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Regular Article |
a Departments of Anatomy and Cell Biology and
b Pathology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, growth factors, placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Proliferative activity of EVT cells in situ declines in a temporal-spatial manner with increasing gestational age, and as EVT cell columns approach the decidua. Examination of proliferation-associated markers in situ shows that proliferative activity is high at the base of the columns, declines progressively thereafter, and disappears after EVT cells have invaded the decidua [3–5]. A progressive withdrawal of cells from the cell cycle has been further demonstrated by in situ immunolabeling for a number of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors [6]. This phenomenon can be explained by one or both of two possibilities: either a progressive differentiation of cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation of proliferation by decidua-derived factors.
So far, a number of laboratories have attempted to address the mechanisms that regulate EVT cell proliferation, migration, and invasiveness in situ with the help of in vitro-designed functional assays that have employed several trophoblast culture models. These models include 1) freshly isolated cytotrophoblast cells that do not proliferate, but they can differentiate along the invasive pathway when plated on matrigel (a reconstituted basement membrane) [7, 8]; 2) EVT cells enriched by laminin-adherence from freshly isolated cytotrophoblasts [9]; 3) explant cultures on plastic of pure EVT cell lines derived from outgrowths of first-trimester chorionic villus [10–14], which retain their proliferative, migratory, and invasive functions in vitro; and 4) cultures of chorionic villus fragments on matrigel [15, 16] or collagen gel in the presence of decidual tissue [17–19], which promote sprouting (a combined function of proliferation and migration) of EVT cells from the villi. Studies with these models have suggested that proliferative, migratory, and invasive functions of EVT cells are stringently regulated in situ in an autocrine/paracrine manner by a variety of factors in the EVT cell microenvironment such as growth factors, growth factor-binding proteins, and proteoglycans; and by components of the extracellular matrix that interact with receptors and binding sites on the EVT cell surface [1, 20–22].
By using in vitro-propagated first-trimester EVT cells, which share phenotypic and functional properties of EVT cells in situ [13, 14], we have shown that transforming growth factor (TGF)-ß, produced primarily by the decidua and, to a smaller extent by the syncytiotrophoblast, is a key negative regulator of EVT cell proliferation [13], migration [23], and invasiveness [12, 24]. The mechanisms responsible for the anti-invasive effect of TGF-ß on EVT cells have also been identified [12, 23–26]. Furthermore, trophoblastic cancer (choriocarcinoma) cells were found to have acquired resistance to both antiproliferative and anti-invasive actions of TGF-ß [27].
We observed that a TGF-ß binding proteoglycan, decorin (also called dermatan sulfate proteoglycan II) is colocalized with TGF-ß in first-trimester decidual extracellular matrix (ECM) in situ. This was shown by a strong and selective immunostaining for both molecules at the same location [28]. Decorin is a member of the small leucine-rich proteoglycan (SLRP) family, which can regulate matrix assembly and cellular growth [29, 30]. The core protein of decorin has TGF-ß binding sites with both high and low affinity [29, 31], and under certain conditions, decorin has been shown to inactivate TGF-ß [32]. Its ability to regulate matrix assembly has been attributed to its ability to bind collagen type I and to retard the formation of collagen fibrils [29]. Decorin has also been shown to inhibit proliferation and migration of certain normal and malignant cells. An antiproliferative action has been reported for carcinoma cell lines [33–36], bone marrow macrophage colony-forming cells [37], and endothelial cells [38]. An antimigratory action has been reported for endothelial cells [39, 40] and an osteosarcoma cell line [41].
We hypothesized that decidua-derived decorin provides a storage mechanism for TGF-ß in the decidual ECM, and that decorin-bound TGF-ß is possibly cleaved and activated in situ by EVT cell proteolytic mechanisms, thus preventing EVT cell overinvasion of the decidua [28, 42]. However, the precise role of decorin in the regulation of EVT cell functions remains unexplored.
The present study sought answers to two questions. First, do EVT cells lose their proliferative ability after the act of invasion? In other words, is the act of invasion an exclusive outcome of terminal differentiation of cytotrophoblast cells along the invasive pathway? And second, what is the functional role of decorin on EVT cells? In other words, does decorin modulate EVT cell proliferation, migration, or invasiveness in a TGF-ß-dependent or TGF-ß-independent manner?
To answer these questions we used in vitro-propagated EVT cells and subjected them to functional assays in vitro. In the first case, we tested proliferative activity with EVT cells that had already invaded and migrated through a matrigel barrier. In the second case, proliferative, migratory, and invasive functions of EVT cells were measured in the presence of decorin, TGF-ß, or their combination. We further examined whether decorin treatment of EVT cells alters the expression of p21WAF1/CIP1 (p21) protein, an inhibitor of cyclin-dependent kinases, and whether choriocarcinoma cells are responsive to decorin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pure decorin, isolated from bovine articular cartilage, was purchased from Sigma Chemical Company (St. Louis, MO). Human recombinant TGF-ß1 was purchased from R&D Systems Inc. (Minneapolis, MN). A TGF-ß-neutralizing monoclonal antibody, 1D11.16.8 (capable of neutralizing both TGF-ß1 and TGF-ß2), was a gift of Wendy Waegell (Celtrix Laboratories, Santa Cruz, CA). Polyclonal goat anti-human Ki-67 antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Biotinylated swine anti-goat immunglobulin G (IgG) was purchased from Cedarlane (Hornby, ON, Canada). Avidin-biotin complex (ABC) was purchased from Vector Laboratories (Burlingame, CA). Monoclonal p21 (WAF-1) antibody was obtained from Oncogene Research Products (Boston, MA). Growth factor-reduced matrigel was purchased from Collaborative Research (Bedford, MA). The cell proliferation kit I (MTT) was purchased from Roche Diagnostics GmbH (Mannheim, Germany).
Cell Lines and Culture
Two EVT cell lines produced earlier in our laboratory were employed in the present study. HTR-8 is a short-lived (it lives 12–15 passages in vitro) EVT cell line derived from a first-trimester chorionic villus explant culture [13]. HTR-8 cells express the markers of EVT cells in situ (e.g., cytokeratin 7, 8, and 18; placental type alkaline phosphatase; high-affinity urokinase type plasminogen activator [uPA] receptors; human leukocyte antigen [HLA] framework antigen W6/32; insulin-like growth factor II [IGF-II] mRNA and protein; a selective repertoire of integrins: 
1, 3, 5, V, and ß1; and the vitronectin receptor, Vß3/ß5) [13, 14]. HTR-8 cells express HLA-G mRNA and protein in the presence of laminin or matrigel [43]. HTR-8 cells at passages 6 to 8 were used in the first component of the study, which evaluated proliferative activity of EVT cells after they had invaded matrigel.
In the second component of the study, to explore the effects of decorin, we also employed the HTR-8/SVneo cell line, an SV40 Tag-immortalized derivative of HTR-8 cells selected on the basis of their neomycin resistance [26]. This cell line shares all the markers of HTR-8 cells and, like HTR-8 cells, it responds to the antiproliferative [26], anti-invasive, and antimigratory [44] signals of TGF-ß as well as migration-stimulatory signals of IGF-II [45] and insulin-like growth factor binding protein (IGFBP)-1 [44]. HTR-8/SVneo cells also share the phenotypic behavior of freshly isolated cytotrophoblast during matrigel invasion, including the expression of HLA-G [46]. These cells were used at passages 70 to 80 during the present study. Because preliminary experiments revealed that both HTR-8 and HTR-8/SVneo cells responded to decorin, TGF-ß, or their combination in a similar manner, only the data derived with HTR-8/SVneo cells are presented here.
In the second component of the study, we also tested the effects of decorin on two choriocarcinoma cell lines, JAR and JEG-3, which we obtained from American Type Culture Collection (Manassas, VA). All cell lines were grown in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Burlington, ON, Canada) at 37°C in a humidified atmosphere of 5% CO2, unless specified otherwise.
Cellularity Assay
Cellular proliferation/survival was measured with cellularity assays using the MTT colorimetric method as reported earlier [44], which exploits the conversion of MTT by the intact mitochondria of living cells to a colored product, formazan, the concentration of which is measured spectrophotometrically. In brief, after defined periods of culture, cells grown in RPMI-1640 complete medium were harvested using a 0.05% trypsin-EDTA solution and resuspended in serum-reduced medium (SRM; RPMI containing 1% FBS). Cell suspensions (2.5 x 104 cells/100 µl) were plated into 96-well plates in SRM with decorin (10–200 nM), TGF-ß1 (10 ng/ml or 400 nM), or a combination of decorin and TGF-ß1. In some experiments, a TGF-ß-neutralizing antibody (25 µg/ml of purified immunoglobulin) was added to wells in the presence of TGF-ß1, decorin, or their combination. The plates were incubated for 24 or 48 h at 37°C prior to the addition of MTT solution, which was followed by spectrophotometric measurement of absorbance at 570 nm. Changes in cell number were deduced from the absorbance data by using the linear part of standard absorbance curves produced with predetermined cell numbers.
Transwell Migration Assay
Cellular migratory function was determined by the ability of cells to migrate through the 8-µm pores of polycarbonate membranes fitted to the bottom of transwell migration chambers, as we reported earlier [44, 45]. In brief, 2 x 105 cells/ml of SRM was plated in the migration chambers (200 µl per transwell), which were immersed in tissue culture wells containing SRM (800 µl/well) under various treatment conditions (no treatment; 10–200 nM decorin, 400 nM TGF-ß1, or a combination of these) applied to both the upper (migration chamber) and lower wells at the same concentration. The number of cells appearing on the undersurface of the polycarbonate membranes was scored visually after a specific time period (24–72 h) and after the cells had been stained with a Hemacolor Stain Set (EM Science, Gibbstown, NJ) in 10 random, nonoverlapping fields at 400x magnification using a light microscope. Each condition was tested in triplicate wells, and the experiments were repeated three times.
Transwell Invasion Assay
The assay conditions were exactly the same as described above for the migration assay, except that the polycarbonate membranes at the bottom of the transwells were coated with a thin layer of growth factor-reduced matrigel, so that the cells had to degrade the matrigel barrier before they migrated to the undersurface of the polycarbonate membranes [47].
Cellular Proliferative Ability Following the Act of Invasion
HTR-8 cells were allowed to invade growth factor-reduced matrigel for 48 h as indicated above, except that the invasion assay was carried out in RPMI-1640 complete medium rather than SRM. Subsequently, cells that had appeared on the undersurface of the polycarbonate membrane were recovered by brief treatment with 0.05% trypsin-EDTA solution followed by a gentle scraping. Cells pooled from multiple (24–32) membranes were washed and resuspended in RPMI-1640 complete medium. Recovered cells was found to be 80%–85% viable, as indicated by trypan blue exclusion. Cells were then split in order to perform two different types of assays; the MTT cellularity assay after 48–72 h of culture as described earlier, or immunolabeling for the Ki-67 proliferation marker. In the latter assay, cells were grown overnight (16 h) on Lab-Tek tissue culture chamber slides (Nalge Nunc International, Rochester, NY). After fixation with 3.7% formaldehyde containing Triton-X 100, cells were sequentially treated with polyclonal goat anti-human Ki-67 primary antibody followed by biotinylated swine anti-goat IgG secondary antibody, and then ABC. Washes with 1% BSA in PBS were performed between treatments. The slides was treated with 3,3'-diaminobenzidine (DAB) and lightly counterstained with hematoxylin for visualization. The same concentration of normal goat immunoglobulin, which was used as a replacement for the primary antibody, was used as a negative control. The percentage of labeled cells (brown staining) was visually screened with a light microscope on the basis of scoring a minimum of 500 cells. The intensity of staining above background (negative control) was recorded as moderate (++) to strong (+++).
Western Immunoblot for p21 Protein
After HTR-8/SVneo cells were grown in RPMI-1640 complete medium for 24 h, they were cultured in serum-free medium supplemented with 1% BSA overnight, and were replenished with 10% serum in the presence or absence of decorin. In brief, the serum-free medium was replaced with 10% serum-containing medium with or without 50 nM decorin for 24 h. At the end of the incubation period, cells were washed twice with ice-cold PBS and lysed with SDS lysis buffer (80 mM Tris-HCl pH 6.8, 2% SDS, and 10% glycerol) containing protease inhibitor mix (Complete Mini tablet, Boehringer-Mannheim, Mannheim, Germany). The whole cell lysates were collected and centrifuged at 13 000 x g for 20 min at 4°C. The supernatant was then collected, and proteins were denatured by boiling for 5 min, followed by chilling on ice. The protein content was detected by using a BCA Protein Assay Kit (Pierce, Rockford, IL).
Equal quantities of protein (100 µg) were separated by SDS-PAGE gradient gel (5%–20%) and transferred to a PVDF membrane (Hybond-P, Amersham, Baie d'Urfé, QC, Canada). After blocking with 5% nonfat milk in 10 mM Tris-buffered saline with 0.05% Tween-20 for 1 h, the membrane was incubated with monoclonal anti-p21 (WAF-1) primary antibody for 2 h at room temperature or overnight at 4°C. After three 5-min washes, the membrane was incubated with a secondary antibody for 1 h. Secondary antibody was sheep anti-mouse IgG conjugated with horseradish peroxidase (Amersham) diluted 1:10 000. Specific signals were detected using an ECL+Plus kit (Amersham) and visualized after exposure to Hyperfilm ECL film (Amersham). Signals were scanned with an Epson scanner (Epson Expression 1680, Long Beach, CA) and quantitated with Scion Image program (Scion Corporation, Frederick, MD).
Statistical Analysis
Data on cellularity, migration, and invasiveness using a single reagent were analyzed by one-way ANOVA followed by the Tukey multiple comparison test. When two reagents were added in combination, to determine any interactive effect, data were log-transformed and analyzed by two-way or factorial ANOVA followed by a least squares means multiple range test. Because the data were not always normally distributed, the Mann-Whitney rank sum test was employed to determine the level of significance in differences in pairs of various treatment groups. All values were expressed as the mean ± SEM. Differences were accepted as significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows cellularity as determined by the MTT assay at 48–72 h after plating HTR-8 cells recovered from the undersurface of polycarbonate membranes at 48 h during the invasion assay (data from two different experiments). For comparison, the same population of control HTR-8 cells placed in routine culture for 48 h, but not subjected to the invasion assay, were employed after lifting them by a method that was similar to obtaining cells from the bottom of the invasion chambers. Although there was a significant reduction (P < 0.01) in the rate of growth (cell proliferation/survival) of postinvasive EVT cells in comparison with control cells, there was still a significant increase (P < 0.01) in cellularity of postinvasive cells (by 40% in experiment 1 and by 60% in experiment 2) during the 48-h period. At 72 h, the number of postinvasive cells in culture remained essentially unchanged, whereas control cells showed a further increase in cell number by 12%–15%. These experiments revealed that proliferation/survival ability of EVT cells after the act of invasion was reduced but not abolished.
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Data derived from Ki-67 immunostaining of the same postinvasive EVT cell population (in experiment 2) that was allowed to attach to chamber slides for 16 h provided independent evidence for their proliferative ability (Table 1). The percentage of Ki-67 positive (S phase) cells was 69% in the postinvasive population, compared with 84% in the control population. The difference was primarily explained by a reduction in the incidence of strongly labeled cells (20% vs. 44% in controls). The Ki-67 labeling data at 16 h combined with the cellularity data at 48–72 h suggest that a high proportion of postinvasive EVT cells enter S phase. However, these cells complete fewer cell cycles in comparison with control cells.
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Suppression of Proliferation/Survival of EVT Cells by Exogenous Decorin in a Dose-Dependent Manner
Figure 2 shows data on cellularity (as measured by the MTT assay) of HTR-8/SVneo cells cultured in the presence of various concentrations of decorin (0–200 nM) for 24 h and 48 h. A decorin dose-dependent reduction in cellularity was significant (P < 0.005) at all concentrations and all time points. At decorin concentrations of 100 nM and 200 nM, the expected temporal increase in cellularity was completely abolished (P < 0.001), indicating a static population. A similar antiproliferative effect of decorin was also noted with HTR-8 cells (the short-lived precursor of HTR-8/SVneo cells; data not shown).
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TGF-ß-Independence of Antiproliferative Effects of Decorin on HTR-8/SVneo Cells
We had earlier shown that TGF-ß has an antiproliferative effect on EVT cells [13]. Because decorin can bind to TGF-ß and inactivate TGF-ß under certain conditions[32], we tested the effects of decorin (50 nM), TGF-ß1 (10 ng/ml or 400 nM), or their combination on cellularity of HTR8/SVneo cells at 48 h. In addition, a TGF-ß-neutralizing antibody (25 µg/ml, shown to neutralize a minimum of 10 ng/ml of TGF-ß1 in earlier studies) was added under certain experimental conditions (Fig. 3). Decorin or TGF-ß1, when added alone, significantly suppressed cellularity at 48 h (P < 0.01). When the two agents were combined, a similar but not more pronounced suppression, was noted. The presence of the TGF-ß-neutralizing antibody completely abrogated the suppressive effects of TGF-ß1. Addition of this antibody to decorin in the presence of TGF-ß1 failed to restore normal cellularity. These results, which were reproduced in two separate experiments, conclusively demonstrate that decorin effects were independent of TGF-ß. However, they do not exclude the possibility that decorin, after binding to TGF-ß, may inactivate TGF-ß, but still retain its TGF-ß-independent antiproliferative effect on EVT cells.
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Migration Inhibition of HTR8/SVneo Cells by Decorin and TGF-ß in an Independent Manner
We had earlier shown that TGF-ß has antimigratory effects on EVT cells [23, 44]. In the present study, we tested the effects of various concentrations (10–100 nM) of decorin alone, TGF-ß1 (400 nM) alone, or their combination on HTR-8/SVneo cell migration at 24 h and 48 h, in three separate experiments.
Because the results were essentially similar at both time points, data on 48-h migration are presented (Fig. 4). TGF-ß1 alone and decorin alone (all concentrations) significantly (P < 0.01) reduced cellular migration. When they were combined, the effects in most combinations were similar to those of decorin alone. Decorin-mediated antimigratory effects could not be entirely attributed to the antiproliferative effects at their respective concentrations of 10 nM and 50 nM (Fig. 2). At these concentrations, migration was reduced by 45% and 85%, respectively, whereas proliferation was reduced by only 15% and 28%, respectively. These results demonstrate that decorin exerts an antimigratory effect on HTR-8/SVneo cells independent of its antiproliferative effect, and independent of TGF-ß1. However, they do not exclude the possibility that decorin may inactivate TGF-ß, but it retains its antimigratory effects on EVT cells in the presence of TGF-ß. In fact, a comparison of the migration indices noted with 400 nM TGF-ß1 alone, 10 nM decorin alone, and their combination, suggests that TGF-ß1 was inactive in the combined treatment. Although TGF-ß1 alone caused a higher (P < 0.01) degree of migration inhibition than decorin alone, antimigratory effects of the combined treatment were identical to those of decorin alone.
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Invasion Inhibition of HTR-8/SVneo Cells by Decorin and TGF-ß in an Independent Manner
We had earlier shown that TGF-ß inhibits EVT cell invasiveness [12, 24]. In the present study we tested the effects of various concentrations of decorin (10–100 nM), TGF-ß1 (400 nM), or their combination on EVT invasiveness at 48 h. Two separate experiments provided similar results. Results from one experiment, presented in Figure 5, revealed that decorin (at all concentrations) and TGF-ß1 independently inhibited EVT cell invasiveness (P < 0.001). The combined effects of TGF-ß1 and decorin were not different from those of decorin alone. Effects of decorin (at 10 nM and 50 nM concentrations) on invasiveness were significantly more pronounced than its effects on cellularity (Fig. 2), indicating that its anti-invasive effect was independent of its antiproliferative action. These results also demonstrate that the decorin block of EVT cell invasiveness was independent of TGF-ß, but they do not exclude the possibility that decorin may inactivate TGF-ß while it retains its anti-invasive effect.
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Resistance of JAR Choriocarcinoma Cells to the Antiproliferative Effect of Decorin
We had earlier shown that JAR and JEG-3 choriocarcinoma cells are resistant to the antiproliferative action of TGF-ß [27]. In the present study, we tested the effects of decorin on cellularity of both cell lines. Because the results were similar, only data on JAR cells are presented (Fig. 6). Under control conditions there was a 3-fold increase in cellularity at 48 h (data not shown). This was unaffected with decorin at 10- to 100-nM concentrations. A significant (P < 0.05) drop in cellularity was noted only at the highest (200 nM) concentrations, but this effect was much smaller than that noted with normal EVT cells (Fig. 2). These results were reproduced in an additional experiment.
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Resistance of JAR Choriocarcinoma Cells to Antimigratory Effects of Decorin or TGF-ß
We tested the effects of decorin (10–100 nM) and TGF-ß1 (400 nM) or their combination on migration of JAR cells at 24 h or 48 h in two separate experiments, both of which provided similar results. Because the effects were similar at both time points, data are presented for 48 h from a single experiment (Fig. 7A). Neither decorin (at any concentration), TGF-ß1, nor their combination exerted any effect on JAR cell migration.
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Resistance of JAR Choriocarcinoma Cells to Anti-Invasive Effects of Decorin or TGF-ß
We have earlier shown that both JAR and JEG-3 choriocarcinoma cells are refractory to anti-invasive effects of TGF-ß [27]. In the present study, we tested the effect of decorin (10–100 nM), TGF-ß1 (400 nM), or their combination on JAR cell invasiveness at 48 h (Fig. 7B). Neither decorin alone (at all concentrations), TGF-ß1 alone, nor their combination failed to exert any effect on JAR cell invasiveness. These results were reproduced in an additional experiment.
Decorin-Mediated Up-Regulation of p21 Protein in HTR-8/SVneo Cells
We tested whether the antiproliferative effect of decorin on EVT cells could be explained by an up-regulation of p21 protein, a well-recognized CDK inhibitor. This was examined in HTR-8/SVneo cells by immunoblot analysis. Cells in the absence of serum became quiescent and expressed an abundance of p21 (Fig. 8). When cells were maintained in 10% serum-containing medium, the expression of p21 was significantly lower (35%) compared with its expression in serum-free medium. However, an additional presence of decorin (50 nM) in the presence of serum up-regulated the expression of p21 by 2.2-fold, compared to a 2.8-fold up-regulation during growth arrest induced by serum starvation. These data suggest that decorin-induced suppression of EVT cell growth can be attributed to an up-regulation of p21 expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Decorin, a member of the SLRP family, is present in a variety of connective tissues. It is composed of a 40-kDa core protein and a covalently linked glycosaminoglycan (GAG) chain, which is tissue-specific (e.g., dermatan sulfate chain in skin, dermatan sulfate/chondroitin sulfate in cartilage [48], and chondroitin sulfate in bone [49]). The nascent core protein of human decorin is 359 amino acids in length [50] and constitutes four domains. Domain I contains a signal peptide, and a propeptide is lost in the mature form. The GAG chain, attached to the N-terminus of domain II, can interact with other GAG chains or core proteins, and has a binding site for thrombospondin. Domain III contains long arrays of leucine-rich repeats, and has binding sites for TGF-ß, thrombospondin, and the heparin-binding domain of fibronectin. The C-terminal region (domain IV) contains binding sites for collagen (types I and II) and for the cell-binding domain of fibronectin [29, 30]. These features of decorin structure explain some of the functions of the molecule (e.g., interactions with TGF-ß, matrix proteins, and regulation of matrix assembly). A major role of decorin in collagen fibrillogenesis is indicated by the phenotype of decorin null mice, which exhibit fragile skin associated with abnormal collagen fibrils in the dermis [51]. Decorin core protein binds to the active but not the latent form of TGF-ß and neutralizes TGF-ß activity, apparently by interfering with TGF-ß binding to type I and type III, but not type II TGF-ß receptors on the surface of target cells. This function is apparently hindered by the presence of GAG chains [31].
This complexity in structural-functional relationship may explain why the TGF-ß-neutralizing function of decorin [32] is not detected under all conditions [52]. Although our present in vitro functional data provided no direct and conclusive evidence in favor or against a TGF-ß-inactivating role for decorin, they are more consistent with decorin-mediated inactivation of TGF-ß, because the combined effects of decorin and TGF-ß were essentially similar to the effects of decorin alone.
Our results conclusively demonstrate TGF-ß-independent functions of decorin in blocking EVT cell proliferation, migration, and invasiveness. The antiproliferative effect of decorin noted on EVT cells appears to be a part of the recognized onco-suppressor role of decorin in controlling cell growth. For example, decorin expression is up-regulated in cells during growth arrest, and this expression is abrogated or suppressed by oncogenic viral transformation in many tumorigenic cell lines [53, 54]. We have identified at least one mechanism for an antiproliferative effect of decorin on EVT cells (i.e., an up-regulation of the CDK inhibitor p21 protein). This finding is in support of previous reports showing that ectopic expression of decorin in a colon carcinoma cell line abrogated its malignant phenotype by causing growth arrest, and which is associated with an up-regulation of p21 [33, 34, 55]. Furthermore, in vitro growth inhibition of a squamous cell carcinoma cell line in the presence of decorin transgene or exogenous decorin [35] and growth arrest of ovarian cancer cells in the presence of exogenous decorin [36] were shown to be associated with an increased expression of p21.
The receptor involved in mediating the antiproliferative signals of decorin remains undefined in the present study. In a chain of elegant studies reported by the laboratory of Renato Iozzo, epidermal growth factor-receptor (EGF-R) has been strongly implicated in mediating decorin action. Decorin-mediated increase in cytosolic Ca2+ [56] and growth suppression [35] in A431 carcinoma cells was shown to be EGF-R dependent. Decorin protein was shown to be a biological ligand for the ectodomain of EGF-R, which bound decorin with relatively low affinity. This binding led to a dimerization of EGF-R [57]. Furthermore, stable expression of a decorin transgene in A431 carcinoma cells caused a sustained down-regulation of EGF-R number and basal EGF-R kinase activity without changing EGF-R mRNA expression [58]. The authors speculated that decorin ligation to EGF-R antagonized EGR-R action by promoting receptor endocytesis and intracellular degradation, resulting in reduced receptor recycling to the cell surface [58]. We had earlier shown that EVT cells express EGF-R and respond to numerous EGF-R ligands such as EGF, TGF-, and amphiregulin by a greater proliferation in vitro [59–61]. Of these ligands, TGF- was shown to act in both autocrine and paracrine manners. Thus, it is possible that decorin functions as an EGF-R antagonist in mediating its antiproliferative effect on EVT cells. This hypothesis remains to be tested. However, the EGF-R-independent effect of decorin on EVT cells, mediated by another receptor, cannot be ruled out at present. For example, decorin-induced cytostasis of bone marrow-derived macrophage colony-forming cells [37] and endothelial cells [38] was found to be independent of EGF-R.
The mechanisms responsible for antimigratory and anti-invasive effects of decorin on EVT cells remain to be identified. These functions may result from the interaction of the decorin core protein or its GAG chain with binding sites on the cell surface or cell-associated ECM. For example, the core protein of decorin interacts with the heparin binding and cell binding domains of fibronectin to inhibit cell adherence properties of this ECM protein [62, 63]. Furthermore, decorin has binding sites in the GAG chain as well as in the core protein for thrombospondin, and can inhibit cell adhesion to thrombospondin-1 [64, 65]. Retrovirally mediated expression of decorin or exogenous decorin was found to have migration-inhibiting effects on macrovascular endothelial cells, and is believed to be due to decorin-mediated modulation of cell-associated fibronectin matrix assembly [39]. Antimigratory effects of decorin on endothelial cells was potentiated in the presence of throbospondin-1 [40]. Furthermore, decorin was shown to impede migration-promoting effects of several matrix molecules (e.g., fibronectin and collagen type I, for which it has binding sites) on an osteosarcoma cell line. And finally, the dermatan sulphate chain of decorin was found to be responsible for decorin-mediated impediment of cellular migration in the presence of fibronectin [41].
In view of these reports it may be suggested that the antimigratory effect of decorin on EVT cells may be mediated by a dual mechanism: an interference of adhesive interactions with fibronectin by the decorin core protein, and an impediment of EVT cell locomotion on its natural substrate, fibronectin, due to binding of the dermatan sulphate chain (of the cartilage-derived decorin used in the present study) to fibronectin. We had shown that EVT cells in vitro make fibronectin [14], and that access to cell surface 5/ß1 integrin (fibronectin receptor) is essential for EVT cell migratory function [23, 44]. The anti-invasive effect of decorin on EVT cells can possibly be explained by its antimigratory function, because migration is an essential step in the cellular invasive function, which also requires matrix degradation. Whether decorin can interfere with the matrix-degrading ability of EVT cells remains to be examined.
We had earlier shown that nascent TGF-ß produced by human first-trimester decidual cells in vitro is secreted in its latent inactive form, and is likely activated by trophoblast-derived proteolytic mechanisms [12]. We can now propose that a colocalization of decorin with TGF-ß reported by us in the decidual ECM [28] has a number of functional implications. First, decorin would provide a mechanism for storage of mature TGF-ß in the decidual ECM, most likely in an inactive state in the decorin-bound form. At the invasive front of the EVT within the decidua, active TGF-ß would then be released by cleavage of the decorin-TGF-ß complex that results from the EVT cell proteolytic function, thereby providing a highly localized mechanism for controlling trophoblast hyperinvasion. This hypothesis is supported by in vitro findings of active TGF-ß1 release from a decorin-TGF-ß1 complex in the presence of matrix metalloproteinases 2, 3, and 7 [66]. Second, decorin, on its own, even if TGF-ß-bound, would serve as an independent negative regulator of EVT cell proliferation, migration, and invasiveness, as shown by the present in vitro experiments. Thus, two decidua-derived factors, TGF-ß and decorin, appear to play a major role in containing tumor-like functions of the placenta, which are required for a healthy utero-placental homeostasis. Decorin has also been isolated from term placental villous tissue, where it is believed to provide antithrombotic function [67].
Unlike normal EVT cells, choriocarcinoma cells were found to be refractory to antiproliferative, antimigratory, and anti-invasive effects of decorin (present study) as well as TGF-ß [27], thus emancipating them from the two important molecular brakes operating in the decidua in situ. We had shown that loss of expression of a key TGF-ß signaling molecule, samd3, by the choriocarcinoma cells may, at least in part, explain their resistance to TGF-ß [68]. Genetic alterations responsible for their refractoriness to decorin remain to be identified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Canadian Institutes of Health Research grant MOP-14646 to P.K.L. and postdoctoral fellowships of the Lalor Foundation Inc. and National Science and Engineering Research Council of Canada to M.-J.G. 
2 Correspondence. FAX: 519 661 3936; pklala{at}uwo.ca
3 These authors contributed equally to this work
Accepted: March 21, 2002.
Received: January 24, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5ß1. Biol Reprod 1999; 60::828-8385ß1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 2001; 86:2484-2493 in the human placenta and decidua: role in trophoblast growth. Biol Reprod 1993; 49:885-894[Abstract] and epidermal growth factor (EGF) on proliferation and invasion by first trimester human trophoblast. Trophoblast Res 1994; 8:455-467This article has been cited by other articles:
|
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 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 B. Gellersen, J. Briese, M. Oberndorfer, K. Redlin, A. Samalecos, D.-U. Richter, T. Loning, H.-M. Schulte, and A.-M. Bamberger Expression of the Metastasis Suppressor KAI1 in Decidual Cells at the Human Maternal-Fetal Interface: Regulation and Functional Implications Am. J. Pathol., January 1, 2007; 170(1): 126 - 139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 S. Goldoni, R. T. Owens, D. J. McQuillan, Z. Shriver, R. Sasisekharan, D. E. Birk, S. Campbell, and R. V. Iozzo Biologically Active Decorin Is a Monomer in Solution J. Biol. Chem., February 20, 2004; 279(8): 6606 - 6612. [Abstract] [Full Text] [PDF]
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