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Receptor Biology Section (S.C.H., B.J.D., K.H., K.S.K.) and Microarray Group (J.C., S.G., C.A.A.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Sylvia Curtis Hewitt, 111 Alexander Drive, MD E-01, Research Triangle Park, North Carolina 27709. E-mail: curtiss{at}niehs.nih.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ERKO) or ERß (ßERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the ßERKO samples were indistinguishable from those of WT samples, whereas the ERKO showed little change in gene expression in response to E, indicating the predominant role for ER in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(1, 2). Early events include nuclear estrogen receptor (ER) occupancy, transcription of early phase genes such as c-fos, fluid uptake (termed water imbibition), hyperemia, and infiltration of immune system cells such as macrophages and eosinophils into the uterine tissue (3, 4). Later phase responses include the transcription of late phase genes such as lactoferrin, increase in uterine wet weight, further accumulation of immune system cells, the development of the epithelial layer into columnar secretory epithelial cells, and subsequent mitosis, which occurs principally in the epithelial layer (5). Although these responses have been well characterized, several questions remain that can be addressed with the available global genomic expression technologies. One such question is whether early and late responses are the result of altered regulation of a single set of genes or whether there are distinct gene clusters characteristic of both the early and late responses. Secondly, whereas a handful of E-responsive genes have been identified and well characterized, the functions associated with these genes are not in themselves sufficient to describe the dramatic biphasic biological responses observed. A more global gene discovery approach might identify novel targets and coordinated regulation of pathways by E and greatly increase our understanding of the biological response of the uterus to ovarian steroids. Finally, in such a study, the relationship between early and late gene clusters could be examined as well as the question of whether the early responses "prime" later responses. Therefore, using microarray technology, we compared the global expression pattern of uterine RNA from ovariectomized (ovx) control animals to those of ovx animals treated with estradiol for various intervals between 30 min and 24 h. Distinct clusters corresponding to early and late phase responses were apparent in the analysis of the relative gene expression in these samples indicating the biphasic physiological response is mirrored by a biphasic genomic response. The genomic response in mice that lacked either ER [ERKO (ER knockout)] or ERß (ßERKO) examined after 2 h (early) or 24 h (late) of E treatment illustrated the requirement for ER in both early and late responses to E.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ERKO, or ßERKO uterine tissue after ovariectomy and treatment with vehicle or estradiol for 2 or 24 h, and then labeled and hybridized pair wise on multiple chip replicates. As we have previously reported, the ERKO uterine tissue is not biologically equivalent to the WT and ßERKO, as the ERKO uterus does not respond to E and remains hypoplastic and immature (6). For this reason, pair-wise hybridizations were carried out as described in Table 2, within each ER genotype, to minimize effects resulting from biological differences in the uterine tissues. Lists of differentially expressed genes at the 99% confidence levels were created from each of the four replicate array hybridizations of each sample pair. Differentially expressed genes were clustered and displayed in dendograms, with red representing up-regulated genes, and green representing down-regulation (Fig. 1). WT and ßERKO samples exhibited distinct clusters of genes whose expression changed at either 2 or 24 h, as well as clusters of genes regulated at both time points, which indicated the gene changes mirror the biphasic nature of the uterine physiological response. Significantly more genes were regulated at both 2 and 24 h in WT and ßERKO samples than in the ERKO samples.
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), and any differences were comparable to the differences observed within the four biological replicate experiments using WT samples (not shown). Interestingly, although there was a minimal genomic response in the ERKO samples (Fig. 1), the cluster analysis indicated that the ERKO response at 2 or 24 h was more similar to that of the WT at the same time point than to the other ERKO time point. Specifically, the ERKO 2 h treatment clustered with WT 2 h rather than with ERKO 24 h (Fig. 1). This indicated that a biphasic genomic response also occurs in the absence of ER, although the response is greatly attenuated relative to the WT and not sufficient for biological responses of uterine growth. In addition to the decreased number of gene changes, the intensity of responsiveness of the genes in the ERKO (depicted by the intensity of the color in the figure) was diminished compared with WT and ßERKO, indicating the need for ER to mount a full response to estradiol. In the tables published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, supplemental Table 1 lists the significant gene changes identified at the 99% confidence level in at least 75% of the hybridizations for the WT, ERKO, or ßERKO samples. The WT list is a compilation of four independent experiments, the ERKO list from two experiments, whereas the ßERKO list is generated from a single experiment.
Treatment of WT, ßERKO, or ERKO mice with the antiestrogen ICI 182,780 (ICI) 30 min before estradiol injection inhibited the genomic responses at both phases, as shown in Fig. 2. Note that the ICI-treated samples were nevertheless most similar to their time point counterparts of E treatment, indicating they retain some responsiveness. The ICI-mediated attenuation was most profound in the WT and ßERKO samples, again illustrating the overwhelming dependence of the genomic response of the uterus to estradiol on classical ER-mediated mechanisms.
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, the majority of the changes in gene expression at the intermediate times occur within the previously identified early (2 h) or late (24 h) clusters, although the intensity of response was greater for certain genes at the intermediate times (indicated by the relative intensity of the colors). The response after 30 min was minimal, including only six genes, some not previously described as E regulated such as I
B [inhibitor of nuclear factor (NF-B)] and p21 (see Table 3 and supplemental Table 1).
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). These included genes involved in chromatin structure, cell cycle regulation, the thioredoxin system, Wnt signaling, keratinization or cornification, and apoptosis (Table 3). For this study, we have initially focused on verifying the observed regulation of cell cycle modulators to gain insight into their roles in the uterine responses to E.
Estradiol Regulates Cell Cycle Modulators
The patterns of transcriptional regulation of several modulators of cell cycle progression by acute E treatment (Table 3) suggested their potential roles in the timing of the observed uterine biological response. For example, transcription of p21, which causes delay of S phase progression (7), was induced rapidly at 30 min, whereas the transcription of cyclin G1, which is important for progression into S phase (8), was increased after 12 h as DNA synthesis is beginning (see Table 1). Real-time RT-PCR analysis was used to verify the regulation of cell cycle genes observed in the microarray study as well as to examine the regulation of other cell cycle modulators known to be expressed in the uterus. As shown in Fig. 4A, RT-PCR analysis, in agreement with the microarray analysis, showed that induction of p21 transcript peaked at 2 h and again at 12 h, and diminished by 24 h. RT-PCR analysis also verified that transcripts of cyclins G1 as well as E1, a G1/S cyclin (9), were induced after acute E treatment (Fig. 4A), but with a timing that lagged compared with that of p21, as these cyclin transcripts continued increasing after the decrease of p21 after 12 h. Transcripts of cyclin D1, although detected in the uterine samples by RT-PCR, showed little variation in levels after E treatment (Table 4), with a 1.6-fold increase at 12 h. Mitotic arrest-deficient, homolog-like 2 (MAD2), which functions in mitotic spindle assembly at the G2/M checkpoint (10, 11), is rapidly and robustly induced, peaking at 2 h with almost 20-fold induction (Fig. 4A), yet later decreases, mirroring the trend observed by microarray analysis (compare with Table 3). Pretreatment with ICI prevented E-mediated induction of all of these cell cycle regulators (Fig. 4B), additionally, none of these transcripts was seen in the microarray analysis of the ERKO samples (see supplemental Table 1) indicating that E regulation of these transcripts involves the classical ER-mediated mechanism. Generally, we observed that the microarray analysis consistently underestimated the degree of regulation when compared with RT-PCR analysis. For example, the microarray analysis indicated that MAD2 transcript level increased 4.3-fold after 2 h of E, whereas RT-PCR analysis indicated the increase was nearly 20-fold. This underestimation has been previously reported as common in comparison of microarray analysis to other methods (12).
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) and peaked at 12 h, with significant expression remaining at 24 h. ICI pretreatment prevented this increase in protein. Although p21 transcription began increasing 30 min following E treatment (Fig. 4A), p21 protein expression did not increase until 6 h (Fig. 5A), but both the transcript and the protein peaked at 12 h. The degree of regulation at the protein level was not as robust, indicating posttranslational regulation may also occur. Cyclin E1 protein could be detected as a weak signal in the 6-h sample (Fig. 5B) and increased in the 12- and 24-h samples, indicating that protein expression mirrored that of the transcript. Pretreatment with ICI reduced the cyclin E1 expression only at 24 h, however. The profile of MAD2 protein expression by Western blot did not correspond to the RNA profile. MAD2 protein was present in ovx uterine samples (Fig. 5C) but increased at 30 min, decreased at 2 h, then began increasing once more at 12–24 h following E treatment. Pretreatment with ICI decreased MAD2 levels below that seen in the vehicle sample. In this case, the timing as well as the degree of the regulation at the protein level did not correlate with that observed at the transcriptional level, indicating that posttranscriptional regulation may occur as well.
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), there was no distinct staining in uterine sections from 6-h samples (not shown). Uterine cross sections from mice that had been treated for 12 h with estradiol exhibited nuclear staining in a portion of luminal epithelium (LE) cells (Fig. 6A, LE, see arrow), as well as in some stromal (Str) cells. This staining was not observed in serial sections stained with secondary antibody only (Fig. 6B), nor in specimens from mice treated for 12 h with ICI and estradiol together (Fig. 6C). Samples from 24-h estradiol treatment showed some faint epithelial staining as well as some positive Str cells (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Analysis of the genomic response from intervening time points illustrated that genes do indeed fall predominantly within the early or late clusters, rather than revealing intermediate clusters (Fig. 3). The relative lack of responses seen in ERKO samples indicates the dependence of most responses on ER, whereas a response in the ßERKO that is comparable to that of the WT illustrates a minimal role for ERß. Similar observations regarding the relative roles of the ER isoforms in kidney and uterine tissues have been reported in microarray analysis employing chronic E treatment models (13, 14). One aspect of acute E dosing that our model has not yet addressed is the infiltration of the uterine tissue by immune cells, which will result in genomic changes in the total tissue RNA samples that represent recruitment of new cell types rather than changes in transcription.
Previously Identified Estrogen Targets Validate Array Analysis
Predictably, the microarray data set included several uterine genes previously characterized as E responsive (see Table 3). The transcription factor c-Fos, which is known to be rapidly induced by estradiol (15), was induced as early as 30 min after estradiol treatment. Lactotransferrin, which is secreted by the epithelial cells into the lumen, is known to be robustly induced by estradiol (16), and was present in the late gene cluster. It was induced beginning 24 h after estradiol injection. The transcription regulator SRY box-containing gene 4 (SOX4) has also previously been shown to be repressed in the mouse uterus after E treatment (17). Cysteine-rich protein 61 (Cyr61), an IGF binding protein (IGFBP)-like protein that has been reported to be E regulated and may play a role in cancer proliferation and progression (18, 19, 20), was also increased by estradiol in our uterine samples. Several components of the IGF signaling pathway were also regulated by estradiol. IGF-1 and IGFBP5 were both up-regulated, and IGFBP3 was repressed by estradiol treatment (Table 3). The E regulation of these components in the mouse uterus has been previously reported (21, 22, 23). The recognition of these previously characterized E targets in our microarray data set serves as an indicator of the validity of our analysis method. Of these genes, only Cyr61 and IGFBP5 were induced in the ERKO uterus (Table 5), indicating that most of these genes are regulated by a conventional ER-mediated pathway. It is especially interesting that, whereas IGFBP5 retained E responsiveness in the ERKO microarray analysis, it was not detected as regulated in the ßERKO microarray analysis. Future studies to verify this observation might indicate whether IGFBP5 requires ERß for regulation, or whether its expression is mediated by another mechanism. Similarly, SOX4, which is repressed by E at both 2 and 24 h in the WT samples, was repressed at 2 but not at 24 h in the ßERKO. Future verification of this observation might indicate whether ERß plays a role in maintenance of repression of this gene as well. Other genes known to be E regulated such as progesterone receptor and creatine kinase B were not represented on this chip [see http://dir.niehs.nih.gov/microarray/chips.htm for a complete list of genes on National Institute of Environmental Health Sciences (NIEHS) Mouse Chip version 1.0].
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). Additionally, because samples from various times after estradiol treatment were included, we were able to evaluate and compare the time course of potentially regulated genes in these pathways. For example several of the E-regulated genes were involved in chromatin structure or modification. Histone H2A mRNA expression increased 6 h following estradiol treatment, whereas histone H1 family member 2 and histone deacetylase 5 (HDAC5) were repressed. HDAC5 is generally associated with transcriptional repression (24, 25), and its suppression by estradiol treatment might alleviate its repressive activity, allowing transcription to proceed. Thus it is interesting that HDAC5 transcription is down-regulated at 6 and 12 h (Table 3), as uterine RNA transcription in general is increasing (Table 1). Although proliferating tissue may in general require increased quantities of histones as the cell number increases, it is interesting that histone H1 family member 2 levels are repressed (microarray data, Table 3; verified by RT-PCR, Table 4). Further verification and investigation of this observation might reveal an important function of chromatin in regulation of the biological response of the uterus to estradiol.
Components of the thioredoxin system, which regulate activities of enzymes and transcription factors by controlling their reduction state (26), were altered by estradiol (Table 3). Thioredoxin, and thioredoxin reductase were both up-regulated beginning 2 h after estradiol treatment, whereas thioredoxin-interacting protein was rapidly repressed.
Several genes involved in Wnt signaling were also E regulated. Axin 2, an inhibitor of ß-catenin (27, 28), and thus of Wnt signaling, is induced 2 h following estradiol treatment. Fz1, a Wnt receptor that is activated by Wnt3a, 3 and 1 (29), is repressed at the same time point, suggesting suppression of Wnt signaling in the early phase of E response. Casein kinase 1, 1, which stabilizes ß-catenin, (30, 31, 32) is also induced after 2 h of E treatment, as is the ras homolog gene family, member U (33), which is reported to be a Wnt responsive gene. The Wnt signaling pathway directs neonatal patterning of the uterus, and Wnts are expressed in the adult mouse uterus (34). Further investigation of Wnt signaling components that were identified as E regulated in this study has uncovered possible roles in uterine physiological response.
A number of genes associated with keratinization or cornification were also E regulated. Small proline-rich protein 1A and small proline-rich protein 2A (cornifin or SPRR 1A and 2A) are both induced, 2A beginning after 12 h, and 1A beginning 24 h after estradiol treatment. Both of these molecules are cross-linked to keratin by transglutaminase (35, 36), which is induced by E beginning at the 6-h time point. Stratifin, which is expressed in keratinizing epithelia (37) and is also involved in G2/M control (38), is induced at 24 h. Keratoepithelin, also called TGFß induced (68 kDa), is expressed in corneal epithelial cells (39), binds collagen I, II, and IV and is repressed by E beginning at 6 h. Several keratin complex molecules are induced by 24 h of E treatment as well. The keratinization of the vagina and cervix in response to E has been well studied (40), and the induction of these genes may reflect the inclusion of some cervical tissue in the uterine samples. It is interesting that keratoepithelin is actually repressed, suggesting a role other than keratinization.
Apoptosis associated components were also E regulated. Bcl-associated death promoter (BAD), which stimulates apoptosis (41), was induced beginning 12 h after E treatment. Thymoma viral proto-oncogene 1, also called AKT1, a cell survival factor (42), showed increased RNA levels on the microarray (Table 3) that was verified by RT-PCR (Table 4), before the increase in BAD. The activity of the AKT 1 protein has been previously shown to be regulated by E (43); however, this analysis is the first to indicate that the transcription of AKT1 is E regulated as well. NF-B plays a critical role in inhibiting apoptosis (44). Interestingly, IB RNA was rapidly induced, and was only detected in the 30 min samples by microarray (Table 3), but remains elevated through 6 h and then declines according to RT-PCR analysis (Table 4). Estrogen withdrawal induces apoptosis in the uterus (45, 46), possibly to allow embryo invasion. The relative regulation of apoptosis genes in our study suggests E might coordinate the timing of apoptosis in the uterus. Future studies will indicate the significance of this finding to uterine physiology.
Receptor (calcitonin) activity modifying protein 3 (RAMP3), a protein that is involved in transport of calcitonin like receptors to the plasma membrane, and in modification of receptors to determine their ligand specificity (47, 48), was robustly and rapidly induced (Table 3), and this induction was verified by RT-PCR (Table 4). This protein has not been previously reported to be E regulated, and its induction suggests a possible role in calcitonin-like ligand signaling in preparation of the uterus for pregnancy, and warrants further investigation.
Estrogen Regulates Cell Cycle Modulators: Potential Role in Synchronous S Phase Progression
For the purposes of this initial report, we have further characterized the genomic response of several genes involved in cell cycle progression after acute E treatment. Regulation of these genes in this uterine model is not surprising, considering the coordinated increased uterine DNA synthesis and mitosis reported to begin 12–24 h following E treatment of ovx mice, indicative of synchronized entry into S phase (5). Thus, uterine epithelial cells must not only proliferate, but must do so at the proper time as the estrous cycle progresses, in preparation for implantation of embryos. Synchronous/coordinated regulation of the components modulating entry into S phase is one mechanism by which the acute exposure to the hormone estradiol might orchestrate this event. Estrogen regulation of some cell cycle modulators in the uterus has been previously reported. For example, E treatment induces nuclear relocalization of cyclin D1 protein and increase in expression of cyclins A and E proteins in the uterine epithelium (49). p21 protein expression has been shown to increase in the uterine LE 1–2 d after E treatment (50). It is especially interesting that p21, which inhibits progression into S phase, is maximally induced at both the RNA and protein levels (Figs. 4 and 5) and is localized to the nucleus 12 h following estradiol treatment (Fig. 6), just before the peak of entry into S phase, suggesting that the increased p21 may prevent S phase progression of the epithelial cells until the proper time, allowing coordinated proliferation of the epithelial cells. Thus, the properly timed increase in p21 may act as a gate, coordinating appropriate S phase progression.
Although p21-deficient mice exhibit normal fertility (51), other cdk inhibitors may compensate for the lack of p21. For example, the cdk inhibitor p27, which is not included on the microarray chip used for this study, is expressed in the uterus but is decreased after E treatment (49). Additionally, p27-deficient female mice are infertile due to both ovarian and uterine deficiencies (52), suggesting an essential role in the uterus. p21 and p18 (ink 4 family) were the only cdk inhibitors included on the microarray chip used in this study, and p18 was not differentially expressed. Although p18 is known to be important in male fertility, p18-deficient female mice are fertile (53). The uterine response of cdk-deficient mice (p21, p18, p27) to acute E dosing in terms of a properly timed coordinated increase in DNA synthesis has not yet been studied.
The moderate increase in MAD2 transcript at 24 h (Fig. 4) and protein at 12–24 h (Fig. 5) as the epithelial cells prepare for mitosis at 16–24 h is consistent with the role of MAD2 in mitotic spindle assembly at the G2/M checkpoint (10). However, the almost 20-fold increase in MAD2 transcript at 2 h as well as the presence of MAD2 protein in the vehicle and 30-min samples, when the cells are mitotically quiescent, is puzzling. Perhaps the early increase in MAD2 acts to delay progression past the G2/M checkpoint until all the molecules necessary for synchronous S phase entry have accumulated. Alternatively, the expression of MAD2 in nonmitotic cells might indicate a novel function for this protein.
Other Uterine Genomic Profiles
The model we applied in this study attempts to mimic the events just subsequent to the preovulatory estradiol surge, addressing a normal biological process. Several other studies of rodent uterine genomic response to E have been published; however, the study designs and biological issues addressed differed from ours. Naciff et al. (54) treated rat embryos with 17-ethinyl estradiol or xenoestrogens on gestational d 11–20 and examined the genomic response in the developing reproductive tract as a model of embryonic exposure to estrogenic compounds. In another study, RNA was prepared from rat uterine RNA after 48 d of chronic treatment with estradiol and compared with vehicle-treated samples to identify E-regulated genes (23). In a third study, ovx rats or mice were treated with vehicle or E for 6 wk and uteri and kidneys collected for analysis to address the effect of chronic E treatment on gene expression (14). These three studies have in common a chronic exposure to E, unlike our study, which addresses a normal biological situation of events just subsequent to the preovulatory estradiol surge. In contrast, Reese et al. (55) attempted to address the biological situation that occurs at the time of implantation in the mouse uterus. As part of their analysis, a delayed implantation model was used whereby the ovaries were removed and replaced with progesterone to delay embryo implantation until estradiol replacement was given. Microarray analysis using these samples identified uterine genes induced 12 h following estradiol administration. Several genes they reported have been previously identified as E-regulated uterine genes or were also seen in our analysis, including lactoferrin, MAD2, small proline rich protein 2A, cyclin E2, keratin complex 1, Histone H2A.1, and Cyr61.
The advent and application here of microarray technology using a study design that mimics the physiological response of the reproductive tract to acute E has illustrated that the genomic response reflects the biphasic biological events. Additionally, application of this approach to transgenic - and ßERKO models indicated the genomic responses primarily require ER. Finally, examination of the identities of other regulated gene families has suggested many new avenues of study that will enhance our understanding of the spectrum of mechanisms that influence the biological response of the uterus to acute E.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ERKO or ßERKO or WT littermates were obtained from Taconic Farms (Germantown, NY), and were ovx and housed for at least 10 d before studies to allow endogenous ovarian steroids to decrease. Groups of animals were treated with sesame oil vehicle (Sigma, St. Louis, MO), or with 1 µg estradiol (Steraloids, Newport, RI) either dissolved in 100 µl sesame oil and injected sc (6-, 12-, and 24-h groups) or 100 µl normal saline and injected ip (30-min and 2-h groups). Some animals were treated with 45 µg ICI (kindly provided by Zeneca Pharmaceuticals, Cheshire, UK) (dissolved in 50 µl dimethylsulfoxide injected ip) 30 min before estradiol injection. Animals were killed at the indicated times using CO2 and the uterus was collected and weighed. In some cases, a portion of the uterus was fixed in 10% formalin (Fisher Scientific, Suwannee, GA), whereas the remainder was snap frozen in liquid nitrogen for later RNA or protein isolation.
Microarray Analysis
Frozen uterine tissue was pooled (at least five uteri per group), pulverized, then homogenized in Trizol (Invitrogen, Carlsbad, CA) and RNA was prepared according to the manufacturer’s protocol. Isolated RNA was then further purified using the QIAGEN (Valencia, CA) Rneasy midi (100–500 mg RNA) or mini prep kit (<100 mg RNA) clean-up protocol.
Hybridizations and Data Analyses
A cDNA Mouse Chip (NIEHS Mouse Chip version 1.0), developed in-house at NIEHS, was used for gene expression profiling experiments. A complete listing of the genes on this chip is available at the following web site: http://dir.niehs.nih.gov/microarray/chips.htm. cDNA microarray chips were prepared according to DeRisi et al. (56). The spotted cDNAs were derived from a collection of sequence verified IMAGE clones that covered the 5' end of the gene and ranged in size from 500-2000 bp (Incyte Genomics, Palo Alto, CA). M13 primers were used to amplify insert cDNAs from purified plasmid DNA in a 100-µl PCR mixture. A sample of the PCR products (10 µl) was separated on 2% agarose gels to ensure quality of the amplifications. The remaining PCR products were purified by ethanol precipitation, resuspended in ArrayIt Spotting Solution Plus buffer (Telechem, San Jose, CA) and spotted onto poly-L-lysine coated glass slides using a modified, robotic DNA arrayer (Beecher Instruments, Bethesda, MD). Each total RNA sample (10 µg) was labeled with Cyanine (Cy) 3- or Cy5-conjugated deoxyuridine triphosphate (Amersham, Piscataway, NJ) by a reverse transcription reaction using the reverse transcriptase, SuperScript (Invitrogen, Carlsbad, CA), and oligo-deoxythymidine primers (Amersham). The fluorescently labeled cDNAs were mixed and hybridized simultaneously to the cDNA microarray chip. Each RNA pair was hybridized to at least four arrays, employing a fluor reversal accomplished by labeling the control sample with Cy3 in two hybridizations and with Cy5 in the other two hybridizations. The cDNA chips were scanned with either an Axon Scanner (Axon Instruments, Foster City, CA) or an Agilent Scanner (Agilent Technologies, Wilmington, DE) using independent laser excitation of the two fluors at 532- and 635-nm wavelengths for the Cy3 and Cy5 labels, respectively.
The raw pixel intensity images were analyzed using the ArraySuite version 2.0 extensions of the IPLab image processing software package (Scanalytics, Fairfax, VA). This program uses methods that were developed and previously described by Chen et al. (57) to locate targets on the array, measure local background for each target and subtract it from the target intensity value, and to identify differentially expressed genes using a probability-based method. The data were filtered to provide a cut off at the intensity level just above the buffer blank measurement values to remove from further analyses those genes having one or more intensity values in the background range. After pixel intensity, determination and background subtraction, the ratio of the intensity of the treated cells to the intensity of the control was calculated using Chen’s method (57). Genes having normalized ratio intensity values outside of a 99% confidence interval were considered significantly differentially expressed. The lists of differentially expressed genes at the 99% confidence levels were created and deposited into the NIEHS Microarray Project System database (58). Any of these genes that indicated fluor bias or high variation were not considered for further analysis, and genes that were represented in 75% of the hybridization replicates were compiled for clustering. Clustering was conducted using the algorithm provided by Eisen (59).
The entire data are available at the web site http://dir.niehs.nih.gov/microarray/hewitt/.
Verification of Microarray Results by Real-Time RT-PCR
Ovx WT mice were treated as described for the microarray analysis, and RNA was prepared from mice treated with vehicle (20 mice) or 1 µg estradiol with (four mice per group) or without (six mice per group) pretreatment for 30 min with 45 µg of ICI and then collected 30 min, 2 h, 6 h, 12 h, or 24 after estradiol treatment. Uteri were frozen in liquid nitrogen, two uteri were pooled and pulverized, and RNA was prepared using Trizol reagent according to the manufacturer’s protocol.
Reverse Transcription
To remove genomic DNA, RNA samples were incubated with 1 U of deoxyribonuclease I (DNaseI) (Invitrogen Corp.) per microgram of RNA for 15 min at room temperature. DNaseI was then inactivated by addition of 2.5 mM EDTA (pH 8.0) and heating at 65 C for 10 min. Reverse transcription of RNA (2 µg) using Superscript II (400 U) was carried out according to the manufacturer’s instructions using oligo-deoxythymidine primers (Invitrogen Corp.). The resulting cDNA was treated with 4 U ribonuclease H (Invitrogen Corp.) for 20 min at 37 C to remove RNA:DNA hybrids. As a negative control, a sample containing RNA but no reverse transcriptase (minus RT) was also included. The resulting samples were diluted to 200 µl with DNase-free water. Ten microliters of this cDNA were used per well in 96-well plates for real-time PCR analysis.
Real-Time PCR Analysis
cDNA levels were detected using real-time PCR with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and SYBR Green I dye. Primers were created using Applied Biosystems Primer Express Software version 2.0 (see supplemental Table 2). For cDNA amplification, 10 µl of cDNA was combined with 40 µl of a mixture containing SYBR Green PCR core reagents (Applied Biosystems, catalog no. 4304886) at the following concentrations: 1x PCR buffer, 4 mM MgCl2, 50 nM each of deoxy (d) ATP, dCTP, dGTP, and deoxyuridine triphosphate, 0.5 U AmpErase uracil-N-glycosylase, 1.25 U AmpliTaq Gold DNA Polymerase, and 200 nM reverse and forward primers. Samples were analyzed in triplicate, and a minus RT sample was included with each plate to detect contamination by genomic DNA. Amplification was carried out as follows: 1) 50 C, 2 min (for uracil-N-glycosylase incubation); 2) 95 C, 10 min (denaturation); 3) 95 C, 15 sec, 60 C, 30 sec (denaturation/amplification). Dissociation curves were also created by adding the following steps to the end of the amplification reaction: 95 C, 15 sec (denaturation), 60 C, 20 sec, then gradually increasing to 95 C over 20 min finally holding at 95 C for 15 sec. Fold expression or repression was determined by quantitation of cDNA from target (E treated) samples relative to a calibrator sample (vehicle). For all samples, the gene for ribosomal protein 7 (rPL7) was used as the endogenous control for normalization of initial RNA levels. To determine this normalized value, 2-
CT values were compared between target and calibrator samples, where Ct = target gene (crossing threshold) Ct - rPL7 Ct, and Ct = Ctcontrol - Cttreatment.
Western Blotting
Protein extracts were prepared by homogenization of a portion of the uterus, treated as described above, in homogenization buffer with added protease inhibitors and phosphatase inhibitors (Sigma) as in (60). Fifty micrograms of protein were loaded into each lane of NuPage (Invitrogen) 10% (p21 and cyclin E1) or 12% (MAD2) gels and proteins were separated and transferred to nitrocellulose membrane using the manufacturer’s protocol and reagents. Membranes were stained with Ponceau S Solution (Sigma) to visualize equal protein loading and transfer (not shown). Mouse monoclonal antibodies against p21 were purchased from BD Pharminigen (San Diego, CA) and used at 1:500. Rabbit polyclonal antibody against MAD2 was generously provided by Dr. Salmon (University of North Carolina, Chapel Hill, NC) and used at 1:2500. Rabbit polyclonal antibody against cyclin E1 was purchased from Upstate Biotechnologies (Lake Placid, NY) and used at 2 µg/ml. Membranes were blocked with 5% milk in Tris-buffered saline (TBS)-Tween 20, then incubated with primary antibody diluted in 5% milk TBS-Tween, and finally in horseradish peroxidase (HRP)-conjugated secondary antibody [antirabbit HRP (Cell Signaling, Beverly, MA; 1:2000), antimouse HRP (Amersham; 1:1000)], also diluted in 5% milk TBS-Tween. Bands were visualized with ECL reagent (Amersham) and Hyperfilm (Amersham).
Immunohistochemstry
Paraffin-embedded sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, then processed in a decloaking chamber (Biocare Medical, Walnut Creek, CA) in Citrate buffer (Biocare) for 3 min. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 10 min. p21 was detected using a mouse monoclonal antibody (sc 6246; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:10 and Vector M.O.M. kit (Vector Laboratories, Burlingame, CA) and accompanying protocol with diethylaminobenzidine substrate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BAD, Bcl-associated death promoter; Ct, crossing threshold; Cy3 or 5, cyanine 3 or 5; Cyr61, cysteine-rich protein 61; DNase, deoxyribonuclease; E, estrogen; ER, estrogen receptor; ERKO, ER knockout; ERKO, mice that lacked ER; ßERKO, mice that lacked ERß; HDAC5, histone deacetylase 5; HRP, horseradish peroxidase; ICI, antiestrogen ICI 182,780; IGFBP, IGF binding protein; IB, inhibitor of NF-B; LE, luminal epithelium; MAD2, mitotic arrest-deficient, homolog-like 2; NIEHS, National Institute of Environmental Health Sciences; NF-B, nuclear factor-B; ovx, ovariectomized; SOX4, SRY box-containing gene 4; Str, stromal; WT, wild-type.
Received for publication April 21, 2003. Accepted for publication July 14, 2003.
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