Molecular Human Reproduction, Vol. 9, No. 8, 465-473,
August 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
The effect of RU486 on the gene expression profile in an endometrial explant model
Submitted on March 3, 2003; accepted on April 3, 2003
1 Reproductive Molecular Research Group, Department of Pathology, University of Cambridge, Cambridge CB2 1QP and 2 Department of Obstetrics and Gynaecology, University of Cambridge Clinical School, The Rosie Hospital, Robinson Way, Cambridge CB2 2SW, UK
3 To whom correspondence should be addressed. e-mail: rc296{at}cam.ac.uk
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Administration of RU486 in vivo during the receptive phase rapidly renders the endometrium non-receptive to the implanting embryo. In order to identify key pathways responsible for endometrial receptivity we have used cDNA arrays to monitor gene expression changes in short-term endometrial explants in response to RU486. Endometrial biopsies from five normal fertile women at mid-secretory phase were cultured in the presence of estradiol and progesterone with or without RU486 for 12 h. cDNA arrays were produced containing
1000 sequence-verified clones which included genes known to be important in angiogenesis, apoptosis, cell signalling, extracellular matrix remodelling and cell cycle regulation. cDNA probes from the paired endometrial samples were hybridized to the arrays and hybridization signals were quantified. A total of 12 genes displayed significant changes in expression; six were up-regulated and six down-regulated following RU486 treatment. For five of these genes this is the first report suggesting that they are regulated by steroids in the endometrium. JAK1 and JNK1 were two of the genes shown by the arrays to be down-regulated in RU486-treated endometrial explants. This was confirmed by real time RT–PCR. JAK1 immunoreactivity was localized to both glandular epithelium and the stroma of normal endometrium and staining was much stronger in the luteal phase of the cycle. These results show that components of two important signalling pathways in endometrium—the JAK/STAT pathway, and the JNK pathway—are altered by RU486. Genes whose expression is controlled by these pathways are likely to be involved in the mechanism by which steroids render the endometrium receptive to the implanting embryo. Key words: cDNA array/endometrium/gene profiling/implantation/steroids
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Introduction |
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In humans the uterus is receptive to blastocyst implantation between 5 and 9 days post-ovulation, a period known as the implantation window (Wilcox et al., 1999). During this period the endometrium becomes functionally receptive to the embryo, which is reflected by its structural and biochemical transformation (Tabibzadeh and Babaknia, 1995). The action of progesterone on an estrogen-primed endometrium results in a particular gene expression profile which renders the endometrium receptive (Giudice et al., 1999; Salamonsen et al., 2001). If the action of progesterone is antagonized, or progesterone levels fall, the endometrium rapidly reverts to a non-receptive state. This is followed by other changes such as focal haemorrhage and degradation of extracellular matrix (ECM) resulting in menstruation (Papp et al., 2000). Based on this paradigm, several approaches have been used to identify genes that are required for a functionally receptive endometrium. The first has been to identify structural and molecular changes that coincide with the transition from a non-receptive state (LH+3), to a receptive state (LH+7). As well as morphological changes, the expression of many proteins alters during this time. These include mucin 1 (Hey et al., 1994) placental proteins 12 and 14 (Julkunen et al., 1986; Rutanen et al., 1986), prolactin (Maslar et al., 1979), various integrins (Lessey et al., 1992), cytokines (Tabibzadeh et al., 1995) and heat shock proteins (Gruidl et al., 1997). A recent microarray study reported >600 genes whose mRNA expression levels altered between these two time-points (Carson et al., 2002). The second approach has been to try to identify genes that are directly regulated by the action of progesterone on endometrium (Kumar et al., 1998; Okulicz and Ace, 1999).
An alternative approach has been to study the effects of antiprogestins such as RU486 (mifepristone). RU486 competes with progesterone for binding to the progesterone receptor and alters its DNA binding characteristics (Jackson et al., 1997). It has both antagonist and agonist activities on the action of progesterone in regulating gene transcription and also exhibits anti-glucocorticoid, anti-estrogenic and anti-androgenic activity (Beck et al., 1993; McDonnell and Goldman, 1994; Hackenberg et al., 1996; Elger et al., 2000). A single dose of RU486 (200 mg) administered to normal cycling women 2 days after ovulation produces peak plasma levels of 0.3–0.4x10–6 mol/l within 2 h (Sarkar et al., 2002). This renders the endometrium non-receptive (Hegele-Hartung et al., 1992; Gemzell-Danielsson et al., 1993). RU486 has an effect on gene expression in the uterus as early as 6 h after oral administration (Critchley et al., 1996). This effect is likely to be due to the direct action of RU486 on endometrium, since there is no significant effect on serum progesterone levels (Swahn et al., 1990). This is supported by the fact that RU486 has been shown to suppress expression of leptin receptor in endometrial explants after only 6 h in culture (Koshiba et al., 2001). Many studies have used antiprogestins to analyse the expression of individual endometrial factors that might be important for implantation (Danielsson et al., 1997; Schatz et al., 1997; Marions et al., 1998; Taylor et al., 1998; Critchley et al., 1999). However, most of these factors have been studied in isolation. To date, no attempt has been made to study the global changes in gene expression caused by RU486 in human endometrium as it changes from a receptive to a non-receptive state.
In order to identify endometrial factors that are important for implantation, we have used an endometrial explant model to identify genes regulated by the action of RU486 on endometrium from the receptive phase. Many previous studies using endometrial explants have shown that they retain their steroid responsiveness in vitro for ≥24 h and respond to steroids in a similar way to endometrium in vivo (Dudley et al., 1992; Ilouz et al., 2000; Koshiba et al., 2001). These results suggest that cultures of endometrial explants are a valid model for studying the effects of steroid treatment in the endometrium. We cultured mid-secretory endometrial explants with RU486 for 12 h at a concentration similar to peak plasma levels after oral administration of 200 mg. Total RNA was isolated and used to probe cDNA arrays containing 1000 genes selected to include the molecular pathways believed to be important for receptivity. These include cell adhesion, apoptosis, signalling, cell cycle regulation, ECM remodelling and angiogenesis. This has enabled the monitoring of early gene expression changes in secretory phase endometrium in response to RU486. The aim was to identify genes whose expression may be involved in the loss of endometrial receptivity.
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Materials and methods |
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Tissue collection
This study took place with the approval of the Cambridge Local Region Ethics Committee. Written informed consent was obtained from all patients. Endometrial biopsies were obtained from seven normally cycling fertile women at the mid-secretory phase (day 19–23) who underwent curettage or hysterectomy for benign gynaecological disorders. The patients were healthy women (aged 28–38 years, with a body mass index between 19 and 25 kg/m2). They had regular menstrual cycles (27–30 days) and had not received hormone therapy for the last 2 months prior to surgery. Each specimen was assessed as normal by histological examination. Endometrial dating was performed using published criteria (Noyes et al., 1950). Biopsies for immunohistology were collected from similar patients throughout the menstrual cycle and confirmed as normal by histological examination.
Explant culture
Endometrial biopsies were washed several times in Dulbecco’s minimum essential medium (DMEM)/Ham’s F-12 without Phenol Red (Life Technologies, UK) to remove blood. The endometrial tissues were chopped into 1–2 mm2 pieces and divided into two equal samples. These were placed in a 4-well plate (Nunc) and incubated with 5 ml media (DMEM/F-12 without Phenol Red), 1% L-glutamine (Life Technologies), 1% penicillin G/streptomycin (Life Technologies) for 1 h to allow recovery, culture medium was then changed (Wang et al., 1987). One part of the sample was treated with 5 ml of DMEM/F-12 (as above) containing 10–9 mol/l estradiol + 10–7 mol/l medroxyprogesterone acetate (all steroids purchased from Sigma, UK). These steroid concentrations have previously been shown to elicit responses in endometrial explant cultures (Koshiba et al., 2001) and closely resemble plasma concentrations reported during the mid-secretory phase of the endometrium (reviewed in Chabbert Buffet et al., 1998). The other half of the endometrial sample was incubated with the same medium to which RU486 (10–6 mol/l) (Sigma) was added. Incubations were performed in a humidified atmosphere at 37°C in 5% CO2. The paired endometrial samples treated with and without RU486 were flash-frozen after 12 h and stored at –70°C.
RNA extraction
Total RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturer’s instructions and treated with RQ1 DNase I (Promega, UK) for 30 min at 37°C, then re-extracted with Trizol. RNA quality was assessed by loading 300 ng of total RNA onto an RNA Labchip (Agilent Technologies) and analysed on an A2100 Bioanalyser (Agilent Technologies, Germany). Two biopsies did not yield sufficient RNA for hybridization to arrays, but were used for the subsequent RT–PCR analysis.
Array production
cDNA arrays were produced containing 1000 sequence-verified clones spotted in duplicate on an 8x12 cm nylon Hybond N+ membrane (Amersham Pharmacia Biotech, UK). The DNA used for spotting was produced by amplification using PCR from the corresponding IMAGE cDNA clones. Specific cDNA clones were chosen that showed little or no homology to other known genes to reduce cross-hybridization and clones were sequenced prior to PCR to confirm the clone identity. To check for any cross-contamination, PCR products were electrophoresed prior to spotting on the array to ensure that each was a single band and 50% of the PCR products used for spotting were resequenced to verify that the correct clone was spotted. The amplified PCR fragments were purified using the Millipore Multi-screen 96 system (Millipore, UK) to a concentration of 500 µg/ml. Approximately 15 ng of each cDNA was contact-printed onto the membrane using a BioRobotics MicroGrid robot (BioRobotics Ltd, UK) with a 96-pin tool with 0.4 mm diameter solid pins to create 2112 individual DNA spots, representing known genes, expressed sequence tags (ESTs) unknown genes and calibration spikes. After spotting, the filters were denatured and neutralized and the DNA was cross-linked using a UV Stratalinker, model 1800 (Stratagene, USA) at 70 000 µJ/cm2. The listing of all the cDNA on the array can be found at http://www.path.cam.ac.uk/angio/.
Array hybridization
Radiolabelled cDNA probes were produced by labelling 5 µg of total RNA from five endometrial samples with [33P]dCTP using the EndoFree RT Kit (Ambion, USA). Unincorporated [33P]dCTP was removed using NICK columns (Amersham, Pharmacia Biotech). The filters were pre-hybridized at 65°C for 3 h using ExpressHyb buffer (Clontech, USA) containing 1 µg/ml Human Cot-1 DNA (Life Technologies), 1 µg/ml Poly dA (Amersham, Pharmacia Biotech) and 5 µg/ml Salmon sperm DNA (Sigma). cDNA probes were denatured then added to the hybridization buffer at 1x106 cpm/ml and hybridized at 65°C for 16 h. The filters were washed twice in 2xstandard saline citrate (SSC), 0.5% sodium dodecyl sulphate (SDS) and twice in 0.1x SSC, 0.1% SDS for 30 min each at 60°C, then dried for 30 min at 60°C. The filters were exposed to low energy storage phosphor screens (Molecular Dynamics, USA) for 48 h and scanned at 50 µm resolution using a STORM 860 Scanner (Molecular Dynamics).
Array analysis
Hybridization signals were quantified using ImaGene v5.0 (BioDiscovery Inc., USA). The mean signal for each spot on the array was determined. The data from each pair of treated and untreated samples from the same patient were then normalized to overcome minor differences in labelling and hybridization efficiency. Normalization was performed by applying intensity-dependent scaling using the ‘loess’ function of the R-statistical software system, in a similar fashion to that used by the SNOMAD protocol (Colantuoni et al., 2002). The normalized transcript abundance data was then compared using a paired t-test to identify transcripts that showed a statistically significant change in expression following exposure to RU486 across all five samples. Statistical significance was defined as P < 0.05. The fold change values for each endometrial biopsy were calculated by taking the background subtracted mean signal for each cDNA spot and dividing the value obtained from the RU486-treated portion of the biopsy by the value of the corresponding spot from the control sample.
Real time RT–PCR
The relative expression of two genes, JAK1 and JNK1, was compared between RU486 and control tissues by real time RT–PCR using an ABI PRISM 7700 sequence detection system (TaqMan) according to the manufacturer’s instructions. Primers and probes were designed using the Primer Express v5.0 software (Applied Biosystems, UK) and were designed within the same region of the cDNA clone as was spotted on the array. Areas in the cDNA that are known to be involved in alternate splicing were avoided. Details of the primers and probes used are detailed below. The probes were labelled with 5'FAM and 3'TAMRA and were purified by high-performance liquid chromatography. JAK1 primers and probes were: 5'-CATGAGAACATTGT GAAGTACAAAGGA-3' (forward primer), 5'-CCCGAAGGCAGAAATT CCAT-3' (reverse primer), 5'-GCTTAATACCATTTCCTCCGTCTTCTGT GCAG-3' (probe); JNK1 primers: 5'-TCAATGGCTCTCAGCATCCA-3' (forward primer), 5'-GCCAAAGTCGGATCTGTTGAC-3' (reverse primer), 5'-CATCGTCGTCTGTCAATGATGTGTCTTCAA-3' (probe). In addition, the endogenous control 18S ribosomal RNA was assayed using primers and probe from Applied Biosystems. Probe and primer optimization and real time PCR were performed using the manufacturer’s recommended conditions. Standard curves were generated by serial dilution of a standard preparation of total RNA isolated from luteal phase endometrium. Data are expressed in arbitrary units relative to the level of the same gene in this standard RNA. cDNA was produced from each endometrial sample by reverse transcription using 5 µg of total RNA with 200 IU Superscript RT (Invitrogen, UK) according to the manufacturer’s instructions. The expression values obtained were normalized against those from the control ribosomal 18S to account for differing amounts of starting material.
Immunohistochemistry
Biopsies for immunohistochemistry were fixed in neutral buffered 10% formalin for 6 h at room temperature and paraffin wax-embedded. Serial sections of 5–7 µm were cut from the paraffin blocks and mounted on APES-coated slides, de-waxed in xylene and rehydrated in graded alcohol. Slides were pressure-cooked at full pressure for 2 min in 10 mmol/l sodium citrate, pH 6.0. After cooling for 5 min, slides were washed in phosphate-buffered saline (PBS), endogenous peroxidase was quenched for 10 min in 0.3% hydrogen peroxide in methanol and blocked in 20% goat serum, 1% bovine serum albumin (BSA) for 30 min. Sections were incubated with rabbit anti-JAK1 (Santa Cruz, Biotechnology, USA) at 4°C for 16 h at a dilution of 1:300 for proliferative phase sections and 1:600 for secretory phase sections in PBS, 5% goat serum, 1% BSA. To confirm specificity of the JAK1 immunoreactivity, we preincubated the anti-JAK1 antibody with a 10-fold excess of the antigenic JAK1 peptide against which the antibody was raised. Slides were then reacted with biotin-labelled goat anti-rabbit IgG and incubated with preformed avidin–biotin–peroxidase complex (ABC kit; Vector Laboratories, USA). Diaminobenzidine (Sigma) was used as a substrate. Sections were counterstained with haematoxylin, dehydrated and mounted.
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Results |
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cDNA array analysis
Endometrium was collected from seven fertile women in the mid-secretory phase of the cycle. Each sample was divided into two equal pieces and cultured for 12 h in estradiol and progesterone with or without the antiprogestin RU486. Total RNA was extracted from each culture and used to probe a custom-made human cDNA array of
1000 sequence-verified clones. Representative genes were chosen to include those important in molecular pathways involved in angiogenesis, apoptosis, cell cycle control, ECM remodelling and cell signalling. We also included markers for specific cell types in endometrium, including cytokeratin (epithelium), vimentin (stroma) and CD45 (leukocytes). These markers provide a means to assess the relative content of each cell type in the paired biopsies from each patient. Total RNA from five pairs of control and RU486-treated endometrial explants was hybridized on individual arrays and then normalized. Typical results of this analysis are shown by a representative scatter plot comparing the signals obtained for each cDNA clone on the array after hybridization to control and RU486-treated endometrium from the same patient (Figure 1A). The hybridization signal values obtained from control and treated tissue samples from five women were normalized in pairs and cDNA with significant changes determined by paired t-test. A total of 12 genes (1.2%) displayed statistically significant changes in expression following RU486 treatment; six cDNA decreased (Table IAIB) and six increased (Table IIAIIB). These cDNA fulfil the following criteria: both duplicate spots on the array changed significantly, and the signal intensity was at least twice that of the backgound. An example of the change seen for the cDNA JNK1 on the cDNA array is shown in Figure 1B. The identity of the 12 genes identified as regulated by RU486, was confirmed by sequencing the actual PCR product spotted on the array corresponding to each regulated cDNA spot.
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TaqMan verification of gene expression determined by array analyses
Real time RT–PCR was used to verify the changes in RNA expression levels indicated by the cDNA analysis. Two genes JAK1 and JNK1, which both apparently decreased >2-fold following RU486 and which were highly expressed, were chosen for verification. cDNA was synthesized from the same five total RNA samples used for the array analysis as well as two additional patient samples. Levels of JAK1 and JNK1 were measured by TaqMan analysis for each cDNA sample relative to a reference RNA and the values were corrected for differences in loading relative to the 18S ribosomal RNA. JAK1 expression decreased by a mean of 2.7-fold and JNK1 expression decreased by a mean of 2.1-fold in the seven RU486-treated samples (Figures 2 and 3). The decrease in both genes was statistically significant (JAK1, P < 0.016 and JNK1, P < 0.047). Therefore the gene list generated by the cDNA array analysis reflects reliable changes in gene expression following RU486 treatment of secretory phase endometrium.
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Immunolocalization of JAK1 in endometrium
JAK1 immunostaining was predominantly in the glands in proliferative endometrium with only faint staining in the stroma (Figure 4A). The intensity of the immunostaining increased considerably in secretory endometrium and staining was seen within the stroma as well as in the glandular and luminal epithelium (Figure 4C). Immunostaining was present throughout the stroma in secretory phase endometrium but was clearly absent from lymphoid aggregates (Figure 4E). Therefore JAK1 immunoreactivity appears to be present within the glands throughout the menstrual cycle, but increases dramatically within the stroma and luminal epithelium at the mid-secretory phase. Staining of sections with JAK1 antibody preincubated with the JAK1 antigenic peptide abolished JAK1 immunoreactivity, indicating that the staining obtained was specific for JAK1 (Figure 4B, D and F).
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Discussion |
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The genes and biochemical pathways believed to be involved in achieving a receptive uterus have been largely derived from animal models (Giudice, 1999; Lessey et al., 2000; Sharkey and Smith, 2003). Other than the progesterone receptor, no genes have yet been formally shown to be essential for implantation in humans. Recently, gene profiling using microarray analysis has been used to identify global expression patterns in endometrium during the menstrual cycle (Kao et al., 2002; Borthwick et al., 2003), the receptive phase (Carson et al., 2002) and in-vitro decidualization (Popovici et al., 2000). Changes in expression of hundreds of cDNA coincides with the transition from the non-receptive to the receptive state, but it is not known which of these molecules are critical for successful human implantation. The current study utilized the antiprogestin RU486 on cultured human secretory endometrium to identify steroid-regulated genes and molecular pathways, which, when disrupted, lead to implantation failure. Custom-made cDNA arrays which included genes known to be important in angiogenesis, apoptosis, cell signalling, ECM remodelling and cell cycle regulation, were used to identify genes which are altered by the action of RU486 on endometrial explants.
The arrays identified 12 genes whose expression was significantly altered after 12 h of treatment with RU486. The gene list represents 1.2% of the genes on the array and three show mean fold changes of ≥2. Since the response to steroids alters over time, this 12 h time-point represents the early responses of the endometrium to RU486. Previous array studies on endometrial tissues and cell lines have revealed a similar number (1.3–5.8%) of genes with a significant change in gene expression (Popovici et al., 2000; Carson et al., 2002; Kao et al., 2002). A fold change of >2 is a frequently adopted cut-off for array profile analysis (Claverie et al., 1999). However, subtle alterations in transcript levels of genes below the 2-fold level should be considered if they are repeatable over a large number of biologically independent replicated experiments, because these smaller gene expression changes may be due to a robust response in a few cells in a complex tissue (Hamadeh et al., 2002). The results of the real time PCR for the two genes JAK1 and JNK1 were in good agreement with the array results and confirm that the array analysis is reliable.
This study employed endometrial explants rather than isolated primary cells. This retains normal epithelial–stromal interactions and such explants have been shown to exhibit normal steroid responsiveness for up to 72 h (Koshiba et al., 2001). Recent reconstitution studies using endometrial tissue from mice deficient for the estrogen or progesterone receptor genes have shown that maintaining epithelial– stromal cell interactions is essential if normal responses to steroids are to occur (DeMayo et al., 2002). The use of explants retains these interactions and also demonstrates the extent of normal patient-to-patient variation in the expression level of individual genes. An example of this is seen in the TaqMan data for JAK1 (Figure 2), where the control samples vary up to 5-fold in the level of JAK1 expression. This variability has several sources including the fact that biopsies from different women vary in their cellular content. Secondly there is considerable variation in gene expression between different women even with fresh endometrial biopsies taken at LH+7 in the cycle (R.D.Catalano and A.M.Sharkey, unpublished data). The only way to overcome this variation is to perform multiple replicates, as we have done in this experiment in order to obtain reliable data.
The 12 genes identified represent genes involved in apoptosis, transcription, stress response, ECM, but predominantly those involved in cell signalling pathways. One of these, cPLA2, has been reported previously to be up-regulated by RU486 (Kol et al., 1998). Others have previously been reported to be expressed in the endometrium, including BAK (Tao et al., 1998), CD94 (Semino et al., 1995), cathepsin L (Jokimaa et al., 2001), GST Pi (Barnette et al., 1999) and NIK (King et al., 2001). To our knowledge there are no previous reports of steroid regulation of the expression of tenascin R, JNK1, JAK1, VAV2 and WISP1 in endometrium.
The gene list and real time RT–PCR data indicate that the components of two major intracellular signalling pathways, the JAK/STAT signalling pathway, and the JNK signalling pathway, are down-regulated by the action of RU486. Janus kinases (JAK) are cytoplasmic protein tyrosine kinases that are activated by binding of cytokines to their receptors. They phosphorylate cellular substrates including the transcription factor family named signal transducers and activators of transcription (STAT) (Schindler et al., 1995; Ihle et al., 1996). JAK1 is recruited by three major cytokine receptor subfamilies, class II cytokine receptors [receptors for interferon (INF)
/ß, INF
and interleukin (IL)-10], cytokine receptors that utilize the c receptor subunit (receptors for IL-2, IL-4, IL-7, IL-9 and IL-15) and receptors that utilize the gp130 subunit (receptors for IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), cilary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1)). The results of this study indicate that signal transduction through all these receptors would be altered by the action of RU486.
Immunohistochemical analysis showed that JAK1 immunoreactivity is seen in the glands and only weakly in the stroma in proliferative endometrium. In mid-secretory endometrium the immunostaining increased, especially in the stroma and luminal epithelium. This staining was abolished by preincubation of the antibody with the JAK1 peptide. The data clearly indicate that JAK1 immunoreactivity is elevated in the stroma, endometrial glands and luminal epithelium in the receptive endometrium. Confirmation that JAK1 protein expression is down-regulated in explants or in vivo by RU486 will require accurate quantification by Western blot analysis. JAK2 expression has been shown to be negligible in the proliferative phase, but is elevated in the glands during the secretory phase with little JAK2 expression in the stroma (Jabbour et al., 1998). Therefore JAK1 appears to have a distinct role compared with JAK2 in the stroma and luminal epithelium in endometrium from the receptive phase.
The JNK family comprises three protein kinases, defined by their phosphorylation of the N-terminal region of c-jun (Pulverer et al., 1991; Kallunki et al., 1996). JNK also phosphorylates the transcription factor ATF2 (Gupta et al., 1995) and the Ets-domain transcription factor Elk-1 (Whitmarsh et al., 1995). The JNK signalling pathways are activated primarily by pro-inflammatory cytokines and stress stimuli. Depending upon the stimulus, JNK activation may be involved in mitogenesis, oncogenic transformation, differentiation, or induction of apoptosis (Minden and Karin, 1997). Prolactin induces activation of both JNK1 and JNK2 and inhibition of JNK1 activation prevents cellular proliferation and induces apoptosis (Schwertfeger et al., 2000). JNK1 was specifically down-regulated in our study and therefore only pathways specifically activated by JNK1 should be affected. Functional differences between JNK1 and JNK2 remain unclear as they share many of the same substrates and are activated by the same kinases. However, the recent development of JNK1 and JNK2 knockout mice shows that they have distinct and specific roles in modulating cell function. JNK1 is known to alter the expression of >30 genes including insulin-like growth factor binding protein 3 (IGFBP-3), vimentin, cytokeratin 18 and junD (Chen et al., 2002). Many of these genes are differentially regulated during the menstrual cycle. JNK1-deficient mice also show defective T-cell differentiation and enhanced Th2 cytokine production (Dong et al., 1998). This effect is seen even in JNK1+/–, JNK2+/– double heterozygotes, indicating that relatively small changes in the levels of JNK gene expression can have significant phenotypic effects (Sabapathy et al., 2001). There are four known isoforms of JNK1 which arise by differential splicing, and whose specificity of binding interaction differs between isoforms. The cDNA probe and TaqMan primers used in this study hybridize to the 3' region common to all isoforms. We have not determined whether expression of all these isoforms is regulated simultaneously.
The action of RU486 on secretory phase endometrium rapidly renders this tissue non-receptive to the implanting embryo (Critchley et al., 1999). This is accompanied by changes in expression of genes such as integrins and leukaemia inhibitory factor, whose expression normally coincides with the receptive state (Ghosh et al., 1998). We have used endometrial explants treated with RU486 to identify genes whose expression is likely to differ between receptive and non-receptive endometrium. cDNA arrays and real time RT–PCR have confirmed that JNK1 and JAK1 are down-regulated in endometrium by RU486 within 12 h. These molecules act in the signal transduction pathways used by cytokines, growth factors and other physiological stimuli to control cell function. These two signalling pathways, together with the other genes we have identified in this study, identify new genes potentially involved in endometrial receptivity, and may provide new targets for the development of novel contraceptive agents.
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Acknowledgements |
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The authors would like to thank the staff and patients at the Rosie Maternity Hospital, Cambridge for their assistance, without which this study would not have been possible. We would like to thank Tom Freeman and Tony Corps for their helpful advice on array analysis. This study was supported by the World Health Organization, and A.S. was supported by The Meres Research Studentship from St John’s College, Cambridge.
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