Departments of 1 Obstetrics & Gynaecology and 2 Pathology, 3 Research Institute Growth and Development (GROW), Academisch ziekenhuis Maastricht and Maastricht University, Maastricht, The Netherlands
4 To whom correspondence should be addressed at: Department of Pathology, Academic Hospital Maastricht, Peter Debyelaan 25, Postbus 5800, 6202 AZ, Maastricht, The Netherlands. e-mail: Patrick.Groothuis{at}path.unimaas.nl
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Abstract |
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Key words: E-cadherin/endometriosis/mesothelial cells/Snail/vimentin
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Introduction |
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The peritoneal lining consists of mesothelial cells which form a monolayer of epithelium-like cells covering the surface of the abdominal cavity. These cells are strongly connected by well developed cellcell junctional complexes, including tight junctions, adherens junctions, gap junctions and desmosomes (Mutsaers, 2002). An undamaged mesothelial architecture provides an important protective barrier against invading microorganisms and dissemination of ectopic cells (Mutsaers, 2002
).
A traumatized or injured peritoneal surface has been shown to result in enhanced peritoneal tumour dissemination and tumour growth (Bouvy et al., 1996; Mathew et al., 1997
; Reymond et al., 1998
; Gutt et al., 2001
). Such damage may be caused by tissue handling and drying out during laparotomy, increased intra-abdominal pressure during CO2 laparoscopy, or port site entry of trochars (Bouvy et al., 1996
; Mathew et al., 1997
; Reymond et al., 1998
; Gutt et al., 2001
). It has also been found that continuous ambulatory peritoneal dialysis is associated with recurrent episodes of peritonitis, which results in mesothelial detachment and in persistent peritoneal denudation (Andreoli et al., 1994
; Yanez-Mo et al., 2003
). This leads to increased intra-abdominal dissemination of cancer cells in these patients compared with non-dialysis patients (Bargman, 2000
).
The early pathogenesis of endometriosis remains to be elucidated with regard to the initial interactions between menstrual endometrium and mesothelial cells. In our previous studies (Demir Weusten et al., 2000; Koks et al., 2000
), shed menstrual effluent has been shown to induce morphological changes in mesothelial cells, which include disruption of intercellular junctions, retraction and exfoliation, and subsequent exposure of underlying extracellular matrix. These morphological changes appeared not to be due to cell death and were likely the result of cellular remodelling (Demir Weusten et al., 2000
). We hypothesize that retrogradely shed menstrual effluent interrupts the mesothelial lining by inducing cellular remodelling. This leads to the exposure of submesothelial extracellular matrix and may facilitate the adherence of endometrium fragments on peritoneum.
Similar phenotypical changes have been described by other investigators and were referred to as epithelialmesenchymal transitions (EMT) (Boyer et al., 2000). This cellular process is a manifestation of epithelial plasticity during embryo and organ morphogenesis, wound healing and tumour progression. During EMT, epithelial cells shift from an epithelial to a mesenchymal phenotype by reorganizing their cytoskeleton. The cellular features of epithelia prior to transformation are loss of polygonal morphology, adhesive cell contacts and cell polarity, development of a fibroblast-like shape with basal cytoplasmic projections, and increased cell motility (Hay, 1995
). At the molecular level the zinc finger transcription factor, Snail, has been recently implicated in the switching mechanism for EMT (Nieto, 2002
). Snail binds to E-boxes in the E-cadherin promoter and directly represses E-cadherin expression (Cano et al., 2000
), resulting in the dissociation of intercellular junctions and an increase in the pool of cytoplasmic
-catenin, a key component of the Wnt signalling pathway. Many transforming epithelia also change their intermediate filaments from cytokeratin to vimentin, a cytoskeletal shift that seems to be mandatory for the start of the transformation process (Perez-Pomares and Munoz-Chapuli, 2002
).
In several cell culture models, EMT are induced by a number of tyrosine kinase receptor binding growth factors, such as epithelial growth factor (EGF), insulin-like growth factors (IGF), fibroblast growth factor (FGF), hepatocyte growth factor/scatter factor (HGF/SF) or transforming growth factor- (TGF-
) (Gavrilovic et al., 1990
; Valles et al., 1990
; Piek et al., 1999
; Morali et al., 2001
; Strutz et al., 2002
). In these cases, several kinase signalling pathways have been implicated, which involve oncogenic Src, Ras, Raf, phosphatidylinositol-3 kinase (PI3K), Akt kinase, extracellular response kinase (ERK) and mitogen-activated protein (MAP) kinase as well as small G proteins (Rho and Rac) (Chan et al., 2002
).
To date, the mechanisms which underlie the effects of menstrual effluent on mesothelial cells are not known. Therefore, we investigated whether the morphological changes in mesothelial cells induced by shed menstrual effluent can be characterized as EMT. To this end, it was evaluated whether the morphological changes are reversible, energy dependent, result from kinase-dependent remodelling of the cytoskeleton (Kellie et al., 1991; Boyer et al., 2000
; Timpson et al., 2001
; Frame et al., 2002
), and involve changes in the expression of Snail, E-cadherin, vimentin and cytokeratin.
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Materials and methods |
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Human omentum (n = 8) was obtained from female patients undergoing abdominal surgery for benign indications. Anterogradely shed menstrual effluent (n = 60) was collected by healthy volunteers (n = 11) who had no history of endometriosis and had a regular ovulatory cycle. The donors collected menstrual effluent in a menstrual cup (Keeper, The Hague, The Netherlands) for 3 h (Koks et al., 1997). Peripheral blood sera were collected from the same individuals.
Mesothelial cell isolation and culture
Isolation of human omental mesothelial cells (HOMEC) and preparation of conditioned media were performed as described in a previous report (Demir Weusten et al., 2000). Briefly, the omental tissue was minced and incubated with collagenase (2 mg/ml, ICN Biochemicals B.V., The Netherlands) in routine medium [Dulbeccos modified Eagles medium (DMEM)/Hams F-12 supplemented with 10% fetal calf serum (FCS), penicillin 100 IU/ml, streptomycin 100 µg/ml and L-glutamine 2 mmol/l, amphotericin 0.25 µg/ml, all from Gibco Life Technologies, The Netherlands] for 20 min at 37°C. The non-digested tissue was removed by a 400 µm stainless sieve (SigmaAldrich Chemie B.V., The Netherlands). Subsequently, the cell suspension was sieved through a 100 µm nylon mesh filter (Micronic, The Netherlands) and a 10 µm polyamide filter (Stokvis & Smits, The Netherlands). The cells retained on both of these filters were resuspended in culture medium [minimum essential medium (MEM)/D-valine supplemented with 10% FCS, 1% ITS (insulin, transferrin and selenium), non-essential amino acids, L-glutamine 2 mmol/l, penicillin 100 IU/ml, streptomycin 100 µg/ml and amphotericin 0.25 µg/ml]. Culture medium was from SigmaAldrich Chemie B.V. Remaining contaminating stromal cells were separated from mesothelial cells by differential plating. After 30 min of incubation at 37°C, the non-adhering cells were collected and placed in a new flask.
The mesothelial cells are cultured in MEM/D-valine medium, which suppresses the growth of fibroblasts. The cells in the cultures are all positive for both cytokeratin and vimentin and negative for the endothelial marker CD34, as was shown previously (Demir Weusten et al., 2000). Confluent HOMEC cultures from passage two were used in the experiments.
Preparation of menstrual serum, conditioned media
After collection, the menstrual effluent was centrifuged at 1200 g for 10 min. The serum was stored and referred to as menstrual serum. The remaining tissue was immediately resuspended in routine medium (1:7 v/v), layered on a Ficoll-Paque gradient (SigmaAldrich Chemie B.V.) and centrifuged at 1200 g for 30 min. Endometrial and inflammatory cells were collected from the interphase, washed and cultured in routine medium for 24 h at 37°C and 5% CO2. After culture, the medium was removed and centrifuged at 1500 g for 10 min. This supernatant was referred to as conditioned medium. Sera and conditioned media prepared from different individuals were pooled (n = 10 and n = 12 respectively), filter-sterilized and stored at 80°C until use. Unless it is indicated, pooled sera or pooled conditioned media were used in all experiments.
Induction of morphological transitions in HOMEC
Mesothelial cells were grown in 24-well plates until confluence and subsequently cultured overnight in one of the above-mentioned sera and media preparations. Mesothelial cells cultured in peripheral blood sera and routine medium served as controls for the changes in cell morphology. Prior to use in the experiments, sera were diluted with routine medium (1:1 v/v). To test whether the morphological changes were reversible, sera and media were replaced with fresh routine medium and the cultures were continued for 4 days. These experiments were repeated 12 times.
Visualization of morphological transition with time-lapse video imaging
Confluent HOMEC monolayers were prepared in 6-well plates which were placed between temperature control plates on a translucent thermo-stage and viewed with a Leica MZFLIII stereomicroscope, equipped with a Donpisha 3-CCD camera. The dish was obliquely transilluminated. Cells were cultured in routine medium or conditioned media. HEPES (45 mmol/l, Gibco Life Technologies) was added to the cultures to compensate for any pH changes in the media. Digital images were prepared every 2 min for a period of 20 h and simulated into a video film using Fast Movie Processor 1.44 software.
MTT assay
Effects of conditioned media on the viability of mesothelial cells were determined using the MTT assay. Viable cells convert the soluble substrate MTT to an insoluble, coloured formazan salt. After culturing confluent HOMEC monolayers overnight with either routine medium or conditioned media, cells were rinsed once with routine medium. Then 400 µl routine medium and 60 µl of MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma-Aldrich Chemie B.V.] were added to each well of a 24-well plate. After 4 h of incubation at 37°C, the supernatant was removed and the formazan salt was solubilized in 200 µl DMSO (Merck, The Netherlands), which resulted in a purple-coloured solution. The amount of formazan formed was quantified by measuring the optical density at 550 nm.
Immunohistochemistry
Mesothelial cell monolayers were incubated overnight in routine medium or conditioned media. The morphological changes in cells were characterized by conventional light and immunofluorescence microscopy. A fluorescein isothiocyanate-conjugated secondary antibody (goat anti-mouse IgG, diluted 1:20; Dako, A/S, Denmark) was used to visualise the primary antibodies, which were directed to cytoskeletal proteins to detect the changes in cell architecture. These antibodies were a pan-cytokeratin antibody against cytokeratins 5, 6, 8, 17, 19 (diluted 1:100, MNF116; Dako A/S), an -tubulin antibody (1:400; SigmaAldrich Chemie B.V.). A direct rhodamine-conjugated phalloidin method was used for fibrillar actin (Friedman et al., 1984
). In the negative controls the primary antibodies were omitted from the incubation solution.
Blocking of energy supply and signalling pathways
To test whether the energy supply of mesothelial cells is related to the morphological changes, sodium azide was added to the routine and conditioned media. Sodium azide inhibits haem-containing proteins, including the cytochromes in mitochondria which are responsible for ATP production, thus blocking the cellular energy supply (Bershadsky and Gelfand, 1981). During the overnight incubation, the routine and conditioned media were supplemented with 0.1% sodium azide.
To evaluate the involvement of kinases in the morphological remodelling of mesothelial cells, HOMEC were incubated overnight in routine or conditioned media in 24-well plates, in the presence or absence of kinase inhibitors. The inhibitors used and their concentrations are listed in Table I. Prior to overnight incubation, HOMEC were preincubated for 30 min in routine medium with vehicle or the inhibitor of interest.
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Real-time RTPCR for Snail, E-cadherin and vimentin expression
Primers and probes for human Snail, E-cadherin, vimentin and cyclophilin A were purchased from the manufacturer as pre-developed assays (PerkinElmer Applied Biosystems, USA). Human cyclophilin A was selected as an endogenous RNA control to normalize for the differences in the amount of total RNA added to each reaction. Human endometrial tissue was used as a positive control in real-time RTPCR amplifications.
All RTPCR reactions were performed using an ABI Prism 7700 sequence detection system (PerkinElmer Applied Biosystems). The thermal cycling conditions comprised an initial decontamination step at 50°C for 2 min, a denaturation step at 95°C for 10 min and 40 cycles at 95°C for 15 s and 60°C for 1 min. Experiments were performed in duplicate for each sample. Quantitative values were obtained from the threshold cycle number (Ct) at which the increase in the signal associated with exponential growth of RTPCR products is first detected with the ABI Prism 7700 sequence detector software (Perkin-Elmer Applied Biosystems). The fold-change in expression was calculated using the Ct method with the cyclophilin A mRNA as an internal control (for details, see user bulletin #2 for the ABI PRISM 7700 Sequence Detection System, available at http://www.uk1. uni-freiburg.de/core-facility/tagman/user_bulletin_2.pdf).
Electrophoresis and Western blot analysis of E-cadherin, vimentin and cytokeratin
Confluent monolayers of HOMEC were cultured overnight, either in routine medium or in conditioned media. After rinsing cells several times with DMEM, cells were lysed in lysis buffer containing 20 mmol/l TrisHCl, pH 8, 137 mmol/l NaCl, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 10% glycerol, 1% Triton X-100, 1 mmol/l phenylmethylsulphonyl fluoride and protease inhibitor cocktail (Boehringer, The Netherlands). Unsolubilized cell rests were pelleted and the supernatants were stored at 80°C for later use. Protein concentrations were determined with the bicinchoninic acid assay (BCA assay; SigmaAldrich).
After the proteins (15 µg/lane) had been electrophoretically separated on a 10% sodium dodecyl sulphatepolyacrylamide gel, they were transferred to a nitrocellulose membrane (Schleicher and Schuell, The Netherlands). The membranes were stained with Ponceau S to evaluate whether the proteins were equally loaded. Non-specific binding sites were blocked with 5% non-fat dry milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST), overnight at 4°C. After washing three times for 5 min with PBST, the blot membrane was incubated with anti-E-cadherin (diluted 1:500; Santa Cruz Biotechnology, USA), anti-vimentin (diluted 1:2000; Cappel, Organon Teknika, USA), and anti-pan-cytokeratin, against cytokeratins 5, 6, 8, 17, 19 (diluted 1:2000, MNF116; Dako A/S), for 1 h at room temperature. At the end of this period the blot membrane was washed three times for 5 min in PBST and then incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG (diluted 1:1000, Dako A/S) for 1 h at room temperature. The antibodies were detected by enhanced chemiluminescene using SuperSignal West Pico Chemiluminescent Substrate (Pierce, USA). The bands were analysed using Kodak X-OMAT film.
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Results |
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Immunostainings with antibodies to pan-cytokeratin, fibrillar actin and -tubulin showed a change in the localization of these cytoskeletal proteins as an effect of conditioned media (Figure 3). In control cultures, cytokeratin filaments were found condensed around the nucleus of mesothelial cells, forming bird nests (Figure 3a), whereas after culture in conditioned media the cytokeratin filaments were concentrated in the spindles and extensions (Figure 3b). Fibrillar actin staining was concentrated at the periphery of the cells in control cultures (Figure 3c). After culturing the cells in conditioned media, fibrillar actin was distributed throughout the cytoplasm (Figure 3d).
-Tubulin staining highlighted the presence of a normal microtubule network that extended throughout the cytoplasm of mesothelial cells which were cultured in routine medium (Figure 3e). After incubation in conditioned media, the microtubule network depolymerized as shown by the homogeneous, non-fibrous nature of
-tubulin staining in morphologically changed cells (Figure 3f).
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Discussion |
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Similarly, reversible changes in cell morphology have been described as a process called cell scattering, associated with EMT (Boyer et al., 1989, 1996; Hay, 1995). The loss of cellcell cohesion, presumably due to damaged intercellular junctions and increased cell motility, strongly suggests concomitant changes in the cytoskeleton and this was indeed observed. The changes in immunofluorescence staining of cytokeratin, actin filaments and
-tubulin demonstrated marked cytoskeletal rearrangements, whereas the MTT assay showed no change in cell viability. Also, the process was found to be reversible. These observations are consistent with our earlier findings (Demir Weusten et al., 2000
), and provide evidence that the morphological alterations in mesothelial cells are not the result of an increase in the number of dying cells, but of cytoskeletal remodelling.
Sodium azide antagonized the EMT-inducing effects of conditioned medium. This compound inhibits haem-containing proteins, such as cytochromes in mitochondria which are responsible for ATP production, thus blocking the cellular energy supply (Vanzetti, 1966; Bershadsky and Gelfand, 1981
). In other studies, sodium azide has also been shown to prevent the induced disassembly of microfilaments and microtubules (Ma et al., 1995
). Apparently, depletion of the ATP pool interferes with ATP-dependent processes such as the phosphorylation of proteins (Wang, 1991
), which will prevent the cytoskeletal reorganization. The characteristics of this process, including the reversibility, are consistent with EMT (Boyer et al., 1989
; Mutsaers, 2002
).
The EMT are a consequence of the activation of signalling cascades through membrane-associated proteins (Boyer et al., 2000). Extracellular matrix components such as collagen, as well as soluble factors, including EGF, HGF/SF, members of the FGF and TGF-
families, have been shown to be directly involved in the induction of EMT (Savagner, 2001
). So far, few studies have been performed on EMT in mesothelial cells, and TGF-
(Yang et al., 2003
), tumour necrosis factor-
(TNF-
) (Zhu et al., 2002
) and HGF/SF (Rampino et al., 2001
) have been implicated in EMT. Yang et al. (2003
) reported similar cytoarchitectural changes of mesothelial cells as a result of continuous incubation for 7 days with TGF-
. The mesothelial cells maintained their fibroblastic phenotype despite the withdrawal of TGF-
. In the current study the changes were reversible and occurred within 18 h. It is therefore not likely that these changes are mediated by TGF-
. TNF-
and HGF/SF are likely candidates and their role in this process is currently under investigation.
The induction of EMT involves the Src tyrosine kinase and Ras signalling pathways. Ras functions by activating MAP kinase and PI3K signalling pathways (Boyer et al., 1997; Potempa and Ridley, 1998
), whereas Src tyrosine kinases phosphorylate adhesion kinase in cellular focal adhesion points, which results in focal adhesion loss during transformation (Fincham and Frame, 1998
; Chaudhary et al., 2002
).
Genistein, a general protein tyrosine kinase inhibitor, fully antagonized the EMT-inducing effects of conditioned media. This indicates that tyrosine kinases are essential mediators in EMT induction. However, the Src family protein tyrosine kinase inhibitor, SU 6656, was not able to fully antagonize the EMT-inducing properties of conditioned medium, indicating that other mechanisms may be involved. This is supported by the fact that Wortmannin, a PI3K inhibitor, was able to partially prevent EMT. These findings indicate that Ras and Src tyrosine kinases signaling pathways are involved in EMT induction in mesothelial cells by menstrual effluent. This is consistent with studies on the characterization of EMT in other cell types (Kellie et al., 1991; Hay and Zuk, 1995
; Thomas et al., 1995
; Gelman et al., 1998
; Timpson et al., 2001
; Frame, 2002
; Frame et al., 2002
).
The transcription factor Snail plays a key role in triggering EMT in Drosophila and cultured epithelia (Alberga et al., 1991; Batlle et al., 2000
; Cano et al., 2000
). Parallel to these reports, the present study provides evidence that the up-regulation of Snail in mesothelial cells that are cultured in conditioned media is associated with the acquisition of a fibroblastoid phenotype. One of the direct targets of Snail is the promoter of E-cadherin. Snail directly represses E-cadherin promoter activity and E-cadherin expression (Batlle et al., 2000
; Cano et al., 2000
). When the mesothelial cells were cultured in conditioned media, E-cadherin mRNA expression was down-regulated and E-cadherin protein could no longer be detected. This will result in a disturbance of epithelial cell organization, probably due to a loss of cellcell contacts. In addition, during this process vimentin expression was strongly increased in the mesothelial cells. This appears to be mandatory to start EMT in mesothelial cells, since it is well known that many transforming epithelia change their intermediate filaments from cytokeratin to vimentin (Greenburg and Hay, 1988
). In the mesothelial cells, cytokeratin expression was also markedly altered.
Recently, transition of peritoneal mesothelial cells from an epithelial to mesenchymal phenotype was shown to be induced in vivo and ex vivo when these cells were subjected to continuous peritoneal dialysis (Yanez-Mo et al., 2003). The authors suggested that long-term exposure of the mesothelial cells to the irritating dialysis solutions may lead to complete transition of the mesothelial cells, which could be responsible for tissue fibrosis and failure in ultrafiltration. Patients undergoing continuous ambulatory peritoneal dialysis have an increased risk of the intra-abdominal spread of tumour cells compared with non-dialysis patients (Bargman, 2000
). This indicates that the mesothelium serves as an effective barrier against the adhesion of cells. Our earlier findings that endometrial fragments preferably adhere to damaged areas with exposed submesothelial structures (Groothuis et al., 1998
; Koks et al., 1999
) confirm this.
Based on the evidence provided in this study, we conclude that the morphological alterations induced by factors released from shed menstrual effluent induce EMT. The implication for the clinic is that larger amounts of retrogradely shed menstrual effluent, and a longer exposure to this effluent, will likely lead to a greater insult for the mesothelium and an increased adhesion of retrogradely shed menstrual endometrial tissue. This is supported by the increasing risk for endometriosis when (retrograde) blood flow is heavier and the menstrual periods are longer (Sanfilippo et al., 1986; Darrow et al., 1993
; Eskenazi and Warner, 1997
).
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Acknowledgements |
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Submitted on March 19, 2003; resubmitted on August 4, 2003; accepted on September 22, 2003.