department of Anatomy and Cell Biology, The Universityof Western Ontario, London, Ontario, Canada, N6A 5C1. Fax: (519)661-3936. E-mail: pklala@julian.uwo.ca.

0014-4827/98 $25.00147Copyright © 1998 by Academic Press

All rights of reproduction in any form reserved.

EXPERIMENTAL CELL RESEARCH 244, 147–156 (1998)ARTICLE NO. EX984195

sesses tyrosine kinase activity and which binds bothIGF-I and IGF-II with equal affinity [16, 17]. However,IGF-II is also capable of binding to the type 2 IGFreceptor (mannose-6-phosphate receptor) which hasgreater affinity for IGF-II than for IGF-I. While therole of this receptor in mediating the biological signalsof IGF-II remains largely obscure [16–19], it is be-lieved to promote the transport of lysosomal enzymesfrom their sites of synthesis to the endosomal/prelyso-somal compartment [17]. Cytotrophoblast cells in situhave been shown to express the mRNA for both types ofreceptor [13].

In addition to binding specific cell surface receptors,IGFs also bind at a high affinity to a family of bindingproteins (BP) present in blood and interstitial fluids.Six IGFBPs (IGFBP-1 to 6) have been identified andcharacterized [20]. Recently, a new family of IGFBP-related proteins which bind IGFs at a lower affinityhave been described. They have been tentativelynamed IGFBP-7 to 10 [21]. High affinity IGFBPs havebeen shown to influence IGF actions in a positive or anegative manner [20]. In certain circumstances, theBPs may enhance IGF-mediated biological actions byretarding the degradation and clearance of IGFs orfacilitating the binding of IGFs to their receptors. Forexample, IGFBP-1 and -2 possess an RGD (arginine-glycine-aspartic acid) sequence which may bind toRGD-binding sites on certain cell surface integrins(e.g., a5/b1) to exert IGF-independent actions or poten-tiate binding of IGFs to their receptors. In other cir-cumstances, IGFBPs may reduce IGF biological ac-tions by sequestering the ligands away from theirreceptors [20, 22]. While immunoreactive IGFBP-1 andIGFBP-2 peptides have both been identified in thehuman placenta [12], in situ hybridization studieshave revealed that mRNA encoding the IGFBPs areexpressed only in the decidual cells [14]. Furthermore,decidual cells in culture release significant amounts ofIGFBP-1 [23], and in situ expression of IGFBP-1mRNA is most abundant in decidual cells adjacent toinvading EVT cells which express IGF-II mRNA [14].These observations suggest that decidua-derivedIGFBP-1 and trophoblast-derived IGF-II may play in-teractive roles in the regulation of invasive trophoblastfunction. Therefore, we examined the effects of IGF-IIand IGFBP-1 on the proliferation, migration, and in-vasiveness of cultured human first-trimester EVTcells, as well as possible mechanisms mediating theseeffects.

MATERIALS AND METHODS

Establishment of first trimester human extravillous trophoblast cellcultures. Human first-trimester EVT cell cultures were establishedaccording to the procedure described earlier from this laboratory [4,24]. Briefly, chorionic villi were collected from elective terminationsof first-trimester pregnancy and rinsed in cold RPMI 1640 medium

(Grand Island Biological Company, Grand Island, NY). Villi werethen mechanically minced and washed in medium, and the villusfragments were cultured as explants in complete medium (RPMI1640 containing 10% fetal calf serum (FCS), 200 mg/ml streptomycin,200 U/ml penicillin, and 0.50 mg/ml amphotericin). After 2–3 days,nonadherent explants and cells were removed and discarded and theremaining adherent explants and cells were expanded for 1–2 weeksprior to passage and characterization. Only those cultures proven tohave the phenotype of pure EVT cells after subsequent passageswere utilized for further experimentation.

Characterization of expanded and passaged trophoblast cell cul-tures with numerous markers was performed as described earlier [4,24]. Prior to each experiment, cells were routinely immunostainedfor cytokeratin to ensure that 100% of the cells were cytokeratinpositive. This confirmation was very important, since morphologyalone was an unreliable guide for cell identity. For example, as thecultures became more confluent, the incidence of spindle-shapedcells increased at the expense of polyhedral and multiangular cells,although every cell remained cytokeratin positive. In addition, asreported earlier [24], concomitant phenotyping revealed that thesecells were immunoreactive for class 1 HLA framework W6/32, IGF-IIpeptide, a1, a3, a5, av, b1 integrin subunits, and the vitronectinreceptor av b3/b5. They were negative for 63/D3 (macrophage mark-er), Factor VIII (endothelial cell marker), and a6, b4 integrins (villouscytotrophoblast markers). These cells also expressed HLA-G mRNAand protein when cultured on laminin or Matrigel (G. Aboagye-Mathiesen, and P. K. Lala, unpublished). This phenotypic profile wasthat of invasive EVT cells in situ [1, 24]. The cells were used at thirdto sixth passages.

Proliferation assay. Cell proliferation was assessed by [3H]thy-midine (3H-TdR) uptake [25] following a terminal 6-h pulse of3H-TdR to serum-starved trophoblast cells grown for 24 h underdifferent experimental conditions. Third to sixth passage first-trimester human EVT cells were cultured in RPMI complete me-dium for 24 h in 96-well microtiter plates (Flow Laboratories,McLean, VA) at a concentration of 104 cells/well. The cells werethen serum-starved for 3 days using 0.2% albumin in place of 10%FCS. On the fourth day, media were removed from the wells andreplaced with serum-free media containing varying doses (5– 625ng/ml) of recombinant IGF-I or IGF-II, or recombinant unphos-phorylated IGFBP-1 (0.5–500 ng), both purchased from UpstateBiotechnology. In other sets of wells, FCS was added to achieveconcentrations of 1 or 10%. Wells were loaded in quadruplicate18 h prior to exposure with 3H-TdR (1 mCi/ml) for 6 h. Followingthe 24-h incubation period, the medium was removed and 100 mlof 0.25% trypsin was added to each well for 15 min. Cells weresubsequently harvested with a Titertek cell harvester, which de-posits cells on strips of filter paper and disrupts them with re-peated distilled water washes to remove unbound water-solubleradioactivity. The filter paper strips were then immersed in scin-tillation fluid and b-counts were taken with a Beckman (Palo Alto,CA) scintillation counter. In our hands this proliferation assaycorroborates the results obtained with measurements of cellular-ity [26].

In vitro matrigel invasion assay. The influence of IGF-II andIGFBP-1 on invasiveness of first-trimester human EVT cells wasassessed by a Matrigel invasion assay [27] as modified in this labo-ratory [8], in which the percentage of cells passing through a Matri-gel barrier at specific intervals provides the invasion index. Thisassay requires that cells are capable of degrading as well as migrat-ing through the Matrigel barrier. Briefly, 200 ml of a 600 mg/mlsolution of Matrigel (Collaborative Research Inc., Bedford, MA) incold RPMI 1640 medium was placed on 6.5-mm-diameter transwellfilters with a pore size of 8 mm (Costar Corp., Toronto, ON) andair-dried for 8 h in a laminar-flow hood. Subconfluent cultures ofthird to sixth passage first-trimester trophoblast cells were incu-bated in the presence of 10 mCi/ml 3H-TdR in RPMI 1640 plus 10%

148 HAMILTON ET AL.

FCS for 72 h. Cells were then trypsinized, washed, and resuspendedin medium, and the number was adjusted to 2.5 3 105 cells/ml. A200-ml sample of the cell suspension in the presence of varying dosesof IGF-II or IGF-I (5 to 625 ng/ml) or recombinant, unphosphorylatedIGFBP-1 (0.5 to 500 ng/ml) was placed in the upper wells. Cells inculture medium alone served as controls and treatments were car-ried out in triplicate. All invasion assays were carried out in serum-reduced (1% FCS) media, since pilot studies [2] showed that thisserum concentration yielded optimum responses with IGF-II andIGFBP-I. A volume of 800 ml of medium was added to lower wells andthe transwells were then placed into the lower wells. After a 72-hincubation period, the media in the upper and lower wells wereremoved and placed in separate tubes. Upper wells were washedonce with PBS and the wash was pooled with the media removedfrom the upper well. To remove cells adhering to the lower well, 800ml of a 0.05% trypsin solution in PBS was placed in the lower wellwith the upper chamber reinserted for 20 min at room temperature.The trypsin solution was then removed and the lower wells werewashed once with 800 ml of PBS and pooled with the incubationmedia from the lower wells. Finally, each membrane was removedfrom the transwells with the aid of a scalpel and placed with thecontents of the upper chamber. Scintillation fluid was then added toeach of the tubes from each transwell and b-counts were taken witha Beckman scintillation counter. The invasion index at 72 h wascalculated as the amount of radioactivity in the lower wells ex-pressed as a percentage of the sum of the total radioactivity in upperand lower wells.

In additional experiments, invasion assays were carried out usingthe combination of IGF-II and IGFBP-1 to test functional interac-tions between these molecules, or receptor-blocking antibodies toassess the importance of accessibility to cell surface IGF type 1receptor and a5b1 integrins (fibronectin receptor). The latter exper-iment included IGF type 1 receptor-blocking antibody (aIR-3, Gen-zyme, purchased through Cadarlane Lab, Hornby ON, 25 mg/ml)with and without IGF-II at 125 ng/ml or integrin a5 b1 receptor-blocking antibody (anti-a5, P1D6, GIBCO; 25 mg/ml) with and with-out IGFBP-1 at 50 ng/ml.

In vitro transwell migration assay. The influence of IGF-II andIGFBP-1 or their combination on the migratory ability of first-tri-mester human EVT was tested under the same conditions as de-scribed above for the invasion assay. The migration assay was thesame as the invasion assay, except for the fact that no Matrigelcoating was provided on the Millipore membrane of the transwell, sothat the EVT cells did not have to degrade and cross the Matrigelbarrier.

Competitive ligand affinity cross-link assay. Third to sixth pas-sage human EVT were plated on 6-well plates (Corning; VWR Can-lab, Mississauga, Canada) containing RPMI 1640 complete mediaand allowed to reach confluence. Cells were then rinsed twice in cold“binding buffer” (0.1 M Hepes, 0.12 M NaCl, 5 mM KCl, 1.2 mMMgSO4, 8 mM dextrose, 10% bovine serum albumin; BSA). Ligandcompetition incubations were initiated by addition of 125I-labeledIGF-I or IGF-II (500,000 cpm/0.8 ml binding buffer) and variousconcentrations of cold IGF-I or IGF-II (0 to 200 ng/ml) or insulin (0 to10 mg/ml). Following overnight incubation at 4°C, wells were rinsedtwice with cold PBS and cells were exposed to cross-link buffer(binding buffer without BSA) containing 10 mM disuccinyl suberatefor 15 min at room temperature. The cross-link buffer was subse-quently removed, cells were lysed in stop solution (1.5 Tris, SDS,glycerol, bromphenol blue, and DTT), and lysates were boiled for 5min and subjected to a gradient (3 to 14%) polyacrylamide gel sep-aration. Gels were dried and exposed to X-ray film for 2 to 14 days.

Zymography. To determine whether IGF-II and/or IGFBP-1 in-fluences the secretion of gelatinases and/or plasminogen activatorsby human EVT cells, culture medium was used in substrate zymog-raphy following conditioning of third to sixth passage EVT cells withIGF-II and/or IGFBP-1. Gelatin was used as the substrate for gela-

tinase zymography while casein was used in plasminogen activatorzymography. Briefly, 20 ml of conditioned culture media was mixedwith denaturing sample buffer containing bromphenol blue dye,placed in the wells of 4% polyacrylamide minigels, and run through10% polyacrylamide separating gels containing either 60 mg/ml gel-atin or 1 mg/ml casein. Gels were electrophoresed at 120–140 V untilthe bromphenol blue bands had run 20 to 30 min beyond the bottomsof the gels. Gels were incubated at 37°C for 24 to 48 h in PBS solution(for gelatinase zymography) or 50 mM Tris-HCl, 0.2 M NaCl, 5 mMCaCl2, 0.5 mg/ml Brij 35, and 0.2 mg/ml NaN2 (for plasminogenactivator zymography). Gels were then stained in Coomassie bluestaining solution and destained in 20% methanol, 7.5% acetic acid.

Statistics and data presentation. Data were analyzed usingANOVA and when appropriate, the significance of differences be-tween treatment groups was determined using Duncan’s multiplerange test. Differences were considered significant at P , 0.05.

Most experiments were reproduced with EVT cells derived fromtwo first-trimester placentas; some experiments were reproducedwith EVT cells from a third placenta. Experiments with EVT cellsderived from a single placenta were reproduced at least twice. Thefollowing data presented for EVT cells from a single placenta arerepresentative of these results, unless indicated otherwise.

RESULTS

Effects of IGFs and IGFBP-1 on First-TrimesterHuman EVT Cell Proliferation

To examine whether IGF-I, IGF-II, or IGFBP-I in-fluenced EVT cell proliferation, assays were conductedwith serum-starved EVT cells cultured alone or in thepresence of a wide range of concentrations of thesemolecules. Addition of serum (1 or 10%) served aspositive controls. As shown in Fig. 1, 3H-TdR incorpo-ration by EVT cells did not change significantly (P .0.05) when the cells were cultured with increasingconcentrations of IGF-I, IGF-II (5 to 625 ng/ml), andIGFBP-1 (0.5–500 ng/ml). Cells were proven to behealthy and capable of proliferative response since pro-liferation was increased two- to fourfold (P , 0.01) byinclusion of FCS at 1 and 10%. These results revealedthat EVT cell proliferation was not influenced in thepresence of IGF-I, IGF-II, or IGFBP-1.

Effects of IGF-II and IGFBP-1 on Matrigel Invasionby First-Trimester Human EVT Cells

We have earlier established that human EVT cellsare a highly invasive cell population and that theirinvasiveness can be further regulated by factors in theEVT cell microenvironment. We examined the effectsof the addition of IGF-I, IGF-II, and IGFBP-1 on theinvasiveness of EVT cells, as measured with Matrigelinvasion assays conducted in serum-reduced medium(1% FCS). Representative results are presented in Fig.2. EVT cell invasion was enhanced (P , 0.05) by IGF-IIat concentrations of 125 and 625 ng/ml in this experi-ment. In some experiments, a significant stimulationwas also seen with a concentration of 25 ng/ml (datanot shown). Treatment with IGFBP-1 also produced

149TROPHOBLAST INVASIVENESS BY IGF-II AND IGFBP-1

significant increases (P , 0.05) in trophoblast invasionat concentrations of 50 and 500 ng/ml (Fig. 2). In otherexperiments, a concentration of 5 ng/ml was also stim-ulatory (as illustrated later in Fig. 3). IGF-I did notcause significant changes in EVT cell invasion whenused at concentrations that were effective for IGF-II(Fig. 2).

To investigate the possible interaction of IGF-II withits binding protein IGFBP-1 during trophoblast inva-sion, invasion assays were performed in the presenceof varying concentrations of IGF-II (0–625 ng/ml),IGFBP-1 (0.5–500 ng/ml), or a combination of IGF-II(125 ng/ml) and varying concentrations of IGFBP-1(0.5–500 ng/ml). In two out of six of these experiments,IGFBP-1 and IGF-II produced synergistic effects oninvasiveness of third and fourth passage EVT cellsisolated from a single placenta. These data are notpresented because of an inability to reproduce thisresponse in the majority of the experiments conductedwith later passage cells of the same placenta or third tosixth passage EVT cells isolated from different placen-tas. However, in all experiments, IGF-II and IGFBP-1produced significant increases (P , 0.05) in tropho-blast invasion when used alone (as shown in Figs. 2and 3). In a majority of experiments, a combination ofthese molecules also stimulated invasiveness, but thedegree of stimulation was not significantly different

from those noted with individual treatments (illustrat-ed in Fig. 2).

Influence of IGF Type 1 and a5b1 Receptor-BlockingAntibodies on Matrigel Invasion by EVT Cells

Receptors on the EVT cell surface responsible forIGF-II or IGFBP-1 effects on cellular invasivenesswere examined by inclusion of the following receptor-blocking antibodies in the invasion assays. IGF type1 receptor-blocking antibody (aIR-3) was used, be-cause IGF type 1 but not type 2 receptor is believedto transduce IGF-II signals in most cell types. SinceIGFBP-1 can bind to RGD-binding sites of some in-tergins such as a5/b1 (fibronectin receptor) presenton EVT cells, we tested whether anti-a5 integrinantibodies could abrogate IGFBP-1 effects. Treat-ment with aIR-3 at concentrations up to 25 mg/ml didnot significantly affect Matrigel invasion by humanEVT when the antibody was used alone (Fig. 3).Similar antibody treatment also failed to reduce theIGF-II enhancement of Matrigel invasion by thesecells (Fig. 3), suggesting that type 1 IGF receptorwas an unlikely mediator of IGF-II action on EVTcells. In parallel experiments conducted in our sisterlaboratory, aliquots of the same antibody wereshown to block IGF-1-mediated stimulation of por-cine cumulus cell expansion in a concentration-de-

FIG. 1. First-trimester human EVT cell proliferation (as determined by 3H-TdR incorporation) after culture in serum-free mediacontaining various concentrations of IGF-I (0–625 ng/ml), IGF-II (0–625 ng/ml), or IGFBP-1 (0–500 ng/ml). Serum was also included in twosets of wells at concentrations of 1 and 10%, respectively. Proliferation was significantly (P , 0.01) affected only by inclusion of FCS at 1 and10%. No significant effect was produced by any other treatment. Points represent the mean (6SEM) of quadruplicate samples.

150 HAMILTON ET AL.

pendent manner, giving a maximum effect at 10ng/ml [28], indicating that this antibody is functionalin blocking IGF-type receptors. IGFBP-1 stimulationof invasiveness was abrogated in the presence ofantibody to the a5 integrin (Fig. 3), indicating thatthe stimulatory effects of IGFBP-1 depended on ac-cess to cell surface a5 integrin.

Effects of IGF-II and IGFBP-1 on First-TrimesterEVT Cell Migration

Since migration is one of the essential steps in cel-lular invasiveness, migration assays were carried outwith similar cell aliquots and subjected to the sameconditions as those used for invasion assays presentedin Fig. 2. Trends in EVT migration paralleled those ofEVT invasion under the same conditions. That is, IGF-II, IGFBP-1, and their combination stimulated EVTcell migration (Fig. 4). These data are a confirmation ofresults of a previous study [5] in which we used amonolayer wound assay to assess migration instead ofthe transwell migration assay described in the presentstudy. In the former assay, cells move along the surfaceof the culture plate, whereas in the latter assay, theymove across the pores of the Millipore membrane at thebottom of the transwell.

Expression of IGF-Binding Molecules on the Surfaceof Human EVT Cells

Our findings of the lack of effect of IGF type 1 recep-tor-blocking antibody, as presented above, came as asurprise, in view of the fact that we had identified thepresence of both IGF type 1 and type 2 receptor mRNAin EVT cells by RT-PCR (data not presented). To elu-cidate the nature of IGF-binding molecules on the EVTcell surface, we conducted affinity cross-link studiesusing 125I-IGF-I and 125I-IGF-II. In autoradiographswe identified a number of cell surface-associated IGF-binding molecules (Fig. 5). While most of the moleculesbound both 125I-IGF-I and 125I-IGF-II, two bands (in-dicated by asterisks in Fig. 5) were identified onlywhen radiolabeled IGF-II was used. One of thesebands, approximately 230 kDa in size, represents theIGF type 2 (mannose-6-phosphate) receptor complexedwith IGF-II. The other band, approximately 75 kDa insize (bottom asterisk), remains unidentified. The IGFtype 1 a chain receptor of 130 kDa was not demon-strated on any of the autoradiographs. Bands equiva-lent to or smaller than 55 kDa in size most likelyrepresent IGF-binding proteins. A number of bindingmolecules on the EVT cell surface appear to have agreater affinity for IGF-II than IGF-I since the inten-sity of these bands was reduced to a much greater

FIG. 2. Representative results of a 3-day Matrigel invasion assay for first-trimester human EVT cells cultured in media containing 1%FCS and various concentrations of IGF-I or IGF-II (0–625 ng/ml) or IGFBP-1 (0–500 ng/ml) or a combination of IGF-II (125 ng/ml) andincreasing doses of IGFBP-1 (0–500 ng/ml). Matrigel invasion was significantly stimulated by treatment with IGF-II or IGFBP-1 (P , 0.05)or their combination but not IGF-I (P . 0.05). Bars represent the mean (6SEM) relative invasion (control group normalized to 100%) for threewells. Bars not sharing a common superscript are significantly different from each other (P , 0.05). That is, a is different from b, but neithera or b is significantly different from ab. C, Control (no treatment).

151TROPHOBLAST INVASIVENESS BY IGF-II AND IGFBP-1

extent by simultaneous treatment with cold IGF-II ascompared to IGF-I (Fig. 5). Furthermore, the two IGF-II-specific bands (asterisks), including the IGF type 2receptor (top asterisk), were enhanced in the presenceof cold IGF-I. This can be explained by the presence ofone or more IGFBPs that can interact with eitherIGF-1 or IGF-II (but not insulin), such that occupationof the IGFBP with excess IGF-1 freed the radiolabeledIGF-II to bind more efficiently to IGF-II-specific pro-teins including the type 2 receptor.

These results open the possibility that IGF-II actionwas mediated by IGF type 2 receptor or a receptorother than type 1 or type 2.

Secretion of Gelatinases and Plasminogen Activatorsby EVT Exposed to IGF-II and IGFBP-1

Whether IGF-II or IGFBP-1 promoted the matrix-degrading ability of EVT cells by stimulating the se-cretion of gelatinases or plasminogen activators wasexamined by zymography of EVT cell supernatants.

When EVT cells were cultured in the presence ofIGF-II and/or IGFBP-1, zymography did not indicateany apparent changes in the secretion of gelatinases orplasminogen activators (Fig. 6). TGFb, known to re-duce trophoblast invasiveness, caused a small reduc-tion in the secretion of plasminogen activators as indi-

cated by a relative reduction in the band intensity onzymograms (Fig. 6).

DISCUSSION

IGF-II is considered to be an important regulator ofembryonic and fetal development [15]. In situ hybrid-ization studies [14] demonstrated a selective abun-dance of IGF-II mRNA in the invasive extravillouscytotrophoblast and IGFBP-1 mRNA in the decidualcells adjacent to the invasive trophoblast, suggestinginteraction of these molecules in regulating invasivetrophoblast function. We tested this possibility in vitro,utilizing highly invasive first normal trimester humantrophoblast cells having a phenotypic profile of EVTcells in situ.

In our study, exogenous IGF-II did not significantlyinfluence the proliferation of first-trimester EVT cellsin culture. IGF-I and IGFBP-1 also proved to be inef-fective in influencing EVT cell proliferation. Thesefindings contrast those reported with villous tropho-blast cells, the proliferation of which was found to bestimulated with both IGF-I [29] and IGF-II [13]. Wefound that exogenous IGF-II and IGFBP-1, but notIGF-I, stimulated EVT cell invasiveness under serum-reduced conditions. In some experiments, the combina-tion of IGF-II and IGFBP-1 enhanced trophoblast in-

FIG. 3. Representative results of another 3-day Matrigel invasion assay with first-trimester EVT cells treated with IGF-I or IGF-II at125 ng/ml with and without monoclonal type I IGF receptor-blocking antibody (aIR-3; 25 mg/ml), or IGFBP-1 (5 ng/ml) with and withoutanti-a5 integrin antibody (25 mg/ml). aIR-3 alone had no significant effect on EVT cell invasion. IGF-II stimulation of invasion was notsignificantly influenced in the presence of aIR-3. IGFBP-1-mediated stimulation of invasion was not demonstrated in the presence of a5

antibody. Bars represent the mean (6SEM) relative invasion (control group normalized to 100%) for triplicate wells. Bars with asterisksalone are significantly different (P , 0.05) from the control.

152 HAMILTON ET AL.

vasion in a synergistic manner. While this synergisticeffect was an interesting finding, the inability to faith-fully repeat the response has made it difficult to exam-ine potential mechanisms for this synergy. We specu-late that the synergistic response may have beenunique to a specific population of early passageEVT cells (3–4 passages) of a single placenta used in

those studies. However, on all occasions, IGF-II andIGFBP-1 independently enhanced the invasiveness ofsimilar or later passage (3–6 passages) trophoblastcells isolated from three first-trimester placentas.

Although IGF-II may interact with both IGF type 1and type 2 (mannose-6-phosphate) receptors, IGF-IIeffects are believed to be transduced predominantly

FIG. 4. Representative results of a 3-day migration assay with first-trimester EVT cells treated with 125 ng/ml IGF II, 5 ng/ml IGFBP-1,or their combination. Bars represent the mean (6SEM) migration index relative to the untreated control (normalized to 100%) for triplicatewells. Bars with asterisks are significantly different (P , 0.05) from the control, but not from one other.

FIG. 5. Representative autoradiograms for trophoblast cell surface-associated IGF-binding molecules. (Left) 125I-IGF-II binding with nocompetition (lane 1) and in the presence of decreasing doses of IGF-1 (200, 100, 50, and 10 ng/ml; lanes 2–5), IGF-II (200, 100, 50, and 10ng/ml; lanes 6–9), or insulin (10 and 1 mg/ml; lanes 10–11). (Right) 125I-IGF-I binding with no competition (lane 1) and in the presence ofdecreasing doses of IGF-I (200, 100, 50, and 10 ng/ml; lanes 2–5), IGF-II (100, 50, and 10 ng/ml; lanes 6–8), or insulin (10 and 1 mg/ml; lanes9–10). Molecular weight markers are indicated at the extreme left of the figure. Asterisks indicate IGF-II specific binding molecules (bandspresent on the left but absent on the right). The top asterisk indicates the type 2 IGF receptor. The type 1 IGF receptor (a chain, 130 kDa)is not evident on either autoradiogram.

153TROPHOBLAST INVASIVENESS BY IGF-II AND IGFBP-1

through the type 1 receptor [16, 17]. In fact, mRNA forboth of these receptors is expressed in the human pla-centa [13] and we have identified their mRNA tran-scripts (by reverse-transcriptase polymerase chain re-action) in cultured EVT (data not presented). Thefailure to identify the IGF type 1 receptor in affinitycross-link studies and the failure of IGF type 1 recep-tor-blocking antibody to influence IGF-II enhancementof invasion suggest that IGF-II action on in vitro prop-agated EVT cells is by a mechanism independent of thetype 1 receptor. A lack of effect of IGF-I (which isknown to act by type 1 receptor) on EVT cell invasive-ness reinforces this suggestion. The abundant expres-sion of IGF type 2 receptor by cultured EVT cells (Fig.5) indicates that it may mediate IGF-II invasion-pro-moting actions most likely by a promotion of cellularmotility. Indeed, type 2 IGF receptor has been shown tomediate IGF-II-induced motility but not mitogenesis ina human rhabdomyosarcoma cell line [30]. Similarly,this receptor has been reported to be responsible forIGF-II and proliferin-mediated stimulation of endothe-lial cell migration [31]. Access to a suitable blockingantibody for the human type 2 IGF receptor shouldfacilitate future functional studies of this receptor withthe human EVT cells.

Competition affinity cross-link studies using coldIGF-I and IGF-II indicated a number of binding mole-cules which appeared to bind more 125I-labeled IGF-II

than 125I-IGF-I and which were more susceptible tocompetition by cold IGF-II than cold IGF-I. These find-ings indicate that the human EVT cells propagated inculture possess a relatively IGF-II-specific and/or IGF-II-preferring repertoire of binding molecules associ-ated with their cell surface. Some of these or perhaps acombination of these binding molecules may contributeto IGF-II (and not IGF-I) enhancement of trophoblastinvasion. The binding molecule of approximately 75kDa in size (lower asterisk, Fig. 5) is of particularinterest, since an IGF-II-specific-binding protein ofsimilar size has also been described for the humanK562 erythroleukemia cell line which did not expresstype 1 IGF receptor, but responded to IGF-II by growthstimulation [32].

Cellular invasiveness, as measured in the presentstudy, depends on numerous steps: the ability of thecells to bind to one or more components of the matrix;the ability of the cells to degrade the matrix; and theability of the cells to migrate through the degradedmatrix. A migration-stimulating action of IGF-II onEVT cells observed in the present and earlier [5] stud-ies, in the absence of significant increases in secretedprotease (gelatinase or PA) activity, suggests thatIGF-II stimulation of EVT cell invasiveness may besolely due to increased migratory activity.

IGF-binding proteins have been shown to both in-hibit and enhance [20, 22] IGF-mediated actions. Theresults of the present study clearly demonstrate anenhancement of invasion by IGFBP-1. Since IGFBP-1possesses a RGD sequence, it may act by interactingwith certain trophoblast cell surface integrins to stabi-lize IGF-II binding to its receptor. The fact thatIGFBP-1 alone (in the absence of exogenous IGF-II)increased trophoblast invasion or migration is not in-consistent with this notion since our laboratory hasshown that these cells contain endogenous IGF-II [24].It is also possible that IGFBP-1 can directly transduceits own signals by binding to trophoblast cell surfaceintegrins whose ligands are known to activate a varietyof intracellular signaling pathways including p125FAK

[33, 34] and integrin-linked kinase [35]. In fact, wehave shown that IGFBP-1 stimulates trophoblast mi-gration and that access to a5/b1 integrin (fibronectinreceptor) is essential for IGFBP-1 stimulation of mi-gration [5]. Consistent with these data, we have shownhere that invasion-stimulating effects of IGFBP-1 wereabrogated in the presence of a5 integrin-blocking anti-body. These findings combined with the fact that IG-FBP-1 had no significant effect on the secretion ofgelatinases or PA by EVT cells suggest that invasionstimulation by IGFBP-1 may primarily result from itsstimulatory effects on trophoblast migratory ability,since migration is an essential step in invasion.

The synergistic effects of IGF-II and IGFBP-1 stim-ulation of EVT cell invasiveness noted in a minority of

FIG. 6. Representative zymograms utilizing gelatin (top) andcasein (bottom) as substrates. Lanes were loaded with serum-re-duced (1% FCS) RPMI 1640 media alone (lane 1; extreme left) andconditioned with human EVT cells exposed to no exogenous factors(lane 2), 125 ng/ml IGF-II (lane 3), 50 ng/ml IGFBP-1 (lane 4), bothIGF-II and IGFBP-1 (lane 5), or 5 ng/ml TGFb (lane 6). For gelati-nase zymograms, the upper band represents 92-kDa type IV colla-genase (gelatinase B) and the lower two bands represent differen-tially activated forms of 72-kDa type IV collagenase (gelatinase A).For plasminogen activator (PA) zymograms (bottom), the upper bandrepresents tissue type PA (tPA) and the lower two bands representdouble and single chain urokinase type PA (uPA).

154 HAMILTON ET AL.

experiments belonging to a single placenta can be ex-plained by independent action of these molecules viatheir respective receptors, as discussed above, as wellas their interdependent actions by stabilizing eachother on the EVT cell surface.

It appears that first-trimester EVT cells are geneti-cally equipped with high migratory and invasive abil-ities, which can be further upregulated by the auto-crine factor IGF-II. Decidua can deliver paracrinesignals which can be stimulatory (e.g., via IGFBP-1 asshown here) as well as inhibitory (e.g., via TGFb) asshown earlier [5, 6]. It remains unknown at presentwhat determines the local activity of these two decid-ua-derived factors with seemingly opposing functions.We had shown that TGFb, which is abundant in theextracellular matrix (ECM) of the early gestationaldecidua, is colocalized with a TGFb-binding and inhib-itory proteoglycan decorin [25], and that TGFb se-creted by the decidua in vitro is in an inactive form [6].It is likely that a local activation of TGFb bound to theECM components; e.g., decorin is required before it canmediate its inhibitory function on the trophoblast insitu. This activation is likely provided by the tropho-blast-derived proteases at the invasive front to controltrophoblast overinvasion of the decidua. For example,there is a highly polarized expression of uPA receptorson the invasive pole of EVT cells in situ [36], which canbind trophoblast-derived uPA capable of activatingplasmin, which in turn, can activate the ECM-storedTGFb.

This study was supported by grants from the Medical ResearchCouncil of Canada (to P.K.L. and V.K.M.H.) and a Lalor FoundationFellowship (to G.S.H.).

REFERENCES

1. Kaufmann, P., and Castelucci, M. (1997). Extravillous tropho-blast in the human placenta (Review). Troph. Res. 10, 21–65.

2. Lala, P. K., and Lysiak, J. J. (1994). Role of locally producedgrowth factors in human placental growth and invasion withspecial reference to transforming growth factors. In “Immunol-ogy of Reproduction” (J. S. Hunt, Ed.), pp. 57–81, Springer-Verlag, New York.

3. Lala, P. K., and Hamilton, G. S. (1996). Growth factors, pro-teases and protease inhibitors in the maternal-fetal dialogue.Placenta 17, 546–555.

4. Graham, C. H., Lysiak, J. J., McCrae, K. R., and Lala, P. K.(1992). Localization of transforming growth factor-b at the hu-man fetal-maternal interface: Role in trophoblast growth anddifferentiation. Biol. Reprod. 46, 561–572.

5. Irving, J. A., and Lala, P. K. (1995). Functional role of cellsurface integrins on human trophoblast cell migration: Regula-tion by TGFb, IGF-II and IGFBP-1. Exp. Cell Res. 217, 419–427.

6. Graham, C. H., and Lala, P. K. (1991). Mechanism of control oftrophoblast invasion in situ. J. Cell. Physiol. 148, 228–234.

7. Graham, C. H., and Lala, P. K. (1992). Mechanisms of placentalinvasion of the uterus and their control. Biochem. Cell Biol. 70,867–874.

8. Graham, C. H., McCrae, K. R., and Lala, P. K. (1993). Molecularmechanisms controlling trophoblast invasion of the uterus.Troph. Res. 7, 237–250.

9. Lysiak, J. J., Han, V. K. M., and Lala, P. K. (1993). Localizationof transforming growth factor-a in the human placenta anddecidua: Role in trophoblast growth. Biol. Reprod. 49, 885–894.

10. Lysiak, J. J., Connelly, I. H., Khoo, N. K. S., Stetler-Stevenson,W., and Lala, P. K. (1994). Role of transforming growth factor-a(TGFa) and epidermal growth factor (EGF) on proliferation andinvasion by first trimester human trophoblast. Troph. Res. 8,455–467.

11. Lysiak, J. J., Johnson, G. R., and Lala, P. K. (1995). Localiza-tion of amphiregulin in the human placenta and deciduathroughout gestation: Role in trophoblast growth. Placenta 16,359–366.

12. Hill, D. J., Clemmons, D. R., Riley, S. C., Bassett, N., andChallis, J. R. G. (1993). Immunohistochemical localization ofinsulin-like growth factors (IGFs) and IGF binding proteins-1,-2 and -3 in human placenta and fetal membranes. Placenta 14,1–12.

13. Ohlsson, R., Homgren, L., Glaser, A., Specht, A., and Pfeifer-Ohlsson, S. (1989). Insulin-like growth factor 2 and short-rangestimulatory loops in control of human placental growth. EMBOJ. 8, 1993–1999.

14. Han, V. K. M., Bassett, N., Walton, J., and Challis, J. R. G.(1996). The expression of insulin-like growth factor (IGF) andIGF-binding protein (IGFBP) genes in the human placenta andmembranes: Evidence for IGF-IGFBP interaction at the fetal-maternal interface. J. Clin. Endocrinol. Metab. 81, 2680–2693.

15. Han, V. K., and Hill, D. J. (1992). The involvement of insulin-like growth factors in embryonic and fetal development. In “TheInsulin-like Growth Factors, Structures and Biological Func-tions” (Schofield, P.N., Ed.), pp. 178–220, Oxford Univer. Press,Cambridge.

16. Jones, J. I., and Clemmons, D. R. (1995). Insulin-like growthfactors and their binding proteins. Endocrine Rev. 16, 3–43.

17. Stewart, C. E. H., and Rotwein, P. (1996). Growth, differentia-tion and survival: Multiple physiological functions for insulin-like growth factors. Physiol. Rev. 76, 1005–1026.

18. Kiess, W., Blickenstaff, G. D., Sklar, M. M., Thomas, C. L.,Nissley, S. P., and Sahagian, G. G. (1988). Biochemical evidencethat the type II insulin-like growth factor is identical to thecation-independent mannose 6-phosphate receptor. J. Biol.Chem. 263, 9339–9344.

19. Mathieu, M., Rochefort, H., Barenton, B., Prebois, C., andVignon, F. (1990). Interactions of cathepsin-D and insulin-likegrowth factor-II (IGF-II) on the IGF-II-mannose-6-phosphatereceptor in human breast cancer cells and possible conse-quences on mitogenic activity of IGF-II. Mol. Endocrinol. 4,1327–1335.

20. Clemmons, D. R. (1997). Insulin-like growth factor bindingproteins and their role in controlling IGF actions. CytokineGrowth Factor Rev. 8, 45–62.

21. Kim, H-S., Nagilla, S. R., Oh, Y., Wilson, E., Roberts, C. T. Jr.,and Rosenfeld, R. G. (1997). Identification of a family of lowaffinity insulin-like growth factor binding proteins (IGFBPs):Characterization of connective tissue growth factor as a mem-ber of IGFBP superfamily. Proc. Natl. Acad. Sci. USA 94,12981–12986.

22. DeMellow, J. S. M., and Baxter, R. C. (1988). Growth hormone-dependent insulin-like growth factor (IGF) binding protein bothinhibits and potentiates IGF-I-stimulated DNA synthesis inhuman skin fibroblasts. Biochem. Biophys. Res. Commun. 156,199–204.

155TROPHOBLAST INVASIVENESS BY IGF-II AND IGFBP-1

23. Clemmons, D. R., Thrailkill, K. M., Handwerger, S., and Busby,W. H. (1990). Three distinct forms of insulin-like growth factorbinding proteins are released by decidual cells in culture. En-docrinology 127, 643–650.

24. Irving, J. A., Lysiak, J. J., Graham, C. H., Han, V. K. M., Hearn,S., and Lala, P. K. (1995). Characteristics of trophoblast cellsmigrating from first trimester chorionic villus explants andpropagated in culture. Placenta 16, 413–433.

25. Lysiak, J. J., Hunt, J., Pringle, G. A., and Lala, P. K. (1995).Localization of transforming growth factor b and its naturalinhibitor decorin in the human placenta and the deciduathroughout gestation: Role in trophoblast growth. Placenta 16,221–231.

26. Khoo, N. K. S., Bechberger, J. F., Shepherd, T., Bond, S. L.,McCrae, K., Hamilton, G. S., and Lala, P. K. (1998). SV 40 Tagtransformation of the normal invasive trophoblast results in apremalignant phenotype. 1. Mechanism responsible for hyper-invasiveness and resistance to antiinvasive action of TGFb. Int.J. Cancer. 77, 429–439.

27. Repesh, L. A. (1989). A new in vitro assay for quantitativetumour cell invasion. Metastasis 9, 192–20.

28. Singh, B., and Armstrong, D. T. (1997). Insulin-like growthfactor-1, a component of serum that enables porcine cumuluscells to expend in response to follicle-stimulating hormone invitro. Biol. Reprod. 56, 1370–1375.

29. Maruo, T., Murata, K., Matsuo, H., Samoto, T., and Mochizuki,M. (1995). Insulin-like growth factor-1 is a local regulator ofproliferation and differential function of the human trophoblastin early pregnancy. Early Pregnancy Biol. Med. 1, 62–66.

30. Minniti, C. P., Kohn, E. C., Grubb, J. H., Sly, W. S., Oh, Y.,Muller, H. L., Rosenfield, R. G., and Helman, L. J. (1992). Theinsulin-like growth factor II (IGF II)/mannose-6-phosphate re-ceptor mediates 16F-II induced motility in human rhabdomyo-sarcoma. J. Biol. Chem. 267, 9000–9004.

31. Volpert, O., Jackson, D., Bouck, N., and Linger, D. I. H. (1996).The insulin-like growth factor II/mannose-6-phosphate receptoris required for proliferin-induced angiogenesis. Endocrinology137, 3871–3876.

32. Zhang, Q., Hall, K., and Tally, M. (1996). Partial purification ofan IGF-II receptor/binding protein from erythroleukemia cellline K562. Mol. Cell. Biochem. 154, 47–54.

33. Kornberg, L., Earp, S., Parsons, J. T., Schaller, M., and Juliano,R. L. (1992). Cell adhesion or integrin clustering increasesphosphorylation of a focal adhesion-associated tyrosine kinase.J. Biol. Chem. 267, 23439–23442.

34. Clark, E. A., and Brugge, J. S. (1995). Integrins and signaltransduction pathways: The road taken. Science 268, 233–239.

35. Hannigan, G. E., Leun-hagsteijn, C., Fitz-Gibbon, L., Coppo-lino, M. G., Radeva, G., Filmus, J., Bell, J. C., and Dedhar, S.(1996). Regulation of cell adhesion and anchorage-dependentgrowth by a new beta 1-integrin-linked protein kinase. Nature379, 91–96.

36. Multhaupt, H. A. B., Mazar, A., Cines, D. B., Warhol, M. J., andMcCrae, K. R. (1994). Expression of urokinase receptors by thehuman trophoblast: A histochemical and ultrastructural anal-ysis. Lab. Invest. 71, 392–40.

Received March 27, 1998Revised version received June 9, 1998

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