Mol. Hum. Reprod. Advance Access originally published online on February 2, 2006
Molecular Human Reproduction 2006 12(2):71-76; doi:10.1093/molehr/gal008
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Transcription factor ETS1 is critical for human uterine decidualization
Department of Pediatrics, University of Cincinnati College of Medicine and Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
1 To whom correspondence should be addressed at: Department of Pediatrics, University of Cincinnati College of Medicine and Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. E-mail: stuart.handwerger{at}chmcc.org
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Abstract |
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The aim of this study was to examine whether the transcription factor ETS1 plays a critical role in the regulation of human decidualization. Decidual fibroblast cells were decidualized in vitro by treatment with medroxyprogesterone, estradiol (E2) and dibutyryl cyclic AMP or prostaglandin E2 in the absence or presence of an ETS1 antisense oligonucleotide (oligo) that blocks the translation of ETS1 mRNA. Control experiments were performed using a control oligo that did not affect ETS1 expression and the induction of specific marker genes for decidualization. The ETS1 antisense oligo markedly inhibited ETS1 protein expression and significantly inhibited downstream targets of ETS1 action. On day 6 of culture, the decidualized fibroblast cells that had been exposed to the ETS1 antisense oligo contained 40–90% less mRNAs for prolactin, insulin growth factor binding protein 1 (IGFBP-1) and other decidualization-specific markers (laminin, tissue inhibitor of metalloproteinase-3 [TIMP3], endometrial bleeding associated factor [EBAF] and decorin) than those of control cells that had not been exposed to the ETS1 antisense oligo. GAPDH mRNA levels, which do not change during decidualization, were unaffected by either the ETS1 antisense or the control oligo. The cells decidualized in the presence of the ETS1 antisense oligo also released significantly less prolactin, EBAF and IGFBP-1 protein, determined by western blot analyses, than the control cells. Taken together, these findings strongly suggest that ETS1 plays a critical role in the induction of human decidualization.
Key words: decidualization/differentiation/gene expression/pregnancy/uterus
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Introduction |
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The ETS proteins comprise a superfamily of wing helix-turn-helix DNA-binding proteins that share a unique DNA-binding domain (ETS domain) that is 85 amino acids in length (Sharrocks et al., 1997
). In the human, there are two members of the gene family, ETS1 and ETS2, which are located on separate chromosomes (Dhulipal, 1997). The transcription factors bind to a GGA(A/T) core motif on the enhancers and/or promoters of target genes and are involved in the control of cellular proliferation, differentiation and apoptosis (Pei et al., 2003). The ETS1 gene is expressed in many tissues, including lymphoid (Bartel et al., 2000) and haematopoietic tissues (Dittmer, 2003), endothelial cells (Lelievre et al., 2001), vascular smooth muscle cells (Naito et al., 1998), astrocytes (Fleischman et al., 1995), extravillous trophoblast cells (Luton et al., 1997), decidual cells (Kilpatrick et al., 1999; Brar et al., 2002) and embryonic cells (Dittmer, 2003). The transcription factor is also produced by many solid tumours, where high levels of expression frequently correlate with a poor prognosis (Takai et al., 2004). ETS1 has been shown to be important in the development of lymphoid tissue and is required for epithelial cells to develop an angiogenic phenotype (Lelievre et al., 2001), a process critical for the development and growth of uterine endometrium. ETS1 also promotes invasive behaviour of endothelial cells, vascular smooth muscle cells and epithelial cancer cells (Hsu et al., 2004). There is evidence that ETS1 also has a role in embryonic development (Kola et al., 1993).
Several lines of evidence suggest that ETS1 plays a role in the regulation of human uterine decidualization. ETS1 protein expression increases 9- to 20-fold in human endometrial stromal cells undergoing in vitro decidualization, in response to progesterone and estradiol (E2) (Brar et al., 2002). In addition, overexpression of ETS1 in human decidual fibroblast cells undergoing in vitro decidualization stimulates the expression of the prolactin gene, a widely recognized marker of decidualization (Brar et al., 2002). The transactivation of the prolactin gene is due to the binding of ETS1 to an ETS motif located at nt –77/–71 of the decidual prolactin promoter (Brar et al., 2002). Furthermore, ETS1 has been shown to regulate the expression of genes involved in extracellular remodelling and vascular reorganization in several cell types (Logan et al., 1996; Trojanowska, 2000), processes that are critical in decidualization. ETS1 has also been shown to stimulate the expressions of MMP1, MMP3, MMP9, uPA, VEGF and the VEGF receptor in several cell types (Dittmer, 2003), all of which are expressed in decidua.
A role for ETS1 in the regulation of decidualization is also supported by DNA microarray studies from our laboratory of human decidual fibroblasts undergoing in vitro decidualization (Brar et al., 2001). An analysis of 6918 genes at frequent intervals during the decidualization process revealed that 121 genes were induced, 110 genes were repressed and 50 genes showed a biphasic pattern of expression. Computer analysis of the most regulated genes revealed that the promoter regions of 23 of the 25 most induced genes and 8 of the 10 most repressed genes have one or more ETS binding sites.
In the present study, we used antisense technology to examine whether inhibition of ETS1 expression attenuates in vitro decidualization. Decidualization was induced in human decidual fibroblasts with medroxyprogesterone acetate (MPA), E2 and dibutyryl cyclic AMP (cAMP) (Richards et al., 1995), and the process of decidualization was monitored by examining the expression of genes known to be induced during the decidualization process (Brar et al., 2001). The expression of ETS1 during decidualization was inhibited by an ETS1 morpholino antisense oligonucleotide (oligo). Morpholino oligos were chosen to block ETS1 expression because of the advantages that these oligos have over other forms of antisense oligos (Morcos, 2000, 2001). In contrast to other types of antisense oligos, morpholino oligos act via an RNAse-H-independent stearic mechanism. Consequently, these oligos are very stable and, unlike other forms of antisense oligos, are not degraded in cells.
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Cell preparations
Permission to obtain human decidual fibroblasts and endometrial tissue was approved by the Institutional Review Boards of the University of Cincinnati and the Children’s Hospital Medical Center, and informed consent was obtained in each instance. The decidual fibroblasts were prepared from term decidua by differential plating as previously described (Richards et al., 1995
). In brief, decidua parietalis was dissected from fetal membranes within 1–2 h after delivery, and the isolated cells were plated on plastic and cultured in Roswell Park Memorial Institute medium containing 10% FBS, 25 µg/ml penicillin, 25 µg/ml streptomycin and 2.5 µg/ml amphotericin B. Twenty-four hours later, the nonadherent cells (which included most of the terminally differentiated decidual cells and bone marrow-derived cells) were freed by agitation and removed by a total exchange of medium. The cells were then grown to confluence and subpassaged. By subpassage 3, essentially all the cells were proliferating fibroblasts. In each instance, the cells were plated at 80% confluency.
Decidualization experiments
The decidual fibroblasts were cultured as described in previous experiments (Frank et al., 1994; Richards et al., 1995). The fibroblasts were plated in 6-well plates and grown to 100% confluency. Decidualization was then induced by treatment with MPA (1 µM), E2, (10 nM) and either dibutyryl cAMP (50 µM) or prostaglandin E2 (PGE2, 1 µM). Earlier experiments from our laboratory demonstrated that the addition of PGE2 to MPA and E2 was as effective as dibutyryl cAMP, MPA and E2 in inducing decidualization. The effect of blocking ETS1 expression on decidualization was determined in experiments in which the cells were decidualized in vitro in the presence or absence of an antisense morpholino oligo (5'-CCGCCTTCATGGTGCCAGGAGTG-3') that prevents the translation of ETS1 mRNA. Control experiments were performed using a control morpholino oligo (5'-ACGTCTTTCCATTTATACACCTTTG-3') that does not block ETS1 expression and the expression of marker genes for decidualization (see Results). The oligos were delivered into the cells using a ‘Special Delivery System’ developed by Gene-Tools, LLC (Philomath, OR, USA), following the manufacturer’s protocol. With this system, the oligo is bound electrostatically to weakly basic ethoxylated polyethylenimine (EPEI), and the complex is efficiently endocytosed by cells. When the pH drops within the endosome, the EPEI more fully ionizes, resulting in the permeabilization of the endosomal membrane and release of the oligo into the cytosol. The cells were exposed to the morpholino/EPEI complex at a final concentration of 1 µM. Three hours later, the morpholino/EPEI medium was removed from the cells and replaced with control medium containing steroids and dibutyryl cAMP or PGE2, as indicated above. In one experiment, decidual fibroblasts were exposed to steroids and PGE2 for 3 days before being exposed to the control and ETS1 morpholino oligos. At the end of the third day of exposure to steroids and PGE2, the cells were exposed to the oligos as described above. The medium was then changed to the decidualizing medium for an additional 24 h.
In a preliminary study to determine the optimal amount of morpholino/EPEI complex that results in maximal delivery of the morpholino oligo without cell toxicity, decidual fibroblast cells were decidualized as above using a fluorescein-labelled control morpholino oligo instead of the ETS1 antisense oligo. Labelled morpholino/EPEI complexes with different molar ratios of morpholino to EPEI (0.25–5.0) were added to the cells, as suggested by the manufacturer. The cells were examined at various time intervals by light and fluorescent microscopies to determine the cellular uptake of the labelled oligo. Subsequent experiments were performed under experimental conditions that resulted in the uptake of the labelled oligo in >95% of the cells.
mRNA analyses
Decidualization was monitored in most experiments by the determination of mRNA levels of several decidualization-specific marker genes by semiquantitative RT–PCR using GAPDH mRNA levels for normalization (Brar et al., 2001). Total RNA was extracted from decidual fibroblasts using Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s directions. One to two micrograms of total RNA was reverse transcribed, and PCR amplification was performed after the addition of 0.14 µl 32P (3000 Ci/mmol) to the reaction mixture, as described previously (Brar et al., 2001). The PCR programme for each primer pair consisted of 25 cycles, which was in the linear range of the amplification curve for each set of primers. Each cycle consisted of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The radiolabelled PCR products were separated by electrophoresis on 6% polyacrylamide gels at 200 V for 3 h. The gels were transferred to 3M paper, dried and quantified using a phosphorimager and Imagequant 1.2 software (Molecular Dynamics, Sunnyvale, CA, USA).
In several experiments, mRNA levels for ETS1, prolactin, IGFBP-1, EBAF, laminin, decorin and GAPDH were measured using the Stratagene MX3000P real-time PCR System (LaJolla, CA, USA). To prevent amplification of genomic DNA, all primer pairs were designed to cross introns. Each primer pair was used at a forward-to-reverse ratio of 1 : 1, except for the GAPDH primers, which were used at a 2 : 1 ratio. The reaction mixture was incubated for 10 min at 95°C with either Brilliant Sybr Green Mastermix + Rox (Stratagene, LaJolla, CA, USA) or QBiogene Real-Time Ready Mix + Rox (ISC Express, Kaysville, UT, USA) containing x0.1 Sybr Green dye (Molecular Probes, Eugene, OR, USA). The PCR was 40 cycles, each consisting of 30 s at 95°C, 60 s at 55°C and 30 s at 72°C. The mixes were used according to the manufacturer’s instructions, except that the quantities of the reagents were changed to allow for the use of a 20 µl final volume instead of the 50 µl volume described. Dissociation/association curves for each reaction were determined after the fortieth cycle. The sequences of the primers used in the RT–PCR and real-time PCR studies are summarized in Table I.
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Western blot analysis
ETS1, EBAF and ß-actin protein levels were assessed in protein extracts of decidual fibroblasts. For the analysis of IGFBP-1 and prolactin levels, 2 ml of medium was concentrated using an Amicon Centrion YM-10 unit (Millipore, Bedford, MA, USA). Total protein was extracted for the ETS1, EBAF and ß-actin studies using HNTG buffer with inhibitors (50 mM HEPES with pH 7.4, 150 mM NaCl, 1% Triton x-100, 10% glycerol, 1.5 mM MgCl2·6H2O, 1 mM EDTA, 10 mM sodium pyrophosphate, 200 mM sodium orthovanadate, 100 mM NaF, 10 mg/ml leupeptin, 10 mg/ml aprotinin and 1 mM phenylmethylsulphonyl fluoride). Aliquots of concentrated medium and cellular extract (25–30 µg protein for prolactin and IGFBP-1, 10 µg protein for ß-actin and EBAF) were then separated by electrophoresis on 12% resolving/5% stacking reducing gels and transferred to Protran nitrocellulose membranes (Schleicher & Schuell Bioscience, Kenne, NH, USA). The membranes were blocked and then incubated overnight at room temperature with the appropriate antiserum. After additional washing and blocking, the blots were incubated with the appropriate horseradish peroxidase (HRP) antiserum at room temperature for 1 h. Chemiluminescence was visualized using the Super Signal West Pico or Femto Chemiluminescent Substrate Kits (Pierce, Rockford, IL, USA), followed by autoradiography on Hyperfilm (Amersham Biosciences, Buckinghamshire, UK). The autoradiographic signals were quantified using Kodak Digital Science ID Image Analysis software (Eastman Kodak, Rochester, NY, USA). Protein concentrations were determined using the BIO-RAD protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The IGFBP-1 studies used a polyclonal goat anti-IGFBP-1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a final dilution of 1 : 200 and a donkey antigoat HRP at a 1 : 5000 dilution (Chemicon, Temecula, CA, USA). The prolactin western analysis used a rabbit antiprolactin at a 1 : 1000 dilution and donkey antirabbit HRP at a 1 : 5000 dilution, both from Chemicon. The ETS1 studies used a polyclonal rabbit anti-ETS1/ETS2 IgG (Santa Cruz Biotechnology) at a 1 : 500 dilution and a donkey antirabbit HRP at a 1 : 5000 dilution (Chemicon). The EBAF western analysis used a goat anti-EBAF serum (R&D Systems, Minneapolis, MN, USA) at a 1 : 500 dilution and a donkey antigoat HRP serum (Chemicon) at a 1 : 2000 dilution. The amount of ß-actin in each lane was determined by western blot analyses using a monoclonal mouse ß-actin antibody (kindly provided by Dr James Lessard, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA) at a 1 : 10 000 dilution and a 1 : 5000 dilution of goat antimouse HRP IgG (Santa Cruz Biotechnology).
Statistical methods
Results are presented as mean ± SEM except where indicated. The statistical differences between sample means were determined by analysis of variance followed by Duncan’s multiple comparison test or the Tukey post hoc test. P-values <0.05 were considered significant.
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Results |
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Preliminary experiments were performed to determine the ratio of EPEI to morpholino oligo that results in the maximal uptake of the oligo into decidual fibroblast cells without apparent toxicity. Decidual fibroblast cells were incubated for 3 h in medium containing a fluorescein-labelled morpholino oligo with EPEI at molar ratios of 0.25–5.0. The medium was then changed to medium containing MPA, E2 and dibutyryl cAMP for 6 days, with daily changes to fresh medium. Maximal uptake of the labelled morpholino oligo without apparent toxicity occurred with 1.0 µM oligo, at an oligo/EPEI molar ratio of 2.5. At the end of day 6 of culture, nearly all the cells (>95%) exposed to the morpholino/EPEI complex at this ratio showed the presence of labelled oligo (data not shown). Similar uptake of the morpholino oligo was also observed after 1, 2, 3 and 4 days of culture. Cells exposed to lower ratios of oligo/EPEI showed less uptake, while cells exposed to higher ratios showed evidence of cell death (data not shown). Since an oligo/EPEI molar ratio of 2.5 resulted in the uptake of the labelled oligo into nearly all cells without apparent cell death, subsequent experiments were performed using this ratio.
Control experiments were next performed to determine whether a control morpholino oligo with a sequence that was not antisense to ETS1 or any known human gene would affect the decidualization of decidual fibroblast cells. As shown in Figure 1, decidual fibroblast cells decidualized with MPA, E2 and dibutyryl cAMP in the presence of the control oligo expressed the same amounts of prolactin, IGFBP-1 and EBAF mRNAs after 6 days of culture as decidual fibroblasts decidualized in vitro in the absence of the oligo. In addition, light microscopy revealed that the morphological appearance of the cells decidualized for 6 days in the presence of the control oligo was not different than the appearance of cells decidualized in the absence of the oligo (data not shown). Taken together, these findings strongly suggest that the control oligo had no effect on the decidualization process.
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Experiments were next performed to examine whether the ETS1 antisense oligo inhibited ETS1 protein levels during decidualization. As shown in Figure 2, decidual fibroblast cells decidualized in the presence of the ETS1 antisense oligo expressed less ETS1 protein than cells decidualized in medium containing the control morpholino oligo or in medium containing no added oligo. ETS1 protein levels in the cells cultured in the presence of the control oligo increased by 2.4- and 2.7-fold on days 3 and 6 of culture, whereas the levels in the cells cultured in the presence of the ETS1 antisense oligo did not increase during decidualization. ETS1 protein levels at the end of the first day of exposure to the ETS1 antisense oligo were 62% less than those of cells exposed to the control oligo and 56% less than those of cells decidualized in control medium alone.
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The ETS1 antisense oligo also markedly blocked the induction of downstream genes that are known to be induced during human decidualization. The left panel of Figure 3 depicts the results of an experiment in which decidual fibroblasts were decidualized for 6 days following exposure to the ETS1 antisense oligo. RNA was collected at the end of the sixth day and analysed for the decidualization marker genes prolactin, IGFBP-1, endometrial bleeding associated factor (EBAF), tissue inhibitor of metalloproteinase-3 (TIMP3), decorin and laminin. GAPDH mRNA levels were used for normalization of the data. Prolactin, IGFBP-1 and EBAF mRNA levels in the cells that had been exposed to the ETS1 antisense oligo were 60–90% less than those of cells exposed to the control oligo. The mRNA levels of TIMP3, decorin and laminin in the ETS1 antisense oligo-exposed cells were 37–41% less than those of cells exposed to the control oligo. The right panel of Figure 3 shows the percentage decrease in prolactin, IGFBP-1, EBAF, TIMP3, decorin and laminin mRNA levels (mean ± standard error) in four separate experiments (the experiment described above and three other experiments performed under identical conditions) in which ETS1 antisense-exposed cells were compared with control oligo-exposed cells at the end of the sixth day of decidualization. The mean decreases in mRNA levels in the ETS1 antisense-exposed cells in the four experiments were comparable with those observed in the representative experiment depicted in Figure 3 (left panel).
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The decidual cells exposed to the ETS1 antisense oligo not only expressed less prolactin, IGFBP-1 and EBAF mRNAs than the cells exposed to the control oligo but also released much less prolactin, IGFBP-1 and EBAF proteins than these cells. The amounts of prolactin, IGFBP-1 and EBAF proteins released by the ETS1 antisense-exposed cells were 53, 99 and 97% less, respectively, than those released by the cells exposed to the control oligo (Figure 4). The amounts of prolactin, IGFBP-1 and EBAF proteins released by the cells decidualized following exposure to the control oligo were nearly identical to those released by control cells that had not been exposed to added oligo before decidualization.
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An additional experiment with the ETS1 antisense oligo was performed in which decidual fibroblasts were decidualized in vitro with MPA, E2 and PGE2 (1 µM) instead of MPA, E2 and dibutyryl cAMP, and mRNA levels were measured by real-time PCR instead of RT–PCR. The cells were exposed to the ETS1 antisense oligo and the control oligo exactly as in the earlier experiments. The cultures were terminated at the end of day 6 and analysed for decidualization marker mRNAs. The cells exposed to the ETS1 antisense oligo expressed less prolactin, IGFBP-1 and laminin mRNAs than the cells exposed to the control oligo (data not shown). The decreases in the amounts of these mRNAs in the PGE2-exposed cells were comparable with the decreases that were noted in the dibutyryl cAMP-exposed cells. The decreases in prolactin, IGFBP-1 and laminin mRNA levels in the PGE2-exposed cells were 65, 86 and 42% less, respectively, than the decreases in the cells exposed to the control oligo.
The ETS1 antisense oligo not only attenuated the induction of decidualization but also inhibited decidualization once the differentiation process had begun. As shown in Figure 5, decidual fibroblast cells that had been exposed to the ETS1 antisense oligo following 3 days of treatment with steroids and PGE2 expressed 58–85% less ETS1, prolactin, IGFBP-1, EBAF, laminin and decorin during the subsequent day than the cells that were exposed to the control oligo.
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Discussion |
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Previous investigations from our laboratory demonstrating that ETS1 protein expression is induced early during in vitro decidualization and that most of the genes that are highly induced during decidualization contain ETS binding sites suggested that ETS1 plays a role in the transcriptional regulation of human decidualization. The results of the present study extend these earlier observations, strongly suggesting a critical and essential role for ETS1 in decidualization. Human decidual fibroblasts decidualized in the presence of an antisense morpholino oligo that blocks the translation of ETS1 mRNA expressed significantly less mRNA for the decidualization markers prolactin, IGFBP-I, EBAF, TIMP3, decorin and laminin than the cells exposed to a control morpholino oligo. In addition, western blot analyses indicated that decidual fibroblast cells decidualized in the presence of the antisense ETS1 oligo released less prolactin and IGFBP-I protein than cells decidualized in the presence of a control oligo that did not inhibit ETS1 expression. In control experiments, the control oligo also had no effect on the induction of decidualization marker mRNAs. The ETS1 antisense oligo not only blocked the induction of decidualization but also inhibited the differentiation process once decidualization had been induced.
While the exact mechanisms by which ETS1 modulates decidualization are unknown, several lines of evidence suggest that ETS1 may interact with the cAMP signal-transduction pathway to induce decidualization. Numerous investigations have shown a critical role for cAMP in the induction of human decidualization (Brar et al., 1997; Gellersen et al., 1997; Gellersen and Brosens, 2003). Factors known to induce cAMP in endometrial stromal cells or decidual fibroblasts have also been shown to induce decidualization. In addition, inhibition of cAMP action in endometrial stromal cells exposed to progesterone and E2 by blocking protein kinase A markedly inhibits decidualization (Brar et al., 1997). The effects of cAMP on gene expression in several cell types are mediated, at least in part, by activation of ETS family members. For example, mutational analyses have shown that the effects of cAMP on the ß-HCG promoter are mediated through an ETS2 enhancer (Johnson and Jameson, 2000). Co-operation of a cAMP response element (CRE) and an ETS motif are important for the function of the FLT1 gene promoter (Wakiya et al., 1996). Greenland and co-workers (Greenland et al., 2000) have also demonstrated that there is crosstalk among ETS1, AP1, cAMP and progesterone-mediated signalling in the regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene. Jayaraman and co-workers (Jayaraman et al., 1999) showed that a cAMP-responsive element-binding protein interacts with ETS1 and ETS2 in the transcriptional activation of the human stromelysin promoter. It is therefore possible that the effect of cAMP on the induction of human decidualization is mediated, at least in part, by ETS1.
Many proteins have been shown to interact with ETS1 on responsive genes, including the estrogen receptor, AP-1, STAT5, GATA3, Sp1 and CBP/p300. In addition, Pit-1/GAF-1 has been shown to interact with the prolactin gene in pituitary cells (Dittmer, 2003; Gellersen and Brosens, 2003). At present, it is unknown whether these or any other proteins interact directly with ETS1 to regulate decidualization. Phosphorylation has been shown to affect the DNA-binding activity of ETS1 (Rabault and Ghysdael, 1994; Kilpatrick et al., 1999; Pufall et al., 2005), but the phosphorylation of ETS1 during in vitro decidualization was not examined in the present study.
In summary, the results of these studies indicate that inhibition of ETS1 expression during in vitro decidualization by an ETS1 antisense oligo causes significant decreases in mRNA and protein levels of genes that are induced during the decidualization process. These findings implicate ETS1 in the regulation of the genetic programme that directs decidualization.
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Acknowledgements |
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We thank Dr James Lessard (Cincinnati Children’s Hospital Medical Center) for providing the ß-actin monoclonal antibody used in the western blot analyses and Dr Edith Markoff for her suggestions. This study was supported by NIH grant NIH-015201 (SH).
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Submitted on September 22, 2005; accepted on December 27, 2005.
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