Molecular Human Reproduction, Vol. 5, No. 12, 1141-1149,
December 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
Regulation of TNF-
mRNA expression in endometrial cells by TNF- and by oestrogen withdrawal
1 Department of Pathology, North Shore University Hospital, Biomedical Science Research Center, 350 Community Drive, Manhasset, NY 11030, 2 Department of OB/GYN, Hershey Medical Center, Hershey, PA 17033, USA, and 3 Department of OB/GYN, Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany
Abstract
During each menstrual cycle, the human endometrium undergoes a series of orchestrated and well controlled changes in anticipation of the arrival of the blastocyst. In the absence of implantation, the endometrium is shed. The underlying basis of the menstrual bleeding is not clear, however, it seems to be related to steroid hormone withdrawal. We showed that tumour necrosis factor-
(TNF-) is released by human endometrium and that endometrial epithelial cells are a major source of TNF- mRNA and protein. We show here that TNF- mRNA shows a specific menstrual cycle-dependent expression. The expression of TNF- is mostly minimal throughout the proliferative, early and mid-secretory phases. Expression of TNF- mRNA, however, is increased in the human endometrium in the late secretory phase and during endometrial bleeding. Such a menstrual cycle-dependent expression suggests that specific signals regulate the expression of TNF- mRNA in the human endometrium. In vitro, the expression of TNF- mRNA in endometrial epithelial cells could be regulated by exogenous TNF-. This induced expression was both time- and dose-dependent. In vitro, the TNF- mRNA expression was not altered by oestrogen, progesterone, or both, in the endometrial epithelial cells under conditions that maintain the steroid hormone receptors. However, in vivo, oestrogen withdrawal led to an enhanced expression of TNF- in endometrial epithelial cells. These findings suggest that the up-regulation of TNF- in human endometrium in the late secretory phase may be related to the falling serum oestrogen concentration at the end of the menstrual cycle as well as the potentiating effect of released TNF- on its own mRNA expression.
endometrium/oestrogen withdrawal/TNF-/TNF- mRNA expression
Introduction
The role of steroid hormones as the primary and systemic force that drives the endometrium through the exquisitely orchestrated phases of the menstrual cycle is well recognized (Markee, 1946
; Tabibzadeh, 1994, 1995, 1996). Bleeding also occurs in the endometrium under such conditions as aberrant follicular maturation, ovulation, or development of the corpus luteum (Blaustein, 1982; Wathen et al., 1995). The underlying basis of endometrial bleeding remained obscure until Markee demonstrated that withdrawal from steroid hormones is responsible for the menstrual shedding of endometrium and bleeding (Markee, 1946). From transplantation of endometrium into the anterior chamber of the eyes of monkeys, Markee concluded that `the stimulus for the bleeding in these transplants must be purely a local one, for the sudden removal of the crystals decreases the concentration of oestrone in that eye, although the systemic level is being raised by the oestrone injected subcutaneously' (Markee, 1946). Several lines of evidence are consistent with the conclusions of Markee that local endometrial factors are implicated in endometrial bleeding. For example, expression of certain members of the metalloproteinase family (MMP), which degrade extracellular matrix, at defined and distinct time periods during the menstrual cycle, is consistent with the role of these local factors in the menstrual bleeding, tissue degradation and tissue reorganization and repair within the endometrium (Rodgers et al., 1993, 1994; Matrisian et al., 1994; Tabibzadeh, 1996). In addition, the expression of genes of various types of MMPs is subject to regulation by steroid hormones, linking these local factors to the systemic signals regulating endometrial function (Rodgers et al., 1994; Schatz et al., 1994; Irwin et al., 1996). Although it has become clear that menstrual bleeding is related to the falling serum concentraion of steroid hormones, by and large, the identity of the local and specific endometrial factors implicated in this process remains largely unrecognized.
Endometrial bleeding and its regulation is a complex process which undoubtedly requires participation of a diverse group of local factors. We identified tumour necrosis factor- (TNF-) as a cytokine which is synthesized and secreted by the human endometrium (Tabibzadeh, 1991; Hunt et al., 1992; Tabibzadeh et al., 1994, 1995a, Tabibzadeh et al., b). The known biological activities and menstrual cycle-dependent expression of the TNF- in human endometrium suggests that it may be implicated in menstrual bleeding and tissue shedding (Tabibzadeh et al., 1994, 1995a,Tabibzadeh et al., b,c). By using in-situ hybridization and immunohistochemcial staining, we showed that the TNF- mRNA and protein are expressed, to a large extent, in the endometrial epithelial cells and, to a lesser extent, in the stromal and lymphoid cells, (Tabibzadeh, 1991; Hunt et al., 1992). TNF- induces haemorrhage in some tumours. However, the effect of this cytokine on the vasculature does not seem confined to tumours. In one study, ultrastructural examination showed that TNF- impaired the blood–retina barrier and induced retinal haemorrhage (Claudio et al., 1994). Administration of TNF- to mice induced vascular damage as well as haemorrhage in endometrium that was indistinguishable from bleeding which occurs during human menstruation (Shalaby et al., 1989). Furthermore, TNF- induces apoptosis in endothelial cells (Robaye et al., 1991). Therefore, we speculated that the disintegration of endometrial vasculature during menstruation may be attributable to the progressive rise in the endometrial tissue level of TNF- during the secretory/menstrual phase. In addition, the role of oestrogen in the regulation of the TNF- mRNA is not well understood. Therefore, this study was carried out to investigate the conditions that regulate the expression of TNF- mRNA in human endometrial epithelial cells.
Materials and methods
Processing of endometria
Endometrial tissues were obtained by biopsy or curettage from hysterectomy specimens of normal fertile women, aged 25–45 years, who underwent this procedure for non-endometrial abnormalities such as ovarian or cervical lesions. Women were healthy and did not have a prior history of disease. Hysterectomy specimens or each endometrial biopsy sample was rapidly processed. The date of the endometrium was determined based on the morphological evaluation of endometrial sections using established criteria (Noyes and Hertig, 1950). Each endometrial sample was divided into aliquots and maintained frozen at –80°C. Escherichia coli recombinant TNF- (specific activity 4.03x107 IU/mg, endotoxin level <0.06 EU/mg by the Limulus amoebocyte lysate assay) was obtained from Genentech (San Francisco, CA, USA). A 1.3 kb cDNA fragment of TNF- and 1.2 kb EcoRI cDNA fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone HHCMC23) were obtained from ATCC (Manassas, VA, USA). A 1.9 kb cDNA of clone 1a was obtained from Dr Richard Lyttle (Wyeth Ayerst Women's Health Research Institute, Philadelphia, PA, USA; Hsu et al., 1988).
Cell cultures
ECC1 is a clonal cell line established from a well-differentiated human endometrial carcinoma transplanted into nude mice (Satyaswaroop et al., 1983, 1987). The cells were grown as previously described (Tabibzadeh et al., 1990). Briefly, the cells were grown in Ham's F-12 medium containing 10% heat-inactivated, mycoplasma- and virus-free fetal bovine serum, antibiotic–anti-mycotic mixture, human insulin (200 IU/l), human transferrin (25 g/l) and glucose (0.4 g/l) in a humidified 5% CO2:95% air atmosphere at 37°C. Cells were incubated with TNF- at the doses and for the durations indicated in the text. Cells were removed from the plate by trypsin–EDTA mixture. For preparation of primary endometrial epithelial cell cultures, the endometrium was first minced under sterile conditions and endometrial glands and stroma were prepared according to a previously described procedure (Satyaswaroop et al., 1979). Endometrial tissue was digested at 37°C in a shaking water bath for 1 h in Dulbecco's modified Eagle's medium (DMEM)/F-12Ham's medium without Phenol Red (Sigma Chemical Corpoation, St Louis, MO, USA) containing 0.2% Zienam&Aelig; (Imipenem und Cilastatin), 0.4% Nystatin&Aelig; (200.000 I.E./l), 10% fetal calf serum (Gibco, Eggenstein, Germany) and 0.153% type I A collagenase (400 IU/mg solid). Separation of stromal and epithelial cells was performed by filtration through a 180 µm nylon membrane (Millipore, Eschborn, Germany) and then through a 40 µm nylon sieve (Becton Dickinson, New Jersey, USA). Endometrial glands were trapped on the second sieve, backwashed and pelleted by centrifugation. Cells were resuspended in medium and were seeded at a density of ~6000 glands/cm2 on 12 mm diameter filters (pore size 0.4 µm) in Millicell CM (Millipore, Eschborn, Germany) inserts, previously coated with 0.1 ml of 1:4 diluted Matrigel without Phenol Red (Becton Dickinson). Inserts were then placed in 24-well tissue culture plates (Falcon, Oxnard, USA), in a dual-chambered system in which medium had access to both sides of the cell layer. Incubation was performed at 37° C in an incubator in the presence of 5% CO2. The medium was changed every 24 h. For stimulation experiments cells were cultured for 4–5 days until 50–90% confluent. Cells were then stimulated with 17-ß-oestradiol or progesterone alone or in combination for 24 h. After 24 h, supernatants were collected and frozen for further evaluation by enzyme-linked immunosorbent assay (ELISA).
Regulation of TNF- in EnCa-101 tumours grown in nude mice
Athymic Balb/C, nu/nu mice (Harlan Sprague Dawley Inc, Indianapolis, IN, USA) were castrated 1 week before tumour transplantation, and maintained in separate barrier facilities used exclusively for these animals. EnCa-101, a histologically well differentiated and oestrogen receptor-positive human endometrial carcinoma grown s.c. in nude mice was used in these studies. About 20 mg of tumour tissue was transplanted s.c. at each of the four sites (infrascapular and lumbar regions of each flank) in castrated nude mice. 17ß-oestradiol pellets (Innovative Research of America, Sarasota, FL, USA) were implanted s.c. in the animals. These pellets are designed to maintain the serum concentration of oestradiol at 200 pg/ml for 60 days (Satyaswaroop et al., 1983, 1987). Pellets were replaced at 55–60 day intervals. When the geometric mean diameter of tumours reached 10 mm, the pellet was removed from some animals and was left in the control group. Tumours were removed at intervals as indicated in the text. Tumours were frozen, pulverized and stored individually in liquid nitrogen for Northern blot analysis of TNF- mRNA.
Isolation of RNA and Northern blotting
The RNA was extracted using acid guanidinium thiocyanate–phenol–choloroform extraction method as described (Chomczynski and Sacchi, 1987). The amount of RNA was assessed spectrophotometrically and the quality of RNA was assessed by evaluating the integrity of ribosomal RNA by electrophoresis of 20 µg of total RNA in 1% formaldehyde–agarose gels in the presence of ethidium bromide. Poly-A RNA was purified by affinity chromatography using oligo (dT) cellulose. Northern blotting was done as described previously (Sambrook et al., 1989). Briefly, 20 µg of total RNA was electrophoresed in a 1.4 % agarose MOPS-formaldehyde gel (Sambrook et al., 1989), transferred to nylon membrane (Hybond N; Amersham, Arlington Heights, IL, USA) by standard capillary transfer in 10x sodium chloride/sodium citrate (SSC). The RNA was fixed to the membrane by UV cross-linking in UV StratalinkerTM (Stratagene, La Jolla, CA, USA). The TNF-, GAPDH and clone 1a cDNA fragments were [32P]-labelled to a high specific activity by random primer labelling and purified by gel filtration. Prehybridization, hybridization and post-hybridization washes were done as described (Church and Gilbert, 1984). The membranes were subjected to autoradiography at –70°C using intensifying screens. For reprobing the membranes for GAPDH or clone 1a, blots were stripped by incubation of the membranes at 50°C in 25 ml of 75% formamide containing 0.1x SSC and 0.2% sodium dodecyl sulphate (SDS). The relative abundance of TNF- mRNA was determined by normalizing the optical densities of TNF- mRNA band as follows. The autoradiograms were scanned by a laser scanning densitometer (Envisions, DynamicPro 30, Burlingame, CA, USA). Then, the relative optical densities of the bands were quantified using SigmaGel (Jandel Scientific, San Rafael, CA, USA). Three independent measurements were made from each band and the mean values of these measurements were calculated. The mean relative optical densities of the TNF- mRNA were normalized by dividing the mean relative optical density values of the TNF- mRNA bands by the mean relative optical density values of the housekeeping genes, GAPDH (Tso et al., 1985, Zentella et al., 1991) or clone 1a (Hsu et al., 1988) from the same sample. The values obtained from controls were regarded as 100% and all other values were calculated as percentages of these values.
Ribonuclease protection assay (RPA)
RPA was carried out according to the instruction of the manufacturer (Pharmingen, San Diego, CA, USA). Briefly, the probe was synthesized and labelled with [-32]-UTP and was separated from unincorporated UTP. The size of the probes were 315 nucleotides for TNF-, 141 nucleotides for L32 and 125 nucleotides for the GAPDH. The size of the protected products were 286 bp for TNF-, 112 bp for L32 and 97 bp for GAPDH. RNAse free total RNAs were hybridized in solution with the radioactively-labelled probe followed by RNAse treatment for removing the unprotected RNAs. After proteinase K treatment, the protected products were separated in a sequencing gel. The separated products were transferred to a nylon membrane and the blot was subjected to autoradiography. In each run, positive and negative controls were also included.
Enzyme linked immunosorbent assay (ELISA)
TNF- was analysed in the cell culture supernatants using a commercially available ELISA kit (Laboserv Diagnostica, Giessen, Germany). TNF- concentrations were measured according to the manufacturer's instruction. The kit detects TNF- in the pg range and did not show cross-reactivity against other cytokines.
Results
We previously showed that the amount of TNF- protein is progressively increased during the secretory phase (Tabibzadeh 1991; Hunt, 1992; Tabibzadeh et al., 1994, 1995a). Here, we examined the TNF- mRNA expression in endometria throughout the menstrual cycle. Total RNA from proliferative and secretory endometria were subjected to Northern blot analysis (Figure 1). Consistent with our previous findings, the TNF- mRNA was not detectable in the proliferative phase and became detectable in the late secretory phase (Figure 1). TNF- was also detected in one case of endometrial bleeding (Figure 1, lane 10). We then reconfirmed these findings by ribonuclease protection assay (Figure 2). From eight proliferative endometria, the TNF- mRNA was detected in two cases and the other cases showed a low level of TNF- mRNA expression (Figure 2). None of the seven early secretory endometria or three mid-secretory endometria exhibited a detectable amount of TNF- mRNA expression (Figure 2). On the other hand, the expression of TNF- mRNA was detected in eight out of eight cases of late secretory endometria (Figure 2). The distinct menstrual cycle-dependent expression of TNF- suggests that specific signals tightly regulate the expression of this gene in human endometrium.
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We further tested the amount of the TNF-
which is produced by the endometrial epithelial cells in vitro (Figure 3). We previously demonstrated that TNF- mRNA and protein, to a large extent, are present in the endometrial epithelial cells (Tabibzadeh et al., 1991; Hunt et al., 1992). We separated endometrial epithelial cells from stroma and examined the amount of TNF- released by these cells (Figure 3). The endometrial epithelial cells were obtained from both proliferative and secretory phase endometria (n = 9). After establishment of the primary cultures, the media were removed and used for the assessment of TNF- concentrations by ELISA. Under our culture conditions, and in the absence of steroid hormones, the endometrial epithelial cells produced a detectable amount of TNF- which was released into the culture medium (Figure 3). This amount was significantly higher in the endometrial epithelial cultures derived from late secretory endometria, when compared with those derived from earlier phases of the menstrual cycle (Figure 3), although a small rise was noted around the time of ovulation.
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; oestradiol 10–9 mol/l 
; progesterone 10–7 mol/l 
). The amount of TNF-We next tested the signals that potentially regulate TNF-
mRNA expression in endometrial cells. The menstrual cycle-dependent expression of TNF- mRNA suggests that this expression may be under the regulation of oestrogen. Therefore, we examined the effect of oestrogen on the TNF- protein which is released from human endometrial epithelial cells in vitro. Glands and single epithelial cells were treated without and with various concentrations of 17-ß oestradiol and progesterone and then the conditioned media were collected after 24 h. Neither oestradiol (10–9 mol/l), nor progesterone (10–7 mol/l) seemed to have any detectable effect on the amount of the TNF- released by the epithelial cells (Figure 3
). Oestradiol at a concentration of 10–8 mol/l and progesterone at a concentration of 10–6 mol/l or a combination of oestradiol (10–9 mol/l) + progesterone (10–7 mol/l) and oestradiol (10–8 mol/l) + progesterone (10–7 mol/l) also did not have any effect on the amount of TNF released into the culture medium (data not shown).
We examined the expression of TNF- in ECC1 cells (an epithelial cell line of endometrial origin) (Satyaswaroop et al., 1983; Clarke et al., 1987). Under the culture conditions, we were unable to show any TNF- mRNA in these cells using Northern blotting analysis (Figures 4 and 5). It was previously reported that TNF- induces TNF- mRNA expression in the breast carcinoma cell line, MCF-7 (Sgagias et al., 1991), and in normal and malignant ovarian cell lines (Wu et al., 1993). Therefore, we examined the effect of exogenous TNF- on the expression of the TNF- mRNA in these cells. TNF- induced TNF- mRNA expression in a time- and dose-dependent manner in these epithelial cells (Figures 4–5). The effect of TNF- on TNF--mediated mRNA expression was not detectable 5 min after the addition of the TNF- (Figure 4). This effect became apparent within 30 min. The TNF mRNA expression showed a progressive increase from 30–360 min (Figure 4). Incubation of cells with TNF for >6 h did not seem to affect the amount of TNF mRNA expression (Figure 5). This effect was still detectable after 72 h of incubation with TNF (Figure 5). The effect of TNF- on TNF--mediated mRNA expression was dose-dependent. The effect of TNF on TNF mRNA expression was not detectable at 1 ng/ml. This effect became apparent, however, with doses of 10 and 100 ng/ml of TNF- (Figure 5).
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In the human endometrium, the expression of TNF-
mRNA becomes apparent at the end of the menstrual cycle when the serum concentration of oestradiol is falling. Therefore, we postulated that TNF- mRNA expression may be positively regulated by oestrogen withdrawal, rather than directly by oestradiol. To test whether oestradiol withdrawal regulates expression of TNF- in vivo, we used a human endometrial carcinoma transplanted into nude mice (Satyaswaroop et al., 1983, 1987; Zaino et al., 1984). Epithelial cells of EnCa-101 tumours exhibit characteristics similar to the normal endometrial epithelium with regard to their responses to steroids, 17-ß-oestradiol and progesterone. Oestradiol enhances growth and progesterone receptor concentrations and progestin inhibits growth in the epithelial tumour cells (Satyaswaroop et al., 1983; Zaino et al., 1985; Clarke et al., 1987). These tumours grow s.c. in these mice only in the presence of oestrogen (Satyaswaroop et al., 1983, 1987; Zaino et al., 1984). Upon the withdrawal of the s.c. oestradiol pellet, the serum concentration of the oestradiol quickly dropped within hours and reached a basal level within 4 h (Clarke et al., 1987). When the oestrogen pellet was removed, the growth of the tumour ceases (Clarke et al., 1987). These findings clearly show that epithelial cells of EnCA-101 tumours are responsive to steroid hormones in vivo. The tumour cells exhibited a low level TNF- mRNA expression when grown in the presence of oestradiol (Figure 6). Upon withdrawal of the oestradiol pellet, a sharp increase in the TNF- mRNA expression was observed in the tumour cells (Figure 6). This expression reached a maximum within 24 h after removal of the oestrogen pellet and then sharply decreased (Figure 6). The amount of TNF mRNA was significantly lower 3 days after oestrogen withdrawal compared with that observed prior to, and after, the first and second days of withdrawal (Figure 6).
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Discussion
In the present report, we showed that the amount of TNF- mRNA in human endometrium varies throughout the menstrual cycle. Northern blot analysis and ribonuclease protection assays both showed that the amount of TNF- mRNA peaks during the late secretory phase and when endometrial bleeding occurs. These findings are consistent with an earlier report (Phillipaeaux and Piguet, 1993). We previously showed that the amount of TNF- protein that is released by endometrium is also low during the proliferative phase. During the secretory phase, the amount of TNF- protein which is released by endometrium progressively rises and peaks in the menstrual phase (Tabibzadeh et al., 1994, 1995a, Tabibzadeh et al., b; Tabibzadeh, 1996). Consistent with these findings, epithelial cells obtained from a late secretory endometrium released more TNF- into the culture medium compared with epithelial cells from other phases of the menstrual cycle. We previously showed, by using in-situ hybridization and immunohistochemcial staining, that the endometrial epithelial cells are the major source of the TNF- in human endometrium (Tabibzadeh, 1991; Hunt et al., 1992). Here, we showed that endometrial epithelial cells when separated from the stroma still maintained in vitro the ability to release TNF- into the culture medium. We identified two separate mechanisms which regulate TNF- mRNA expression in endometrial epithelial cells. We found that TNF- induces the expression of TNF- mRNA in a dose- and time-dependent manner in endometrial epithelial cells. These findings are consistent with previous results on the up-regulation of TNF- mRNA by TNF- in breast (Sgagias et al., 1991) and ovarian (Wu et al., 1993) epithelial cell lines. The effect of TNF on its own mRNA expression is initiated within minutes after exposure to TNF and lasts for several days in the continuous presence of TNF. Menstrual cycle-dependent TNF mRNA expression and release of TNF- by endometrium suggested that the expression of this cytokine is subject to regulation by steroid hormones. Consistent with this hypothesis, we found three half palindromic oestrogen response elements at positions (–658) to (–654), (–971) to (–967) and (–993) to (–989), in the promoter region of the TNF- gene. This regulation, however, is complex and may consist of a negative regulatory effect by oestrogen as well as a positive regulation by its withdrawal and may be cell-lineage specific. For example, oestrogen down-regulated expression of TNF- in human peripheral blood mononuclear cells (Loy et al., 1992; Shanker et al., 1994) and inhibited its release from these cells (Ralston et al., 1990). The amount of TNF- released by human endometrium peaks at a time during the menstrual cycle when the amount of serum oestrogen has fallen. We could not find evidence for the effect of oestradiol without progesterone on the amount of TNF- which is released by endometrial epithelial cells when these cells were cultured under conditions that allow maintenance of the steroid hormone receptor (Classen-Linke et al., 1997). The experiments in the nude mouse, however, are consistent with a role for oestrogen in the regulation of TNF- mRNA. In this model, oestrogen withdrawal led to up-regulation of TNF- mRNA expression. Therefore, the up-regulated expression of TNF- mRNA in human endometrium at the end of the menstrual cycle may be related to two simultaneous events. The drop in the serum concentration of oestrogen may lead to up-regulation of TNF- mRNA. The increased amount of TNF- released by the epithelial cells, in turn, may lead to a further increase in TNF- mRNA expression. TNF may not be the only local factor implicated in endometrial bleeding. Other factors seem to be involved in this process. For example, progesterone withdrawal reduces tissue factor (TF) and type-1 plasminogen activator inhibitor (PAI-1) expression and increases urokinase-type plasminogen activators (tPA and uPA respectively), MMPs, and endothelin-1 (ET-1) expression (Lockwood and Schatz, 1996). Therefore, it is thought that such changes may account for the haemorrhage, enhanced fibrinolysis, ECM degradation, and ischaemic spiral arterial vascular injury characterizing menstruation (Lockwood and Schatz, 1996).
TNF- may have several target cells in the human endometrium. The endometrial epithelium exhibits immunoreactivity for both receptors of TNF- (TNF-RI, TN-RII) suggesting that it is able to respond to TNF- (Tabibzadeh et al., 1995a). Therefore, TNF- may exert its effect in an autocrine fashion on the epithelial cells or may act in a paracrine fashion on the endometrial endothelial cells. We have shown here that TNF- can up-regulate TNF- mRNA expression in endometrial epithelial cells, confirming the autocrine role of TNF- in endometrial cells. The increased amount of TNF- may be sufficient to lead or contribute to the inhibition of proliferation and apoptosis in endometrial epithelial and endothelial cells. TNF- exerts an anti-proliferative effect on the epithelial cells (Pusztai et al., 1993). In endometrial epithelial cells, this cytokine induced features which are characteristic of menstrual glands during menstruation. This included induction of apoptosis (Tabibzadeh et al., 1994), cell–cell dissociation (Tabibzadeh et al., 1995c), aberrant expression of adhesion molecules (Tabibzadeh et al., 1995c) and conversion of filamentous (F)-actin to globular (G)-actin (Tabibzadeh et al., 1995c). TNF- induces apoptosis in endometrial epithelial cells (Tabibzadeh et al., 1994). On the other hand, the development of stromal oedema and haemorrhage in the human endometrium, may be attributable to the effect of TNF- on the endometrial vessels. TNF- may be contributory to the oedema that is seen during the proliferative phase in endometrium. Consistent with this hypothesis, two of the eight proliferative cases exhibited TNF- mRNA expression detectable by the ribonuclease protection assay. TNF- impairs endothelial cell–cell binding and results in increased permeability of endothelial linings to the macromolecules and lower molecular weight solutes (Brett et al., 1989; Robaye et al., 1991; Partridge et al., 1993) and thus induces tissue oedema. These effects are temporally associated with changes in the cytoskeleton and cell shape, development of intercellular gaps (Brett et al., 1989; Goldblum et al., 1993), and conversion of F-actin to G-actin (Goldblum et al., 1993). The diverse effects of TNF- on epithelial and endothelial cells are reminiscent of the changes that occur during the menstrual phase such as loss of expression of adhesion molecules at the cell–cell borders of epithelial cells (Tabibzadeh et al., 1995b), dissociation of epithelial cells leading to glandular fragmentation (Tabibzadeh et al., 1995b), loss of filamentous actin (Tabibzadeh et al., 1995b), and development of a significant number of apoptotic cells in the epithelium (Tabibzadeh et al., 1994). In addition, menstruation is associated with disintegration and disruption of vasculature and bleeding (Ferenczy, 1979). Taken together, the available data suggest that TNF- may be contributing to diverse reactions in human endometrium such as the oedema, bleeding and glandular fragmentation seen during the menstrual process (Tabibzadeh, 1996). Even though these arguments support a role for TNF- in menstrual bleeding, certain questions remain unanswered. For example, it is not clear why continuous release of TNF- during the secretory phase is not associated with tissue dissolution and endometrial bleeding. One possibility is that the effect of TNF- on the endometrium may be dose-dependent. For example, TNF- seems to have dual effects on the fate of endothelial cells. In low amounts, TNF- supports growth of endothelial cells; in larger amounts, however, it inhibits proliferation of these cells (Frater-Schroder et al., 1987; Saegusa et al., 1990; Fajardo et al., 1992). Given these findings, it is possible that the diverse effects of TNF- are exerted in the endometrium in a cell- and dose-dependent manner. If up-regulation of TNF is indeed responsible for uterine bleeding, then it is possible to regulate endometrial bleeding by factors that inhibit its expression.
Acknowledgments
This work was supported by grants CA46866 to ST and CA62211 to PGS.
Notes
4 To whom correspondence should be addressed 
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Submitted on March 16, 1999; accepted on September 10, 1999.
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