Molecular Human Reproduction, Vol. 6, No. 10, 921-927,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
Differential expression of the 
2-macroglobulin receptor and the receptor associated protein in normal human endometrium and endometrial carcinoma
1 Perinatal Research Centre, Department of Perinatal Medicine, Royal Women's Hospital, Carlton, Victoria 3053, and 2 Department of Obstetrics and Gynaecology, University of Melbourne, Parkville 3052, Australia
Abstract
Extracellular matrix degradation, mediated by the activation of receptor-bound proteolytic enzymes, is essential to the process of cellular invasion. Many normal physiological functions such as endometrial remodelling are reliant on the activation of these surface associated proteolytic enzymes, as are pathological functions such as cancer-cell invasion. The internalization of proteolytic complexes is mediated by the multi-functional clearance receptor, 
2-macroglobulin receptor/LRP. The role of LRP and its ligand binding inhibitor, the receptor-associated protein (RAP), in the advancement of invasive endometrial carcinoma is unknown. The aim of this study was to compare the expression of LRP and RAP mRNA in normal endometrium (n = 14) and endometrial carcinoma (n = 33) by semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR). Expression of LRP mRNA in normal endometrium was significantly increased in the secretory phase when compared with proliferative phase endometrium (P < 0.05). The expression of LRP in all carcinomas examined was significantly reduced to about 20% of the amount in normal endometrium (P < 0.05), whereas RAP expression was not significantly different between endometrium and carcinoma. No significant difference in the level of LRP or RAP expression was observed between carcinoma grades or stages. In conclusion, we have shown that LRP expression is differentially regulated in the normal endometrium during the menstrual cycle and is decreased in invasive endometrial carcinomas.
endometrium/endometrial neoplasm/gene expression/LDL receptors
Introduction
The low density lipoprotein receptor-related protein (LRP) mediates the endocytosis of a variety of structurally diverse ligands, most of which bind to LRP at distinct sites. LRP is a member of a family of receptors related to the low density lipoprotein (LDL) receptor, including the very low density lipoprotein receptor and gp330/megalin (Herz et al., 1988
). The 15 kb mRNA transcript translates to a 600 kDa protein, comprising an 85 kDa transmembrane domain non-covalently associated with a 515 kDa domain which contains the ligand recognition sites (Williams et al., 1992; Nykjaer et al., 1994). A 39 kDa receptor associated protein (RAP) which co-purifies with LRP, is predominantly an endoplasmic reticulum associated protein which acts not only as a chaperone molecule but also ensures correct intra-domain folding of LRP (Bu and Rennke, 1996). RAP is an LRP receptor antagonist which inhibits intra-cellular binding of all known ligands to LRP (Herz et al., 1991; Warshawsky et al., 1995).
Ligands of LRP include 2-macroglobulin complexed with cytokine or growth factors, (Dickson et al., 1981; Van Leuven et al., 1981), lipoprotein lipase, ß-very low density lipoproteins enriched with apolipoprotein E (Kowal et al., 1990; Kristensen et al., 1990), Pseudomonas exotoxin A, lactoferrin (Kounnas et al., 1992), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI) type 1 and 2 complexes (Orth et al., 1992; Andreasen et al., 1994; Conese et al., 1995). Normally LRP is abundantly expressed in the liver (hepatocytes), central nervous system (neurones and astrocytes), monocytes–macrophages, smooth muscle cells, fibroblasts and also placenta (Moestrup et al., 1992). The diversity of ligands and the variety of cell types expressing LRP suggests a role for this protein in a number of important physiological processes ranging from lipoprotein metabolism to tissue remodelling and cell migration.
Tumour cell invasion into the surrounding tissue is dependent upon the cell's ability not only to free itself from cell–cell and cell–substratum constraints, but also to cross the basement membrane, interstitial stroma and intercellular junctions that separate tissue compartments. The activation of cell surface associated proteolytic enzyme systems like the plasminogen activation cascade, is a pre-requisite for the process of cell invasion. The broad substrate range enzyme, plasmin not only degrades many components of the extracellular matrix but is also responsible for the activation of other enzymes such as matrix metalloproteinases. Under normal physiological conditions, components of the plasminogen activation cascade play an important role in cell migration, for the purpose of tissue repair and remodelling, inflammation, angiogenesis (Sandberg et al., 1998) and trophoblast implantation (Hoffman et al., 1994). Under patho-physiological situations, the plasminogen activation cascade is important for cancer metastasis. The overexpression of uPA, and its receptor uPAR and inhibitor PAI-1, in the late stages of a variety of diverse tumours, including gynaecological cancers, has been well documented (Kohler et al., 1997; Danerinavarro et al., 1998). Despite this, little is known about the expression of LRP and RAP, which are directly involved in the clearance of these inactivated protease complexes from the surface of cancer cells.
Although LRP has been identified in various tumour cell lines, the regulation of LRP mRNA expression in vivo is not clearly defined. Recent in-vitro studies suggested cell culture density and oestrogen may stimulate LRP expression in an oestrogen receptor-positive breast cancer cell line (Burgess and Stanley, 1997; Li et al., 1998). In contrast, in-vivo expression of LRP in rat liver was down-regulated in oestradiol-treated rats (Szanto et al., 1992). Regulation of LRP is evident in certain cell types, however the mechanism by which this is achieved is not clearly understood. A decrease in the level of LRP expression in vivo, in melanoma (DeVries et al., 1996) and hepatocellular carcinomas (Gonias et al., 1994) has been previously described. However, expression of LRP in endometrial carcinomas has yet to be established.
Endometrial carcinoma is the most commonly diagnosed cancer of the female gynaecological tract, with thousands of new cases diagnosed each year. Carcinoma of the endometrium is commonly diagnosed in both menopausal and post-menopausal women, with only a small percentage of cases presenting prior to menopause. Despite its frequency, the mechanisms that induce transformation from a normal to neoplastic cell phenotype and contribute to the progression of metastatic disease remain to be identified. In this study, we determined the level of mRNA expression of two genes, LRP and RAP, which may be involved in processes important in the development of invasive endometrial carcinoma. The expression levels of LRP and RAP mRNA were compared in normal endometrium and endometrial carcinomas of varying clinical stage and grade of differentiation by semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis.
Materials and methods
Patients and samples
This project was approved by the Royal Women's Hospital Research and Ethics Committees and written informed consent was obtained from participating patients. Clinical specimens were obtained at the time of surgery from pre-menopausal and post-menopausal women undergoing total abdominal hysterectomy and bilateral salpingo-oophorectomy for gynaecological malignancies. The samples were snap frozen in liquid nitrogen and stored at –80°C. A total of 14 normal endometrial samples were obtained from pre-menopausal patients (secretory phase endometrium, n = 6; proliferative phase endometrium, n = 6) and post-menopausal patients (inactive post-menopausal endometrium; n = 2). Routine pathology and histological assessment was performed on all samples in this study and all carcinomas were classified using the International Federation of Gynecology and Obstetrics (FIGO) classification. In all, 33 endometrial adenocarcinomas were obtained from post-menopausal (n = 30) and pre-menopausal patients (proliferative phase, n = 3). The carcinomas were classified into three grades of differentiation: grade 1 (well-differentiated, n = 14); grade 2 (moderately-differentiated, n = 8); and grade 3 (poorly-differentiated, n = 11). The carcinomas were also divided into groups according to surgical stage (FIGO): stage IB (invasion to the inner half of the myometrium, n = 12); stage IC (invasion to the outer half of the myometrium, n = 6); stage IIA (endocervical gland involvement, n = 4); stage III+ (IIIA-uterine serosa involvement, IIIB-vaginal metastases and IIIC-nodal involvement and IV- intra-abdominal metastases, n = 11).
RNA isolation and cDNA preparation
Total RNA was extracted from frozen tissue biopsies as described previously (Chirgwin et al., 1979). The RNA was incubated with Proteinase K (10 IU/µg RNA, 37°C for 30 min) and subsequently DNAse 1 (10 IU/µg RNA, 37°C for 15 min) to remove any residual protein and genomic DNA. The concentration of total RNA extracted was determined by spectrophotometric analysis using a scanning spectrophotometer (UV-2101/3101 PC, Shimadzu Corporation, Kyoto, Japan). The optical density of a 2 µl aliquot in 1 ml was measured to determine the concentration and quality of the RNA. The RNA integrity was also determined visually by non-denaturing gel electrophoresis on a 1% agarose gel run in 1x TBE running buffer (1 mmol/l Tris-base; 0.1 mmol/l boric acid; 2 mmol/l EDTA, pH 8.0). The RNA was stored at –40°C.
To allow a comparable rate of efficiency in the reverse transcription step, all test reverse transcription (RT) reactions were aliquoted from a general mastermix that contained all reagents except for the test RNA. Control reverse transcription reactions were aliquoted from a separate mastermix that contained all reagents except for the test RNA and reverse transcriptase. Reverse transcription was carried out on total RNA (500 ng), using an oligo dT primer (T13) in a 20 µl final reaction containing 50 mmol/l Tris pH 8.3, 50 mmol/l KCl, 4 mmol/l MgCl 2, 10 mmol/l dithiothreitol (DTT), 0.2 mmol/l of each dNTP, 2 µmol/l of primer and 13 IU of Maloney murine leukaemia virus (MMLV) reverse transcriptase (Gibco/BRL, Life Technologies, New York, NY, USA). Reverse transcription was performed at 37°C for 1 h, after which the enzyme was inactivated by heating the reactions to 95°C for 5 min.
Semi-quantitative RT–PCR
RT–PCR was performed using two specific primers for each of the three mRNA transcripts (Table I). Three 2 µl aliquots from each reverse transcription reaction were added to three separate PCR tubes containing one of either three sets of specific primers (LRP or RAP or GAPDH). Primer sequences (Table I) were determined using PRIMER software available at WebANGIS (http://www.angis.org.au). To allow a comparable rate of efficiency in the PCR step, an aliquot of a primer-specific general mastermix was added to each test reverse transcription aliquot and corresponding control reverse transcription aliquot in separate tubes. PCR reactions were in a final volume of 25 µl containing 10 mmol/l Tris pH 8.3, 10 mmol/l KCl, 2 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 1 µmol/l each of forward and reverse primer and 2 IU of Taq DNA polymerase (Gibco/BRL). Thermocycling was performed with an Omni-E Thermocycler (Hybaid, UK) using a high stringency thermal profile which consisted of 94°C for 30s, 60°C for 30s and 72°C for 1 min for either 30 cycles (LRP and RAP PCR) or 24 cycles (GAPDH PCR). Cycle number was determined by the establishment of a standard curve of PCR product amount versus PCR cycle number for LRP, RAP and GAPDH PCR. The cycle number chosen was deemed to be the point at which PCR product amount was optimal and within the linear portion of the curve, well before saturation point. PCR negative controls for each sample contained aliquots of non-reverse-transcribed RNA. PCR products were not detected in these controls.
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cDNA probe isolation and preparation
LRP, RAP and GAPDH cDNA sequences were each separately cloned into the PCRscriptTM plasmid (Stratagene, La Jolla, USA) and sequenced using a PE Applied Biosystems 377 DNA sequencer and a Dye terminator reaction kit (Perkin Elmer, Norwalk, USA) to confirm sequence composition. Specific probes were isolated from the three recombinant plasmids following restriction enzyme digestion and purification from agarose following electrophoresis. The cDNA was excised and purified from the agarose gel using a DNA purification resin column (Promega, Wisconsin, USA). The cDNA was random labelled with
-32P (3000 Ci/mmol) dCTP (Amersham, UK) in a 50 µl reaction containing 250 mmol/l Tris–HCl, pH 8.0; 25 mmol/l MgCl2, 10 mmol/l DTT, 1 mol/l HEPES; pH 6.6 and 1 IU of DNA polymerase 1, Large (Klenow) fragment (Promega), and used as a probe to confirm the specificity of the PCR by Southern Blot analysis.
Southern Blot analysis
PCR products (10% of final PCR volume) were resolved on 2% agarose gels prepared in 1x TAE buffer with pGEM DNA markers (Promega) to confirm PCR product sizes. The gels were prepared for Southern transfer by sequential soaking in 0.5 mol/l NaOH/1.5 mol/l NaCl solution for 40 min and 1 mol/l Tris/1.5 mol/l NaCl for 40 min. The amplicons were transferred to Hybond-N membrane (Amersham) by capillary blotting with 20x SSC and were immobilized by UV cross-linking. Membranes were incubated for 1 h at 42°C in hybridization buffer (50% formamide, 6x SSPE, 0.5% sodium dodecyl sulphate (SDS), 5x Denhardt's solution (0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone) and 0.025% skimmed milk powder) and then hybridized with the radio-labelled probe overnight at 420C. The membranes were washed in 1x sodium chloride/sodium citrate (SSC)/0.1% SDS at 420C for 15 min followed by 0.1x SSC/0.1% SDS at 55°C for 15 min. The membranes were overlaid with Kodak XOMAT-AR film for 2 h. The autoradiograms were scanned and the PCR products were quantified using laser densitometry (Molecular Dynamics, USA). For semi-quantitative analysis, the intensity of hybridization signal for either LRP or RAP amplicons was expressed as a ratio of a positive control on the membrane and was normalized against the GAPDH amplicon signal.
Statistical analysis
The data were analysed from homogeneity of variance using Levene's test. Analysis of homogenous data was performed using one-way analysis of variance (ANOVA) and group means were compared using Duncan's multiple range test. Non-homogenous data sets were analysed using non-parametric statistics (Kruskal–Wallis ANOVA, Mann–Whitney U-test). LRP data are expressed as median values with 25% and 75% quartiles. RAP data are expressed as mean ± SEM. P < 0.05 was considered to be statistically significant.
Results
Semi-quantitative RT–PCR analysis
Initial quantification of LRP mRNA by Northern blot analysis was problematic when examining the expression of LRP in endometrial tissues. The enormous size of the transcript and other influential factors such as the presence of ribonucleases and sample collection time may have affected the mRNA stability and overall total RNA quality. The limited amount of collectable tissue and the limited success of Northern analysis in the detection of LRP mRNA led to the application of PCR-based methodology for the quantification of LRP mRNA in these tissues. In this study, the level of LRP mRNA expression in normal endometrium and endometrial carcinomas was determined by semi-quantitative RT–PCR. Reactions with specific LRP or RAP primers were performed separately with optimal sub-saturation cycle number, 30 cycles (Figure 1) to generate a 301 bp amplicon (LRP, Figure 2A) and 316 bp amplicon (RAP, Figure 2B). From each PCR, a single band was observed which corresponded to the predicted PCR product size for the target sequence and which subsequently hybridized with a sequenced cDNA probe of identical sequence. Intensity of the GAPDH PCR product (805 bp) confirmed the integrity of the RNA samples and also the RT activity.
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LRP mRNA expression in normal endometrium and endometrial carcinomas
LRP mRNA was detected in all of the normal endometrial samples (n = 14) examined. Expression of LRP mRNA in proliferative, secretory and menopausal endometrium was examined within the normal group and the carcinoma group and normalized with respect to GAPDH expression (Figure 3
). Expression of LRP mRNA in secretory phase endometrium was significantly increased when compared with proliferative phase endometrium (P < 0.05, Kruskal–Wallis ANOVA). No significant difference in expression was observed between secretory phase and post-menopausal endometrium or between proliferative phase and post-menopausal endometrium. The relative expression of LRP mRNA in the carcinoma group was significantly decreased when compared with either the proliferative, secretory or post-menopausal endometrium (P < 0.05, Mann–Whitney U-test).
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The expression of LRP mRNA was significantly higher in each normal endometrium group when compared with each grade of tumour differentiation (P < 0.05, Mann–Whitney U-test). Expression of LRP mRNA did not alter significantly with decreasing degree of differentiation in grade 1 (n = 14) and grade 2 (n = 8) nor grade 3 (n = 11) carcinomas (data not shown). LRP mRNA expression was also significantly higher (P < 0.05, Mann–Whitney U-test) in each group of normal endometrium, when compared with each of stage 1B (n = 12), stage IC (n = 6), stage IIA (n = 4) and stages III and IV (n = 11). No significant difference between each stage of invasion was observed (data not shown).
RAP mRNA expression in normal endometrium and endometrial carcinomas
RAP mRNA was detected in all tissue samples (n = 47) examined. The data were normalized with respect to GAPDH expression and the relative expression of RAP mRNA was compared between the normal and carcinoma tissues (Figure 4). RAP mRNA expression did not alter significantly between the normal proliferative, secretory and post-menopausal endometrium and the carcinoma group. The level of RAP expression did not change significantly between each normal group and each carcinoma group divided on the basis of clinical stage or grade of differentiation.
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Discussion
The aim of the present study was to determine the expression of LRP and RAP mRNA in normal secretory and proliferative phase endometrial tissues and endometrial carcinomas of varying clinical stage and grade of differentiation. The relative abundance of LRP and RAP, with respect to GAPDH mRNA expression, was determined by semi-quantitative RT–PCR. LRP transcripts were clearly detected in all normal endometrium and in ~ half of the carcinoma tissues examined. This is the first time the level of LRP and RAP mRNA expression has been determined in normal human endometrium and endometrial carcinoma tissues.
Proteolytic degradation of extracellular matrix and basement membrane surrounding tumour cells is a fundamental process of cell invasion and also metastasis (Stahl and Mueller, 1994; Raos et al., 1994), as is the rapid removal of inactivated protease complexes from the cell surface. LRP controls extracellular protease activity through the internalization of cell surface receptor bound-uPA and inhibitor complexes. Previous studies have shown that components of the plasminogen activation cascade are up-regulated in endometrial, ovarian and cervical carcinomas (Kobayashi et al., 1994; Kohler et al., 1997; Sier et al., 1998). In this study, the mRNA expression of LRP, the receptor known to be involved in the endocytosis of inactivated receptor bound uPA–inhibitor complexes, was down-regulated in all clinical stages of endometrial carcinoma. Our finding that LRP mRNA expression decreased with the development of endometrial carcinoma was unexpected as according to the proposed model this would supposedly slow the rate of removal of inactive receptor bound uPA and thus suppress focalized uPA mediated proteolysis and reduce the potential for cellular invasion.
The decrease in LRP mRNA expression in these endometrial carcinomas indicates that LRP may not be the major clearance receptor of inactive protease complexes from the surface of endometrial carcinoma cells. This suggests that clearance receptors, other than LRP, may be involved in the internalization of receptor-bound uPA-serpin complexes from the surface of endometrial cancer cells. LRP belongs to a large family of endocytic clearance receptors including the very low density lipoprotein receptor and gp330/megalin, which share many ligands with LRP, including inactivated PA system components (Heegaard et al., 1995; Stefansson et al., 1995). A previous report describing the pattern of LRP, RAP and gp330 protein expression in the normal endometrium established that LRP was prominently and uniformly expressed in the stroma (Sayegh et al., 1995). The morphological changes associated with endometrial carcinoma development shift the balance of stromal and glandular cells, in favour of glandular cells. This change in the ratio of LRP expressing stromal cells to glandular cells, which express lower levels of LRP, may account for the observed decrease in LRP mRNA expression in the endometrial tumours examined. The endocytic receptor, Gp330/megalin has been previously localized to the glandular component of normal secretory and proliferative endometrium and may be involved in the clearance of inactive PA components from the surface of endometrial carcinoma cells. The results of this study propose that LRP may not play a significant role in the progression of invasion in this tumour type through directional uPA-mediated proteolysis.
In concordance with this study, previous investigations examining the expression of LRP and RAP in highly invasive sub-clones derived from human prostate, melanoma and breast tumour cell lines identified a decrease in LRP mRNA and a constant level of expression of RAP mRNA (Kancha et al., 1994). Similarly, immunohistochemical analyses of human melanocytic lesions showed a decrease in LRP and RAP protein expression as the tumour stage progressed from benign to metastatic melanoma (de Vries et al., 1996). Our study, while confirming LRP mRNA expression was down-regulated in carcinoma tissues, showed that RAP mRNA expression was unaltered.
The decrease in the relative abundance of LRP mRNA in endometrial tumours in this study may be the result of transcriptional down-regulation or a decrease in mRNA stability. The factors that may regulate LRP transcription in the endometrium are yet to be clearly established. The decrease in the expression of LRP mRNA in these tumours may be an effect caused by cancer cell production of cytokines and growth factors, which have been shown to regulate LRP expression in vitro. Previous reports of LRP transcription in macrophages showed interferon-
down-regulated LRP mRNA in macrophages (Hussaini et al., 1996). A recent in-vitro study examining the regulation of LRP in neoplastic astrocytomas and non-neoplastic astrocytes also suggested that EGF mediated a significant down-regulation of LRP mRNA transcription (Hussaini et al., 1998). Modulation of LRP gene expression in endometrial cancer cell invasion may be mediated by epidermal growth factor which has also been associated with the normal endometrial tissue remodelling processes, e.g. decidualization (Bany et al., 1998).
The expression of LRP mRNA in this study was significantly higher in secretory endometrium when compared to proliferative endometrium. In contrast to these findings, a previous study localising the expression of immunoreactive LRP in the endometrium could not identify any change in the level of expression between the secretory and proliferative phase endometrium (Sayegh et al., 1995); however this may be due to the nature of the methodology employed. The differential expression of LRP in the endometrium could be due to the increase in stromal cell maturation (pre-decidualization) during the secretory phase or may even be the result of steroid hormone changes associated with this stage of the cycle. At specific points in the secretory phase of the cycle, oestrogen concentrations drop off after ovulation and then surge again in the middle of this phase, while circulating concentrations of progesterone surge after ovulation and then gradually decline until menstruation. To clearly establish the mechanism by which LRP is regulated in the endometrium during the menstrual phase it would be necessary to accurately date the endometrium. This specific information, which was unavailable in our sample set, may determine whether progesterone acting on oestrogen-primed early secretory phase endometrial cells increases LRP transcription or even if the mid luteal-phase surge in oestrogen concentrations in combination with progesterone may contribute to an increase in LRP transcription in the latter half of the phase. It is also important to note that cellular differentiation in the form of decidualization does occur in the late secretory phase in the endometrial stromal cells that surround the spiral arteries in the endometrium. This differentiation of the stroma, which is generally initiated after the mid-luteal surge in oestrogen concentrations, may contribute to the increased expression of LRP observed in secretory phase endometrium.
The expression of LRP is believed to be increased in differentiated cell types, therefore LRP mRNA expression, in this study, was correlated to the standard prognostic variable of grade of differentiation. The relative abundance of LRP mRNA decreased in the progression from the normal endometrial cell phenotype to well-differentiated grade 1 carcinomas, whereas RAP mRNA levels were constant. No significant change in expression was identified between well-differentiated, moderately-differentiated and poorly-differentiated tumours. No significant change in expression was observed between endometrial carcinomas and carcinomas with alternative differentiation (special variant) which are seen to be far more aggressive than the endometrial tumours (data not shown). These data suggest that LRP mRNA expression may be down-regulated in the early stages of transformation from a normal to a neoplastic phenotype. While transcriptional regulation of LRP and RAP in vivo has not been clearly established, in-vitro studies have shown LRP expression is markedly increased in differentiated cell types. LRP expression was increased in the spontaneous differentiation of isolated cytotrophoblast cells into syncytiotrophoblast in culture (Coukos et al., 1994). Similarly, LRP expression was up-regulated in the maturation of monocytes into macrophages and has been proposed as a monocyte differentiation antigen (Moestrup et al., 1990).
The relative abundance of the 39 kDa inhibitor, RAP did not alter in any clinical stage or grade of endometrial carcinoma when compared with the normal group, whereas the relative abundance of LRP mRNA was significantly decreased in the progression from a normal to neoplastic cell phenotype. LRP expression was also differentially regulated in different phases of the menstrual cycle, with secretory phase endometrium expressing the highest levels of LRP. The decrease in LRP mRNA relative abundance in the endometrial carcinomas examined in this study may be due to factors associated with gene transcription down-regulation or a decrease in LRP mRNA stability. Future studies using patient matched normal and carcinoma tissues may also determine whether the observed decrease of LRP mRNA expression in the carcinoma group may be due to age-related factors since the majority of carcinomas examined were post-menopausal as compared with the normal group which was mostly pre-menopausal. Our data however, indicate that LRP may not play a significant role in directional uPA-mediated proteolysis in invasive endometrial tumours, thus suggesting that other clearance receptors may be involved in the clearance of these proteolytic enzymes. This study also revealed that LRP mRNA is differentially expressed in normal secretory phase endometrium, and this may be the result of steroid hormone action on endometrial stromal cell differentiation.
Acknowledgments
The present investigation was supported by the National Health and Medical Research Council of Australia (G.E.Rice). G.E.Rice is in receipt of a National Health and Medical Research Council of Australia Principle Research Fellowship. We thank Mrs Julene Harro for tissue sample collection.
Notes
3 To whom correspondence should be addressed at: Perinatal Research Centre, Department of Perinatal Medicine, The Royal Women's Hospital, 132 Grattan St, Carlton 3053, Victoria, Australia. E-mail: gerice{at}ariel.its.unimelb.edu.au 
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Submitted on January 31, 2000; accepted on June 23, 2000.
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