Mol. Hum. Reprod. Advance Access originally published online on August 6, 2004
Molecular Human Reproduction 2004 10(10):719-728; doi:10.1093/molehr/gah097
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DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection
1Department of Gynecology, Polyclinique de l'Hôtel-Dieu, CHU, Clermont-Ferrand, France, 2Department of Obstetrics & Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan, 3INSERM UMR 384, Faculté de Médecine, Clermont-Ferrand, 4Department of Pathology, Centre Jean Perrin, Clermont-Ferrand and 5Department of Pathology, Hôtel-Dieu, CHU, Clermont-Ferrand, France
6 To whom correspondence should be addressed at: Department of Gynecology, Polyclinique de l'Hotel-Dieu, CHU clermont-Ferrand, Bd. Léon Malfreyt 63058, Clermont-Fearrnd, Cedex 1, France. Email: sachikoma{at}aol.com
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Abstract |
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Endometriosis, a common gynecological disorder that causes infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. However, despite extensive studies its etiology and pathogenesis are not completely understood. Differentially expressed genes were investigated in epithelial and stromal cells from deep endometriosis and matched eutopic endometrium using cDNA microarrays and laser capture microdissection. Validation of results of several up- and down-regulated genes was performed by quantitative real-time RT–PCR. Our data showed that platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta1 (PKC beta1) and janus kinase 1 (JAK1) were upregulated, and Sprouty2 and mitogen-activated protein kinase kinase 7 (MKK7) were downregulated in endometriosis stromal cells, suggesting the involvement of the RAS/RAF/MAPK signaling pathway through PDGFRA in endometriosis pathophysiology. In addition, two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were downregulated in endometriosis epithelial cells, which might result in increased local production of estrogen in endometriosis epithelial cells. Furthermore, three potential candidate genes that might be involved in endometriosis related pain were identified: tyrosine kinase receptor B (TRkB) in endometriosis epithelial cells, and serotonin transporter (5HTT) and mu opioid receptor (MOR) in endometriosis stromal cells were all upregulated. One of the candidate genes, MOR, may be involved in a defective immune system in endometriosis. This study has provided new insights into endometriosis pathophysiology.
Key words: cDNA microarray/endometriosis/endometrium/laser capture microdissection
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Endometriosis, a common gynecological disorder responsible for infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites (Clemeny, 1994
). It affects 
10% of women of reproductive age (Eskenazi and Warner, 1997). In addition, there is increasing evidence that endometriosis is a polygenically inherited disease of complex multi-factorial etiology (Olive and Schwartz, 1993). To date, a number of candidate genes have been studied (Zondervan et al., 2001). However, despite extensive studies its etiology and pathogenesis are not fully understood. A complete cure for patients with endometriosis awaits new targets and strategies. The long-term goal of our project is to identify endometriosis gene expression markers that can be used for noninvasive diagnosis and for development of strategies for prevention and more effective targeted therapies, and to gain a better understanding of the pathophysiology and disease etiology.
Identifying these genetic markers by conventional methods such as Northern blots, differential display, and serial analysis of gene expression has been labor intensive or nonsystematic. A more promising approach for analyzing multiple genes simultaneously is the hybridization of entire cDNA populations to nucleic acid arrays. This technology has a wide range of applications, including investigating normal biological and disease processes, profiling differential gene expression, and discovering potential therapeutic and diagnostic drug targets (Schena et al., 1995; Brown and Botstein, 1999). Gene expression studies performed on endometriotic tissue homogenates have yielded results reflecting mRNA abundance in a mixture of cell types (including epithelial cells, stromal cells, fibrotic tissue and muscle tissue). Therefore, a method for quantifying gene expression separately in individual cell populations is essential for identifying genetic markers. A relatively new technique for obtaining pure populations of cells from heterogeneous tissues is laser capture microdissection (LCM) (Emmert-Buck et al., 1996; Bonner et al., 1997).
Endometriosis is a heterogeneous disease based on location and clinical outcome. The different theories of histogenesis proposed for the different forms of endometriosis are based on location (Nisolle and Donnez, 1997). Correlation of basic science with a well-defined clinical population is the key to successful translational research that will lead to the development of new diagnostic and targeted therapeutic approaches (Murphy, 2002). Thus, in this study, we focused on patients with deep endometriosis, which is one of the severe forms of endometriosis. Deep endometriosis is defined as endometriosis deeper than 5 mm under the peritoneum peritoneal surface.
For the first step of our project, we investigated genes that were differentially expressed between deep endometriosis, and matched eutopic endometrium within the same patients using cDNA microarrays and laser capture microdissection. Validation of expression results was performed for a subset of up- and down-regulated genes by real-time PCR.
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Materials and methods |
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Patients
Patients undergoing laparoscopy and/or laparotomy for deep endometriosis (rectovaginal nodules) were recruited for this study from May 2001 in the polyclinique de l'Hotel Dieu, CHU Clermont-Ferrand, Clermont-Ferrand, France. Six patients (three patients during the proliferative phase and three during the secretory phase) were recruited for further microarray analysis. Furthermore, to independently validate our microarray results, an additional six patients (three patients during the proliferative phase and three during the secretory phase) were recruited for further real-time RT–PCR analysis. Clinical characteristics of the 12 patients are shown in Table I. No patients received hormonal treatments, such as gonadotropin-releasing hormone agonist (GnRHa) or sex steroids, nor did they use intrauterine contraception for at least 6 months prior to surgery. Recruited patients had regular menstrual cycles (between 26 and 32 days), had their menstrual history confirmed, and serum 17ß estradiol and progesterone levels measured. The endometrial dating criteria as described by Noyes et al. (1950)
and the menstrual history were utilized to assess the menstrual cycle phase. Endometrial tissue biopsies were performed just before operation, using an endometrial suction catheter (Pipelle, Laboratoire CCD, Paris, France). The depth of deep endometriosis was measured using a scale just after collection. Samples of deep endometriosis and the matched eutopic endometrium were divided into two portions. The first tissue portion was fixed in 20% formalin-acetic acid and embedded in paraffin for histopathological examination. The second portion was immediately collected in RNA later (Ambion, Cambridgeshire, UK) and stored at –20°C until further analysis was performed. All tissue samples were obtained with full and informed patient consent. The research protocol was approved by the Consultative Committee for Protection of Persons in Biomedical Research (CCPPRB) of the Auvergne region.
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Laser capture microdissection (LCM) and RNA extraction
From each fresh frozen tissue, 8-µm thick frozen sections were prepared from endometriotic tissues and 10-µm thick frozen sections from the matched eutopic endometrium. The sections were mounted on positively charged slides (Super flost Plus, Menzel GmbH, Braunschweig, Germany). H&E staining on frozen sections was performed using the NCI protocol (www.cgap-mf.nih.gov/Protocols/index.html) with some minor modifications.
Briefly, slides were fixed in 70% ethanol for 15s, stained with H&E, followed by dehydration in two 15s washes in 95% ethanol, two 60s washes in 100% ethanol, and a final wash in xylene for 3 min twice. Slides were air-dried for 5 min and stored in a dessicator for no more than 1 h. Hematoxylin and eosin were used at 10% of their standard concentrations. Since the slides were microdissected without a coverslip, the tissue is not index-matched and substantial light scattering occurs, typically producing ‘dark’ images. Thus, both image quality and molecular recovery can be improved by decreasing stain concentrations. Glandular epithelial cells and stromal cells from endometrial or endometriotic tissues were isolated from the slides using the PixCell II LCM System (Arcturus, Plaisir, France) according to the manufacturer's instructions. A 7.5 µm and 15 µm beam diameter was utilized for epithelial cells and for stromal cells, respectively. Microdissected tissues were collected on optically transparent LCM Macro caps for endometrium and LCM HS caps for endometriosis (Arcturus).
RNA extraction and quantification
After LCM, RNA extraction was performed using the Picopure RNA extraction kit (Arcuturus). The caps were placed in microcentrifuge tubes (Eppendorff, le Pecq, France) containing lysis buffer and incubated at 42°C for 30 min. After centrifugation, the caps were removed and RNA was isolated using the Picopure RNA extraction protocol. To eliminate potential genomic DNA contamination, RNA samples were treated with DNaseI (15 U; DNaseI, Courtaboef, Qiagen, France) at RT for 15 min. Finally, total RNA was resuspended in 11 µl of RNase-free water and kept at –80°C until needed. RNA quantities were measured with the Ribo-Green RNA Quantitation Kit (Molecular Probes Europe BV, Leiden, The Netherlands). All procedures were performed according to the manufacturer's instructions.
T7 based linear amplification
Because of the insufficient quantity of total RNA from microdissected tissues for microarray analysis, T7 based linear amplification starting from 50 ng of total LCM-extracted RNA was performed in one round, using the protocol by Baugh et al. (2001) with some minor modifications. Briefly, total LCM-extracted RNA was mixed with 100 ng of T7T24 primer (MWG Biotech, Courtaboeuf, France) in RNase-free water to a total volume of 7.8 µl. The RNA/primer mixture was denatured at 70°C for 4 min, then chilled on ice. Next, RT was performed in a 12 µl reaction, containing 1 µl of 10x RT buffer, 1.2 µl of 25 mM MgCl2, 1 µl of 0.1 M DTT, 0.5 µl of 10 mM dNTPs and 0.5 µl of Superscript II (Invitrogen, Cergy Pontoise, France). After 1 h incubation at 42°C, the reaction was inactivated by a 65°C incubation for 15 min. Then, second-strand cDNA synthesis was performed by adding 45.5 µl of RNase-free water, 15 µl of 5x second-strand reaction buffer (Invitrogen), 1 µl of 10 mM dNTPs, 5 U of DNA ligase (Invitrogen), 20 U of DNA polymerase (Invitrogen) and 1 U of RNase H (Invitrogen). After incubating the second-strand cDNA reaction for 2 h at 16°C, 10 U of T4 DNA polymerase (Invitrogen) was added, followed by incubation at 16°C for 15 min. The reaction was inactivated by a 70°C incubation for 10 min. Double-stranded cDNA was purified by phenol:chloroform:isoamyl alcohol extraction using a phase-lock-gel (Eppendorf) and precipitation with 2.8 µl of 5 M NaCl, 20 µg of glycogen (Ambion) and 2.5 volumes of 100% ethanol at –20°C overnight. After precipitation, the cDNA was resuspended in 8 µl of RNase-free water and used as a template to transcribe unlabeled antisense RNA (aRNA) using T7 RNA polymerase and the Megascript kit (Ambion). The reaction was incubated for 5 h at 37°C and the resulting aRNA was purified using RNeasy spin columns (Qiagen). Eluted aRNA was precipitated by adding 0.1 volume of 7.5 M ammonium acetate, 20 µg of glycogen and 2.5 volumes of 100% ethanol. The resulting aRNA was resuspended in 9 µl of RNase-free water.
Probe labeling and hybridization
aRNA was primed with 1 µg of random hexamers (Invitrogen) and labeled in a reverse transcription reaction with [
-32P]dCTP (Perkin Nen Life Science, Paris, France). Labelled cDNA was separated from unincorporated nucleotides using a QUIAquick Extraction kit (Quiagen). The Clontech AtlasTM human 1.2 cDNA expression array (Clontech, Palo Alto, CA) is a positively charged nylon membrane (8 x 12 cm) that is spotted with 200–600 bp cDNA fragments representing 1176 genes and nine housekeeping genes. The 1176 genes were arrayed in six quadrants with genes of like function grouped together geographically. Membranes were prehybridized at 68°C for 30 min in ExpressHyb solution (Clonetech) to which 0.1 mg/ml of salmon sperm DNA (Invitrogen) had been added. cDNA probes were hybridized to the arrays at 68°C overnight. Membranes were washed four times with solution 1 (2xSSC and 1% SDS) and once with solution 2 (0.1x SSC and 0.5% SDS) for 30 min at 68°C in all cases and exposed to X-ray film at –80°C for 3 days. Exposed X-ray film was scanned with Adobe Photoshop software for further image analysis.
Gene array analysis
Quantification was performed with NIH image analysis 1.51 using the default external background. Data from each different array experiment were normalized by the median value to eliminate variability due to sample labeling or to exposure duration. The validity of this normalization step was examined by showing that gene expression quantification gave identical values when a blot was exposed for various times. However, housekeeping genes such as GAPDH or ß-actin produced very strong signals that saturated rapidly (<12 h) in NIH image analysis, leading to artifactual values for these genes (data not shown). Thus, housekeeping genes were excluded from the analysis. The adjusted signal intensity (signal intensity minus background) for a gene is considered to be positive only if it is over twice the background. A 2-fold difference was applied to select up-regulated and down-regulated genes (Claverie, 1999). Finally, genes that were regulated with a fold change >2 in at least two out of three samples during the same phase of the menstrual cycle were selected as differentially expressed genes.
Validation of gene expression data by quantitative real-time RT–PCR
Quantitative real-time RT–PCR with a Light cycler was performed on non-amplified total RNA from microdissected tissues. Total RNA (10 ng) was subjected to an RT reaction using Superscript II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed in a Light Cycler System using the Fast Start DNA master SYBR green I kit as recommended by the manufacturer (Roche, Mannheim, Germany). In a total volume of 20 µl, each reaction contained 2 µl SYBR green I reaction mix (consisting of Taq DNA-polymerase reaction buffer, dNTP mix, SYBR green I, MgCl2 and Taq DNA polymerase), 0.3–0.5 µM of each primer, 3–4 mM MgCl2 and 2 µl cDNA, standard or nuclease-free water as a negative control. Primer sets are shown in Table I and IITable I and II. Quantification of the targets in the unknown samples was performed using a relative quantification method with external standards. The target concentration is expressed relative to the concentration of a reference housekeeping gene, GAPDH. After each run, melting curve analysis was performed to verify the specificity of the PCR reaction.
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Statistical analysis
The Statview 4.5 program (Abacus concepts Inc., Berkeley, CA) was used for statistical analysis. The Wilcoxon sign rank test was performed to compare differences in gene expression levels in paired eutopic and ectopic endometrial samples. Statistical significance was defined as a P-value of <0.05.
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Results |
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Microdissection: Figure 1
Our preliminary experiments showed reproducible results among three cDNA microarray experiments, using T7 based linear amplified aRNA synthesized from 50 ng of microdissected total RNA (R2=0.9). Thus, after LCM and RNA extraction, deep endometriosis samples were selected, which contained >7.2 ng/µl of total microdissected RNA.
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Microarrays
There were no genes that were up-regulated or down-regulated in both epithelial cells and stromal cells from the samples obtained from all six patients.
Epithelial cells
Up-regulated and down-regulated genes in endometriosis compared to the matched eutopic endometrium are shown in Tables III and IV, respectively. Eight and seventeen genes were identified as upregulated and downregulated, respectively. Out of the 17 downregulated genes, PKC alpha, PKC zeta, MTTF1 and Inhibin beta A were upregulated in samples S1, S2, P2 and P3, respectively.
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Stromal cells
Upregulated and downregulated genes in endometriosis compared to the matched eutopic endometrium are shown in Tables V and VI, respectively. Eleven genes were identified as up-regulated and down-regulated in each sample type. Out of the 11 upregulated genes, Granzyme A was downregulated in sample P3.
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Quantitative real-time RT–PCR
The expression of a subset of upregulated genes, which included HSP90A, TRkB, PDGFRA, JAK1 and MOR, 5HTT, PKC beta 1 and a subset of downregulated genes, which included PGE2 EP3, COUP-TF2, MKK7 and Sprouty2, was validated by quantitative real-time RT–PCR. The results are shown in Table VII. Expression levels of TRkB, JAK1, MOR, 5HTT, PKC beta 1, PGE2 EP3 and COUP-TF 2 were significantly different between endometriosis and matched eutopic endometrium during the proliferative (P<0.03, P<0.03, P<0.03, P<0.03, P<0.03, P<0.03, P<0.03, respectively) and secretory phases (P<0.03, P<0.03, P<0.03, P<0.03, P<0.03, P<0.05, P<0.03, respectively) (Table VII). We detected significant differences in expression levels of HSP90A, PDGFRA, MKK7 and Sprouty2 during the secretory phase (P<0.03, P<0.03, P<0.03, P<0.03, respectively) (Table VII).
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Discussion |
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In this study, we investigated differentially expressed genes both in microdissected epithelial and stromal cells between endometriosis and matched eutopic endometrium. Although a pure population of epithelial cells can be obtained by LCM, a variety of cell types populate the microdissected stromal compartment, including immune cells, inflammatory cells and vascular cells. Furthermore, the proportions of these different cell types may be different between endometriosis and eutopic endometrium (Jones et al., 1998
). However, direct interactions of epithelial and stromal cells and their microenviroments are necessary both for normal and pathological tissue development and growth (Aboseif et al., 1999; Liotta and Kohn, 2001). There is growing evidence that the tumor microenvironment is indispensable in the neoplastic process (Liotta and Kohn, 2001). Therefore, investigation of gene expression profiling both in epithelial and stromal cells, using cDNA microarrays and LCM techniques, could provide more important information for endometriosis pathophysiology and targeting novel therapies. Although further studies are necessary to focus on each distinct cell type in stroma, we decided to investigate differentially expressed genes in stromal cells as well as in epithelial cells as the first step of this project. In this study, we could not evaluate RNA quality extracted from LCM tissues, because of insufficient quantity. However, a study demonstrated that RNA from LCM tissues was of high quality, using the same protocol for LCM as the present (Luzzi et al., 2001).
Because of sequence-dependent hybridization characteristics and variations inherent in any hybridization reaction, any microarray data should be considered as semiquantitative. In addition, in this study we used a T7 based RNA amplification method to generate probes for microarray analysis. Thus, it is necessary to validate all of the microarray results with quantitative RT–PCR (QRT–PCR) on non-amplified microdissected samples. Although only a subset of upregulated and downregulated genes was validated in this study, the results of the microarray analysis were comparable to those of QRT–PCR in accordance with previous studies that have confirmed the correlation between microarray and QRT–PCR data (Iyer et al., 1999; Luo et al., 1999). Other studies have also demonstrated the fidelity and reproducibility of array results utilizing amplified material (Wang et al., 2000; Luzzi et al., 2003). Furthermore, because of the better quality of the array data, routine mRNA amplification is recommended for cDNA microarray-based studies (Feldman et al., 2002).
This study provided novel findings of differentially expressed genes between endometriosis and matched eutopic endometrium. However, few genes were identified that have been previously reported in the literature. There are several possible explanations for these results. Primarily, this is the first study to investigate gene expression of epithelial and stromal cells in endometriosis, using the combined methods of cDNA microarrays and LCM. Although cell cultures provide pure populations of epithelial and stromal cells, the genetic information derived from cultured cells may not accurately reflect the molecular events taking place in the actual lesion. Molecular analysis of RNA from microdissected cells may provide more accurate information regarding in vivo molecular events. Until now, many data analysis techniques have been applied to DNA microarray data, but this field is still evolving. We might miss many ‘truly differentially expressed genes’ because of false-negative errors. In addition, the Atlas Human Array 1.2 contained relatively small numbers of genes on the cDNA microarray. Because there are so few studies focusing on deep endometriotic tissues, these findings might be specific to deep endometriosis. Since different theories of histogenesis are proposed for the different forms of endometriosis based on location (Nisolle and Donnez, 1997), it is necessary to investigate if potential candidate genes identified in this study are also differentially expressed in other endometriosis forms, such as ovarian endometriosis.
This study demonstrated that PDGFRA and members of the downstream RAS/RAF/MAPK signaling pathway were differentially expressed in stromal cells between endometriosis and matched eutopic endometrium. Our cDNA microarray analysis identified upregulation of the PDGFRA, PKC beta1 and JAK1, and downregulation of Sprouty2 and MKK7 in endometriosis stromal cells. However, QRT–PCR analysis detected no significant differences in expression levels of PDGFRA, Sprouty2 and MKK7 during the proliferative phase, probably due to individual differences. Further studies should be necessary to investigate expression of these genes in larger samples throughout the menstrual cycle.
PDGFRs are members of a large family of receptor tyrosine kinases (RTK) possessing intrinsic cytoplasmic enzymatic activity, which are essential components of signal transduction pathways that affect cell proliferation, differentiation and migration (Marshall, 1995). Uncontrolled activation of receptor tyrosine kinases (RTKs) is implicated in cancer cell proliferation (Porter and Vaillancourt, 1998). Therefore, tight regulation of RTK cascades is critical for eliciting an appropriate type and level of response to external stimuli. This study identified upregulation of PKC beta1, which contributes to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway, by recruiting the Grb-2/Sos complex to the plasma membrane (Kawakami et al., 2003). PKC beta1 also contributes to ERK2 activation (Cao et al., 2001). Furthermore, we identified downregulation of Sprouty2, which is now established as a receptor tyrosine kinase-induced modulator of the RAS/RAF/MAPK pathway (Wong et al., 2002; Yusoff et al., 2002). A negative regulator of cell proliferation, MKK7, was also downregulated. MKK7 is a MAPK kinase that specifically activates only the c-JUN N-terminal protein kinase (JNK) family of enzymes (Wolter et al., 2002). Several studies have demonstrated that PDGFRA activation transduces both positive and negative signaling for cell growth and that PDGFRA-activated JNK1 plays an antagonistic role for PDGF-induced transformation (Yu et al., 2003). Downregulation of MKK7 might enhance positive signaling through PDGFRA in endometriosis stromal cells. In addition, we identified up-regulation of JAK1 in endometriosis stroma cells, which is involved in the JAK–STATs signaling pathway. Although until now there has been no direct report that JAK1 expression is upregulated in endometriosis, there is much evidence that several cytokines and growth factors, such as IL-6, IFN-gamma and oncostatin M, which activate JAK1, are upregulated in endometriosis and may play important roles in endometriosis pathogenesis (Harada et al., 2001). Furthermore, there is a link between the JAKs and the Raf/MAPK signaling pathways since JAK1 is required for Raf-1 activation by both IFN-gamma and Oncostatin M (Stancato et al., 1997).
A recent microarray study clearly demonstrated that PDGFRA and the RAS/RAF/MAPK pathway are involved in the metastatic phenotype of meduloblastoma (MacDonald et al., 2001). Both PDGFRA and JAK 1 have been shown to support its growth in androgen-independent prostate cancer bone marrow metastases (Chott et al., 1999). Although endometriosis is a benign disease, studies suggested that the invasive phenotype in endometriosis shares aspects with tumour metastasis (Gaetje et al., 1995; Zeitvogel et al., 2001). Clinical observations and in vitro experiments imply that endometriotic cells are invasive and able to metastasize (Gaetje et al., 1995; Zeitvogel et al., 2001). The PDGFRA signalling pathway may be involved in the metastatic phenotype of endometriosis. Activation of PDGF receptor-transduced signaling pathways is also crucial for blood vessel stability in newly formed vessels (Heldin and Westermark, 1999; Cao et al., 2003). It is now believed that angiogenesis is essential in the development and growth of endometriosis (Healy et al., 1998). A kinase inhibitor incorporating selectivity for PDGFRs is shown to block further growth of end-stage tumors, eliciting detachment of pericytes and disruption of tumor vascularity (Bergers et al., 2003). Although further studies are necessary to clarify the functional roles and in which cell type PDGFRA is upregulated in endometriosis stromal cells, one of the potential novel strategies for more effective treatment may be to target the signal transduction of PDGFRA in endometriosis stromal cells.
The development and growth of endometriosis is estrogen-dependent. In human estrogen-dependent neoplasmas, such as breast, endometrioid endometrial and surface epithelial–stromal ovarian carcinomas, intratumoral aromatase is considered to play an important role in converting circulating androgens derived from adrenal cortex and/or ovary to estrogens (Sasano and Harada, 1998). Although endometriosis is not a neoplasma, it is now believed that in situ estrogen production via aromatase plays a critical role in its pathophysiology (Noble et al., 1996; Zeitoun et al., 1999). Our study identified downregulation of two potential negative regulators of aromatase expression in endometriosis epithelial cells, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 (PGE2) receptor EP3 subtype (PGE2EP3). Zeitoun et al. (1999) demonstrated that aromatase expression in endometriosis stromal cells was regulated by two transcription factors, steroidgenic factor-1 (SF-1), a stimulatory transcription factor, and COUP-TF, an inhibitory factor, which competes for the same binding site in the aromatase promoter II. Using whole tissues of endometrium and endometriosis, they demonstrated that COUP-TF was expressed ubiquitously in both eutopic endometrium and endometriosis. However, our study identified the downregulation of COUP-TF in epithelial cells of endometriosis compared to those of matched eutopic endometrium. Furthermore, we identified another potential negative regulator of aromatase expression in endometriosis epithelial cells, PGE2EP3. PGE 2 was found to be the most potent known inducer of aromatase activity in endometriosis (Noble et al., 1997). This PGE2 effect was mediated via the cAMP-inducing PGE2EP2 receptor subtype (Noble et al., 1997). There are four main prostanoid receptors to which PGE2 binds: EP1, EP2, EP3 and EP4 (Breyer et al., 2001) and EP3 is the only inhibitory receptor identified to date (Breyer et al., 2001). A recent study demonstrated that aromatase expression may be regulated through two distinct PGE2 receptor subtypes, EP2 and EP3 (Richards and Brueggemeier, 2003). Our findings suggested that downregulation of these two genes might result in increased aromatase expression in endometriosis epithelial cells.
In addition, we identified upregulation of Hsp90A, which actively participates in steroid-induced signal transduction (Segnitz and Gehring, 1997), in endometriosis epithelial cells. Hsp90 occurs in two isoforms (alpha and beta) and Hsp90 alpha expression is primarily inducible in humans (Bohen and Yamamot, 1994). A recent study demonstrated that Hsp90-specific inhibitors, gelandamycin and radicicol, destabilize estrogen and progesterone receptors in a ligand-independent manner in breast cancer both in vitro and in vivo (Bagatell et al., 2001). Because Hsp90 is also known to activate Raf-1 (Schulte et al., 1995), Hsp90 alpha may merit attention as a potential new target. Further investigation of the effects of Hsp90 inhibitors both in vivo and in vitro will clarify the biological significance of upregulation of Hsp90 alpha in endometriosis.
Pain symptoms are a major clinical problem for endometriosis patients. Endometriosis is diagnosed in 30–50% of women undergoing laparoscopy for pelvic pain (Eskenazi and Warner, 1997). Particularly, deep endometriosis is strongly associated with pelvic pain, severe dysmenorrhea and deep dyspareunia (Koninckx and Martin, 1994). However, despite the strong clinical correlation, the basic mechanism of endometriosis-related pain is unclear. This study identified three potential candidate genes involved in molecular mechanisms of endometriosis-related pain. Anaf et al. (2000) demonstrated that perineurial and intraneurial invasion by epithelial and stromal cells was associated with pain symptoms in deep endometriosis. Strong expression of nerve growth factor (NGF) has been demonstrated both in epithelial and stromal cells of deep endometriosism, and its specific receptor, TrkA, is localized in nerves within deep endometriosis (Anaf et al., 2002). Our study identified upregulation of TrkB, which is a specific receptor of brain-derived neurotrophic factor (BDNF) in endometriosis epithelial cells. Interestingly, a recent study suggested that upregulation of autocrine expression of NGF and BDNF in epithelial cells might be concomitant with transformation to a malignant phenotype capable of invasion along the perineural space in prostate cancer (Dalal and Djakiew, 1997). Upregulation of TrkB in epithelial cells may be involved in molecular mechanisms of perineurial and intraneurial invasion in deep endometriosis.
Another potential candidate gene is serotonin transporter (5HTT). 5HTT plays a central role in serotonin neurotransmission (Kress and Reeh, 1996). There is evidence linking such inflammatory mediators, like bradykinin, PGE2 or serotonin (5-HT), to the generation of pain and hyperalgesia (Kress and Reeh, 1996). In 5-HT (serotonin) transporter-deficient mice (5-HTT–/– mice), 5-HT levels in the injured peripheral nerves were reduced and correlated with diminished behavioral signs of thermal hyperalgesia, a pain-related symptom caused by peripheral sensitization (Vogel et al., 2003). Our study identified upregulation of 5-HTT in endometriosis stromal cells, suggesting that 5HT/5HTT may also be involved in the molecular mechanism of endometriosis-related pain. The upregulation of mu opioid receptor (MOR) in endometriosis stromal cells was found in our study. Opioid receptors (ORs) and their mRNA are present in the central and peripheral nervous systems and in different peripheral tissues (Satoh and Minami, 1995). The endogenous opioid system is activated during inflammation as a physiological feedback mechanism to attenuate inflammatory pain (Stein et al., 1993). Some studies have demonstrated that MOR mRNA in intestinal mucosa significantly increased during intestinal inflammation and MOR is induced by factor/s or mediators related to inflammation (Pol et al., 2001). Tumor necrosis factor (TNF), a proinflammatory cytokine, induces mu opioid receptor gene transcription both in neuronal and immune cells (Kraus et al., 2003). Although it is not clear if endometriosis-related pain is caused by inflammation, there is strong evidence that proinflammatory cytokines, including TNF, may be involved in endometriosis pathophysiology (Harada et al., 2001). MOR is also expressed on cells of the immune system. It has been demonstrated that mu-opioids possess broad immunomodulatory activity, including the inhibition of NK cell activity, macrophages and polynuclear cell function (Sibinga and Goldstein, 1998). There is numerous evidence that endometriosis is associated with a defective immune system (Lebovic et al., 2001). A defective immune system may support the attachment, persistence and progression of ectopic endometrial tissue in endometriosis patients (Lebovic et al., 2001). Apoptosis of T lymphocytes by the Fas/Fas-L system may produce a local immunotolerant environment for the development of ectopic implants (Selam et al., 2002). Interestingly, a recent study demonstrated that the mu opioid receptor is involved in stress-mediated immunosuppression by lymphocyte apoptosis (Wang et al., 2002). We speculate that upregulated MOR in endometriosis stromal cells may be involved in both endometriosis-related pain and a defective immune system. Further investigation should be performed to clarify in which cell type, nervous cells, immune cells, or both, MOR is upregulated in endometriosis stromal cells.
In conclusion, this study has provided new insights on endometriosis pathophysiology for endometriosis patients. Further studies are necessary to confirm protein expression levels of potential candidate genes.
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Acknowledgements |
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We are grateful to all the staff at the Polyclinique of l'Hotel Dieu, CHU Clermont-Ferrand; particularly to all the residents and staff in the operating room. Without their invaluable assistance, this study would never have been completed. Also, we are indebted to all the staff in the Department of Pathology, l'Hotel Dieu, CHU Clermont-Ferrand and in the INSERM, UMR 384, Faculté de Médecine. In addition, we thank Dr A.Tchirkov (CHU Clermont-Ferrand) for his technical assistance during the quantitative real-time RT–PCR analysis, Dr L.R.Baugh (Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA) for his helpful discussion and Dr T.Taylor (Arcturus, Mountain View, CA) for her generous gift of a test sample of a Picopure RNA extraction kit and for helpful discussions. This study was supported in part by grants PHRC 2002 and PHRC 2003 of CHU Clermont-Ferrand.
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References |
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Submitted on June 9, 2004; resubmitted on July 5, 2004; accepted on July 11, 2004.
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