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Endocrinology Vol. 138, No. 11 4921-4930
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of the Expression of the Angiogenic Enzyme Platelet-Derived Endothelial Cell Growth Factor/ Thymidine Phosphorylase in Endometrial Isolates by Ovarian Steroids and Cytokines1

Lyna Zhang, Ian Z. MacKenzie, Margaret C. P. Rees and Roy Bicknell

Molecular Angiogenesis Group (L.Z., R.B.), Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, and Nuffield Department of Obstetrics and Gynaecology (L.Z., I.Z.M., M.C.P.R.), University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

Address all correspondence and requests for reprints to: Roy Bicknell, Molecular Angiogenesis Group, Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. E-mail: bicknelr{at}icrf.icnet.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The angiogenic enzyme platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) was strongly expressed in the endometrial glands in the luteal and menstrual, but not the proliferative, phases of the cycle. The converse was seen in the stroma, where expression was strong in the proliferative, but not the luteal or menstrual, phases. Inflammatory cytokines induced PD-ECGF/TP expression in primary cultures of human normal endometrial epithelial (NEE) and normal endometrial stromal cells. The profile of cytokine induction of PD-ECGF/TP was cell dependent. Thus, in NEE cells, PD-ECGF/TP expression was strongly induced by the combination tumor necrosis factor-{alpha} and interferon-{gamma}. In contrast, in normal endometrial stromal cells, interferon-{gamma} gave, by far, the strongest induction of PD-ECGF/TP. Expression of the enzyme was not regulated by ovarian hormones alone. Although treatment of NEE cells with a physiological concentration of progesterone (5 x 10-8 M) or transforming growth factor-ß1 (10 ng/ml) alone had no effect on PD-ECGF/TP expression, when delivered together at the same dose they induced a 48-fold increase in expression. This expression correlates with cyclic changes in progesterone and transforming growth factor-ß1 levels in the uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOME 7 yr ago, platelet-derived endothelial cell growth factor (PD-ECGF) was identified as the sole mitogenic activity toward endothelial cells present in platelets (1). It is now known to be the intracellular enzyme thymidine phosphorylase (TP) (2, 3, 4). We and others have shown PD-ECGF/TP to be strongly angiogenic in a range of angiogenesis assays that include the chick chorioallantoic assay, the rat sc sponge model, and a cryo-injured wound healing model (1, 5). The enzyme activity has been shown to be essential for angiogenic activity (5). PD-ECGF/TP catalyses the phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose-1-phosphate. The latter may then be dephosphorylated to 2-deoxy-D-ribose. Haraguchi and co-workers (6) have reported in a letter that 2-deoxy-D-ribose was angiogenic in the chick chorioallantoic assay but gave few details.

Human endometrium has the unique property of undergoing benign angiogenesis, a process otherwise restricted to a few other physiological processes, such as ovarian follicular development and during wound healing. The endometrium is a mucosa supplied by a microvascular blood supply that develops cyclically under the influence of sequential estradiol and progesterone (P), secreted by the ovary during each menstrual cycle. In this study, we have examined a possible role of PD-ECGF/TP in endometrial angiogenesis. We have developed techniques that permit the preparation and long-term culture of pure isolates of normal endometrial epithelial (NEE) and stroma. The isolates are known as NEE and normal endometrial stromal (NES) cells. In view of strong in vivo expression of PD-ECGF/TP in these cells in the luteal endometrium we have attempted to identify what regulates this expression by using the cells in culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
RL95–2 (CRL 1671) cells were from the American Type Culture Collection, Bethesda, MD. Ishikawa cells were a gift of Dr. J. White, Royal Postgraduate Medical School, Hammersmith Hospital, London. All cell culture media were prepared at the Imperial Cancer Research Fund Clare Hall Laboratories, London. Anti-TP monoclonal antibody (PGF44C) was raised against Escherichia coli- expressed recombinant PD-ECGF/TP (7). T3 or T7 RNA polymerase and ribonuclease (RNase)-free deoxyribonuclease I were from Boehringer Mannheim. A stock solution of RNase A of 20 mg/ml was prepared by dissolving pancreatic RNase A (Sigma, Poole, UK) in 10 mM Tris-HCl (pH 7.5) and 15 mM NaCl and heating to 100 C for 15 min to inactivate contaminating deoxyribonuclease. Prestained, low molecular weight-range protein markers were from BioRad. [{alpha}-32P]-cytidine triphosphate (400 Ci/mmol) and enhanced chemiluminescence detection kits from Amersham plc, Amersham, UK. Ultrapure deoxynucleotide triphosphates, di-deoxy-nucleotide triphosphates, and ribonucleotide triphosphates were from Pharmacia. Endothelial cell growth supplement, collagenase type 1A, dextran-coated charcoal (DCC)-stripped FCS, 17-ß-estradiol (E), and P were from Sigma. RU 486 was a gift from Hoechst Marion/Rousel Ltd., Broadwater Park, Uxbridge, UK. Human recombinant tumor necrosis factor-{alpha} (TNF-{alpha}), human recombinant interleukin 1-{alpha} (IL-1{alpha}), human recombinant interferon-{gamma} (IFN-{gamma}), and human recombinant transforming growth factor-ß1 (TGF-ß1) were from R. and D. Systems, Abingdon, UK. All routine laboratory chemicals were supplied by Sigma or BDH, unless otherwise stated.

Cell culture and cytokine and/or steroid treatment
All cell cultures were maintained at 37 C in a humidified atmosphere of 5% CO2/95% air and, by regular screening, shown to be mycoplasma free. NEE and NES cells were isolated and cultured as described (8). Other cells were routinely cultured in HEPES-buffered DMEM supplemented with 10% FCS, penicillin (100 units/ml), streptomycin (10 units/ml), and glutamine (2 mM). For gene expression experiments with steroids and/or cytokines, NEE or NES cells were cultured to confluence in 15-cm Petri dishes and then left to quiesce for 1 week in estrogen-free medium [phenol red-free (PRF)-DMEM with 10% DCC-stripped FCS]. Cells were then treated with fresh PRF-DMEM/10% DCC-stripped FCS containing the cytokines or steroids under study. Endometrial carcinoma cells were seeded at low density, cultured in estrogen-free medium to confluence, and then treated with steroids and/or cytokines as described as above. Total RNA was prepared either 6 h (RL95–2) or 24 h (NEE and NES) after treatment. Cell lysates were prepared 30 h (NEE or NES) after treatment.

Preparation of total cellular RNA
Isolation of total cellular RNA was carried out by acid guanidinium-thiocyanate-phenol chloroform extraction (9) or by the CsCl ultracentrifugation method described by Ausubel et al. (10). RNA concentrations were calculated from the OD at 260 nm (A260 of 1 = 40 mg/ml). Purity of RNA was assessed from the A260/A280 ratio and by agarose gel electrophoresis.

RNase protection analysis
RNase protection analysis was performed as described by Ausubel et al. (10). The template for GAPDH has been described (11). To prepare a template for PD-ECGF/TP, a 241-bp fragment (corresponding to nt 817-1058 of the coding region of PD-ECGF/TP) was released from plasmid PL-5 (1) and cloned into the EcoRV/HindIII sites of pBluescript KS+. The resulting construct was linearized with HindIII and transcribed with T3 polymerase to obtain a template. Twenty nanograms of total cellular RNA was used in each protection.

Immunoblotting
After treatment with cytokines and/or steroids, confluent cells in either 10- or 15-cm Petri dishes were washed twice with ice-cold PBS, scraped into 100 µl of ice-cold Tris-buffered saline and lysed by mild sonication (Soniprep 150). Insoluble debris was removed by centrifugation. Supernatants were then concentrated using an Amicon-30 concentrator (Amicon, Inc., Beverly, MA). Before electrophoresis, 100 µg of soluble cell lysate was boiled in SDS sample buffer (0.125 M Tris-HCl (pH 6.8), 1% SDS, 1% 2-mercaptoethanol, 5% glycerol, 0.005% bromophenol blue). After SDS-PAGE, immunoblotting was performed as detailed by Ausubel et al. (10), with minor modifications. Briefly, SDS-PAGE gels were soaked in transfer buffer [25 mM Tris base, 150 mM glycine, 15% methanol (pH 8.3–8.4], and proteins subsequently transferred onto Immobilon-P membrane (Millipore, Bedford, MA) by electroblotting at 110 V for 1 h or 15 V overnight in a Bio-Rad minitransblot apparatus. Membranes were blocked for 2 h at room temperature with blocking buffer (0.1% Tween-20 and 5% Marvel fat-free milk powder in PBS). Monoclonal anti-TP (PGF44c) was diluted to 16 µg/ml in blocking buffer and incubated with the filter for 1 h at room temperature. Membranes were then washed for 15 min in blocking buffer (x3) before visualization by ECL.

Densitometry
To obtain values of fold-induction, messenger RNA (mRNA) abundance (determined by RNase protection assay) or protein abundance (from immunoblot analysis) were quantitated from the signal on autoradiographic film by scanning laser densitometry using a Millipore Bioimage Analyzer.

Immunohistochemistry
Normal human endometrial (n = 30) and carcinoma tissue (n = 30) was obtained from the archival files of the Histopathology Department of the John Radcliffe Hospital. Specimens of normal tissue were from premenopausal women with documented normal menstrual cycles. The phase of the menstrual cycle was identified from the patients menstrual history, and histologically by an independent histopathologist (12, 13). Of the 30 samples examined, 10 were proliferative, luteal, or menstrual. Paraffin-embedded human endometrial sections were immunostained with anti-PD-ECGF/TP monoclonal antibody (PGF44c) (7). Antibodies were visualized by the alkaline phosphatase antialkaline phosphatase method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical staining of PD-ECGF/TP expression in human endometrium and endometrial adenocarcinoma
Expression of PD-ECGF/TP in human endometrium was first examined by immunohistochemistry. PD-ECGF/TP was detected in endometrium throughout the menstrual cycle. Immunostaining was most intense in the glandular epithelium of the late luteal and menstrual phases (Fig. 1Go, A, B, and E). The intensity of epithelial staining was greatest in the basalis adjacent to myometrium, and diminished toward the endometrial surface (Fig. 1Go, A and B). Staining of endometrial stroma was seen in the proliferative phase (Fig. 1Go, C and D). Strong staining of PD-ECGF/TP also was detected in endothelium and in the walls of arterioles and venules (Fig. 1EGo). Vascular staining was menstrual cycle independent (data not shown). Unexpectedly (see Discussion), PD-ECGF/TP staining was not detected in 30 cases of human endometrial adenocarcinoma.



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  		Figure 1. Immunohistochemistry of PD-ECGF/TP expression in the human endometrium. PD-ECGF/TP was detected in glandular epithelial cells in the luteal (A and B) and menstrual phase (E), in stromal cells of the midproliferative (C and D) and in endothelial cells here shown (E) in a section from the menstrual phase of the cycle. A control luteal section (F), in which the primary antibody was replaced with mouse IgG. All sections are x 100. m, Myometrium; g, gland; s, stroma; la, lymphoid aggregate. Anti-PD-ECGF/TP monoclonal antibody PGF44c was used (7).

 
Regulation of PD-ECGF/TP expression in NEE cells
In view of strong immunohistochemical staining of PD-ECGF/TP in luteal and menstrual epithelium, it was of interest to identify the factors that regulate this expression. It has previously been shown that the inflammatory cytokines TNF-{alpha}, IL-1{alpha}, and IFN-{gamma} induce PD-ECGF/TP expression in several carcinoma cell lines but not in normal human fibroblasts (14, 15). The effect of inflammatory cytokines on PD-ECGF/TP expression in NEE cells was examined first. Figure 2AGo shows a RNase protection analysis of PD-ECGF/TP mRNA in NEE cells after treatment with TNF-{alpha}, IL-1{alpha}, IFN-{gamma}, or various combinations of the three. Basal expression of PD-ECGF/TP in cultured NEE cells was not detectable. It is seen that treatment with no single factor alone affected PD-ECGF/TP expression but that a combination of TNF-{alpha} and IFN-{gamma} induced strong expression (205-fold). PD-ECGF/TP induction was unique to this particular combination of the three cytokines; thus, neither TNF-{alpha} + IL-1{alpha} or IL-1{alpha} + IFN-{gamma} induced PD-ECGF/TP expression. A strong induction also was seen on treatment with all three cytokines, but no greater (140-fold) than on treatment with TNF-{alpha} + IFN-{gamma} (205-fold) (Fig. 2AGo).



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  		Figure 2. The effect of cytokines and ovarian steroids on PD-ECGF/TP expression by NEE cells in vitro. A, RNase protection analysis of the effect of cytokines on PD-ECGF/TP expression; top, the protected fragment of PD-ECGF/TP and the internal loading control GAPDH; bottom, the normalized optical density of PD-ECGF/TP expression to that of GAPDH; lanes: 1, TNF-{alpha}; 2, IL-1{alpha}; 3, IFN-{gamma}; 4, IFN-{gamma} + TNF-{alpha}; 5, TNF-{alpha} + IL-1{alpha}; 6, IFN-{gamma} + IL-1{alpha}; 7, a mixture of all three; 8, control. Cytokines were used at the following concentrations: TNF-{alpha} (100 u/ml), IL-1{alpha} (100 u/ml), and IFN-{gamma} (75 u/ml). Cells were quiesced for 1 week in PRF-DMEM/10% DCC-stripped FCS and then exposed to cytokines for 24 h before harvest. B, RNase protection analysis of the effect of ovarian steroids on PD-ECGF/TP expression, either alone or in combination with a mixture of TNF-{alpha}, IL-1{alpha}, and IFN-{gamma}. Lanes: 1, control; 2, E (5 x 10-10 M); 3, P (5 x 10-8 M); 4, three cytokines: [TNF-{alpha} (100 u/ml), IL-1{alpha} (100 u/ml), and IFN-{gamma} (75 u/ml)]; 5, E + three cytokines; 6, P + three cytokines. Cells were treated as described in A. n = 3, mean + SD. *, P < 0.05.

 
The second set of RNase protection analyses examined the effect of the ovarian steroids E and P on PD-ECGF/TP expression either basally or in the presence of the three cytokines TNF-{alpha}, IL-1{alpha}, and IFN-{gamma}. Maximum physiological concentrations (outside of pregnancy) of E (5 x 10-10 M) and of P (5 x 10-8 M) were used. E and P alone were without effect on PD-ECGF/TP expression. Preliminary (single-point) assays suggested that E and P may modulate the induction of PD-ECGF/TP by a combination of the three cytokines TNF-{alpha}, IL-1{alpha}, and IFN-{gamma}. However, a repeat of the analysis in triplicate (i.e. treating three dishes of cells and extracting and analyzing the RNA separately) showed this modulation not to be statistically significant (Fig. 2BGo).

Regulation of PD-ECGF/TP expression in the endometrial adenocarcinoma cell line RL95–2
Figure 3AGo shows that PD-ECGF/TP expression was induced in the RL95–2 cell line by treatment with either TNF-{alpha} alone (12-fold); a mixture of the three cytokines: TNF-{alpha}, IL-1{alpha}, and IFN-{gamma}; or of these three + TGF-ß1. IL-1{alpha}, IFN-{gamma}, and TGF-ß1 alone had no detectable effect on PD-ECGF expression (Fig. 3AGo). As in NEE cells, the basal expression of PD-ECGF/TP mRNA was barely detectable in RL95–2 cells (Fig. 3AGo).



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  		Figure 3. Effect of cytokines and ovarian steroids on PDECGF/TP expression by RL-95 endometrial carcinoma cells. A, RNase protection analysis of the effect of the cytokines TNF-{alpha}, IL-1{alpha}, IFN-{gamma}, or TGF-ß1, alone or in combination, on PD-ECGF/TP expression. Lanes: 1, control; 2, TNF-{alpha}; 3, IL-1{alpha}; 4, IFN-{gamma}; 5, TGF-ß1 (10 ng/ml); 6, TNF-{alpha} + IL-1{alpha} + IFN-{gamma}; 7, a mixture of all four. The concentrations of cytokines were as follows: TNF-{alpha} (100 u/ml), IL-1{alpha} (100 u/ml), IFN-{gamma} (75 u/ml), and TGF-ß1 (10 ng/ml). B, RNase protection analysis of the effect of E and P on PD-ECGF/TP expression by RL-95 cells, either alone or in combination with TNF-{alpha}. Lanes: 1, control; 2, E (5 x 10-10 M); 3, P (5 x 10-8 M); 4, TNF-{alpha}; 5, E + TNF-{alpha}; 6, P + TNF-{alpha}. TNF-{alpha} was used at a concentration of 100 u/ml. Cells were treated as described in Fig. 2Go, with the exception that they were exposed to cytokines for 6 h before harvest. n = 3, mean + SD. **, P < 0.05 above control; *, P < 0.05 above the value for TNF-{alpha}.

 
Late passage (as used here) RL95–2 cells lack estrogen but retain functional P receptors (16). E had no effect on PD-ECGF/TP expression, but exposure to 10-8 M P induced a modest (4.2-fold, P < 0.05, n = 3) increase in expression (Fig. 3BGo).

Regulation of PD-ECGF/TP expression in NES cells
Figure 4AGo shows that NES cells exhibit a different cytokine PD-ECGF/TP mRNA induction profile than NEE cells. Thus, TNF-{alpha}, IL-1{alpha}, and TGF-ß1 alone had no effect on expression, but IFN-{gamma} strongly induced PD-ECGF/TP by 260-fold (P < 0.01, n = 3). A mixture of all three or four factors also increased expression, but not markedly, more than that seen on treatment with IFN-{gamma} alone (data not shown).



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  		Figure 4. Effect of cytokines and ovarian steroids on PD-ECGF/TP expression by NES cells. A, RNase protection analysis of PD-ECGF/TP expression in NES cells, in response to cytokines, showing strong induction by IFN-{gamma}. No induction of expression was seen with TNF-{alpha} (up to a dose of 100 u/ml) or IL-1{alpha} (up to a dose of 100 u/ml). Lanes: 1, control; 2, TNF-{alpha} (12.5 u/ml); 3, TNF-{alpha} (100 u/ml); 4, IL-1{alpha} (12.5 u/ml); 5, IL-1{alpha} (100 u/ml); 6, IFN-{gamma} (15 u/ml); 7, IFN-{gamma} (75 u/ml). Cells were treated as described in Fig. 2Go. B, RNase protection analysis of PD-ECGF/TP expression in NES cells, in response to ovarian steroids, either alone or in combination with IFN-{gamma}. Lanes: 1, control; 2, E (5 x 10-10 M); 3, P (5 x 10-8 M); 4, IFN-{gamma} (75 u/ml); 5, E + IFN-{gamma}; 6, P + IFN-{gamma}. Cells were treated as described in Fig. 2Go. n = 3, mean + SD. *, P < 0.05 above control.

 
The effect of E and P on PD-ECGF/TP expression in NES cells, basally or in combination with IFN-{gamma}, was then examined. The effect of E and P alone on PD-ECGF/TP mRNA expression in NES cells was similar to that in NEE cells, namely, a modest induction by E (5 x 10-10 M, 5.6-fold, P < 0.05, n = 3) (Fig. 4BGo) or P (10-8 M, 3-fold, P > 0.05, n = 3). Similar effects were seen at the protein level by immunoblotting (data not shown). When mixed with IFN-{gamma}, neither E or P had a statistically significant effect on PD-ECGF/TP expression (Fig. 4BGo).

The effect of P and transforming growth factor-ß1 on PD-ECGF/TP expression in NEE cells
Treatment of NEE cells with a physiological dose of either P (10-8 M) or TGF-ß1 (10 ng/ml) had no effect on PD-ECGF/TP mRNA expression. In marked contrast, when administered together, P and TGF-ß1 induced a 48-fold increase in PD-ECGF/TP mRNA expression (P < 0.05, Fig. 5AGo). Immunoblotting confirmed that the P + TGF-ß1-stimulated expression of PD-ECGF/TP mRNA also occurred at the protein level (Fig. 5BGo). This response was, by far, the strongest induction of PD-ECGF/TP by an ovarian steroid. Further, the response was unique to the combination of P with TGF-ß1; thus, no induction of PD-ECGF/TP expression was detected on treatment with combinations of P with TNF-{alpha}, IL-1{alpha}, or IFN-{gamma}. Figure 5BGo shows that the induction by P + TGF-ß1 is efficiently blocked by the P antagonist RU486. Finally, an identical treatment of NES cells with P and TGF-ß1 had no effect on PD-ECGF/TP mRNA or protein expression.



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  		Figure 5. A physiological dose of P and TGF-ß1, in combination but not alone, strongly induces PD-ECGF/TP expression in NEE cells. A, RNase protection analysis of PD-ECGF/TP expression. Lanes: 1, control; 2, E (5 x 10-10 M); 3, P (5 x 10-8 M); 4, TGF-ß1 (10 ng/ml); 5, E + TGF-ß1; 6, P + TGF-ß1. n = 3, mean + SD. **, P < 0.05. Cells were treated as described in Fig. 2Go. B, Immunoblotting analysis of PD-ECGF/TP expression. Lanes: 1, 5 ng human recombinant PD-ECGF/TP; 2, control; 3, P; 4, TGF-ß1; 5, RU486; 6, P + TGF-ß1; 7, P + TGF-ß1 + RU486. The concentrations were, in each case, as follows: TGF-ß1 (10 ng/ml) and P and RU486 (5 x 10-8 M). Bottom panel gives the optical density of the bands in the autoradiograph of the Western analysis, quantitated by laser densitometry.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The angiogenic activity of PD-ECGF/TP has now been clearly demonstrated in a range of in vivo assays (1, 2, 5, 17). Despite this, the role of PD-ECGF/TP as a regulator of blood vessel formation at sites such as endometrial development during the menstrual cycle remains unclear. The present study has shown that PD-ECGF/TP is widely expressed in human endometrium throughout the menstrual cycle, in a range of cell lineages that include glandular epithelium, stroma, and endothelium. Marked menstrual cycle-related changes in expression were evident from the 30 specimens of normal endometrium examined.

The menstrual cycle-related changes in PD-ECGF/TP expression show a shift in the cell type, in that expression moves from stroma to epithelium as the cycle progresses. This would be consistent with expression being under the control of ovarian steroids. A mixture of the inflammatory cytokines (TNF-{alpha}, IL-1{alpha}, and IFN-{gamma}) has been reported to enhance PD-ECGF/TP expression in several carcinoma lines but not in normal fibroblasts (14). It is known that these same cytokines are present in human endometrium throughout the menstrual cycle (18, 19), and thus, they are potential mediators of hormonally modulated PD-ECGF/TP expression in human endometrium. Expression of TNF-{alpha} and IFN-{gamma} in the endometrium does not vary throughout the cycle. The establishment of isolated human epithelial and stromal endometrial cell cultures (8) has now permitted an exploration of the mechanisms underlying the regulation of PD-ECGF/TP expression in endometrial isolates.

The data presented here clearly demonstrate that expression of PD-ECGF/TP is regulated by cytokines and ovarian steroids in human endometrial isolates. The pattern of cytokine induction of PD-ECGF/TP was cell dependent. This suggests that different mechanisms of regulation of expression occur in the epithelium and stroma. In stromal (NES) cells, IFN-{gamma} gave, by far, the strongest induction. In epithelial (NEE) cells, no single cytokine alone significantly induced PD-ECGF/TP mRNA expression; however, a combination of IFN-{gamma} and TNF-{alpha} strongly induced PD-ECGF/TP expression. The combined presence of IFN-{gamma} and TNF-{alpha} often results in an enhanced or diminished biological response. For example, IFN-{gamma} increases production of TNF-{alpha} and expression of the cell surface receptors for TNF-{alpha} in a variety of different cell types (20, 21, 22, 23). In turn, TNF-{alpha} stimulates IFN-{gamma} production by natural killer cells (22, 23, 24, 25).

The strongest ovarian hormone induction of PD-ECGF/TP occurred with a combination of P and TGF-ß1 in NEE but not in NES cells. In a recent paper, an induction of PD-ECGF/TP in NES cells by P was reported (26), although a fold induction was not given. We have found this induction in NES cells to be small (3-fold) and not to be enhanced when combined with TGF-ß1. These authors also showed that PDECGF/TP is expressed in the human endometrium predominantly in the stroma, and not in the glands, during the early secretory phase of the cycle. The reason for this discrepancy between the work of Osuga et al. (26) and ours is not known but may arise as a result of using different antibodies, specimens, and staining techniques.

Expression of TGF-ß1 mRNA and protein in human endometrium occurs in the luminal and glandular epithelial cells and varies during the menstrual cycle, with the highest level in the late proliferative and early to midluteal phases of the cycle (27). P receptor expression is high in the glands of the proliferative phase of the cycle and then falls (28). P receptor expression on the stromal component of the endometrium is largely cycle independent (28). P alone stimulates TGF-ß1 and ß2 expression in endometrial epithelium in ovariectomized rats (29) and TGF-ß1 expression in human endometrial stroma (30). Serum P levels increase after ovulation and peak in the midluteal phase of the menstrual cycle. Thus, the increase in the expression of PD-ECGF/TP in glandular epithelium, seen on immunohistochemistry, parallels the appearance of P and TGF-ß1 in endometrium. We postulate from the in vitro experiments that it is this unique combination of P and TGF-ß1 that is principally responsible for PD-ECGF/TP expression in the endometrium. This would explain why the expression of PD-ECGF/TP was not detected in endometrial adenocarcinomas, which in all cases examined, were from postmenopausal women in whom P is absent.

Lymphoid aggregates are a common feature in the endometrial basalis during the luteal and menstrual phases of the cycle. These aggregates secrete the cytokines (amongst others) that have been shown to induce PD-ECGF/TP expression, and it was not surprising to find strong immunostaining of PD-ECGF/TP in the epithelium and stroma adjacent to such aggregates.

PD-ECGF/TP is highly expressed in the normal human endometrium but not in endometrial carcinoma; this is in stark contrast to expression in other tissues, where increased expression has been found in neoplastic tissue, compared with that in normal tissue (31, 32). Expression is particularly strong in the mammary carcinoma, compared with normal breast tissue (5, 7). Curiously, despite the lack of PD-ECGF/TP expression in endometrial carcinoma described here and postulated to be caused by the absence of P in those patients, this is clearly not the case in breast carcinoma, where most of the cases we previously examined were from postmenopausal women (5). It is unclear, at present, what mechanism(s) is responsible for these dramatic differences in PD-ECGF/TP expression in the breast and endometrium, especially in that both tissues are influenced by the ovarian steroids.

In summary, this study has demonstrated that PD-ECGF/TP is present in the human endometrium throughout the menstrual cycle and that its expression is differentially regulated by cytokines and ovarian steroids. In vitro studies point to the regulation of PD-ECGF/TP expression in this tissue by a unique combination of P and TGF-ß1; this is in accord with the immunohistochemistry. These findings are consistent with PD-ECGF/TP playing a critical role in the regeneration of microvasculature during endometrial development.


   Footnotes
 
1 This work was funded by the Sir Jules Thorn Charitable Trust and the Imperial Cancer Research Fund. Back

Received April 4, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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