JOURNAL OF CELLULAR PHYSIOLOGY 150:33&343 (1992)

Subtypes of Betaglycan and of Type I and Type II Transforming Growth Factor+ (TCF-P) Receptors With Different Affinities for JGF-P1 and TCF-P2 Are Exhibited by Human Placental Trophoblast Cells E. JANEMITCHELL,* LINDA FITZ-GIBBON, AND MAUREEN D. O'CONNOR-McCOURT Receptor Group, Biotechnology Research Institute, National Research Council Canada, , H4P 2R2 Montreal, Q u e b ~ Cdnadd Transforming growth factor+ is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 'L'I-TGF-P1 or "'I-TGF-P2 and bis(sulfosuccinimidy1) suberate (BS') to characterize TGF-P binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type Ill TGF-P receptor) and relatively low levels of the Type I and Type II TGF-P receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-P1 and TGF-P2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-P1 and TGF-P2, yet has a preferential affinity for TGF-P2; the second subtype has a higher capacity and lower affinity and binds TGF-P1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-p isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-P2 than for TGF-PI, that we had observed previously on term placental membranes, i s actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-P receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 'LSI-TCF-P1was used as the radiotracer, the Type I and II TGF-P receptors show a much higher affinity for TGF-P1 than for TGF-P2, as observed with other cell types. However, when '"I-TGF-P2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-PI and TGF-P2 were also revealed.

TGF-P is a multifunctional regulatory peptide which is involved in the growth and differentiation of almost all cell types, the deposition of the extracellular matrix, and immunosuppression (for reviews see Roberts and Sporn, 1989; Massague, 1990). The human TGF-P family consists of three isoforms of which TGF-P1 and TGF-P2 have been isolated from tissue sources, whereas TGF-P3 has been produced by recombinant DNA technology (ten Dijke e t al., 1990a). These isoforms are highly homologous, and although in many assays their biological potencies are indistinguishable, several reports have now indicated that these isoforms do in fact have different physiological functions (Ohta et al., 1987; Rosa et al., 1988; Jennings et al., 1988; ten Dijke e t al., 1990b; Merwin et al., 1991). Chemical cross-linking techniques with 1251-TGF-f3 have been used to identify three types of high affinity binding components on most cell types (for reviews see 0 1992 WILEY-LISS, INC.

Segarini, 1989; Massague et al., 1990; Boyd et al., 1990).These affinity-labelled complexes are recognized as the Type I receptor (Mr -65,000), the Type I1 receptor (Mr -85,000) and betaglycan, formerly called the Type I11 receptor (Mr -250,000-350,000). The Type I and I1 components are glycoproteins that are currently believed to be true signalling receptors since they are frequently lost in TGF-P resistant mutants (Boyd and Massague, 1989; Laiho et al., 1990). The betaglycan component is a chondroitin-sulfate and heparan-sulfate containing proteoglycan, that exists in both membraneanchored and soluble forms. It is currently thought to

Received July 9,1991; accepted September 20, 1991

"To whom reprint requestsicorrespondence should be addressed.

TGF-fi RECEPTORS ON PLACENTAL TROPHOBLASTS

serve non-signalling roles such as in the sequestration or the clearance of TGF-P (Andres et al., 1989). In general, the predominant populations of Type I and I1 TGF-P receptors on cells display much higher affinities for TGF-P1 than -pa, whereas the predominant population of betaglycan components show equal affinities for TGF-PI and -P2. However, subtypes of these betaglycan components which have a higher affinity for TGF-P2 than for -Pl have now been recognized(Segarini et al., 1987; Mitchell and O’Connor-McCourt, 1991). Also, subtypes of Type I and I1 TGF-P recep-tors which have a high affinity for TGF-P2 have recently been identified (Cheifetz et al., 1990). The identification of these subtypes for each of the three types of TGF-P binding components (I, 11, and beta-glycan) should help clarify the discrepancies between the biological potencies and the receptor binding affinities of TGF-P1, TGF-p2, and TGF-P3. We have recently identified a betaglycan component in human placental membrane preparations which exhibits a higher affinity for TGF-P2 than for TGF-61 (Mitchell and O’Connor-McCourt, 1991). This finding has led us to determine which placental cell types display this unique class of betaglycan. The most abundant cell types in placenta are trophoblasts and fibroblasts (Laga et al., 1973). Trophoblast cells are the epithelial-like covering of the chorionic villae a t the fetal-maternal interface, Receptors for polypeptide hormones/growth factors on human trophoblast cells have been studied with primary trophoblast cell shortterm cultures (Deal and Guyda, 1983; Lai and Guyda, 1984),trophoblast cell lines derived from primary culture (Goustin et al., 1985) and with trophoblastic cell lines, such as BeWo, derived from choriocarcinomas (O’Connor-McCourt and Hollenberg, 1983; Deal and Guyda, 1983; van der Ende et al., 1990). Trophoblasts have crucial roles in the exchange of nutrients and wastes, the physical attachment of the placenta to the uterus, the production of hormones such as hCG and hPL and the regulation of the maternal immune response toward the fetus. The roles of growth factors in these placental functions are poorly understood (for reviews see Ohlsson, 1989; Blay and Hollenberg, 1989; Pollard, 19901, although they are assumed to mediate activities through autocrine and paracrine circuits. The activities of TGF-P, notably in extracellular matrix deposition, growth inhibition and immunosuppression, point to potential roles for TGF-P, respectively, a s a mediator of trophoblastic invasion of the endometrium, trophoblast differentiation, and the maintenance of pregnancy (Tamada e t al., 1990; Lala and Graham, 1990; Morrish et al., 1991; Clark et al., 1990; Altman et al., 1990). Recently, the TGF-P2 isoform in particular has been implicated a s having roles both in placental function and in embryonic development in various murine studies (Miller e t al., 1989; Pelton et al., 1989; Clark et al., 1990; Altman et al., 1990; Kelly et al., 1990). In order to understand the mechanism of action of the different TGF-(3 isoforms in these functions, characterization of the placental TGF-P receptors is necessary. In this report, we have identified and characterized the TGF-P binding components on trophoblast cells in culture. We have demonstrated that the betaglycan subtype which has a greater affinity for TGF-p2 than

335

for TGF-P1, observed previously with placental membrane preparations, is exhibited by trophoblast cells. These results have been presented in preliminary form a t the Annual meeting of the American Society for Cell Biology in San Diego, California, December 1990 (Mitchell et al., 1990 and see reference list).

MATERIALS AND METHODS Preparation of primary placental cells Cultures enriched for trophoblast cells were established essentially according to the method of Branchaud et al. (1983) using successive 10 min trypsinizations of fresh villous tissue obtained after therapeutic abortions at 12-17 weeks gestation (kind gifts from Drs Charlotte Branchaud and Cynthia Goodyer, Montreal Children’s Hospital). Villous tissue was carefully dissected away from membranes and washed extensively with Mg++/Ca++-freeD-PBS. Approximately 20 g tissue was added to approximately 100 ml Mg++/Ca’ ’ free D-PBS containing 0.25% trypsin (DIFCO, Detroit, MI) and 500 U DNAase 1 (Sigma, St. Louis, MO). The digestion was allowed to proceed a t 22°C with hand agitation every 5 min. The supernatants collected from above the settled tissue from the first three digests of 15 min each were discarded. Cells from the fourth through tenth 10 min trypsinizations of villous tissue were collected in centrifuge tubes containing 1 ml of fetal bovine serum (in order to inactivate the trypsin) and were harvested by centrifugation. The cells obtained from the fourth and fifth trypsinization steps contained a fairly morphologically homogeneous population of cells when viewed by light microscopy. Flow cytometric analysis confirmed that this population was highly enriched in HLA- cells characteristic of villous trophoblast cells (see below). The cells obtained from the sixth through tenth trypsinization steps were a heterogeneous population that included some trophoblasts, fibroblasts and other cells presumably of mesenchymal origin. Flow cytometric analysis confirmed that this population was highly enriched in HLAt cells. Thus this population was termed mesenchymal cellenriched. The trophoblast-enriched or mesenchymal cell-enriched cell populations were pooled and treated with 10 ml of ice-cold ammonium chloride-phosphate buffer (0.83% NH,C1 in PBS, pH 7.2) for 10 min in order to lyse red blood cells (Branchaud et al., 1983).Following lysis, cells were again placed into a centrifuge tube containing 3 ml of fetal bovine serum and collected by centrifugation. These cells were resuspended in culture medium (RPMI) supplemented with 10% (YV) fetal bovine serum and antibiotics, Penicillin-G (200 IU/ml) and streptomycin (200 IUlml), and plated a t confluent density in 12-well plastic tissue culture plates. As pointed out by Branchaud et al. (1983), attempts to plate cells according to cell number were not satisfactory as the viability of cells following plating was somewhat variable from culture to culture. However, care was taken to use wells a t the same degree of confluency for experiments. The cultures were incubated a t 37°C in a humidified atmosphere of 5% CO, in air. The following day, medium was removed and the cultures were gently rinsed with fresh medium to remove any debris. The trophoblast cultures appeared to be differentiated into syncitia by the second to third day in culture.

336

MITCHELL ET AL TABLE 1. Flow cytometric analysis of a representative placental cell preparation after labelling with Mab W6/32 (anti-HLA) and FITC-conjugated goat anti-mouse IgG ?6 cell population fluorescently labelled

Cell population derived from Fourth and fifth trypsinization of villous tissue Sixth through tenth trypsinization of villous tissue Immuno-magnetic separation in which cells from fourth-fifth trypsinization steps that were reactive with Mab WW32 were removed Mock immuno-magnetic separation in which cells from fourth-fifth trypsinization steps were treated with SP/O myeloma supernatant

Purification of trophoblast cells by immuno-magnetic separation For some experiments, trophoblast cells derived from the fourth and fifth trypsinizations of the villous tissue were further purified using a n immuno-magnetic separation technique similar to that described by Douglas and King (1989). Following collection by centrifugation, cells were incubated a t approximately l x lo7 cellsiml with 2% (%) horse serum in D-PBS for 30 min a t 4°C to block Fc receptor sites. Cell supernatants either from the hybridoma secreting the Mab W6132 (specific for HLA class I antigens) (Barnstable e t al., 1978) or the myeloma line SPiO (for control) were then added (approximately 100 p1 supernatant per 10” cells) and the cells were incubated for 30 min a t 4°C (both of these cell supernatants were kindly provided by A. Marcil, Biotechnology Research Institute, Immunology Section). Cells were carefully washed three times using 2% (Yv)horse serum in D-PBS, followed by incubation with magnetic goat anti-mouse IgG (Advanced Magnetics, Inc., Cambridge, MA) a t a ratio of 10 particles per cell, for 30 min a t 4°C. The HLA+ cells, having bound the magnetic particles, were separated by applying the BioMag Magnetic Separator (Advanced Magnetics, Inc.) alongside the cells contained in a plastic tissue culture flask and carefully removing the HLA- cells to another flask, then applying the magnetic separator again to remove any remaining HLA+ cells. The purified HLAtrophoblast cells were plately a t confluent density in 12-well plates in supplemented medium a s described above.

25 88 0 31

with D-PBS containing 0.1% (w/v) BSA for a total of 30 minutes a t 4°C. They were then incubated with either 1251-TGF-plor Iz5I-TGF-p2,in the absence or presence of unlabelled TGF-P1 or TGF-P2 as indicated, in a final volume of 300 p1 per well for 3 h r a t 4°C with continuous gentle agitation. Monolayers were washed three times with ice-cold D-PBS and incubated with 1 mM BS3 (Pierce, Rockford, IL) in D-PBS for 10 min, a t 4°C. The cross-linking reaction was stopped by the addition of 75 pl of 500 mM glycine and a further 5 min incubation. Cells were washed once with D-PBS, then incubated with 100 pl of solubilization buffer containing 1% (Yv) Triton-X-100; 10% (Yv)glycerol; 1 mM EDTA; 20 mM Tris-HCI, pH 7.4; 1mM PMSF; 20 pg/ml aprotinin; 20 pgiml leupeptin; 20 pgiml STI; 25 mM benzamidine, for 30 min at 4°C. Solubilized material was recovered from each well with a pipette tip after placing the tissue culture plate at a slight angle. One-fifth volume of 5 x electrophoresis sample buffer: 0.25 M Tris-HC1, pH 6.8, 5% (%) SDS, 10% (YV) P-mercaptoethanol, 50% (%) glycerol, 0.0004% bromophenol blue, was added to each sample followed by heating a t 100°C for 5 min. SDS-polyacrylamide gel electrophoresis and autoradiography were performed a s described previously (Mitchell and O’Connor-McCourt, 1991). (YV)

RESULTS Primary placental cell culture Our previous affinity-labelling studies with membrane preparations derived from human term placentae indicated the presence of a novel betaglycan subtype that exhibits a higher affinity for TGF-62 than for Flow cytometric analysis TGF-P1 (Mitchell and O’Connor-McCourt, 1991). By Flow cytometric analysis was carried out using a Pro- mass, the two most abundant cell types in placenta are file I1 Analyzer (Coulter, Hialeah, FL) configured for fibroblasts and trophoblasts (Laga et al., 1973) and fluorescein. Cells were preincubated for 16 hr a t 4°C therefore should be proportionately represented in the with cell supernatants from either the hybridoma se- membrane preparations. In this report, we have further creting the Mab W6/32 or from the myeloma line SPiO characterized placental TGF-P binding components on (control), followed by treatment with FITC-labelled placental cells in culture by affinity-labelling experigoat anti-mouse IgG for 30 rnin a t 4°C (Becton-Dickin- ments with ‘251-TGF-pl and lZ5I-TGF-p2 in order to son, Mountain View, CA). For control monitoring of the determine which placental cell types exhibit the betacell populations derived from placental villous tissue, glycan component that preferentially binds TGF-(32. the cells were also treated with either Anti-Leu-4 Primary placental cultures enriched in trophoblast (CD3), or Anti-HLA-Dr (also from Becton-Dickinson) to cells were derived from the villous tissue of 12-17 week human placenta by selective trypsinization. The fourth check for the presence of T and B lymphocytes. and fifth trypsinization steps yielded cell populations Affinity-labelling of cells in monolayer that contained 25%, or less, HLAf cells, when analyzed TGF-P1 and TGF-P2 (from porcine platelets) were by Flow Cytometry, whereas the sixth through tenth purchased from R & D Systems (Minneapolis, MN) and trypsinization steps yielded a different population that were iodinated as previously described (Mitchell and consisted of approximately 90% HLA’ cells (Table 1). O’Connor-McCourt, 1991). Monolayers of primary pla- The HLA antigen was used a s a marker because villous cental cells (in 12-well plates) were washed three times trophoblast cells are characteristically HLA- (Bulmer

TGF-B RECEPTORS ON PLACENTAL TROPHOBLASTS

A.

B.

'251-TGF-P1 10 25 50 100200400800

200-

337

1251-TGF-p2 10 25 50 100 200 400 800

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9768-

Fig. 1. Receptor saturation patterns of TGF-61 and TGF-p2 in trophoblast-enriched placental cell cultures. Cells were affinity-labelled with the indicated concentrations (pM) of '"I-TGF-pl (A) or 12s1TGF-p2 (B). Shown here are autoradiograms of Triton-X-100 extracts of affinity-labelled cells. The extracts were treated with sample buffer (final concentration of P-mercaptoethanol was 2% ("A)),and electrophoresed on 3.10%' linear gradient polyacrylamide gels. Different in-

200-

+ +

97-

setensities seen within the betaglycan band are indicated by arrows. Type I and I1 receptors are not evident after this autoradiographic exposure time of 10 days. The specific activities for "'I-TGF-pI and '"II-TGF-p2 in this experiment were 3.2 pCiipmo1 and 3.4 KCiipmol, respectively. The molecular mass markers are indicated in kilodaltons. Similar results were obtained with cultures derived from placentas from eight individuals.

nent was saturated at concentrations of 100-200 pM (Fig. 1B). Furthermore, at saturation, the intensity of labelling of the betaglycan component with TGF-Pl was much higher than that observed with TGF-P2. We therefore propose that there are distinctive subtypes of the betaglycan component exhibited by trophoblast cells. While we cannot exclude the possibility of cooperativity, the simplest explanation is that one of the subtypes has a lower affinity, though higher capacity for TGF-P1, whereas a second subtype has a higher affinity but a lower capacity for TGF-p2. The marked split in labelling intensity that is seen in Figure 1 (arrows), more distinctly with TGF-P2 but also with TGF-P1, varied between individual placental cell preparations Receptor saturation patterns with p r i m a r y and may reflect variations in carbohydrate modificaplacental cells tion. In order to confirm t h a t these distinctive betaglycan These placental cultures were affinity-labelled by incubating them with increasing concentrations of lZ5I- subtypes were expressed on the trophoblast cells and TGF-P1 or '251-TGF-P2,followed by treatment with the not on the small percentage of contaminating cells, we bifunctional cross-linking reagent BS3. Such affinity- carried out affinity-labelling saturation experiments labelling assays have a n advantage over normal equi- (Fig. 2) on primary cultures that were enriched in meslibrium binding assays and Scatchard analysis because enchymal cell types (Table 1). As described above, these each binding component can be analyzed independently cultures were obtained from the sixth through tenth a s a labelled band on SDS-PAGE. Saturation experi- trypsinization steps of placental villous tissue and conments on the tro hoblast-enriched cultures revealed tained approximately 90% HLA+ cells. In saturation that binding of '"I-TGF-Pl to the betaglycan compo- experiments with these cultures, the labelling of the nent was not saturated a t concentrations up to and betaglycan component with either 1251-TGF-plor lZ5Iincluding 800 pM (Fig. 1A).In other experiments when TGF-p2 was similar (Fig. 2A,B). This result is suggeshigher concentrations of 1251-TGF-pl were used, we tive of the presence of only one betaglycan subtype, found that the betaglycan component was saturated a t which can bind the two TGF-P isoforms with similar approximately 2000 pM (data not shown). In contrast, affinities and capacities, typical of that seen on most the binding of '"I-TGF-P2 to the betaglycan compo- cell types. This observation supports the conclusion

and Johnson, 1985). We thus defined the cell population derived from the fourth and fifth trypsinization steps as trophoblast-enriched. This population corresponds to that studied by Branchaud et al. (1983). The cell population derived from the later trypsinization steps contained a much larger proportion of HLAf cells including fibroblasts and other mesenchymal cells, hence we designated these cultures as mesenchymal cell-enriched. Antibodies that recognize the human antigen markers known as CD3 and HLA-DR were also used in the Flow Cytometric analysis and indicated that there were no detectable T or B lymphocytes present (data not shown).

MITCHELL ET AL.

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A.

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- TGF-01

'251-TGF-f12 50 100 250 500 750

50 100 250 500 750 \

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Fig. 2. Receptor saturation patterns of TGF-p1 and TGF-p2 in mesenchymal cell-enriched placental cell cultures. Cells were affinitylabelled with the Indicated concentrations (pM) of '""ITGF-p1 (A) or 'L511-TGF-p2 (B) as described in Figure 1. Autoradiograms following polyacrylamide gel electrophoresis of Triton-X-100 extracts of affinity-labelled ceIIs are shown following a 4 week exposure time. The

68-

43-

specific activities for 'z51-TGF-pland '251-TGF-p2in this experiment were 3.7 IJ-Ciipmoland 3.2 pCiipmo1, respectively. The Type I and I1 receptors and betaglycan component and the molecular mass markers in kilodaltons are indicated. Similar results were observed with three separate cultures.

higher range of concentrations of the competing TGF-P1 and TGF-02 was used, there was a small but detectable amount of 1251-TGF-plbinding to betaglycan that was displaceable by TGF-P1, but not by TGFp2, even with more than a 1,000-fold excess (Fig. 3B). This observation, together with those from the saturation experiments (Fig. l),are consistent with the concept of the existence of two subtypes of the betaglycan component. We propose that the one subtype which has a relatively higher affinity and lower capacity binds to both TGF-pl and -p2, and yet has a preferential affinity for TGF-p2; the second subtype which has a relatively lower affinity and higher capacity, in fact, binds only to TGF-01. According to this proposal, a t the low concentration of radiotracer used ,ii.e., 35 pM in Fig. 3B), only a small percentage of lL"I-TGF-pl would be associated with the exclusive TGF-(31subtype which has a lower affinity. Most of the '"I-TGF-Pl would be Receptor competition patterns with primary associated with the other higher affinityilower capacity placental cells subtype that binds both TGF-PI and TGF-(32. Thus, Competition experiments with either 12511-TGF-P1 or only a small percentage of the occupied betaglycan sites '"I-TGF-P2 and varying concentrations of unlabelled should be displaceable by TGF-P1 but not by TGF-P2 in competition experiment. Indeed, this is TGF-Pl or TGF-02 were also performed with the pri- the 1251-TGF-(31 mary trophoblast-enriched cultures. In the representa- what we observed (Fig. 3B) after a long radiographic tive '"I-TGF-Pl affinity-labelling experiment shown exposure of the gel. The betaglycan binding that is not in Figure 3A, it can be seen that the betaglycan compo- displaceable by TGF-82 likely represents the TGF-P1 nent exhibits a 5-lo-fold greater affinity for TGF-P2 bound to the betaglycan subtype that binds TGF-P1 than for TGF-P1. Here it is seen that approximately 0.5 exclusively. Figure 3C illustrates a competition experiment on nM TGF-P2 blocks the binding of 50 pM 1251-TGF-plto the betaglycan component by the same extent as 5 nM trophoblast cells in which '"I-TGF-P2 was used as the TGF-P1. This distinctive betaglycan component shows radiotracer. The betaglycan component again exhibited the same preferential affinity for TGF-PS a s we had a preferential affinity for TGF-P2 as compared to TGFobserved with human term placental membrane prepa- P l . Moreover, in these experiments there appeared to rations (Mitchell and O'Connor-McCourt, 1991). When be the same extent of maximal displacement with autoradiograms were exposed for longer times (5weeks TGF-P1 or TGF-P2. This result agrees with our profor Fig. 3B compared with 1 week for Fig. 3A) and a posal since the higher affinity betaglycan subtype,

that the distinctive betaglycan subtypes observed with the trophoblast-enriched cultures are not contributed by any contaminating mesenchymal cells. Furthermore, it indicates that the different amounts of labelling with TGF-P1 and TGF-P2 seen with the trophoblast-enriched cultures (Fig. 1) cannot be due to differences in cross-linking efficiencies between the two isoforms. The levels of the Type I and I1 TGF-P receptors relative to the level of the betaglycan component appear to be higher in the mesenchymal cell-enriched cultures as compared with the trophoblast-enriched cultures. These low M, receptors on the mesenchymal cells were preferentially labelled with 125I-TGF-P1 (Fig. 2A) as compared to 1Z51-TGF-p2 (Fig. 2B), indicating that they exhibit a higher affinity for TGF-P1 than for TGF-P2 as has been typically observed on most cell types.

TGF-p RECEPTORS ON PLACENTAL TROPHOBLASTS

A.

TGF-81 0 0.1 0.5

6.

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'zsl-TGF-fll

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TGF-p2 20 4 0

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4

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20 4 0

(6OpY)

TGF-PI

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1 2 0 4 0

1 4 2 0 4 0

Fig. 3. Receptor competition patterns of TGF-p1 and TGF-p2 in trophoblast-enriched placental cell cultures. Cells were affinity-labelled with 50 pM (A) or 35 pM (B) '"I-TGF-pl or 60 pM (C) '"I-TGF-p2 in the presence of the indicated concentrations (nM) of TGF-p1 or TGFp2. Autoradiograms following polyacrylamide gel electrophoresis of

Triton-X-100 extracts of affinity-labelled cells are shown. The autoradiographic exposures were 1week (A)and 5 weeks (B,C).The positions of the Type I and I1 TGF-P receptors and the betaglycan component as well as the position of the molecular mass markers in kilodaltons are indicated.

which would be preferentially detected with low picomolar concentrations of '"I-TGF-P2, also binds TGFp l , albeit with a somewhat (5-10 fold) lower affinity. In competition experiments on the mesenchymal cellenriched cultures (data not shown), the betaglycan component showed equal affinity for TGF-p1 and TGFp2, in agreement with the saturation data (Fig. 2). In comparative experiments, the affinity of the mesenchyma1 cell betaglycan component for TGF-P1 and $2 was equal to that of the trophoblast betaglycan component for TGF-Pl, that is approximately 5-10-fold lower than the trophoblast subtype for TGF-P2. The Type I and Type I1 TGF-P receptors were also revealed in competition experiments with the trophoblast-enriched cultures when the autoradiograms were exposed for longer times (Fig. 3B). These receptors were preferentially competed for by TGF-P 1, indicating that they exhibit a higher affinity for TGF-p1 than for TGF-P2 which is characteristic of the Type I and I1 receptors on most cell lines. The low level of the Type I and I1 TGF-p receptors relative to the predominant betaglycan is consistent with our previous observations on term placental membrane preparations (Mitchell and O'Connor-McCourt, 1991). There was no observable difference in the pattern of the TGF-P complexes, i.e., the Type I and I1 and betaglycan, with the time of the cells in culture (2 days up to 6 days, data not shown). The Type I and I1 TGF-P receptors that normally exhibit a marked preferential affinity for TGF-P1 would be expected to bind little TGF-p2 in the '251-TGF-P2competition experiment. However, it is evident that both the Type I and I1 receptors not only bind lZ5I-TGF-P2but also show similar extents of competition with either TGF-p1 or -p2 (Fig. 3C). This suggests that there are low abundance subtypes of both the Type I and I1 receptors on trophoblast cells, detected only when 1251-TGF-p2is used as the radiotracer, that have similar affinities for TGF-p1 and TGF-p2. These Type I and I1 subtypes are masked in the competition experi-

ment with lZ51-TGF-pl (Fig. 3B) by the predominant Type I and I1 subtypes which have a preferential affinity for TGF-p1. These subtypes were also recently detected on certain cell lines (Cheifetz et al., 1990). Results with the mesenchymal cell-enriched cultures suggests there may be a small proportion of these Type I and I1 TGF-P receptor subtypes present (data not shown). However, we cannot exclude that they are contributed by contaminating trophoblast cells.

Immuno-magnetic purification of trophoblast cultures Because the levels of the Type I and I1 TGF-p receptors were low relative to the betaglycan component, we wanted to rule out the possibility that they were contributed by the small percentage of non-trophoblastic cells in our cultures. As described above, the trophoblast-enriched cell populations derived from the fourth and fifth trypsinizations of villous tissue contained 25%, or less, HLA+ cells (Table 1). The remaining HLAt cells were removed from the trophoblast-enriched cell population using a n immuno-magnetic separation technique similar to that described previously (Douglas and King, 1989). HLAi cells were separated from the HLA cells using a magnet following incubation with the Mab W6l32, which recognizes HLA class I antigens, and a magnetic goat anti-mouse IgG as the secondary antibody. Table 1 indicates that this procedure effectively removed the HLAt cells as determined by Flow Cytometry. These highly purified HLA trophoblast cells were affinity-labelled with '"I-TGF-PI (Fig. 4)and all three TGF-P binding components, betaglycan and the Type I and I1 receptors, were present at levels comparable to the control cultures which were mock purified using the SPiO myeloma supernatant. These results indicate that the Type I and I1 TGF-6 receptors are indeed exhibited by the trophoblast cells. ~

~

MITCHELL ET AL.

340

SP/O

WW32

c BG

4-11 -1

Fig. 4. Receptor patterns of TGF-p1 labelled trophoblast cultures purified by an immuno-magnetic technique. The cells were purified as described in Materials and Methods following treatment with supernatants from either the parental myeloma cell line SPiO (control)or the hvbridoma W6132 which produces a Mab that recognizes HLA class 1 antigens. Shown are autoradiograms of polyacrylamide gels containing Triton-X-100extracts ofcells affinity-labelled with 100 pM ""I-TGF-p1. Type I and I1 receptors and betaglycan component are seen in both cell populations.

DISCUSSION Identification of betaglycan s u b t y p e s on trophoblast cells in culture The purpose of this study was to determine which placental cell types display the unique class of betaglycan component that exhibits a higher affinity for TGF-P2 than for TGF-P1, recently observed by us in affinity-labelling competition experiments on term placental membranes (Mitchell and O'Connor-McCourt, 1991). This betaglycan component is distinctive because, with the exception of one earlier report (Segarini et al., 19871, the betaglycan component observed on cells in culture shows equal affinity for TGF-P1 and -p2 (Segarini, 1989; Massague et al., 1990; Boyd et al., 1990). The identification of the placental cell type(s) that exhibit this unique betaglycan component is important for the understanding of the functional role(s) of TGF-P, particularly TGF-P2, in placenta. In addition, further characterization of this distinct betaglycan will aid in a better understanding of the molecular basis of the specificity of the interaction between betaglycan and the TGF-P isoforms. Here, using affinitylabelling studies, we show that trophoblast-enriched primary placental cultures display this betaglycan subtype which exhibits a preferential affinity for TGF-P2. In addition to affinity-labelling competition experiments, in the present study we have also carried out affinity-labelling saturation experiments on trophoblast-enriched primary cultures. These saturation experiments showed different amounts of labelling of the betaglycan component with 1251-TGF-pland ""I-TGF-

pa, suggesting the presence of at least one other subtype of the betaglycan component. In addition to the subtype that exhibits a preferential affinity for TGFp2, trophoblast cells appear to have a second betaglycan subtype that has a high capacity and binds TGF-P1 exclusively. In contrast, the results of saturation and competition experiments with the mesenchymal cellenriched cultures suggested the presence of a single betaglycan subtype that binds both TGF-P1 and 432 equally, typical of that reported for most cell types. We therefore propose that the simplest explanation is that the trophoblast cells exhibit two distinctive betaglycan subtypes. The one betaglycan subtype, which we initially observed in competition experiments with placental membrane preparations (Mitchell and O'Connor-McCourt, 1991), has a relatively higher affinity and a lower capacity, binds both TGF-P1 and -p2, yet preferentially binds TGF-p2. This subtype accounts for the betaglycan component observed in the l"I-TGF-P2 saturation experiment and is preferentially detected in the IZ51-TGF-pland 1251-TGF-f32competition experiments that are carried out with low picomolar concentrations of radiotracer. The second betaglycan subtype has a higher capacity and lower affinity and binds TGF-p1 exclusively. These sites are best seen in the high concentration range in the '"I-TGF-pl saturation experiment (Fig. lA), but also likely account for the betaglycan component that is competable by TGF-P1 but not by TGF-p2 in the 1251-TGF-plcompetition experiment (Fig. 3B). At present, we cannot rule out the possibility that the betaglycan component on trophoblast cells exhibits cooperative binding effects. A more quantitative analysis of these betaglycan subtypes was difficult to achieve using primary cultures because of inherent variations including cell density and differentiation. Recently, we have found that the BeWo trophoblastic cell line appears to have betaglycan subtypes with characteristics similar to those described here for the primary trophoblast cells (Mitchell, O'Connor-McCourt, Fitz-Gibbon and Lee, manuscript in preparation). We are currently using BeWo cells to quantitatively characterize these betaglycan subtypes, both by affinity-labelling and by equilibrium binding studies. This established trophoblastic cell line is a more uniform model in which to study these placental TGF-P receptor subtypes as compared with the primary cells. However, it is essential to know that the receptor types observed in the trophoblastic choriocarcinoma cell lines are indeed similar to those seen in the corresponding primary trophoblast cells. This latter cell type is expected to more closely resemble the in vivo state. By comparison of our affinity-labelling data for primary trophoblasts to our more quantitative results for the BeWo trophoblastic cell line, we estimate approximately 5,000-10,000 TGF-P receptors per primary trophoblast. BeWo cells appear to express approximately 10-fold more TGF-P receptors (estimated a t 70,000 receptors per cell) than the primary trophoblasts. Recently, Lala's group have developed a methodology for maintaining pure cytotrophoblasts in long-term culture and this may be more amenable to quantitative TGF-P receptor analysis as compared to the short-term cultures described in the present study (Lala and Graham, 1990).

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directly by controlling TIMP. Lala's group (personal communication) have also recently found that exogenous TGF-P added to trophoblast cultures enhances TIMP mRNA expression. TGF-P was recently shown to suppress the invasiveness of tumour cells by increasing the expression of TIMP (Kubota et al., 1991). Furthermore, the observation by Lala and Graham (1990) that the invasiveness of trophoblast cells could be enhanced above control levels suggests that the trophoblasts themselves secrete TGF-p. Studies by this group also Identification and characterization of the low M, suggest that TGF-P may indirectsly control trophoblast TGF-p receptors on placental cells in culture invasiveness by stimulating differentiation to syncIt has been paradoxical that the Type I and I1 TGF-P tiotrophoblast (Lala and Graham, 1990). An important receptors that are currently believed to be true signal- next step in these studies, that will tie in with our ling receptors exhibit a much higher affinity for identification of TGF-P receptors on trophoblast cells, TGF-P1 than for TGF-p2, since the biological potencies will be the distinction of these effects between TGF-P1 of TGF-PI and TGF-PB are similar in many assays. and TGF-P2. The trophoblast is also the key player in the prevenHowever, in the present study we have detected subtypes of the Type I and I1 TGF-P receptors that exhibit tion of allograft-type rejection of the pregnancy by the similar affinities for TGF-P1 and -P2 on the tropho- mother. It is now well established that villous trophoblast-enriched cultures in competition experiments in blast cells fail to express Class I HLA antigens (Bulmer which 1251-TGF-p2was the radiotracer. These results and Johnson, 1985). Indeed, we and others (Douglas are in agreement with those of Cheifetz et al. (19901, and King, 1989) have taken advantage of this in order who have proposed that these subtypes of the Type I to monitor and purify HLA- villous trophoblast cells and I1 TGF-P receptors are revealed only in experi- for primary culture using the Mab W6132 that binds ments in which '251-TGF-P2 is used as the radiotracer. HLA class I antigens. Recently, a novel class I molecule However, these subtypes were not detected on all cell HLA-G has been identified on cytotrophoblasts (Kovats types. These Type I and I1 receptors with a high affinity et al., 1990),although its significance is not yet underfor TGF-P2 are considered to be true TGF-P-signalling stood. Very recently two groups have implicated the receptors (Cheifetz et al., 1990) based on the fact that immunosuppressive capacity of TGF-P, in particular cell-specific differences in the level of this subtype with TGF-P2, to be important for the maintenance of preghigh affinity for TGF-p2 correlates with cell specific nancy (Clark et al., 1990; Altman e t al., 1990).Neutraldifferences in responsiveness to this isoform. We are izing antibodies against TGF-p2 were shown to incurrently characterizing these Type I and I1 subtypes as crease the rate of spontaneous abortion in mice (Clark well as a novel 50 kDa TGF-P binding component on et al., 1991). Moreover, this group has shown that this BeWo cells (Mitchell, O'Connor-McCourt, Fitz-Gibbon TGF-P2-like molecule is secreted by decidual cells imand Lee, manuscript in preparation). It was not clear plying a paracrine immunoregulatory mechanism. Altfrom our studies whether the primary mesenchymal man et al. (1990), however, found very low levels of cell-enriched cultures exhibited these Type I and I1 sub- TGF-p2 mRNA expression by Northern analysis in detypes. Further studies possibly using long-term pri- cidua. This group has discovered a TGF-p2-like molemary cultures of pure placental mesenchymal cells are cule that appears to be complexed with a-fetoprotein in murine amniotic fluid. These studies, taken together needed. with our identification of a trophoblast betaglycan comBiological significance of TGF-P receptors on ponent and trophoblast Type I and I1 TGF-p receptors trophoblast cells that have high affinity for TGF-P2, support the notion TGF-P is a multifunctional regulatory factor whose of autocrine and paracrine TGF-P (particularly TGFactivities in extracellular matrix remodelling, growth p2) loops operating a t the fetal-maternal interface. Imand differentiation, and immunosuppression suggest munocytochemical evidence suggests that TGF-P1 and that it is a likely candidate as a mediator of trophoblas- TGF-@2are present intracellularly within murine trotic invasion, trophoblast differentiation and tropho- phoblasts and extracellularly in the mesenchymal comblastic immuno-regulatory events via autocrinel partment (Thompson et al., 1989; Flanders e t al., 1990). paracrine mechanisms. Trophoblast invasion of the Preliminary studies of human tissue show similar patendometrium appears to involve reorganization of the terns (P.K. Lala, personal communication). TGF-(32 has also been implicated in murine pregextracellular matrix which is controlled by proteases and their inhibitors (for review see Lala and Graham, nancy by Northern analysis and in situ hybridization 1990). Both metalloproteinases (Fisher e t al., 1989) and studies that indicated that TGF-P2 mRNA expression serine proteases (Lala and Graham, 1990) are impli- was exceptionally high in placental mesenchymal tiscated. It was recently shown that conditioned medium sue (Miller et al., 1989; Pelton et al., 1989). These refrom decidual cells blocks the invasiveness of cultured sults have been recently challenged by Schmid e t al. trophoblast cells. (Lala and Graham, 1990). This activ- (1991) and Altman et al. (19901,who, by using different ity was mimicked by TGF-p and enhanced above con- probes and hybridization conditions, did not detect high trol levels by neutralizing antibodies against either levels of TGF-PB mRNA in mouse placenta. However, TGF-P or TIMP (tissue inhibitor of metalloproteinase), Altman et al. (1990) did report that very high levels of implying that TGF-(3 inhibits trophoblast invasion in- TGF-p2 mRNA expression occurred in the mouse

At present, it is difficult to speculate on the significance of this unusually high affinity for TGF-p2 in a betaglycan component which is thought to serve a nonsignalling role. However, together with the recent finding of Danielpour and Sporn (1990) that a2-macroglobulin also preferentially binds to TGF-P2, it suggests that TGF-P1 and TGF-P2 are differentially controlled. It is possible that betaglycan transfers the TGF-p2 to a signalling receptor.

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uterus at day 15 of gestation, suggesting a strict temporal control of its expression. Finally, a recent report indicated that TGF-P2 is important in differentiation of the mouse embryo itself (Kelly et al., 1990). Together, these studies underscore the importance of understanding the roles of TGF-@,particularly TGF-62, in placental functions and embryonic development during pregnancy. ACKNOWLEDGMENTS We are grateful to Drs Charlotte Branchaud and Cindy Goodyer of The Montreal Children’s Hospital for providing human placenta. We also wish to thank Lucie Bourget for the Flow Cytometric analysis, Chantal Stonehouse for assistance with the manuscript preparation, and Drs. Maria Debanne, Luis Martin, and David Thomas for their critical comments and suggestions on the manuscript. This is NRC Publication 32794. LITERATURE CITED Altman, D.J.,Schneider, S.L., Thompson, D.A,, Cheng, H.-L. and Tomasi, T.B. (1990) A transforming growth factor p2 (TGF-p2)-like immunosuppressive factor in amniotic fluid and localization of TGF-p2 in the pregnant uterus. J. Exp. Med., 172t1391-1401. Andres, J.L., Stanley, K., Cheifetz, S., and Massague, J . (1989) Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-p. J . Cell Biol., 109r31373145. Barnstable, C.J., Bodmer, W.F., Brown, G., Galfre, G., Milstein, C., Williams, A.F., and Ziegler, A. (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens.-New tools for genetic analysis. Cell, 14t9-20. Blay, J., and Hollenberg, M.D. (1989) The nature and function of polypeptide growth factor receptors in the human placenta. J. Dev. Physiol., 12:237-248. Boyd, F.T., Cheifetz, S., Andres, J.,Laiho, M., and Massague, J. (1990) Transforming growth factor-p receptors and binding proteoglycans. J . Cell Sci. [Suppl.], 13t131-138. Boyd, F.T., and Massague, J . (1989) Transforming growth factor-p inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J . Biol. Chem., 264:2272-2278. Branchaud, C.L., Goodyer, C.G., and Lipowski, L.S. (1983) Progesterone and estrogen production by placental monolayer cultures: effects of Dehydroepiandrosterone and luteinizing hormone-releasing hormone. J . Clin. Endocrinol. Metab., 56t761-766. Bulmer, J.N., and Johnson, P.M. (1985)Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance. Placenta, 6t127-140. Cheifetz, S., Hernandez, H., Laiho, M., ten Dijke, P., Iwata, K.K., and Massague, J. (1990) Distinct transforming growth factor-p (TGF-8) receptor subsets as determinants of cellular responsiveness to three TGF-p isoforms. J . Biol. Chem., 265t20533-20538. Clark, D.A., Flanders, K.C., Banwatt, D., Millar-Book, W., Manuel, J., Stedronska-Clark, J., and Rowleyh, B. (1990) Murine pregnancy decidua produces a unique immunosuppressive molecule related to transforming growth factor p2. J . Immunol., 144t3008-3014. Clark, D.A., Lea, R.G., Podor, T., Daya, S., Banwatt, D., and Harley, C. (1991) Cytokines determine the success or failure of pregnancy. Ann N.Y. Acad. Sci. 626526536. Danielpour, D., and Sporn, M.B. (1990) Differential inhibition of transforming growth factor p l and p2 activity by a2-macroglobulin. J. Biol. Chem., 265t6973-6977. Deal, C.L., and Guyda, H.J. (1983) Insulin receptors of human term placental cell and choriocarcinoma (JEG-3) cells: characteristics and regulation. Endocrinology, I12r1512-1523. Douglas, G.C., and King, B.F. (1989) Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J . Immunol. Methods, 119t259-268. Fisher, S.J., Cui, T.-Y., Zhang, L., Hartman, L., Grahl, K., Guo-Yang, Z., Tarpey, J., and Damsky, C.H. (1989) Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J . Cell Biol., 109:891-902. Flanders, K.C.. Cissel. D.S.. Mullen, L.T., Daniebour,. D... SDorn. M.B.. . and Roberts; A.B. (1990) Antibodies t o transforming growth fac:

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