Hum. Reprod. Advance Access originally published online on October 6, 2007
Human Reproduction 2007 22(12):3148-3158; doi:10.1093/humrep/dem310
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Estrogen metabolizing enzymes in endometrium and endometriosis
1 Research Institute GROW, University Hospital Maastricht/University Maastricht, Peter Debyelaan, The Netherlands 2 Department of Pathology, University Hospital Maastricht/University Maastricht, Peter Debyelaan, The Netherlands 3 Department of Obstetrics and Gynaecology, University Hospital Maastricht/University Maastricht, Peter Debyelaan 25 HX 6229, The Netherlands 4 Department of Gynaecology, Université de Catholique de Louvain, Brussels, Belgium 5 Solvay Pharmaceuticals Research Laboratories, Hannover, Germany
8 Correspondence address. E-mail g.dunselman{at}og.unimaas.nl
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Abstract |
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BACKGROUND: Estradiol (E2) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent.
METHODS: In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17
-hydroxysteroid dehydrogenases (17-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)] in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures.
RESULTS: Aromatase and 17-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17-HSD, EST and STS were readily detectable. Only 17-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies.
CONCLUSIONS: In endometriosis lesions, the balance is tilted in favor of enzymes producing E2. This is due to a suppression of types 2 and 4 17-HSD, and an increased expression of aromatase and type 1 17-HSD in ectopic endometrium.
Key words: endometriosis/steroidogenic enzymes/aromatase/estrogen metabolism/endometrium
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Introduction |
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An estimated six percent of women suffer from endometriosis and have complaints such as chronic pelvic pain and some are even infertile because of this disease (Olive and Schwartz, 1993
; Vessey et al., 1993; Kjerulff et al., 1996). Endometriosis is defined as the presence of endometrial glands and stroma in extra-uterine sites, mostly the pelvic peritoneum, ovaries and rectovaginal space (Olive and Schwartz, 1993; Zeitoun et al., 1999; Bulun et al., 2000, 2002a).
Endometriosis is an estrogen-dependent disease (Dizerega et al., 1980; Zeitoun et al., 1998). The primary source of estradiol (E2) is the ovary, and therapies aimed at suppressing ovarian function or antagonizing the actions of estrogens, have proven to be effective. It has long been known that the endometrium itself is a source of E2, estrone, estrone sulfate and estrone-3-sulfate (Tseng et al., 1982; Tseng, 1984b) and that elevated aromatase activity is associated with malignancies of the endometrium (Tseng et al., 1982; Yamaki et al., 1985). It took however, another 10 years for investigators to suspect that endometriotic lesions may be self-supporting as evidenced by aromatase overexpression (Yamamoto et al., 1993; Noble et al., 1996,1997). Aromatase overexpression has now indeed been confirmed in numerous reports at the transcript level (Noble et al., 1996; Bulun et al., 1997; Matsuzaki et al., 2006; Smuc et al., 2007), at the protein level (Kitawaki et al., 1997,1999; Murakami et al., 2006; Velasco et al., 2006), as well as at the activity level (Kitawaki et al., 1997; Noble et al., 1997; Murakami et al., 2006). The clinical relevance of these observations was illustrated by the fact that aromatase inhibitors were effective in the treatment of endometriosis, particularly in those cases where GnRH agonist treatment failed (Takayama et al., 1998; Ailawadi et al., 2004), supporting the notion that significant estrogen production continues at extraovarian sites, including adrenals, adipose tissue, skin and endometriotic lesions.
The net production of 17-oestradiol is the result of a delicate balance between the synthesis and the inactivation of E2. Next to aromatase, the production of E2 is also mediated through the 17-hydroxysteroid dehydrogenases (HSD) type 1, 3, 5, 7 and 12, as well as steroid sulfatase (STS), which converts the sulfated estrogens to biologically active estrogens (Matsuoka et al., 2002; Moeller and Adamski, 2006). Gene transcripts for types 1 and 7 17-HSD and estrogen sulfatase (Fusi et al., 2005; Smuc et al., 2007) were found to be overexpressed in endometriotic tissues when compared with normal endometrium. In the human, aromatase produces mainly estrone and not E2, which would support a key role for the type 1 17-HSD as well.
Another key protein, which is believed to be involved in a rate-limiting step in steroid biosynthesis, is the steroidogenic acute regulatory protein (StAR). Even though StAR is not an enzyme, it is responsible for the transport of the substrate for steroid synthesis, cholesterol, across the mitchondrial membrane. StAR is highly overexpressed in endometriotic lesions (Tsai et al., 2001).
The conversion of E2 into less active metabolites in endometrium tissue is believed to be mediated for a large part by 17-HSDs types 2, 4 and 8, which form by an oxidative reaction androstenedione and estrone (Miettinen et al., 1996; Husen et al., 2001; Luu-The, 2001; Mensah-Nyagan et al., 2001; Yang et al., 2001), and by the estrogen sulphotransferase (EST), which conjugates sulfate groups to the 3-hydroxyl position (Qian and Song, 1999).
The 17-HSDs types 4 and 8 are constitutively expressed in normal human endometrium (Husen et al., 2001; Punyadeera et al., 2003). No information is available with regard to the expression levels of these two genes in endometriosis. EST is also expressed in normal endometrium (Utsunomiya et al., 2004). It is expressed in the secretory phase only, suggesting that it is regulated by progestins. In the study of Smuc et al. (2007) no differences were observed in the expression of EST in normal endometrium and ovarian endometriosis.
The type 2 17-HSD is believed to be one of the most important E2-inactivating enzymes in the endometrium (Tseng and Gurpide, 1974,1975). There is clear evidence that transcription of the 17HSD2 gene in normal endometrium is regulated by progesterone (Tseng and Gurpide, 1975; Kauppila, 1984; Casey et al., 1994; Mustonen et al., 1998; Zhang et al., 2006), even though some inconsistent findings have been reported (Kitawaki et al., 2000; Matsuzaki et al., 2006).
Such contrasting reports were also published with regard to the expression of 17-HSD type 2 in eutopic and ectopic endometrium of endometriosis patients. The general consensus is that the expression of type 2 17-HSD is reduced or absent in eutopic and ectopic endometrium of endometriosis patients (Husen et al., 2001; Bulun et al., 2002a), and that 17-HSD type 2 gene expression is insensitive to progestins (Vierikko et al., 1985; Zeitoun et al., 1998), indicating that endometriotic tissue may have reduced capacity to inactivate E2. Kitawaki et al. (2000), however, described exactly the opposite: in endometrium of patients 17-HSD type 2 expression was increased in secretory endometrium of endometriosis patients. Also Smuc et al. (2007) did not observe differences in type 2 17-HSD expression levels between normal endometrium and endometriosis. Similar discrepancies were reported for aromatase. Kitawaki et al. (1997,1999) and Matsuzaki et al. (2006) found no aromatase expression in endometrium of disease-free women, whereas Tseng and coworkers (1982,1984b) clearly showed aromatase activity in normal cycling endometrium.
The inconsistent findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls and endometriosis patients and endometriotic lesions, can be attributed to a variety of factors including the use of different technologies, the quality of the samples (i.e. is the endometriotic tissue contaminated with normal tissue from the local environment) and the lesion types studied. More efforts are needed to address these issues.
Most studies have focussed on adenomyosis, leiomyomas and ovarian and peritoneal endometriosis, but little information is available about steroidogenic enzymes in the severe deep-infiltrative type of endometriosis. In the current study, we evaluated the expression of aromatase, STS, EST and the types 1, 2 and 4 17-HSD in eutopic endometrium and endometriotic tissues of patients with deep-infiltrative endometriosis. In addition, expression was assessed in endometria from disease-free women. Hormonal regulation of the expression of these enzymes was also investigated in explant cultures of normal human endometrium.
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Materials and Methods |
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Tissues
Endometrial tissue was collected from 48 women of 26–52 years of age with regular menstrual cycles, who underwent surgery for benign indications other than endometriosis. The tissue was collected from hysterectomy specimens or by pipelle biopsies during laparoscopy (Pipelle catheter, Unimar Inc., Prodimed, Neuilly-Enthelle, France). The women were documented not to be on any kind of steroid medication. All women signed an informed consent, as required by the protocol approved by the Medical Ethical Committee of the University Hospital Maastricht.
Forty-eight biopsies of normal human endometrium were collected. Twelve were collected in the menstrual phase (M phase), 18 were collected in the proliferative phase: early proliferative phase, n = 9; late proliferative phase (LP phase), n = 9. Another 18 were collected in the secretory phase: early secretory phase (ES phase), n = 8; mid-secretory phase (MS phase), n = 8; late secretory phase, n = 2. After macroscopic inspection of the uteri by a pathologist, endometrium tissue was collected. The endometrium was dated according to clinical information with respect to the start of the last menstrual period, which was reconfirmed by histological examination of the tissue (Noyes et al., 1975).
Endometrium and endometriotic lesions were collected in 14 women of 22–39 years of age who underwent laparoscopic surgery to remove rectovaginal endometriosis. The endometrium was collected by pipelle biopsies during the operation (Pipelle catheter, Unimar Inc.). The collected tissues were frozen in Tripure Isolation Reagent TM (Roche, Basel, Switzerland) at –80°C until RNA isolation.
From each biopsy, part of the tissue was fixed in 10% buffered formalin for histology and immunohistochemistry and another part of the tissue was snap frozen in lysis buffer (Tripure Isolation ReagentTM, Roche) and stored at –80°C for RNA isolation.
Explant culture
Endometrium tissues from M phase (n = 8) and LP phase (n = 8) biopsies were collected and transported to the laboratory in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium on ice. The tissue was minced into pieces of 2–3 mm3. Twenty-four explants were placed in Millicell-CM culture inserts (pore size of 0.4 µm, 30 mm diameter, Millipore, Billerica, MA, USA) which were placed in 6-well plates containing phenol red-free DMEM/Ham's F12 medium (1.5 ml) (Life Technologies, Grand Island, NY). The medium was supplemented with L-glutamine (1%), penicillin and streptomycin (1%). The tissue was cultured for 24 h. At the end of the experiment, part of the explant was collected in formalin and embedded in paraffin, the rest was collected in lysis buffer (SV Total RNA Isolation Kit, Promega, Madison, WI, USA) and stored at –80°C until RNA-isolation.
Previous experiments have shown that collagenase activity remains very low in proliferative endometria during the first 24 h of culture (Cornet et al., 2002), and that the tissue viability is not affected after 24 h of culture (Marbaix et al., 1992). Treatments included: control (0.1% ethanol); E2 (1 nM); progesterone (1 nM) and E2 and progesterone (1 nM each). The steroid hormones were gifts from Organon pharmaceuticals (Oss, The Netherlands).
RNA isolation from normal endometrium
Total cellular RNA from explants and uncultured endometrium was extracted using the SV total RNA isolation kit (Promega) according to the manufacturer's protocol, with slight modifications. The concentration of DNase-1 during DNase treatment of the RNA samples was doubled and the incubation time was extended by 15 min in order to completely remove genomic DNA. Total RNA was eluted from the column in 50 µl RNase-free water and stored at –80°C until further analysis. The quality of the RNA samples was determined with a spectrophotometer and agarose gel electrophoresis. All the samples analysed gave RNA to DNA ratios higher than 1.5. Although primer/probes were designed to overlap exon boundaries, a PCR for a housekeeping gene glyceraldehyde-3-phosfate dehydrogenase (GAPDH) was performed on the RNA samples to verify that the samples were free of genomic DNA.
RNA isolation from endometriotic tissue
Prior to RNA isolation, the tissue was thawed at 4°C, cut into small pieces and homogenized twice for 30 s with an Ultra-Turrax homogenizer (Rose Scientific, Edmonton, Alberta, Canada). The samples were centrifuged 10 min at 12 000 g. To the supernatant 200 µl chloroform was added. The samples were vortexed 15 s and incubated at room temperature for 10 min. The samples were centrifuged 15 min at 12 000 g. The aqueous upper phase was collected and 500 µl isopropanol was added to precipitate the RNA. The samples were vortexed 10 s and incubated 10 min at room temperature. The samples were centrifuged 10 min at 12 000 g. The pellets were washed with 1 ml 75% ethanol. After air-drying, the pellets were collected in RNase-free water and incubated for 10 min at 60°C.
cDNA synthesis
For complementary DNA (cDNA) synthesis, total RNA (1 µg) was incubated with random hexamers (1 µg/µl, Promega) at 70°C for 10 min. The samples were chilled on ice for 5 min. To this mixture, a reverse transcriptase (RT)-mix consisting of 5x RT-buffer (4 µl), 5 mM dNTP mix (2 µl) (Pharmacia, Uppsala, Sweden), 0.1 M dithiothreitol (2 µl) (Invitrogen, Breda, The Netherlands) and superscript II reverse transcriptase (200 U) (Invitrogen) was added and the samples were incubated at 42°C for 1.5 h, after which the reverse transcriptase was inactivated by heating the samples at 95°C for 5 min. The cDNA was stored at –20°C until further use. In each real-time PCR reaction 50 ng of cDNA template was used.
Real-time PCR
Primers and probes for 17-HSD-1 (Hs00166219-g1), 17-HSD-2 (Hs00157993-m1), 17-HSD-4 (Hs00264973-m1), STS (Hs00165853-m1), EST (Hs00193690-m1) and aromatase (Hs00240671-m1) were purchased from Perkin-Elmer Applied Biosystems as pre-developed assays. Human cyclophylin A (Hs99999904-m1) was selected as an endogenous RNA control in order to normalize for the differences in the amount of total RNA added to each reaction. A pool of total RNA obtained from uncultured human endometrium tissues was included as positive control. All PCR reactions were performed using an ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised an initial decontamination step at 50°C for 2 min, a denaturing step at 95°C for 10 min and 40 cycles of 15 s at 95°C followed by 1 min at 60°C. Experiments were performed for each sample in duplicate. Quantitative values were obtained from the threshold cycle number (Ct) at which the increase in signal associated with the exponential increase of the number of PCR products was first detected with the ABI Prism 7700 sequence detector software (Perkin-Elmer, Foster city, CA). The fold-change in expression was calculated using the 
Ct method, with cyclophylin A mRNA as an internal control (Livak and Schmittgen, 2001). For detailed description of the procedure please refer to the ABI user manual. (http://www.uk1.unifreiburg.de/core/facility/tagman/user_bulletin_2.pdf)
Immunohistochemistry and western blotting
Novel polyclonal antibodies (pAb) were generated against 17-HSD type 1, type 2 and STS (prepared at Pineda Antikörper-Service, Berlin, Germany, on behalf of Solvay Pharmaceuticals). The pAb against type 1 17-HSD (protein accession number NP_000404
[GenBank]
) was generated by injecting rabbits with a recombinant 17-HSD type 1 peptide (h17HSD) which lacked 42 amino acids at the C-terminus (generously provided by Christina Fischer, University of Frankfurt, Germany). The pAb against type 2 17-HSD (protein accession number NP_002144
[GenBank]
) was generated by injecting a cocktail of the three synthetic peptides, NENGPGAEELRRTC (peptide A), CNIAGTSDKWEKLEKD (peptide B) and CAKRHFGQDKPMPRALR (peptide C). The pAb against STS (protein accession number NP_000342
[GenBank]
) was generated by injecting a cocktail of the three synthetic peptides, CLWEAESHAASRPNII (peptide A), CAHVEEVSSKGEIHGGS (peptide B) and CSSKGEIHGGSNGIYK (peptide C). The peptides were conjugated to a protein carrier (KLH). Three rabbits were injected with the peptide-conjugate cocktails for each antigen, and from each rabbit preimmune serum was collected. The generated antibodies were affinity purified using the immunizing peptides.
Specificity of the pAbs was demonstrated by western blotting and immunohistochemistry.
To confirm specificity of the 17-HSD type 1 pAb, gels were loaded with 10 µl (1 µg/µl) HSD1-containing tumor cytosol (Fig. 1, lane 1), 20 µl homogenate (0.55 µg/µl) from a HSD1-positive-cell line (Fig. 1, lane 2) and 10 µl homogenate (1 µg/µl) from a HSD1-negative-cell line (Fig. 1, lane 3; for general characteristics of cells and tumors see Husen et al. 2006), and subsequently probed with the preimmune serum, the pAb or the pAb after pre-incubation with the immunizing peptides. Antibody binding was visualized with an alkaline phosfatase-conjugated anti-rabbit immunoglobulin G (1:10 000, Sigma-Aldrich, St. Louis, MO, USA) and incubation with NBT/BCIP-substrate solution (Sigma-Aldrich). Specific binding of the pAb against type 2 17-HSD was shown by loading the individual immunizing peptides, as well as lysates prepared from placenta. The pAb and preimmune serum were applied at a 1:5000 dilution. Antibody binding was visualized with anti-rabbit horse-radish peroxidase (1:10 000; Dako, Glostrup, Denmark) and incubation in a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, USA). Specific binding of the anti-STS pAb was demonstrated by loading lysates prepared from endometrium and placenta and purified arylsulfatase C (Sigma-Aldrich) on the gels. The affinity purified antibody was applied at a dilution of 1:1000. Antibody binding was visualized in the same way as for the type 2 17-HSD.
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For immunostaining, paraffin sections of 5 µm were cut from uncultured and cultured explants and from the deep-invasive endometriotic lesions and uterus from endometriosis patients. In addition, sections were prepared from placenta and breast cancer tissue to serve as positive tissue controls. The sections were deparaffinized 2 x 5 min in xylene and 2 x 5 min in 100% ethanol. The endogenous peroxidases were blocked by incubating 30 min in 0.3% H2O2 in methanol. For antigen retrieval, the sections were boiled in Tris–EDTA buffer (pH 9.0) in a microwave oven for 20 min. In the preliminary stainings, a 1:1000 dilution was used for the anti-17
-HSD type 1 pAb and preimmune serum. The Dako-Envision protocol (Dako Diagnostika, Hamburg, Germany) was used to visualize antibody binding. The preimmune serum and pAbs against 17-HSD type 2 and STS were diluted 1:2000 and 1:1000, respectively. Antibody binding was visualized with the ChemateTM Envision kit (Dako Diagnostika).
After assessing specificity, the immunohistochemistry procedure was further optimized. The conditions used to stain the clinical specimens were the following. The anti-17-HSD type 1 pAb and preimmune serum were used at 1:4000 and sections were incubated for 1 hr at room temperature. The anti-17-HSD type 2 pAb and preimmune serum were incubated for 2 h at room temperature at a dilution of 1:2000. The anti-STS polyclonal antibody and preimmune serum were diluted 1:1000 and sections were incubated overnight at 4°C. Antibody binding was visualized by incubating 30 min with ChemateTM Envision (Dako Dako Diagnostika) and staining with diaminobenzidine solution. The reaction was stopped in water. The sections were briefly counter-stained in haematoxylin, dehydrated and sealed in Entellan (Merck, Whitehouse Station, NJ, USA).
Staining intensitiy was scored in epithelial and stromal cells separately on a 5-point scale: negative (0); weak (1); weak-moderate (2); moderate (3); moderate-strong (4) and strong (5). In addition, the percentage of postively stained cells within each compartment was assessed.
Statistical tests
Statistical tests were carried out using the Statistical Package for the Social Sciences 11 (SPSS Inc., Chicago, IL) statistical analysis package. To evaluate whether expression levels varied significantly throughout the menstrual cycle, the non-parametric unpaired Mann–Whitney U-test was used to test for differences between these expression levels versus the expression level in the M phase. This was done for both the mRNA and protein data. This test was also used to test for differences in the expression of the estrogenic enzymes in normal endometrium versus eutopic and ectopic endometrium of endometriosis patients. The non-parametric Wilcoxon signed-rank test was used to test for differences between steroid-treated explants and controls at a confidence level of 95%.
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Results |
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Validation of the antibodies against 17
-HSD types 1 and 2 and STSWith immunoblotting, a specific band at the expected molecular weight of 37 kDa was visible in the cytosol of the tumor cell line and cells known to express 17
-HSD type 1, which was not visible with the preimmune serum, and which disappeared after pre-incubation with the 17-HSD type 1 fusion protein (Fig. 1A). Immunostaining in paraffin sections of breast cancer tissue also disappeared after pre-incubation of the pAb with excess h17HSD fusion protein (Fig. 1B) indicating the specificity of the generated pAb.
Only one of the three pAbs generated against type 2 17-HSD showed cross reactivity with one of the three peptides (NENGPGAEELRRTC, peptide A; Fig. 2A). A specific band was also present in the placenta lysate at the expected molecular weight of 43 kDa. The other pAbs only gave a non-specific band in placental lysates around 75 kDa, which was also seen with the preimmune serum (results not shown). These antibodies also showed strong staining in the syncytiotrophoblast, which could not be displaced by any of the three peptides (Fig. 2B, polyclonal 2). The candidate antibody, however, showed immunostaining mostly in the vessels, which was completely abolished by pre-incubation with the immunizing peptides (Fig. 2B, polyclonal 1).
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To check specificity of the STS pAb, western blot analysis was performed on purified microsomal steroid sulfatase (arylsulfatase C; Fig. 3A). Besides the expected bands at 63 kDa, other bands (
40 and 50 kDa) were visible as well. A similar pattern was observed when incubated with the preimmune serum (Fig. 3A) and lower dilutions of the pAb (results not shown), and these bands could not be blocked with the immunizing peptides. In lysates of endometrium and placenta tissues also the 63 kDa band is visible (Fig. 3B), however, the 40 and 50 kDa bands were more abundant. Based on the known size of the active enzyme and the post-translational modifications that could occur, it is not possible that the 40 and 50 kDa bands represent STS. In contrast, immunohistochemical staining was completely prevented after pre-incubation of the pAb with the immunizing peptide A, but not peptides B and C (Fig. 3C). This antibody appears therefore to be suitable for immunohistochemistry and not for western blotting.
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Expression of E2 synthesizing and metabolizing enzymes throughout the menstrual cycle
The expression of type 2 17
-HSD mRNA increased in normal endometrium during the secretory phase of the menstrual cycle compared with the proliferative phase of the cycle (Fig. 4). Transcript levels increased in the ES phase and remained high throughout the secretory phase. At the protein level, however, 17-HSD type 2 expression was observed mostly in the glandular epithelium (Fig. 5). Levels peaked during the MS phase, the implantation window (Fig. 6). Little staining was also observed in the stroma, but expression in the stroma did not change throughout the menstrual cycle.
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Aromatase gene transcript levels in normal endometrium were near the detection limit, and no cyclical variations were observed.
No expression of 17-HSD type 1 mRNA was observed (results not shown). Surprisingly, however, prominent expression was observed at the protein level (Figs 5 and 6). These levels did not vary between stages of the cycle (Figs 5 and 6).
Transcript levels of 17-HSD type 4 and STS were similar to that of 17-HSD type 2, but the levels did not increase in the secretory phase (results not shown). Expression of STS protein did also not change during the menstrual cycle accept for the LS phase were it was significantly downregulated compared to the menstrual phase (Figs 5 and 6). STS protein was expressed in both epithelial and stromal cells.
EST transcript levels were lower (P < 0.05) than that of 17-HSD types 2 and 4, but expression levels increased significantly in the secretory phase (Fig. 4). Despite these differences in expression levels, a highly significant correlation was found between the cyclic variations in mRNA expression of 17-HSD type 2 and EST (correlation 0.79, P < 0.0001).
Steroid regulation of E2 synthesizing and metabolizing enzymes
The production of aromatase, the 17-HSD types 1 and 4 and STS mRNA in cultured endometrium explants prepared from M-phase and LP-phase explants was not affected by E2 or progesterone (data not shown).
The levels of EST and 17-HSD type 2 gene transcripts were significantly induced by progesterone in both, M-phase and LP-phase explants versus control (Fig. 7). Treatment with E2 alone had no effect on the 17-HSD type 2 and EST mRNA expression in M-phase and LP-phase explants. Tissues collected from the LP phase were significantly (P < 0.05) more responsive then M-phase tissue to the action of progesterone for both EST and 17-HSD type 2.
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Steroid regulation at the protein level in these short-term cultures was not confirmed with the semi-quantitative immunohistochemistry.
E2 synthesizing and metabolizing enzymes in endometriotic tissues
Compared with normal endometrium, transcript levels of 17-HSD type 1 and aromatase were significantly higher in the endometrium (P < 0.01) and endometriotic lesions (P < 0.01) of endometriosis patients compared with normal endometrium (Fig. 8). Aromatase mRNA levels were found also to be significantly higher (P < 0.05) in the endometriotic lesions compared with the endometrium of endometriosis patients (Fig. 8). The 17-HSD type 1 protein is expressed in all cells, but staining intensity is significantly lower in epithelial (P < 0.01) and stromal cells (P < 0.01) of the eutopic and ectopic endometrium of the endometriosis patients compared with the normal endometrium (Figs 9a and 10).
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Transcript levels of 17
-HSD type 2 were significantly lower (P < 0.01) in the endometriotic lesions than in the endometrium of patients and controls. No significant differences were found between the normal endometrium and the eutopic endometrium of patients (Fig. 8). The 17-HSD type 2 protein is expressed mostly in epithelial cells. However, the number of positive stromal cells was slightly increased (P < 0.05) in the eutopic endometrium of patients, whereas staining intensity was significantly reduced (P < 0.05) in stromal cells of the lesions compared with the normal endometrium (Figs 9b and 10).
The mRNA levels of 17-HSD type 4 were significantly (P < 0.01) lower in the lesions and endometrium of endometriosis patients when compared with the normal endometrium (P < 0.01). No difference was found between the lesions and the endometrium of endometriosis patients (Fig. 8).
No differences were observed between the STS mRNA levels of endometrium and lesions of endometriosis patients and endometrium of patients without endometriosis (Fig. 8). STS protein was expressed both by stroma and epithelium. STS protein levels were significantly (P < 0.05) lower in epithelial cells of endometrium of endometriosis patients compared with those of normal endometrium (Fig. 9c and 10). No difference in STS protein levels was observed between epithelial cells of the lesions and the normal endometrium (Fig. 9C).
EST mRNA levels were significantly (P < 0.05) higher in endometriotic tissue when compared with normal endometrium (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryFundingReferences |
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In this study, we have investigated the expression of six enzymes involved in the synthesis and inactivation of E2 in eutopic and ectopic endometrium of patients with deep-infiltrative endometriosis. Three pAb were generated to study types 1 and 2 17
-HSD and STS protein expression in the tissues. We show that there are parallels between deep-infiltrative endometriosis and the reports on adenomyosis, ovarian and peritoneal endometriosis, with regard to the expression of steroidogenic enzymes, but we also report some novel findings.
With regard to the E2 synthesizing enzymes, we found very low levels of aromatase and 17-HSD type 1 mRNA in normal human endometrium, which is in line with earlier reports (Bulun et al., 1993; Matsuzaki et al., 2006). Aromatase and 17-HSD type 1 expression was not responsive to E2 and progesterone in the explant cultures. This is in contrast with finding of Tseng (1984a) who showed that aromatase activity in isolated endometrial stromal cells was induced up to 40-fold by progesterone, which was further enhanced 20–100-fold by E2. Whether this is due to the fact that in the tissue context, estrogen metabolism may be different than in isolated cell compartments, or that prolonged exposure is needed to induce aromatase expression remains to be elucidated.
In contrast to the low levels of 17-HSD type 1 mRNA in the normal human endometrium, we did however detect 17-HSD type 1 protein. The fact that we were unable to detect 17-HSD type 1 mRNA transcripts in the endometrium of disease-free women, suggests that these transcripts either degraded quickly or are synthesized in low quantities. Reports on the expression of 17-HSD type 1 in normal endometrium are conflicting. Miettinen et al. (1996) reported low expression of 17-HSD type 1 mRNA in endometrium, whereas Casey et al. (1994) found no expression of 17-HSD type 1 mRNA in normal human endometrium, and Utsunomiya et al. (2001) found no 17-HSD type 1 protein expression in normal endometrium. In this study, we used a new pAb and we clearly demonstrated expression of type 1 17-HSD in normal endometrium. Steroidal regulation, however, was not observed in the explant cultures. The presence of type 1 17-HSD protein agrees with the observed E2 conversion into estrone in normal proliferative endometrium (Delvoux et al., 2007).
Aromatase and type 1 17-HSD mRNA levels were higher in eutopic and ectopic endometrium of endometriosis patients than in the endometrium of controls, which is in agreement with the findings of others (Noble et al., 1996; Matsuzaki et al., 2006). Surprisingly, however, 17-HSD type 1 protein levels were lower in epithelial and stromal cells of endometrium and lesions of endometriosis patients compared with the normal endometrium. For 17-HSD type 1 mRNA, two isoforms have been described, 1.3 and 2.3 kb (Miettinen et al., 1996). Only the 1.3 kb mRNA is correlated with 17-HSD type 1 protein expression and activity (Miettinen et al., 1996). In normal human endometrium, the 1.3 kb 17-HSD type 1 mRNA expression is low in normal human endometrium, but the 2.3 kb mRNA is abundantly present (Miettinen et al., 1996). Expression of the type 1 17-HSD gene may therefore not necessarily reflect changes at the functional protein level. The type 1 17-HSD is still considered a rate-limiting enzyme in the production of E2, since the major product of the aromatase activity in humans is estrone and not E2 (Simpson et al., 1994), and is therefore still an interesting alternative target for therapy.
At the IXth World Congress on Endometriosis in Maastricht, The Netherlands (2005), Fusi et al. (2005) showed that both endometrium and endometriotic tissues have high STS activity, which suggests that these tissues can generate high levels of estrone from the serum pool of estrone-3-sulfate. This agrees with the report of Benedetto et al. (1990), who also showed STS activity in human endometrial cells, but is not in line with the finding of Utsunomiya et al. (2004), who showed immunohistochemically that STS protein is not present in normal human endometrium using another pAb directed against STS purified from placenta. In this study, however, we demonstrated STS gene expression in disease-free endometrium, but in contrast to the finding of Smuc et al. (2007) in ovarian endometriosis, we did not observe higher STS mRNA levels in endometriotic lesions. Using our own pAb, we clearly demonstrated immunostaining in the endometrium. In contrast to Benedetto et al. (1990) who showed that STS activity increased in the presence of E2 and medroxyprogesterone acetate, we did not observe steroid responsiveness in our short-term explant cultures. This may be due to the fact that prolonged incubation (8–15 days) was required to evoke this response, which would not be visible in the 24 h-explant cultures.
The differences we observed in the mRNA expression levels of aromatase, type 1 17-HSD and STS between eutopic and ectopic endometrium from endometriosis patients and normal endometrium are significant, but small. In deep-infiltrative endometriosis, it is possible that differences in gene expression may be masked due to the presence of normal tissue from the local environment, which does not express steroidogenic enzymes (Noble et al., 1996). This also explains why Matsuzaki et al. (2006) found much higher levels of aromatase in the ectopic than the eutopic endometrium, because the ectopic endometrium was isolated from its environment with laser capture microdissection. Despite the relatively small effects, we still observed significantly higher levels of aromatase and type 1 17-HSD in deep-infiltrative endometriosis.
With regard to the E2-inactivating enzymes we confirmed that type 2 17-HSD mRNA levels are significantly lower in endometriotic lesions compared with levels in normal endometrium (Zeitoun et al., 1998; Matsuzaki et al., 2006). We also show for the first time that the type 4 17-HSD is expressed in endometriotic lesions and that the mRNA levels are lower than in normal endometrium. EST mRNA levels were not different between the groups, which agrees with the findings of Smuc et al. (2007). Using a new antibody, however, we could not confirm that type 2 17-HSD protein is expressed at lower levels in endometriotic tissue, which is in contrast to the observation of Zeitoun et al. (1998).
We showed in our steroid-responsive explant cultures that of all enzymes investigated, only the 17-HSD type 2 and EST were responsive to progesterone which agrees with the findings of others (Tseng and Gurpide, 1974; Liu and Tseng, 1979; Pack et al., 1979; Maentausta et al., 1991; Casey et al., 1994; Husen et al., 2001). The lower expression of 17-HSD type 2 in the ectopic endometrium in patients was postulated to be the result of reduced sensitivity to progesterone (Bulun et al., 2000,2002a,b). We observed, however, that in contrast to type 2 17-HSD, expression levels of EST were not reduced in the endometriotic lesions, whereas in normal endometrium the expression patterns of 17-HSD type 2 and EST were strongly correlated. This suggests that the expression of 17-HSD type 2 is selectively suppressed.
It is clear that enzyme activities are different in the epithelial and stromal compartments, as is the expression of the different steroidogenic enzymes. Aromatase activity was shown to be present in both stroma and epithelium, but activity in the stroma was 3–4 times higher (Tseng, 1984b). For this reason, most in vitro studies have been performed with stromal cells isolated from the endometriotic lesions. Aromatase mRNA (Matsuzaki et al., 2006) and protein (Kitawaki et al., 1997), however, were found to be expressed mostly in the epithelial cells. The importance of intercellular communication with regard to the regulation of steroidogenesis was illustrated by for instance the paracrine regulation of type 2 17-HSD expression in epithelial cells by factors released by the stroma (Cheng et al., 2007), and the presence of progesterone receptor in the stromal cells (Yang et al., 2001). Endometrial cell-specific production of metabolites was demonstrated for tibolone (Groothuis et al., 2006). Stromal cells were shown to contain 3
- and -aldoketoreductases which converted tibolone into 3- and 3-hydroxytibolone, whereas in the epithelial cells tibolone is directly converted into 4-tibolone. It is therefore important to consider that the net production of E2 synthesis is also influenced by cellular interactions, which has to be accounted for in the experimental designs, i.e. by using co-cultures or explant cultures.
Finally, it has to be noted that even though much attention was given to the expression and activities of individual enzymes, there is no information with regard to the actual output of E2 by the ectopic endometrium. An important aspect in this regard is the fact that the intracellular redox state of cells is an important determinant of the enzymatic conversions by HSDs (Sherbet et al., 2007). The nicotinamide cofactors required for the reduction and oxidization activities are abundant in cells, reaching millimolar concentrations. In cells the major reducing cofactor is NADPH (NADPH/NADP+ ratio 100:1) and the major oxidizing cofactor is NAD+ (NADH/ NAD+ ratio 1:1000; Krebs, 1973; Reich and Sel'kov, 1981). In addition, it was shown that low micromolar concentrations of NADPH potently inhibits NAD+-dependent oxidative reactions (Steckelbroeck et al., 2004). Collectively, this suggests that in vivo the reductive activity is dominant (Steckelbroeck et al., 2004). This would for instance mean that the type 1 17-HSD activity would dominate type 2 activity. In addition, P450 enzymes like aromatase also need NADPH and molecular oxygen and will irreversibly oxidize the steroid nucleus (Miller, 2005). These observations indicate that increases in the expression of E2-generating enzymes will have a greater impact on the net production of E2 than a reduction in the expression of E2-inactivating enzymes.
The endometrium is also a source of drug metabolizing enzymes usually present in the liver. Reddy et al. (1981) showed that human endometrial tissue was able to metabolize E2 into the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol. It has now been shown that the human endometrium expresses CYP3A4, known to convert estrone into 16-hydroxyestrone and E2 into 2-hydroxyestradiol (Hukkanen et al., 1998), and CYP1B1, known to convert E2 into 4-hydroxyestradiol (Tsuchiya et al., 2004). Furthermore, the human endometrium expresses catechol-O-methyltransferase, which converts 2-hydroxy- and 4-hydroxyestradiol into 2- and 4-methoxyestradiol (Salih et al., 2007). Another group of potential E2-inactivating enzymes was also identified in the endometrium, the uridine diphospho-glucuronosyltransferases (UGTs) (Lepine et al., 2004). The endometrium appears to be a rich source of UGTs, which are able to conjugate both estrogens and catecholestrogens on different positions. However, their relevance with regard to the development of endometrial pathologies is not known.
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We confirm that aromatase and 17
-HSD type 1 expression is higher in deep-invasive endometriosis than in eutopic and normal endometrium. Even though no 17-HSD type 1 gene transcripts were detected in normal endometrium, we did observe a clear expression of the 17-HSD type 1 protein, which was not elevated in the ectopic endometrium. Expression of the STS was prominent at both the mRNA and protein level, but was not elevated in endometriotic tissues. Type 2 17-HSD gene transcript levels were lower in deep-invasive endometriosis. Type 2 17-HSD protein levels, however, were not lower in lesion compared with the eutopic or normal endometrium. We show for the first time that the E2-inactivating enzyme 17-HSD type 4 is expressed in deep-invasive endometriosis, and that it is also down-regulated in endometriosis. Together with the continuous presence of aromatase and the substantial levels of STS, this may result in an increase in the local production of E2 in the endometriotic lesions. Inventarization of the net metabolic conversions in the whole endometriotic tissue, would aid in the selection of the appropriate combination of enzyme inhibitors to optimize medical therapy of endometriosis.
In normal endometrium both the 17-HSD type 2 and EST were responsive to progesterone. The fact that in deep-infiltrative endometriosis only the expression of type 2 17-HSD was suppressed and not that of EST, suggests local and selective dysregulation of type 2 17-HSD expression.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryFundingReferences |
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This study was funded by Solvay Pharmaceuticals Research Laboratories, Hannover, Germany.
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6 Present address: Department of Molecular Diagnostics, Philips Research, High Tech Campus 11, 5656 AE Eindhoven, The Netherlands
7 Present address: Department of Pharmacology, N.V. Organon, Postbus 20, 5340 BH Oss, The Netherlands
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Submitted on February 26, 2007; resubmitted on July 12, 2007; accepted on July 24, 2007.
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