Primary cultures of human endometrial stromal cells expressed a single class of specific high-affinity binding sites for urokinase plasminogen activator (UPA) ...
Molecular Human Reproduction vol.4 no.6 pp. 585–593, 1998
Degradation of urokinase plasminogen activator (UPA) in endometrial stromal cells requires both the UPA receptor and the low-density lipoprotein receptor-related protein/α2-macroglobulin receptor Bertil Cassle´n1,3, Barbro Gustavsson1, Bo Angelin2 and Mats Gåfvels2 1Department
of Obstetrics and Gynecology, Lund University Hospital, S-221 85 Lund and 2Center for Metabolism and Endocrinology, Department of Medicine and Molecular Nutrition Unit, Center for Nutrition and Toxicology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden
3To
whom correspondence should be addressed
Primary cultures of human endometrial stromal cells expressed a single class of specific high-affinity binding sites for urokinase plasminogen activator (UPA) with a dissociation constant KD 1.0 nmol/l and saturation at 2.0 nmol/l. Similar binding data and number of free binding sites, about 200 fmol/mg protein, were found for UPA in complex with its inhibitor plasminogen activator inhibitor-1 (PAI-1). These binding data agree with those reported for the specific cell surface receptor for UPA, and stromal cell expression of UPA receptor mRNA was identified in Northern blots. Cell surface-bound UPA was degraded at 37°C. Degradation of complexed UPA was more efficient than that of free UPA. Degradation of free UPA did not require prior binding to endogenous PAI-1. Degradation of both free and complexed UPA was reduced by 70% by colchicine, chloroquine and methylamine, indicating that degradation involved both internalization and lysosomal enzymes. Furthermore, degradation was independently inhibited by about 70% with anti-UPA receptor antibodies and receptor-associated protein, indicating that the UPA receptor as well as one or more receptors of the low-density lipoprotein (LDL) receptor supergene family were involved in the degradation process. Receptor-associated protein ligand blotting demonstrated a major band co-migrating with the LDL receptorrelated protein or glycoprotein 330/megalin, and a minor band co-migrating with the very low-density lipoprotein receptor. Immunoblotting positively demonstrated expression of LDL receptor-related protein, but not glycoprotein 330. Key words: internalization/plasminogen activator inhibitor-1/receptor-associated protein/LDL receptor related protein/VLDL receptor
Introduction Urokinase plasminogen activator (UPA) initiates a proteolytic cascade, which degrades extracellular matrix during tissue growth and remodelling (Danø et al., 1994). The secreted proform of UPA is activated after binding to a specific highaffinity cell surface receptor. Secreted UPA is exposed to rapid inactivation in the pericellular space by plasminogen activator inhibitor-1 (PAI-1), which is stored in the extracellular matrix bound to vitronectin (Cubellis et al., 1989; Loskutoff, 1991). Active UPA is a two-chain molecule, identified either as high molecular weight UPA (high MW UPA), or low molecular weight UPA (low MW UPA). Low MW UPA lacks the aminoterminal fragment and is thus unable to bind to the receptor. It has, however, the active site, and is thus capable of forming a complex with PAI-1. Complexed UPA is internalized and degraded by certain cells after binding to the UPA receptor (Cubellis et al., 1990; Jensen et al., 1990; Olson et al., 1992). Since the UPA receptor is attached to the cell membrane with a glycosyl-phosphatidylinositol domain and thus has no transmembrane peptide sequence signalling for endocytosis in clathrin-coated pits, it was postulated that the UPA receptor would mediate only initial binding of the ligand, whereas internalization would © European Society for Human Reproduction and Embryology
proceed via a separate receptor. The low-density lipoprotein (LDL) receptor-related protein/α2-macroglobulin receptor has been shown to mediate degradation of UPA receptor-bound ligands (Herz et al., 1992; Nykjaer et al., 1992). This hypothesis is, however, subject to controversy since, in hepatic cells, LDL receptor-related protein bound and internalized pro-UPA without preceding binding to the UPA receptor (Kounnas et al., 1993). The receptor-associated protein is a 39-kDa protein, which co-purifies with LDL receptor-related protein and prevents binding of all described ligands to this receptor (Strickland et al., 1994). Receptor-associated protein, as well as the UPA:PAI-1 complex, also binds to other members of the LDL-receptor supergene family, like glycoprotein 330 and the very low-density lipoprotein (VLDL) receptor (Kounnas et al., 1992; Moestrup et al., 1993b; Battey et al., 1994; Argraves et al., 1995; Heegaard et al., 1995). The physiological role of receptor-associated protein is probably to act as a chaperone during intracellular processing of receptors in the LDL-receptor superfamily, or to prevent premature ligand binding (Willnow et al., 1996). Endometrial tissue has a unique property of responding to oestradiol with proliferation in the proliferative phase of the menstrual cycle, and to progesterone with differentiation and inhibition of proliferation in the secretory phase. This functional 585
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shift, which is initiated by progesterone, involves a concomitant adaptation of the proteolytic enzymes. The release of UPA from endometrial tissue is at peak during maximal growth in the end of the proliferative phase in vivo as well as in vitro (Cassle´n et al., 1981; Cassle´n and Åstedt, 1981; Cassle´n and Åstedt, 1983). Inhibition of proliferation by progesterone in the secretory phase also involves lower UPA activity. This can be reproduced in culture by the addition of progesterone to endometrial tissue explants (Cassle´n et al., 1986). Reduced U-PA activity was at least partly explained by the induction of PAI-1 by progesterone (Cassle´n et al., 1992b; Sandberg et al., 1997). Cell membranes prepared from endometrial tissue express specific high-affinity binding sites for UPA, and the number of binding sites was higher in secretory than in proliferative endometria (Cassle´n and Gustavsson 1991). It has been shown recently that reduced UPA activity in endometrial stromal cell cultures stimulated with progesterone resulted not only from increased expression of PAI-1 but also from increased degradation of UPA, and that an increased number of available UPA-receptor binding sites was a prerequisite for this process (Cassle´n et al., 1995). The purpose of this study was to analyse possible degradation pathways for free UPA and UPA:PAI-1 complex, in primary cultures of human endometrial stromal cells.
Materials and methods Materials Medium 199 and Hanks’ buffered salt solution (Hanks’ BSS) without phenol red, fetal calf serum (FCS), and L-glutamine were from Gibco (Paisley, Scotland). Crude collagenase, DNase, tosyl-L-lysinechloromethyl-ketone, penicillin-streptomycin-fungizone, bovine serum albumin (BSA), lactoperoxidase, p-aminobenzamidine, bromphenol blue, ethidium bromide, formaldehyde, formamide, 2-mercaptoethanol, MOPS, trizma base, colchicine, chloroquine, methylamine and mannose-6-phosphate were obtained from Sigma (St Louis, MO, USA). Ukidan™ was from Serono (Geneva, Switzerland). Nitex™ nylon meshes with pore size 350 µm and 35 µm were obtained from Tetko (Elmsford, NY, USA). DEAE Sephadex, Sephadex G-100, Sephadex G-25 PD10, CH-Sepharose 4B, Sephadex G-50 DNA grade, and fibrinogen were from Pharmacia AB (Uppsala, Sweden). Megaprime DNA labelling system, 32P dCTP, 125I and ECL Western blotting detection reagents were from Amersham (Solna, Sweden). High-range protein molecular weight standard, sodium dodecyl sulphate (SDS) and polyacrylamide were from Bio-Rad (Richmond, CA, USA). Tissue culture plates, Spin-X™ centrifuge filter units, IsoTip filter tips, Multi Safeseal™ microcentrifuge tubes were from Costar (Broadway-Cambridge, MA, USA), and RNeasy™ total RNA preparation kit was from Qiagen (Hilden, Germany). Bacto agar, tryptone and yeast extract were from DIFCO (Detroit, MI, USA), and competent cells, Wizard Minipreps DNA purification system and blue/orange loading dye were obtained from Promega (Madison, WI, USA). Glycogen and restriction enzymes were from Boehringer Mannheim Scandinavia AB (Bromma, Sweden), and molecular size standards for DNA and RNA were from Gibco BRL (Bethesda, WA, USA). Polyclonal rabbit anti-human vitronectin immunoglobulin G (IgG) (#31054) was from Technoclone (Vienna, Austria), and rabbit nonimmune IgG (#55867) was from Cappell (Durham, NC, USA). Monoclonal antibodies to human vimentin (#7020), cytokeratin (#821)
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and Aspergillus niger glucose oxidase (#X0931) together with normal rabbit serum, biotinylated rabbit anti-mouse IgG antibodies (#E354), biotinylated multi-link swine anti-rabbit IgG antibodies (#E453) and avidin–biotin–peroxidase complex were from Dako AS (Glostrup, Denmark). Diaminobenzidine was obtained from Saveen Biotech AB (Malmo¨, Sweden). Recombinant 39-kDa receptor-associated protein and monoclonal antibodies to LDL receptor-related protein were kindly provided by Dr P.H. Jensen (Aarhus, Denmark). Monoclonal antibodies to the UPA receptor for experiments (Rønne et al., 1991) were provided by Dr G. Høyer-Hansen (Copenhagen, Denmark). Rabbit anti-receptorassociated protein, anti-glycoprotein 330/megalin and anti-LDL receptor-related protein IgG were gifts from Dr D. Strickland (Rockville, MD, USA). Polyclonal anti-VLDL receptor antibodies were obtained by immunizing rabbits with a fusion protein obtained by expression in Escherichia coli as described by Multhaupt et al. (1996). The cDNA probe for UPA receptor was generously provided by Dr L.R. Lund (Copenhagen, Denmark). 125I UPA:PAI-1 complex, with specific radioactivity 1.2 MBq/µg, was a kind gift from Dr P. Andreasen (Aarhus, Denmark).
Tissue processing Endometrial tissue was obtained from uteri removed for benign nonendometrial pathology, i.e. cervical dysplasia, dysmenorrhoea, uterine prolapse and fibromyomas. All patients were parous, 32–45 years of age. Permission to use part of the endometrium was granted by the University Review Board for studies on human subjects. Endometrial pathology was subsequently excluded by histopathologic examination of the formalin-fixed portion of the endometrium. Endometrial tissue was gently scraped off from the upper part of the uterine cavity immediately after removal of the uterus, and transferred to the laboratory in sterile Hanks’ BSS. Separation of the endometrial tissue in epithelial and stromal cells was performed as previously described (Cassle´n et al., 1990). Briefly, the tissue was transferred to dissociation solution, cut in 1-mm3 pieces and incubated at 37°C in a shaking waterbath for 45–60 min. The dissociation solution was made up with Hanks’ BSS containing collagenase 2.5 g/l, DNase 0.2 g/l, and tosyl-L-lysine-chloromethyl-ketone 0.2 µmol/l. After digestion, the suspension was filtered through a 350-µm nylon mesh to remove undigested fragments, and then passed through a 35-µm nylon mesh to collect the glands. Stromal cells were then collected by centrifugation at 1200 g for 5 min and resuspension of the pellet in culture medium. The purity of stromal cell preparations, analysed with immunocytochemistry using antibodies to vimentin and cytokeratin, was 96–99% in cultures used for experiments (Cassle´n et al., 1995). Tissue culture Stromal cells were plated 105 cells/well in 24-well plates, and the cultures rinsed with Hanks’ BSS after one day. Cells were grown in medium 199, supplemented with 10 % FCS, glutamine 2 mmol/l, penicillin 105 IU/l, streptomycin 100 mg/l and Fungizone 0.25 mg/l, and incubated in humidified air with 5% CO2 at 37°C. The medium was changed every second day, and the cultures usually reached confluence in 4–6 days. Purification and radiolabelling of UPA and the complex UPA was purified from Ukidan™ by affinity chromatography on a benzamidine-Sepharose column as previously described by Cassle´n et al. (1992a). The active enzyme fraction was further separated into high MW UPA (Mr50 kDa) and low MW UPA (Mr33 kDa) by gel filtration on Sephadex G-100. Immunoblotting of the peak low MW UPA fractions revealed trace amounts of high MW UPA, estimated
Receptors for urokinase in endometrial cells to 1%. The high MW UPA fraction, which was 98–99% pure, was used for 125I labelling using the lactoperoxidase method (Thorell and Johansson, 1971). Specific radioactivity was in the range 0.4–0.6 MBq/µg of labelled UPA. 125I UPA:PAI-1 complex was prepared by incubating 125I UPA 1 µg/mL with SDS-activated PAI-1 10 µg/mL. The complex was subsequently isolated from excess PAI-1 via affinity purification in an anti-UPA IgG column. The complex migrated as a single Mr 94,000 band in SDS polyacrylamide gel electrophoresis (SDS–PAGE) (Jensen et al., 1990).
Binding of radiolabelled UPA Confluent stromal cell cultures were incubated with serum-free medium for 2 h at 37°C. Binding experiments were subsequently performed on ice for 2 h with radiolabelled UPA in Hanks’ BSS containing BSA 10 g/l. UPA was either in its free form or in complex with PAI-1. After removal of the experimental buffer, cells were washed four times with ice-cold Hanks’ BSS and subsequently lysed with NaOH 1 mol/l. Radioactivity of the lysate was counted in a 1260 Multigamma counter (Pharmacia AB, Uppsala, Sweden). Nonspecific binding was assayed in the presence of 100-fold molar excess of unlabelled UPA, and specific binding calculated as the difference between total and non-specific binding. Cellular degradation of surface-bound radiolabelled UPA After binding of radiolabelled ligands for 2 h at 0°C, cultures were washed four times at 0°C, and then incubated with serum-free medium at 37°C. The experiment was stopped at given time points. The media were collected and the cultures washed once with fresh medium. Incubation media and washing media from each well were pooled, and trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) was added to 10%. Soluble radioactivity was counted in the supernatant and insoluble in the sediment after centrifugation at 3000 g for 20 min. The cultures were subsequently incubated with trypsin 1 g/l in Hanks’ BSS on ice for 30 min, in order to solubilize surfacebound radiolabelled UPA. The use of cold trypsination instead of acid treatment was preferred because at this stage of the experiment the cells tended to detach more easily after acid treatment. In experiments where detached cells actually were detected after exposure to trypsin, the trypsin solution was centrifuged at 1200 g for 5 min to remove cells before counting the radioactivity. In order to validate the cold trypsination technique, dissociated radioactivity from the cells as well as remaining radioactivity on the cells after treatment with either trypsin or acidification were compared. In two experiments the mean percentage of dissociated radioactivity was found to be 74% after trypsination, and 68% after acidification. Northern blot analysis of UPA receptor mRNA. Total RNA was prepared from cell pellets using the RNeasy™ total RNA purification kit. RNA aliquots (5 µg) were size-separated on 1% agarose gels containing 2.2 mol/l formaldehyde. The RNA was then transferred to Gene Screen Plus nylon filters (Du Pont, Boston, MA, USA) (Thomas, 1980). Human UPA receptor cDNA, nucleotides 497-1081, was subcloned into the Bam HI site of pBluescript KS1 (Roldan et al., 1990). The probe was radiolabelled with 32P dCTP using the random labelling method Megaprime DNA labelling systems (Feinberg and Vogelstein, 1984). Filters were hybridized in 0.25 mol/l sodium phosphate, 7% SDS, 1 mmol/l EDTA at 65°C for 12 h. After hybridization, filters were washed in 0.02 mol/l sodium phosphate, 1% SDS for 3310 min at 65°C. Autoradiography was performed for 12 h. 39-kDa receptor-associated protein ligand blotting Tissue extracts for ligand blotting were prepared from endometrial stromal cells, CHO cells (transfected ldl-a7 cell strain obtained from
Dr Monty Krieger, MIT, Cambridge, MA, USA) overexpressing the human VLDL receptor (Gåfvels, et al., 1993; Battey et al., 1994), mouse F9 carcinoma cells (American Type Culture Collection, Rockville, MD, USA) treated with retinoic acid 0.1 µmol/l and dibutyryl cAMP 0.2 µmol/l (Stefansson et al., 1995), human myocardium, and third trimester placenta. Cells in culture were detached from culture dishes by scraping into 1 x phosphate buffered saline, 1 mmol/l phenylmethylsulphonyl fluoride (Sigma), and cell pellets obtained after centrifugation were frozen at -70°C before processing. The cell extracts were prepared by homogenizing cell pellets and tissue pieces by the use of a Polytron (Kinematica, Luzern, Switzerland). Cells and tissue pieces were homogenized in 250 µl of 50 mmol/l HEPES (Boehringer Mannheim), 0.5 mol/l NaCl, 0.05% Tween 20, 1% Triton X-100 containing 1 mmol/l phenylmethylsulphonyl fluoride and 0.02 g/l leupeptin (Sigma). The homogenates were centrifuged at 14 000 g for 10 min, and supernatants were recovered for analysis of protein concentration (Bradford, 1976). Cell proteins were subjected to SDS– PAGE on 4–15% gradient gels, and electrophoretically transferred to nitrocellulose membranes. The procedure of receptor-associated protein ligand blotting was performed exactly as described by Battey et al. (1994). Receptor-associated protein blotting as well as Western blotting were visualized by enhanced chemiluminescence.
Statistical methods Results are given as the means and, when more than two experiments, 6 SEM. Wilcoxon’s signed rank test was used to compare groups with different treatments. Differences between curves obtained after different treatments were analysed as follows: the logarithm of each contributing value was calculated, and curves derived from these data were considered straight lines. Regression coefficients for these lines were subsequently compared using Wilcoxon’s signed rank test.
Results Binding of 125I-labelled UPA to endometrial stromal cells in culture indicated a single class of high-affinity binding sites, dissociation constant KD 1.0 nmol/l and saturation at 2.0 nmol/ l (Figure 1, left panel). The number of free available binding sites was estimated to about 200 fmol/mg protein. Binding was almost completely blocked by 100-fold excess nonlabelled UPA, suggesting very low non-specific binding of UPA. Binding of the labelled UPA:PAI-1 complex had similar kinetic data, suggesting binding to the same site as noncomplexed UPA (Figure 1, right panel). These data suggest the presence of high-affinity UPA receptors. Northern blotting using a cDNA probe demonstrated UPA receptor mRNA (Figure 2). Non-specific binding of radiolabelled UPA:PAI-1 complex, but not of free UPA, was obvious at concentrations above saturation of the high-affinity binding sites (Figure 1, right panel), and was apparently related to the PAI-1 part of the complex. Knowing that PAI-1 binds to extracellular vitronectin (Loskutoff, 1991), the effect of antibodies to vitronectin on binding of the labelled complex in the presence of excess unlabelled UPA was studied. Binding was inhibited by 50% in the presence of 0.1 µmol/l of anti-vitronectin IgG, but not by control IgG (data not shown). Membrane-bound radiolabelled UPA disappeared quickly from the membranes at 37°C (Figure 3). The radioactive ligands disappeared rapidly from the membranes during the 587
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Figure 1. Binding of 125I urokinase plasminogen activator (UPA) at indicated concentrations to monolayers of endometrial stromal cells. Radiolabelled UPA was either in its free form (left panel) or in complex with plasminogen activator inhibitor-1 (PAI-1) (right panel). Specific binding (-·-·-·-·-) was calculated by subtracting non-specific binding (-----), i.e. binding in the presence of 100-fold excess nonlabelled UPA, from total binding (———) (mean of 2 experiments). Inset: Scatchard diagrams. Binding data indicated KD 1 nmol/l, saturation at 2 nmol/l, and 200-fmol binding sites/mg protein for both free and complexed UPA.
Figure 2. Urokinase plasminogen activator (UPA) receptor mRNA was demonstrated by Northern blotting of total RNA extracted from three different cultures of endometrial stromal cells. Filters were hybridized with a 32P-labelled cDNA probe for U-PA receptor. The size of the transcript corresponded to 1.4 kb.
first hour in 37°C, but the remaining radioactivity, 20–30%, was fairly stable. The rate of disappearance of radioactivity from the cell surface was not significantly different between free and complexed UPA (Figure 3A). TCA-insoluble radioactivity of the medium, i.e. dissociated not degraded radiolabelled ligand, increased rapidly during the first hour, but was then stable at about 60% (Figure 3B). TCA-soluble radioactivity, which resulted from degradation of the radiolabelled ligand, accumulated gradually over the 4-h period. The rate of accumulation was higher after binding of complexed than after binding of free 125I UPA (P 5 0.04) (Figure 3C). Thus, after 1 h, only about 30% of initially bound radioactivity remained on the cell surface; subsequent disappearance of radioactivity from the cell surface was slow. TCA-insoluble radioactivity accumulated rapidly in the medium during the first hour, indicating dissociation of about 50% of the radiolabelled ligand from the cell surface. Radiolabelled UPA:PAI-1 complex disappeared slightly faster than free UPA, but the difference was not statistically significant. Only about 20% remained membrane bound after 1–2 h. Significantly more TCA-soluble radioactivity accumulated in the medium after binding of complexed than after binding of free UPA, indicating more efficient degradation of complexed UPA than of free UPA; non-complexed UPA was degraded by the cells at a slower rate. 588
Figure 3. The fate of cell surface-bound radioactivity, after binding of 125I urokinase plasminogen activator (UPA) at 0°C, and subsequent incubation of the cells at 37°C, was analysed as remaining cell surface-bound radioactivity (A), trichloroacetic acid (TCA)-insoluble radioactivity (B), and TCA-soluble radioactivity (C) (5 independent experiments). Radiolabelled UPA (1.5 nmol/L) was either in its free form (———) or in complex with PAI-1 (----). All results were expressed as per cent of surface-bound radioactivity at 0 h, i.e. the time of transfer to 37°C.
It was considered to be important to exclude the possibility that non-complexed UPA had to form complex with endogenous PAI-1 prior to being endocytosed. For this purpose 10fold excess low MW UPA was included during both binding
Receptors for urokinase in endometrial cells
Figure 4. Binding of 125I urokinase plasminogen activator (UPA) (1.5 nmol/l) and subsequent degradation of the cell surface-bound ligand, measured as trichloroacetic acid-soluble radioactivity after 2 h at 37°C, was assayed in the absence (open bars) and presence (filled bars) of 10-fold excess low MW UPA (mean of two experiments). Since low MW UPA competes with 125I UPA for binding to plasminogen activator inhibitor-1 (PAI-1) but not to the receptor, the result indicates that degradation of non-complexed UPA does not require prior binding to endogenous PAI-1.
at 0°C and internalization at 37°C. Low MW UPA has the active site, which binds to PAI-1, but lacks the receptorbinding site. However, presence of low MW UPA did not reduce degradation of radiolabelled UPA. This indicates that non-complexed UPA is internalized and degraded as such, i.e. does not require preceding complex formation with PAI-1 in these cells (Figure 4). In order to show that UPA was internalized and degraded by lysosomal hydrolases, and not by extracellular proteases, cells with surface-bound radiolabelled UPA or UPA:PAI-1 were incubated at 37°C in the presence of inhibitors of endocytosis (colchicine) or lysosomal hydrolases (chloroquine and methylamine). These inhibitors reduced accumulation of TCA-soluble radioactivity by about 70% for both ligands (Figure 5). Blocking antibodies to the UPA receptor, which were present both during binding of the radiolabelled ligands at 0°C and during subsequent incubation at 37°C, were as effective as the inhibitors to reduce degradation. The finding indicates that the UPA receptor has a crucial role in the process of UPA degradation. Furthermore, receptor-associated protein, used in the same way as the antibodies, was equally effective in blocking degradation. This clearly indicates a central role also for one or more of the receptor-associated protein binding receptors of the LDL-receptor supergene family. Free and complexed UPA were equally sensitive to both blocking agents, suggesting that both ligands were internalized along the same pathway. In order to identify the receptor-associated protein binding receptor(s), stromal cell extracts and placenta were analysed with ligand blotting. The main receptor species with highaffinity receptor-associated protein binding co-migrate with LDL receptor-related protein or glycoprotein 330/megalin at a molecular weight well above 200 kDa (Figure 6). Binding
of receptor-associated protein in endometrial stromal cell extracts was also demonstrated to a band co-migrating with the 115-kDa VLDL receptor, also shown in human myocardium, which is known to highly express the VLDL receptor, and in extracts from cells overexpressing the unspliced variant of the human VLDL receptor. In some cases a 90-kDa band was identified that may represent the precursor form of the 115-kDa VLDL receptor, or a VLDL receptor variant missing the O-linked sugar domain (Sakai et al., 1994). Western blotting using antibodies to LDL receptor-related protein and glycoprotein 330/megalin showed that endometrial stromal cells express significant amounts of LDL receptor-related protein, whereas glycoprotein 330/megalin was not demonstrated (Figure 7). Degradation was also assayed in the presence of mannose6-phosphate 2 mmol/l in order to exclude involvement of the mannose-6-phosphate receptor/insulin-like growth factor II receptor in internalization of the UPA:UPA receptor complex. TCA-soluble radioactivity, measured after 2 h, in the absence versus presence of mannose-6-phosphate was 21 versus 23 fmol/mg protein, respectively, after binding of 125I UPA to the cells, and 54 versus 65 fmol/mg protein, respectively, after binding of 125I UPA:PAI-1 complex (not shown). These data suggest that this receptor is not involved in internalization of the UPA:UPA receptor complex in endometrial stromal cells.
Discussion This study identified a single class of saturable, high-affinity binding sites for UPA on human endometrial stromal cells, indicating specific cell surface receptors. A receptor with similar KD has previously been found in a number of cell lines, as well as in normal cells (Danø et al., 1994), and this group has reported similar KD for UPA binding sites in isolated membranes of endometrial tissue as well as in cultured stromal cells (Cassle´n and Gustavsson 1991; Cassle´n et al., 1995; Sillem et al., 1997). Our group has also demonstrated UPA receptor mRNA in endometrial stromal cells by Northern blots. Complexed UPA bound to the cells with similar KD as that of free UPA, which is in agreement with an earlier report on receptor affinity for complexed and free UPA (Cubellis et al., 1989). Additional binding of the labelled UPA:PAI-1 complex, but not of labelled free UPA, appeared at concentrations exceeding the saturation level for the receptor. Such binding was inhibited by antibodies to vitronectin, a secreted protein with high affinity for PAI-1, which is integrated in the extracellular matrix and serves to store PAI-1 in this position (Loskutoff, 1991). Binding of the complex to heparin-like molecules or laminin-nidogen is also possible, but is more likely to involve UPA (Stephens et al., 1992a,b). More than 60% of the membrane-bound radioligands disappeared within the first hour at 37°C. The majority of this portion was, however, retrieved undegraded in the medium. After the first hour, however, the radiolabelled ligand disappeared very slowly from membranes. Other authors reported that receptor-bound UPA was internalized very slowly, and remained fully active at the cell surface with a 4–6 h half-life at 37°C (Cubellis et al., 1990; Estreicher et al., 1990; Olson 589
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Figure 5. Degradation of surface-bound 125I urokinase plasminogen activator (UPA) 1.5 nmol/l and 125I UPA:PAI-1 1.5 nmol/l was studied in the presence of blocking antibodies to the UPA receptor (auPAR) 10 mg/l, receptor-associated protein (RAP) 150 nmol/l, an inhibitor of endocytosis colchicine (Col) 10 mmol/l, inhibitors of lysosomal hydrolases chloroquine (Chl) 0.1 mmol/l, and methylamine (Met) 10 mmol/ l. Preceding binding of the radiolabelled ligands at 0°C as well as the following incubation at 37°C for 2 h included these agents. Trichloroacetic acid-soluble radioactivity of three independent experiments was expressed as per cent of the control incubation without the inhibitors. Original data, expressed as fmol/mg protein, were used for the statistical analysis. Degradation of both free and complexed 125I UPA was inhibited by about 70% by all five agents (P , 0.05).
Figure 6. Scanning for some receptors of the low-density lipoprotein (LDL) receptor supergene family by 39-kDa receptorassociated protein (RAP) ligand blot performed on cell extracts from very low-density lipoprotein (VLDL) receptor overexpressors (lanes 1 and 6: 12.5 µg protein), human myocardium (lane 2: 50 µg protein), endometrial stromal cells (lanes 3 and 4: 25 and 12.5 µg protein, respectively), and human third trimester placenta (lane 5: 50 µg protein). A high-range protein molecular weight standard was used. Endometrial stromal cells had a strong band, which appeared to co-migrate with LDL receptor-related protein (LRP) or glycoprotein 330, and a weak band which co-migrated with the VLDL receptor band. Anti-RAP also detected endogenously expressed 39-kDa RAP.
et al., 1992). In this study the cells were loaded at 1 nmol/l of radiolabelled UPA, and the 60% that were released unchanged to the medium may represent an excess. The remaining 20–30% that stayed on the membranes for at least 4 h may, on the other hand, represent UPA binding to a stable pool of receptors. An alternative explanation is derived from the observation by Ragno¨ et al. (1993), who showed that soon after binding to the UPA receptor, UPA:PAI-2 complexes are cleaved into two fragments. The smaller, which consists of the aminoterminal fragment of UPA, remains bound to the 590
Figure 7. Identification of receptor-associated protein (RAP)binding receptors in endometrial stromal cells by RAP blotting and Western blotting. Membrane proteins extracted from human third trimester placenta 250 µg (lane 1), mouse F9 cells stimulated with retinoic acid 100µg (lane 2), Chinese hamster ovary cells overexpressing VLDL receptor 75 µg (lane 3), and endometrial stromal cells 75 µg (lane 4) were fractionated and blotted as described in Materials and methods. Identical filters were probed with 39-kDa RAP for ligand blotting (left panel), and with antibodies to glycoprotein 330 (gp330)/megalin (middle panel) and LDL receptor-related protein (LRP) (right panel) for Western blotting.
UPA receptor, while the larger, which consists of the low MW UPA:PAI-2 complex, appears to be either endocytosed or released. Although possible cleavage of UPA into low MW UPA and the aminoterminal fragment after binding to the receptor was not addressed in this study, it is a possibility that
Receptors for urokinase in endometrial cells
the 30% radioactivity, which remains cell surface bound throughout the experiment, represents the aminoterminal fragment of UPA, since 30% radioactivity corresponds to the relative size of the aminoterminal fragment. Free UPA can potentially be internalized either in its native form, or after complex formation with endogenous PAI-1. To test whether such complex formation was necessary, excess low MW UPA was added, which does not bind to the UPA receptor but forms a complex with PAI-1. Thus having neutralized endogenous PAI-1, it was found that subsequent degradation of labelled non-complexed UPA was not decreased, and it was concluded that complex formation was not required for UPA to be internalized and degraded. Binding and internalization of pro-UPA via the LDL receptor-related protein has been demonstrated previously in hepatic cells (Kounnas et al., 1993), and our data in endometrial stromal cells indicate that internalization of UPA does involve both the UPA receptor and the LDL receptor-related protein or some other receptor of the LDL-receptor supergene family (Kounnas et al., 1992; Moestrup et al., 1993; Heegaard et al., 1995). Internalization and degradation of complexed UPA was first reported to involve only the UPA receptor (Cubellis et al., 1990; Estreicher et al., 1990; Olson et al., 1992), but was subsequently found to involve also the LDL receptor-related protein receptor in certain cells (Herz et al., 1992; Nykjaer et al., 1992). It was suggested that binding of the ligand to the UPA receptor preceded endocytosis mediated by the LDL receptor-related protein. The results of this study show that degradation of complexed, as well as free, UPA was inhibited by antibodies to both the UPA receptor and receptor-associated protein, indicating involvement of both the UPA receptor and a receptor-associated protein inhibitable receptor. Thus, the results in endometrial stromal cells agree with the previously proposed model, suggesting that initial binding of UPA involves the UPA receptor, whereas subsequent internalization involves the LDL receptor-related protein or some other member of the LDL-receptor supergene family. Also, our data support the view that the UPA receptor in endometrial stromal cells has functional and presumably a spatial relationship to this multiligand receptor. This study indicated that the LDL receptor-related protein, and not glycoprotein 330/megalin, represents the major high-affinity binding receptor for the 39kDa receptor-associated protein in endometrial stromal cells, whereas the VLDL receptor was expressed at low levels. Since, however, both these receptors have been shown to mediate cellular catabolism of UPA:PAI-1 complex (Kounnas et al., 1992; Moestrup et al., 1993), future studies using specific inhibition of these receptors should indicate the individual contribution by each pathway in the clearance of UPA in endometrial stromal cells. Proteolytic activity in the endometrium apparently has important implications in the reproductive process, since plasminogen activator (PA) activity varies with the ovarian cycle. Low PA activity of the endometrium in the secretory phase assures minimal degradation of extracellular matrix and minimal risk for endometrial bleeding, and thus presents optimal conditions for successful implantation of the fertilized ovum. In-vitro studies of endometrial tissue explants and
primary cultures of endometrial stromal cells have shown that reduced UPA activity of the medium in response to progesterone was secondary to a decrease of UPA as well as an increase of PAI-1 (Cassle´n et al., 1986, 1995). The reduction of extracellular UPA was secondary to increased internalization and degradation of UPA, mainly in complex with PAI-1. As also shown in this study, such degradation involves the UPA receptor. Thus, up-regulation by progesterone of the number of receptor-binding sites appears to be a crucial step in the control of the level of UPA. Furthermore, the results in this paper imply that functional clearance via the LDL receptorrelated protein is crucial in this process. This receptor system has been implicated in other reproductive processes, e.g. transplacental lipid transport of the developing embryo (Gåfvels et al., 1992; Herz et al., 1992; Coukos et al., 1994). Apart from localizing UPA activity and mediating UPA clearance, several reports have lately implicated that the UPA receptor initiates intracellular tyrosine phosphorylation and signal transduction following ligand binding (Dumler et al., 1993; 1994). This effect is independent of the proteolytic capacity of UPA, but requires the receptor-binding site. These effects can thus be elicited by the aminoterminal fragment of UPA, but not by low MW UPA. Cellular effects, which reportedly are stimulated by binding of UPA to its receptor, include proliferation (Rabbani et al., 1990), adhesion (Nusrat and Chapman 1991; Waltz et al., 1993), and migration (Odekon et al., 1992). Paracrine signals between different endometrial cell types may involve UPA and be mediated by UPA receptor signalling, although this has not been studied. It has, however, been observed that UPA stimulates migration of endometrial stromal cells in a dose-dependent way, and that this effect was inhibited by antibodies to the UPA receptor (Sandberg et al., 1997b).
Acknowledgements The technical assistance of Tatiana Egereva is gratefully acknowledged. This study was supported by grants from the Swedish Cancer Fund (2693-B96-07XBB), Swedish Medical Research Council (11549), the Lund University Medical Faculty, the Lund University Hospital Fund for Cancer Research, the Gunnar, Arvid, and Elisabeth Nilssons Fund for Cancer Research, the Foundations of Axel Ax:son Johnson, Åke Wiberg, Tore Nilsson, and Magnus Bergvall, and the Swedish Heart and Lung Foundation.
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