ToxSci Advance Access originally published online on March 9, 2007
Toxicological Sciences 2007 97(2):467-490; doi:10.1093/toxsci/kfm046
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Uterine Temporal Response to Acute Exposure to 17
-Ethinyl Estradiol in the Immature Rat
The Procter and Gamble Company, Miami Valley Innovation Center, Cincinnati, Ohio 45253
1 To whom correspondence should be addressed at The Procter and Gamble Company, Miami Valley Innovation Center, PO Box 538707 #805, Cincinnati, OH 45253-8707. Fax: (513) 627-0323. E-mail: naciff.jm{at}pg.com.
Received January 19, 2007; accepted February 20, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17
-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 µg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p 
0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response. Key Words: estrogens; microarray; female reproductive tract; temporal gene expression; uterotrophic response.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Immediately before ovulation serum estrogen levels rise rapidly, and in response to this surge the uterus exhibits well-characterized physiological and biochemical responses (Clark and Mani, 1994
). Among the morphological and physiological changes induced in the uterus after the estrogen surge, it has been established that there is an increase in water imbibition in the uterine endometrium as a result of hyperemia and vasodilation of uterine capillaries. This process leads to swelling of the uterus with a consequent significant decompaction of stromal cells. This water then moves from the epithelium into the lumen, leading to a decrease in viscosity of uterine luminal fluid and an increase in uterine wet weight. The secretory capacity of the uterus is highly increased by estrogen stimulation, mainly due to the development of the epithelial layer into columnar secretory epithelial cells, and subsequent mitosis, principally in the epithelial layer. At the same time, there is an increase in the vascularization of the uterus, which increases the amount of blood in this organ (hyperemia), accompanied by infiltration of immune system cells such as macrophages and eosinophils. This event mimics an acute inflammatory response. There is also an increase in the number and size of specific cell types (hypertrophy and hyperplasia), which also contributes to the large increase in uterine weight that can be detected between the 16 and 30 h after estrogen stimulation ("classical" uterotrophic response).
Given the importance of estrogen in the physiology of multiple tissues during development and adulthood, one of the highest priorities in research in this field is to better understand the molecular mechanism of estrogen action. There is also concern about exposure to chemicals with possible endocrine activity that could affect estrogen action. The predominant biological effects of estrogens are mediated through at least two distinct intracellular receptors: estrogen receptor (ER) alpha and ER-beta (Klinge, 2001; Nilsson et al., 2001), which are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Both types of ERs are physiologically important, and they have distinct and nonoverlapping functions in different tissues and organs of the body (Britt and Findlay, 2002; Couse and Korach, 2001; Couse et al., 2001; Curtis and Korach, 2000). The ERs function as ligand-activated transcription factors which in combination with multiple regulatory proteins are able to modulate the expression of specific genes, by activation or repression. It is believed that through this mechanism, estrogens are able to regulate the expression of genes whose products are involved in development, differentiation of specific cellular types, reproduction, and other functions.
Since regulation of gene expression is integral to the signal transduction pathway for estrogens, global analysis of the gene expression changes in estrogen-sensitive tissues offers the opportunity to understand the underlying molecular effects that lead to physiological and pathophysiological responses after exposure to active levels of estrogens. Although multiple estrogen-regulated genes have been identified thus far (see, e.g., Daston and Naciff, 2005; Klinge, 2001; Kwekel et al., 2005; Moggs et al., 2004; Naciff et al., 2002, 2003, 2005; Waters et al., 2001) the number and identity of the gene expression changes induced by estrogens are still limited. In particular, our understanding of the temporal characteristics of those gene expression changes is still fragmented (Currie et al., 2005; Moggs et al., 2004; Moggs 2005; Kwekel et al., 2005).
In order to better understand the temporal relationship between the changes in gene expression and the phenotypic changes induced by estrogen exposure, we have expanded our initial analysis of the estrogen response in the immature rat uterus by identifying the temporal gene expression profile induced by a single dose of 17-ethinyl estradiol (EE), 10 µg/kg, in the rat uterus (from postnatal day [PND] 20 to day 24). We chose the prepubertal rat since at this developmental stage the concentration of 17ß-estradiol, the endogenous hormone, is consistently low (Noda et al., 2002), but exposure to estrogens during the prepubertal period can induce an uterotrophic response. The morphological and physiological changes that comprise the uterotrophic response can be used to phenotypically anchor the gene expression changes induced by estrogen exposure. Our approach will help to better understand the quantitative relationships between changes in gene expression and manifestations of estrogenicity and will assist to generate a mechanistic understanding of the mode of action of chemicals with estrogenic activity. At the same time, this information will be valuable to refine the molecular signature for chemicals with estrogenic activity that we have identified in previous studies (Naciff et al., 2003, 2004, 2005). It will also help to generate a robust transcript profile to be used as the basis for screening chemicals for estrogenicity, either in vivo as a refinement to current tests or in vitro, using human-derived cell lines which genomic response to estrogen exposure shares similarities to the in vivo response.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Chemicals.
EE and peanut oil were obtained from Sigma Chemical Company (St Louis, MO).
Animals and treatments.
Fifteen-day-old female Sprague Dawley rats were obtained (Charles River VAF/Plus) in groups of 10 pups per surrogate mother. This rat strain was chosen in order to be consistent with previous gene expression studies conducted in our laboratory. The rats were acclimated to the local vivarium conditions (24°C; 12-h light/12-h dark cycle) for 5 days. Starting on PND 20 all rats were singly housed in 20 x 32 x 20 cm plastic cages. The animals were fed a casein-based diet, essentially phytoestrogen free (Purina 5K96; Purina Mills, St. Louis, MO) from PND 16 onward, to override any possible effects of the standard rodent diet (Purina 5001) used by the animal supplier. The casein-based diet consistently contains less than 1 ppm aglycone equivalents of genistein, daidzein, and glycitein (Purina Mills). The experimental protocol was carried out according to Procter and Gamble's approved animal care protocols, and animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
On PND 20, the animals were randomly assigned to 14 groups of 10 animals each (seven control and seven EE-treated groups, respectively) and dosed once by sc injection, with 0 or 10.0 µg EE/kg in peanut oil. Animals received 5 ml/kg body weight of dose solution. The dose was administered to each animal in a staggered fashion, within the group and between groups, to ensure that the time of exposure was within 5% of the reported exposure length. Body weights (nearest 1.0 g) were recorded daily and before sacrifice. The animals were sacrificed by CO2 asphyxiation at 1, 2, 8, 24, 48, 72, or 96 h after dosing. The body of the uterus was cut just above its junction with the cervix and leaving the ovaries attached to it, carefully dissected free of adhering fat and mesentery, and weighed as a whole. Then the ovaries were dissected free, and the uterine and ovarian wet weight were recorded. The individual uteri were then placed into RNAlater (50–100 mg/ml of solution; Ambion, Austin, TX), at room temperature.
Histology.
Reproductive tissues from two animals in each dose and time group were fixed in 10% neutral buffered formalin immediately after weighing, dehydrated, and embedded in paraffin. Serial 4- to 5-µm cross-sections were made through equivalent regions on the oviducts and uterine horns and stained with hematoxylin and eosin. To evaluate the morphological changes induced by EE exposure in the uterus, we focused on the proliferative state of the endometrial stroma and luminal epithelium along the uterine horns. Tissue sections from the mid-region of each uterine horn were evaluated for epithelial cell height in five different areas of the epithelium lining the lumen, using a light microscope (Zeiss Axioplan) interfaced with an AxioCam MRc5 high-resolution digital camera (Carl Zeiss Vision GmbH, München-Hallbergmoos, Germany).
Expression profiling.
Total RNA was extracted from RNAlater-treated uteri of individual animals using TRI-REAGENT (Molecular Research Center, Inc., Cincinnati, OH), 24 h after tissue collection. Total RNA was further purified by RNeasy kit (Qiagen, Valencia, CA). Ten micrograms of total RNA from each tissue sample (individual animals) was converted into double-stranded cDNA using SuperScript Choice system (GIBCO BRL, Rockville, MD) with an oligo-dT primer containing a T7 RNA polymerase promoter. The double-stranded cDNA was purified by phenol/chloroform extraction and then used for in vitro transcription using ENZO BioArray RNA transcript labeling kit (Enzo Life Sciences, Inc., Farmingdale, NY). Biotin-labeled cRNA was purified by RNeasy kit (Qiagen), and a total of 20 µg of cRNA was fragmented randomly to 
200 bp at 94°C for 35 min (200mM Tris-acetate, pH 8.2, 500mM KOAc, 150mM MgOAc). Labeled cRNA samples were hybridized to the Affymetrix GeneChip Test 3 Array (Affymetrix Inc., Santa Clara, CA) to assess the overall quality of each sample. After determining the target cRNA quality, samples from five individual females (replicates) from each treatment group with high-quality cRNA were selected and hybridized to Affymetrix Rat Genome 230A high-density oligonucleotide microarrays for 16 h. The microarrays were washed and stained by streptavidin-phycoerythrin (SAPE) to detect bound cRNA. The signal intensity was amplified by second staining with biotin-labeled anti-streptavidin antibody and followed by SAPE staining. Fluorescent images were read using the Hewlett-Packard G2500A Gene Array Scanner.
Real-time reverse transcriptase–polymerase chain reaction.
In order to corroborate the relative changes in gene expression in selected genes identified by the oligonucleotide microarrays, we used a real-time (kinetic) quantitative reverse transcriptase–polymerase chain reaction (QRT-PCR) approach, as described (Naciff et al., 2003). Table 1, in supplementary data, shows the nucleotide sequences for the primers used to test the indicated gene products. Preliminary experiments were done with each primer pair to determine the overall quality and specificity of the primer design. To confirm the amplification specificity from each primer pair, the amplified PCR products were size fractioned by electrophoresis in a 4% agarose gel in Tris borate ethylene diamine tetracetic acid buffer and photographed after staining with ethidium bromide. After QRT-PCR, only the expected products, at the correct molecular weight, were observed.
Data analysis.
Potential interindividual variability was addressed by using independent samples of each dose group (n = 10 or 5 for uterine weight or transcript profile, respectively) for analysis. For the uterine weight determination, we evaluated 10 animals per dose group (Fig. 1). For gene expression analysis, scanned output files of Affymetrix microarrays were visually inspected for hybridization artifacts and then analyzed using Affymetrix Microarray Suite (version 5.0) and Data Mining Tool (version 3.0) software, as described (Lockhart et al., 1996; http://www.affymetrix.com/index.affx). Arrays were scaled to an average intensity of 1500 units and analyzed independently. The Affymetrix GeneChip Rat Expression 230A microarrays used in this study have 15,923 probe sets corresponding mostly to well-annotated rat genes, although it has some expressed sequence tags (ESTs). For a full description of the Rat 230A array content see http://www.affymetrix.com/products/arrays/specific/rat230.affx. Each gene or EST is represented by 11 pairs of 25-mer oligonucleotides that span the coding region. Each probe pair consists of a perfect match sequence that is complementary to the cRNA target and a sequence that is mismatched by a single base change at the middle of the nucleotide, a region critical for target hybridization. The mismatched oligonucleotide serves as a control for nonspecific hybridization. For the entire analysis, each probe set was analyzed as an individual entity, based upon its Affymetrix ID number, regardless of the multiplicity of probe sets representing any given gene product, and considered as representing an individual gene until the completion of the analysis. Distinct algorithms made an absolute call, present/marginal/absent, for each transcript, and calculated the average difference between perfect match and mismatch probe pairs (signal value). The mathematical definitions for each algorithm are described in the Affymetrix Microarray Suite User's Guide, version 5.0.
|
The gene expression values derived from The Microarray Analysis Suite (MAS 5.0; Affymetrix Inc.), hereafter termed signal, were evaluated for quality based upon both overall measures of the microarray quality and simple outlier detection methods. Four microarrays, one 2-h control, one 2-h treated, and two 24-h treated, were consistently flagged as outliers in a simple outlier detection analysis. These analyses led to the removal of these four samples (microarrays) from analyses for treatment effect.
The samples (microarrays) that were retained from the QC analysis were then analyzed for treatment and time effects using a linear model for the log signal value that is a function of both treatment and time, where time is treated as a nominal factor. For each probe set (gene) separately, treatment effect was assessed by comparing the model that estimates signal uniquely for each combination of time and treatment to the model that estimates signal for each time, but with no allowance for any treatment effect. This approach makes no assumptions about the functional form of the time course of response, which is unlikely to be of a uniform magnitude for all time points. For the purposes of summarization, follow-up analyses that evaluate treatment effect at each time point were also conducted. An analysis of the effect of time was also conducted on control samples only, using a standard single-factor analysis of variance (ANOVA) on the log signal response. In this case, each set of control samples from each time point were compared to the control samples of the 1-h group. Probe sets (genes) for which any of the tests had p 0.0001 was taken as evidence that the expression of these probe sets (genes) was modified by EE treatment at the time being tested. This procedure was done for each time point versus control, and for the full group of study results (vehicle vs. EE treated, at all time points). Fold-change summary values for genes were calculated as a signed ratio of mean signal values (for each time point group compared with the appropriate control). Because fold-change values can become artificially large or undefined when mean signal values approach zero, all the values < 100 were made equal to 100 before calculating the mean signal values that are used in the fold-change calculation. Note that all statistical analyses use the measured signal values, even if they were smaller than 100 units. After selecting all the significant probe sets, for any given time being evaluated, for vehicle control and EE-treated samples, we remove redundant probe sets representing the same gene product, leaving only one probe set (with the most significant p value) per gene listed for each group.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
In order to identify the gene expression changes associated with the histological response of the uterus, juvenile female rats (20 days of age), 10 per time point, were given a single sc injection of EE (10 µg/kg) or vehicle control (peanut oil). The temporal uterotrophic response as well as the gene expression pattern elicited in the uterus were evaluated 1, 2, 8, 24, 48, 72, and 96 h after dosing.
Histological Changes Induced by a Single Dose of EE
The time course of the uterine weight change after EE exposure is shown in Figure 1A. As can be seen, a clear uterotrophic response is elicited by 10 µg EE/kg 8 h after treatment and is maximal at 24 h. The increase in uterine weight is significant even after 96 h of exposure. The increase in uterine weight is not accompanied by significant changes in the body or ovarian weights (Figs. 1B and C, respectively).
The histological changes induced by EE exposure are shown in Figure 2, in supplementary data, and are concordant with the gravimetric data. The uterine weight increase observed at 8 h is associated with thickening of the stromal endometrium (Fig. 2, at 8 h, in supplementary data), which is believed to result from a modest uptake of fluid (water imbibition). The largest increase in uterine weight at 24 h (Fig. 2, in supplementary data) is the result of hypertrophy and hyperplasia (Anderson et al., 1974) as well as accumulation of fluid in the lumen. Hypertrophy and hyperplasia are particularly evident in the uterine luminal epithelium. In control animals the uterine epithelium consists of two to three layers of cuboidal cells (Fig. 2, controls, in supplementary data). After EE treatment, these cells change from cuboidal- to cylindrical-shaped, vacuolated cells (Fig. 2, EE at 8–96 h, in supplementary data). The thickness of the luminal epithelium increases to six to eight layers of cells, and the luminal epithelial cell height is increased significantly (p 0.001) compared with vehicle-treated controls. Hypertrophy of stromal and myometrial cells, thickening of the stromal layer, and some stromal inflammatory reaction are evident between 24 and 48 h and can still be seen after 96 h after treatment (Fig. 2, in supplementary data). After 72 and 96 h of EE exposure, apoptotic cell bodies are detectable in the stroma and glandular and luminal epithelia, consistent with the decrease of the uterine weight.
Temporal Gene Expression Changes Induced by EE
A single dose of EE induced statistically significant changes in the expression of 3837 genes of the uterus, at least at one time point (p 0.0001 and 
1.5-fold change as compared to the appropriate control, up- or downregulated). Although there are quantifiable gene expression changes at each time point evaluated, the largest number of genes being affected by EE occurs 8 h after treatment. Of the 15,923 rat annotated genes and ESTs evaluated, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes is modified after 1, 2, 8, 24, 48, 72, or 96 h of exposure to 10 µg/kg EE, respectively (p 0.0001, t-test, and 1.5-fold change as compared to the appropriate control, up- or downregulated).
The temporal transcriptional response of the affected genes is shown in an Eisen diagram (Fig. 3, in supplementary data), in which the relative expression levels of the 3837 probe sets showing the most robust response are compared at the different times of EE exposure. The genes are ordered by similarity of temporal expression through a "dendrogram" diagram. Each cell of the data table is represented as a color-coded rectangle in which the color indicates the relative expression value (normalized fold change) of unaffected (white), upregulated (red), or downregulated (blue) genes.
The temporal expression pattern suggests that the uterine response to EE involves changes in the transcriptional status of genes that could be classified as immediate-early (before the first 8 h of EE exposure), immediate-late (between 8 and 24 h of EE exposure), late (after 24 h of EE exposure), and sustained responsive genes. As an example of the specificity that EE exerts on the expression of individual genes, the temporal response to EE exposure of eight selected genes is shown in Figure 4, in supplementary data. As can be appreciated in this figure, the biological variability, in the majority of the cases, is minimal. The relevance of the temporal analysis of the gene expression changes induced by activation of the ER pathway by a single dose of EE is clearly illustrated in the eight genes shown.
In order to help identify associations between the estrogen-responsive genes and specific biological processes affected by EE treatment, we used the Gene Ontology (GO) terms associated with these genes. The probe sets representing ESTs were not included for this analysis since there is no annotation associated to them. The GO (http://geneontology.org/) is a detailed description of molecular, cellular, and organismal biology that is organized into a data structure that links individual genes to biological function categories. In order to assign a unique GO term to individual probe sets identified as statistically showing expression changes induced by EE exposure, we mapped the probe sets selected from the statistical analysis of the data (p 0.0001, t-test) to their corresponding annotations in the GO using the three major branches of the GO: biological process, cellular component, and molecular function. Redundancies due to multiple probe sets representing the same gene were eliminated for this analysis. The probe set selected showed the most statistically robust response. Each gene was assigned to all possible biologically descriptive GO terms and processed to identify clusters of genes representing unique GO terms. The clusters in the ontology were identified using the algorithm of Joslyn et al. (2004) in combination with Fisher's exact test (Beissbarth and Speed, 2004) to estimate the statistical significance of the mapping. This analysis indicated that EE elicits changes in the expression of genes associated with 100 unique GO biological processes, 14 unique GO molecular functions, and 24 unique GO cellular components. The clusters associated with the unique GO biological process are shown in Table 2 (in supplementary data). The GO annotation of some of the genes affected by EE treatment indicates biological processes that at first glance may seem irrelevant to describe the uterotrophic response, e.g., development, morphogenesis, and organogenesis. However, these are parent GO terms that encompass multiple biological processes, some of which are relevant to understand the underlying mechanism of action of estrogens in the uterus. For example, the GO term development encompasses cell growth and angiogenesis, among others. Further, the GO terms listed on the table also indicate that genes linked to these terms must encode products for which functions are linked to the uterine response to estrogen but which have not yet been characterized.
The GO molecular functions affected by EE exposure are the following: threonine endopeptidase activity, ligase activity, forming phosphoric ester bonds, signal transducer activity, receptor activity, major histocompatibility complex class II receptor activity, DNA binding, transcription factor activity, translation initiation factor activity, protein binding, tubulin binding, calcium ion binding, cyclin-dependent protein kinase regulator activity, and transcription regulator activity. The majority of the products encoded by the genes whose expression is regulated by estrogen are associated with intracellular components. However, a significant number of these products are also associated with membranes or with components of the extracellular space.
Further, the genes identified as EE responsive at the different times were also mapped against a set of 141 canonical pathway maps from the Kyoto Encyclopedia of Genes and Genomes (KEGG). Most of these pathways represent small-molecule metabolism, and a few describe portions of signal transduction and/or gene regulation networks (http://www.genome.jp/kegg/). From the metabolic pathways mapped, the ones that are significantly regulated by estrogen exposure include steroid and bile acid metabolism, fatty acid metabolism, glutathione metabolism, glycolysis/gluconeogenesis, oxidative phosphorylation, one-carbon pool metabolism, purine metabolism, terpenoid biosynthesis, and amino acid metabolism (Table 2, in supplementary data). From the pathways representing signal transduction and/or gene regulation networks that were mapped, the ones that showed significant regulation by EE exposure include vascular endothelial growth factor (VEGF), canonical Wnt pathway (WNT), Janus kinase (JAK)/STAT, (signal transducer and activator of transcription), transforming growth factor (TGF-ß), and mitogen-activated protein kinase (MAPK). The expression of the majority of genes that mapped to the indicated pathways was affected mostly between 8 and 48 h after EE exposure.
A list of selected genes (according to the highest fold change induced by treatment) whose expression was modified by EE at the indicated times, along with their accession number, gene symbol, and the average fold change induced by EE (average of five independent samples, calculated by comparing treatment vs. control), is shown in Tables 1–7. In these tables, we have included the GO term from the biological process or the molecular function (when applicable), associated with each gene listed, in order to get a better insight of the potential functional implications for each individual gene affected by EE exposure.
|
|
|
|
|
|
|
Immediate-early genomic response.
As indicated, the exposure to EE results in a rapid response of the uterus at the gene expression level. Within the first 2 h of exposure, EE modulates the expression of genes encoding members of multiple intra- and intercellular signaling pathways, as well as transcriptional regulators (Tables 1 and 2). The immediate-early genes affected by EE exposure include those that encode growth factors (BMP2, BMP4, EGR1, EGR2, WNT4, VEGF, TMEPAI, OXT, NR4A2, and NR4A1), proteins involved with angiogenesis (RHOB, IGF1, VEGF, and VEGFA TNFRSF12A), signal transduction (AXIN2, RGS7, BTG2, CASP2, CDH11, CRY2, CXCR4, DAB2, EDG5, ENC1, FST, FZD1, FZD2, GPR85, GRPR, GUCY1B3, HPCAL1, IL15, IL1B, NMUR2, MT1A, MADH7, LITAF, KAZALD1, and RASD1), kinases (CKB, SPHK1, SNF1LK, SGK, PVR, PLK2, and PIM3), phosphatases (DUSP1, DUSP5, and SYNJ2), DNA processing (GADD45A, TOP1, and KPNA1), transcriptional regulators (CEBPB, CITED2, CITED4, DSCR1, ETS2, FOXP1, HOXA10, ICSBP1, IFRD1, IRF8, JUN, JUNB, NCOA1, TWIST2, TOB1, TLE3, TCF21, SOX4, SMAD7, SMARCA2, SESN1, PRRX2, PER2, NFIL3, MSX2, MAFK, MAF, KLF4), ion transporters (SLC31A2, SLC25A30, SLC16A1, KCNC3, and KCNA1), and metabolism (CNP1, CYP51, EEF2K, EIF4A1, ETF1, FKBP4, GOT1, IDI1, PECI, PDE5A, ODC1, OAZIN, NXN, NARS, MPG, MAT2A, LIAS, and LDHD). By far, the largest number of early EE-responsive genes encode proteins implicated in transcriptional regulation and signal transduction.
Immediate-late genomic response.
The genomic response of the rat uterus 8–24 h after EE exposure is tremendously amplified as compared to the immediate-early or -late responses. The number of genes whose expression is affected by the ER agonist is maximal within the first 8 h of exposure (3620 genes affected, p 0.0001); however, a high level of response is maintained at 24 h (2445 genes affected, p 0.0001). A partial list of the immediate-late genes responding to EE is shown in Tables 3 and 4 (only the genes with the highest fold change induced by treatment are shown). The products of these genes are involved in multiple molecular pathways. Using the available GO annotation, we determined that 2278 genes (from both 8 and 24 h combined) can be associated with a particular molecular function. From these functions identified as being affected by EE exposure, those with the larger number of genes affected are as follows: signal transduction (238 and 168), receptor activity (139 and 96), transcription factors and transcriptional regulators (180 and 142), and translation (24 and 14; genes affected after 8 and 24 h of exposure, respectively). The number of immediate-late genes affected by EE exposure mapped to a particular molecular process is shown in Table 2 (in supplementary data). As can be seen in this table, between 8 and 24 h of exposure to EE, the uterus response engages changes in the expression of 474 genes, whose products are associated only with regulation of transcription and transcription; 145 with translation and its regulation; 141 genes associated with cell differentiation, 31 with cell growth, 186 with cell cycle, and 131 with regulation of cell cycle; and 158 are associated with cell proliferation. In this group of genes affected by EE, there are also genes whose products have been implicated in apoptosis such as: programmed cell death 4 and 8 (PDCD4 and 8); programmed cell death 6–interacting protein (PDCD6IP); caspase 1, 2, 11, 12 (CASP1, 2, 11, and 12); Bcl2-associated X protein (BAX); BCL2-like 1 (BCL2L1); BCL2/adenovirus E1B 19-kDa interacting protein 3 like (BNIP3L); Bcl2-associated athanogene 1 (BAG1); BCL2-antagonist/killer 1 (BAK1); signal transducer and activator of transcription 1 (STAT1); and eukaryotic translation initiation factor 2, subunit 1 alpha (EIF2S1), among others. During this period after EE exposure, there are also multiple genes affected, whose products encode proteins related to various aspects of metabolism, e.g., 334 genes are related to regulation of metabolism and 254 with cellular catabolism.
Late genomic response.
After 48 h of EE exposure, the expression of 620 genes is modified in a statistically significant manner, while after 72 h the expression of 724 genes was affected. After 96 h of EE exposure, the genomic response of the immature uterus is still very robust, and the expression of 176 genes remains affected. A partial list of the EE late-responsive genes is shown in Tables 5–7. These genes encode proteins implicated in multiple cellular processes (Table 2, in supplementary data). However, there is some enrichment in genes involved in cell growth and cell differentiation (53 genes), mitosis and cell division (216), tissue regeneration (14 genes), cholesterol, lipid, sterol, and steroid biosynthesis (169 genes), and various modalities of metabolism (212 genes), as well as DNA replication (66 genes), immune response (103 genes), complement activation (26 genes), and regulation of endocytosis and phagocytosis (16 genes).
The analysis of the effect of time on gene expression was also conducted, using a standard single-factor ANOVA on the log signal response of the control samples only. In this case, each set of control samples from each time point were compared to the control samples of the 1-h group. From this analysis we identified that the expression of 47 genes was modified at different times of sampling. However, with the exception of the sulfotransferase (SULT1A1) gene, none of the other 46 genes were associated with the estrogenic response. The expression of SULT1A1 is downregulated by time, reaching a maximum downregulation at 96 h (with a – 2.3-fold change compared with the response at 1 h). However, EE promoted the downregulation of SULT1A1 at lower levels and at earlier times (Table 3), e.g., at 8 h the expression of SULT1A1 is downregulated by – 5.3-fold compared to its own 8-h control.
Quantitative reverse transcriptase–polymerase chain reaction.
The reliability of the microarray data was independently corroborated by QRT-PCR analysis of selected genes in samples from the same batch of RNA, from each sample, used for microarray analysis. The relative expression level of complement component 3 (C3), intestinal calcium–binding protein or calbindin 3 or S100 calcium–binding protein G (S100G), progesterone receptor (PGR), 11-beta-hydroxylsteroid dehydrogenase type 2 (HSD11B2), F-box and leucine-rich repeat protein 20 (FBXL20), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2), interleukin 4 receptor (IL4R), matrix metallopeptidase 7 (MMP7), and metallothionein 3 (MT3) was evaluated by QRT-PCR. The relative expression level of C3, S100G, PGR, HSD11B2, FBXL20, MMP7, and MT3 mRNAs was similar as that determined by microarray analysis (Table 10, in supplementary data). The expression values of C3 and MMP7, both highly induced by EE, were higher as measured by QRT-PCR mostly due to the optimization of this approach for each individual gene and the abundance of the corresponding mRNA. No significant change in the expression levels of a housekeeping gene, peptidylprolyl isomerase B (cyclophilin B, PPIB), was identified by QRT-PCR or microarray analysis.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
The uterine response to an estrogen requires changes in the expression level of genes whose products regulate successive and functionally interlinked cellular processes that culminate in an uterotrophic response. Those genes can be classified as immediate-early, immediate-late, late, and sustained responsive genes. The temporal transcript profile induced by EE here described is concordant with, and expands, those reported for the uterotrophic response of immature, ovariectomized mice after exposure to EE (Fertuck et al., 2003
) or of immature intact mice after exposure to 17ß-estradiol (Hewitt et al., 2003; Moggs et al., 2004). Data from this study agree with the genomic response determined at a single time point and multiple estrogen doses in the rat and mouse (Kwekel et al., 2005; Naciff et al., 2003, 2005). In addition, our data also concur with data from other studies, where the expression of a limited number of genes was evaluated by microarray analysis after estrogen exposure (Green et al., 2001; Hong et al., 2004; Watanabe et al., 2002; Wu et al., 2003), or with studies where the expression of individual genes has been evaluated (see, e.g., Daston and Naciff, 2005). The temporal expression pattern of well-established estrogen-responsive genes, such as complement C3, progesterone receptor, matrix metalloproteinase 7, calbindin 3 or S100 calcium–binding protein G (S100G), creatine kinase b, arginosuccinate synthetase, transferrin, insulin-like growth factor (IGF)–binding protein 3, bone morphogenetic protein 2, scavenger receptor class B, member 1, carbonic anhydrase 11, growth-associated protein 43 and glutathione S-transferase, and mu 2, among others, was clearly identified in our study and further supports the reliability of our approach. The earliest changes observed after EE treatment were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by the expression of genes implicated in mRNA and protein synthesis, cell cycle regulation, DNA replication, cell proliferation and differentiation, apoptosis, tissue remodeling, and immunological responses. The compendium of genes presented gives an exceptional level of detail of the molecular events that first elicit and at the same time limit the extent of the uterine response to estrogen exposure. This information will be useful to better define the estrogen mechanism of action, as well as to refine a gene expression–based assay to screen chemicals for estrogenic activity.
Early responses of the mammalian uterus to estrogen exposure include an increase in the vascularization of the uterus, which increases the amount of blood in this organ (hyperemia), and it is accompanied by infiltration of immune system cells such as macrophages and eosinophils into the uterine tissue, increased calcium influx, histamine release, increased RNA and protein precursor uptake, and enhanced metabolic capabilities, including an increase in the glucose oxidation capabilities a well as increased RNA and protein synthesis (Batra, 1987; Catelli and Baulieu, 1976; Clark and Mani, 1994; Cocchiara et al., 1992; Muller and Knowler, 1984; Yamada and Nagata, 1993). The early uterine genomic responses that we have identified denote some of the molecular precursors of these events. For example, the expression of VEGF is rapidly (between 2 and 8 h) increased by EE (at 8 h it is 1.8-fold), while the expression of the C-terminal–truncated VEGF receptor-2FLK-1 (KDR or FLK-1/KDR) is also induced 8 h after EE exposure (1.7-fold). The induction of VEGF expression by estrogen in many target cells, including epithelial cells, fibroblasts, and smooth muscle cells, and a role for this hormone in the modulation of angiogenesis and vascular permeability have been established (reviewed by Koos et al., 2005). VEGF induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the activation of its receptor tyrosine kinase (FLK-1/KDR). Recently, Herve et al. (2006) reported data that suggest that estrogen upregulates FLK-1/KDR expression in endothelial cells mainly through the modulation of VEGF by a paracrine mechanism. It is also known that reactive oxygen species derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation. Both the small GTPase RAC1 and endothelial-type gp91-phox gene (CYBB) are critical components of the endothelial NAD(P)H oxidase complex. The expression of the genes encoding these two components was increased by EE. The expression of the EST-BI285447
[GenBank]
was induced by EE after 8 h, and it peaks at 24 h (with a 2.8-fold increase). Its sequence is similar to the angiopoietin 1 (ANGPT1) gene, which encodes a peptide with a potentially unique role among the vascular growth factors by acting to enlarge blood vessels without inducing sprouting (Thurston et al., 2005). Another gene for which expression was stimulated within the first 2–8 h of EE exposure is cysteine-rich protein 61 (CYR61), a known estrogen-responsive gene (Hewitt et al., 2003). CYR61 can promote endothelial cell growth, migration, adhesion, and survival in vitro, and it has been proposed that it acts as a regulator of angiogenesis and endothelial cell function by regulating the production and/or activity of other angiogenic molecules (e.g., bFGF [basic fibroblast growth factor] and VEGF) as well molecules that affect the integrity or stability of the extracellular matrix (e.g., collagen, matrix metalloproteases, and their tissue inhibitors) (Brigstock, 2002). Thus, the increased expression of VGEF, its receptor FLK-1/KDR, EST-BI285447
[GenBank]
(angiopoietin 1), and CYR61, among other gene expression changes induced by EE in the uterus may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue.
Furthermore, it is known that nitric oxide (NO) and prostacyclin (PGI2) production by endothelial cells changes in response to extracellular ligands such as bFGF, epidermal growth factor (EGF), VEGF, ATP, and estrogen. These ligands elicit a rise in intracellular calcium and/or activate kinases that in turn activate endothelial NO synthase (eNOS) and cytosolic phospholipase A2 (cPLA2). In blood vessels, eNOS produces NO which acts as a potent vasodilator and platelet aggregation inhibitor. Exposure to EE promoted the expression of endothelial nitric oxide synthase 3 (NOS3 or eNOS), but only within the first 8 h, then is downregulated between 24 and 48 h to finally return to basal levels after 72 h. It has been observed that estrogen treatment in vivo stimulates cGMP accumulation in the rat uterus, and it has been established that chronic estrogen treatment in prepubertal and/or ovariectomized models increases nitric oxide (NO) production (Buhimschi et al., 2000). However, in our experiments, we determined that the expression of guanylate cyclase 1, soluble, alpha 3 (GUCY1A3) is rapidly decreased after EE exposure, reaching maximal downregulation after 8 h and returning to control levels after 72 h. Very similar effects have been determined in the uterus of the immature rat after exposure to estradiol (Krumenacker et al., 2001). Many of the NO-dependent cell-signaling events that regulate important physiological processes are mediated through activation of GUCY1A3. After production, NO can diffuse freely and bind to GUCY1A3, enhancing the production of cGMP and subsequently stimulating cGMP targets, such as cGMP-dependent protein kinases, cyclic nucleotide phosphodiesterases, and ion channels. Thus, the decreased expression of GUCY1A3 induced by EE ensures that the NO-cGMP cascade is not overamplified. Our data suggest that the increase in cGMP is not the result of increased expression of the enzyme responsible for its synthesis, but rather by the decrease in the level of expression of cGMP-specific phosphodiesterases. EE induced the early (between the first 2–8 h of exposure) downregulation of the gene encoding the cGMP phosphodiesterase delta subunit, the cGMP-specific phosphodiesterase 5A (PDE5A), which was mildly induced after 2 h of EE exposure but by 8 h is downregulated and remained so until after 48 h. EE also promoted the expression of the natriuretic peptide receptor 2 (NPR2), the primary receptor for C-type natriuretic peptide (CNP), which upon ligand binding exhibits greatly increased guanylyl cyclase activity (reviewed by Schulz, 2005). The NO-cGMP cascade is also regulated by EE through the increased expression of argininosuccinate synthetase (ASS1), which has a role in preventing autotoxicity from nitric oxide overproduction (Hao et al., 2004).
The hyperemia and increased fluid retention in the uterus are associated with changes in ion transport (Clemetson et al., 1977). We determined that within the first hours of EE treatment the expression of the genes encoding the ATPase, Na+/K+ transporting, alpha 1 polypeptide (ATP1A1); ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 (ATP2A2); voltage-dependent anion channel 1 (VDAC1); calcium channel, voltage-dependent, T-type, alpha 1G subunit (CACNA1G); solute carrier organic anion transporter family, member 2a1(SLCO2A1); and aquaporin 1 and 8 (AQP1 and AQP8) is increased, reaching a maximum at 8 h. The expression of potassium inwardly rectifying channel, subfamily J, member 8 (KCNJ8); solute carrier family 22 (organic cation transporter), member 17 (SLC22A17); ATPase, Ca2+ transporting, plasma membrane 1 and 2 (ATP2B1 and 2); ATP-binding cassette, subfamily C (CFTR/MRP); member 9 (ABCC9); sodium channel, voltage-gated, type 6, alpha polypeptide (SCN6A); solute carrier organic anion transporter family, member 2b1 (SLCO2B1); solute carrier family 8 (sodium/calcium exchanger); member 1 (SLC8A1), FXYD domain–containing ion transport regulator 1, 3, 4, 6, and 7 (FXYD1, 3, 4, 6, and 7); solute carrier family 21 (organic anion transporter), member 5 (SLC21A5); and aquaporin 5 (AQP5) was downregulated by EE exposure.
The increase in protein synthesis observed at 8–24 h after EE exposure is associated with a number of genes. For example, EE induced changes at the 8-h time point in the expression of 24 genes which encode proteins with translation initiation factor activity (EIF2S1, EIF4E, EIF5, EIF-5A, EIF3 P66, EIF3 P36, EIF3 P110, EIF3S6, EIF4G1, etc.) and 50 genes whose products are associated with protein translation in general (CCT3, FKBP1A, FKBP4, KPNB1, KPNB3, KPNA2, IPO13, TCEB1, TCEB2, TGIF, EEF1E1, EEF1B2, EEF2K, ETF1, RRBP1, RRBP1, RAMP4, etc.). EE also induced changes in the expression of 102 genes whose encoded products are associated with the endoplasmic reticulum (e.g., HSPA5, RPN1 AND 2, CANX, SSR3, RCN, CALR, RTN1, LMAN1, PLOD, AARS, GARS, NARS, SARS1, WARS2, etc.). This cluster of genes associated with the endoplasmic reticulum is also particularly enriched after 24 h of EE exposure, with 99 genes identified at this time. The associated annotation with these genes indicates that their products participate in the assembly of the general transcription machinery, the ribosome, or act at transcription initiation, elongation, and termination; protein folding; protein maturation; and protein nuclear import and export. The effect of EE on most of these genes is upregulation. Concurrent with these gene expression changes, EE induces the expression of genes encoding components of the ubiquitin-proteosome proteolytic pathway, such as CDC34, NEDD8, PSMA2, 3, 5, 6 AND 7, PSMB1, 2, 3, 4, 5, 6, 8 and 9, UCHL3, UBE2D3, UFD1L, and USP10, among others. Clearly, the uterine genomic response to estrogen stimulation ensures that the timely and irreversible degradation of critical regulators, essential for normal cellular function, occurs at the appropriate time by sequential regulation of the expression of the various components of this ubiquitin-proteosome proteolytic pathway.
The infiltration of the uterine tissue by immune cells, particularly the presence of eosinophils in the endometrium and stroma of rodents during the estrous cycle or after estrogen administration to ovariectomized animals is well documented, and it has been equated to an inflammatory response (Koshi et al., 2005). An example of the close correlation between the histological changes and gene expression changes induced by EE exposure is the recruiting of eosinophils to the uterine epithelia and the stroma as part of the edematous response observed at 8 h associated with an increase in the expression level of small inducible cytokine A11 (SCYA11, previously known as eotaxin). The expression of this gene was promoted by EE after only 1 h and reached a maximum at 8 h. SCYA11 is the estrogen-regulated chemotatic factor responsible for recruiting eosinophils into the stroma of the pubertal and cycling uterus of the rat (Gouon-Evans and Pollard, 2001). Other genes upregulated by EE and whose products have a role in the recruitment of immune cells to the uterus are small inducible gene JE (SCYA2) or monocyte chemoattractant protein 1 (CCL2); small inducible cytokine A6 (CCL6); macrophage migration inhibitory factor (MIF); suppressor of cytokine signaling 1, 2, and 3 (SOCS1, 2, and 3); interleukin 4 receptor (IL4R); polymeric immunoglobulin receptor (PIGR); chemokine (C-X3-C motif) ligand 1 (CX3CL1); chemokine (C-X-C motif) receptor 4 (CXCR4), which encodes a CXC chemokine receptor specific for stromal cell–derived factor 1; chemokine (C-X-C motif) receptor 4 (CXCR4); chemokine orphan receptor 1 (CMKOR1); and IL1B, IL6ST, IL15, and IL18.
Estrogen exposure of immature or ovariectomized rodents elicits a synchronized wave of DNA synthesis and mitosis in the uterus, beginning between 12 and 24 h after treatment (Cheng et al., 1985; Clark and Mani, 1994; Kiss et al., 1987; Pollard et al., 1987). This uterine response to estrogen involves a considerable increase in uterine weight, which in the case of EE exposure, peaked after 24 h (Fig. 1), as well as a large increase in the thickness of the luminal epithelium (Fig. 2, in supplementary data). We determine that within the first 24 h of EE exposure, there is a significant increase in the level of transcripts encoding proteins involved with DNA synthesis and chromosomal replication, such as polymerase (DNA directed), alpha 2 (POLA2), polymerase (DNA directed), epsilon(POLE), replication protein A2 (RPA2), DNA primase, p49 subunit (PRIM1), polymerase (DNA directed), delta 1, catalytic subunit (POLD1), origin recognition complex, subunit 6 homolog like (yeast) (ORC6L), flap structure–specific endonuclease 1 (FEN1), proliferating cell nuclear antigen (PCNA), minichromosome maintenance deficient 4 and 6 (Saccharomyces cerevisiae) (MCMD4 and 6), MCM3 minichromosome maintenance deficient 3 and 7 (S. cerevisiae) (MCM3 and 7), and CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae) (CHTF18).
EE exposure also modified the expression of genes whose products are involved in cell cycle control. In the cluster of early-responsive genes are growth arrest and DNA damage–inducible 45 alpha and gamma (GADD45A and G). The expression of GADD45A was induced 2 h after EE exposure, was still elevated by 8 h, but was downregulated by 24 h. The product of this gene is a nuclear protein involved in maintenance of genomic stability, DNA repair, and suppression of cell growth through interaction with nuclear elements, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and PCNA (Gramantieri et al., 2005). The expression of CDKN1A was also upregulated by EE, reaching a maximum between 8 and 24 h after exposure to EE. CDKN1A encodes a potent cyclin-dependent kinase inhibitor and inhibits the activity of cyclin-CDK2 or -CDK4 complexes and thus functions as a regulator of cell cycle progression at G1 (Munoz-Alonso et al., 2005). CDKN1A can interact with PCNA (upregulated by EE at 24 h and then downregulated at 72–96 h), a DNA polymerase accessory factor, and plays a regulatory role in S-phase DNA replication and DNA damage repair. GADD45G was induced immediately after EE exposure, remained elevated at 24 h, and returned to basal levels by 48 h. GADD45B and GADD45G are CDC2/cyclin B1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints and are induced by genotoxic stress (Vairapandi et al., 2002). The expression of cyclin D1 (CCND1) was upregulated between 8 and 24 h after EE exposure. This cyclin forms a complex with and functions as a regulatory subunit of CDK4 (mildly upregulated after 8–24 h of EE exposure) or CDK6, whose activity is required for cell cycle G1/S transition. Recent studies have demonstrated a critical link between estrogen's mitogenic effects and cell cycle progression, particularly at the G1 to S transition where key effectors of estrogen action are v-myc myelocytomatosis viral oncogene homolog (avian) (C-MYC) and CCND1, which converge on the activation of cyclin E–CDK2 (reviewed by Butt et al., 2005). The expression of another cyclin (cyclin D3, CCND3), which also functions as a regulatory subunit of CDK4 or CDK6 (reviewed by Sherr, 1995), was downregulated by EE but only between 48 and 72 h. A more complex pattern of expression was determined for cyclin B (CCNB1) which was consistently upregulated at 24–48 h but then was downregulated both at 72 and 96 h. The protein encoded by this gene is a regulatory protein involved in mitosis, complexing with p34 (CDC2) to form the maturation-promoting factor (MPF) (Pines and Hunter, 1992). Genes that encode proteins required for maintaining genome integrity, such as checkpoint kinase 1 and 2 (CHEK1 and 2), mediator of DNA damage checkpoint 1 (MDC1), and CDC28 protein kinase 1B (CKS1B), were also regulated by EE exposure. Further, the expression of some of the genes whose products have been associated with maintaining cells in growth-arrest state were also regulated by EE exposure. For example, cyclin-dependent kinase inhibitor 1B (CDKN1B or P27, KIP1), the inhibitor of the activation of cyclin E–CDK2 or cyclin D–CDK4 complexes, was downregulated between 8 and 24 h, as was mitogen-activated protein kinase kinase 6 (MAP2K6) gene, which encodes a member of the dual-specificity protein kinase family and acts as a MAPK kinase. However, its substrate, MAPK6, which acts as an integrator point for multiple biochemical signals, is upregulated after 8–24 h of EE exposure.
Some of the genes whose expression is modified by EE are master genes since they encode products capable of regulating multiple pathways. One of them is STAT3, which was upregulated within the first 2 h after exposure to EE, reaching a maximum at 8 h and then returning to basal levels by 48 h. STAT family members are phosphorylated by the receptor-associated kinases (in response to cytokines and growth factors) and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators (reviewed by Stephanou and Latchman, 2005). In particular, STAT3 is activated in response to various cytokines and growth factors including interferons, EGF, IL5, IL6, hepatocyte growth factor, leukemia inhibitory factor, and BMP2. STAT3 mediates the expression of a variety of genes, such as C-MYC, cyclin D1, P21WAF1, BCLII, and BCL-XL, in response to cell stimuli and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase RAC1 has been shown to bind and regulate the activity of STAT3 (Debidda et al., 2005), while its activity is specifically inhibited by the PIAS3 protein (Long et al., 2004). While the expression of RAC1 was affected by EE exposure at 24 h (being upregulated), the expression of the PIAS3 gene was downregulated within the first hours after EE exposure, reaching a maximum downregulation at 8 h. STAT3 seems to play a very important role in the mammalian uterus. Recently, Catalano et al. (2005) determined that the inhibition of STAT3 activation in the endometrium prevents implantation. Our data suggest that the changes in the expression level of STAT3, RAC1, and PIAS3, induced by EE exposure, are part of the preparation of the uterus, induced by estrogen, for a successful implantation.
Another master gene rapidly induced after EE exposure is IGF1. The expression of IGF1 was fairly robust 2 h after EE exposure, peaks at 8 h, but remains elevated at 48 h. It has been postulated that at least some of the physiological effects of estrogen are mediated by locally produced IGF1 (revised by Moyano and Rotwein, 2004). In humans, it has been determined that IGF1 is involved in the regulation of endometrial growth (O'Toole et al., 2005), playing an important role in cell proliferation and differentiation. Further, the expression of one of its inhibitors, IGFBP3, is downregulated by EE exposure. In fact, in the mammalian uterus the growth stimulatory action of estradiol is associated with suppression of IGFBP3 expression (Hung and Pollak, 1995). The biological functions of IGFBP3, aside from being the major binding protein for IGF1, are complex and remain poorly understood. However, IGFBP3 is known to inhibit cell proliferation by interfering with the interaction of IGF1 and its receptor and also modulates cell growth and survival independent of IGF, presumably via interactions with cellular proteins such as TGF-ß receptor (Huang and Huang, 2005; Lee and Cohen, 2002). In the mammalian uterus, IGF1-signaling pathways involve MAPK and phosphatidylinositol-3 kinase (PI-3 kinase) (Inoue et al., 2005). As indicated (above) the MAPK pathway was regulated not only by EE exposure but also the PI-3 kinase pathway. The expression of PI-3 kinase, regulatory subunit, polypeptide 2 (PIK3R2) was downregulated by EE, showing a maximal downregulation at 8 h.
EE is a potent ER agonist, and it has been used as a chemical reference of the natural hormone estradiol in multiple studies. Thus, the gene expression changes described here provide the molecular foundation for understanding how other chemicals with estrogenic activity elicit a uterine response. The information we have gathered will be also useful to better define an assay to evaluate the potential estrogenicity of chemicals, based upon this specific mechanism of action (using specific gene expression changes as biomarkers of estrogenicity), as well as to refine the classical uterotrophic assay. The reliability of this assay can be increased by evaluating the uterine expression level of estrogen-responsive genes, such as the ones identified here.
Our data showed that exposure to a single dose of a potent ER agonist induces a full uterotrophic response within the first 24 h of exposure, but remains detectable until 96 h after estrogen exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes in a program that involves multiple molecular pathways. The earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses, among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.
|
SUPPLEMENTARY DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Affymetrix image files for the 70-chip hybridizations and the absolute analysis results of each time point group (available at ArrayExpress-EBI; accession no. E-MEXP-999); nucleotide sequences for primers used to verify the array-based gene expression changes induced by EE in the immature uterus of the rat by QRT-PCR (supplementary Table 1), the corresponding expression values (supplementary Table 10), the summary of the clustering of EE-responsive genes using the GO terms and KEGG pathways map (Table 2), the representative uterine histology from equivalent regions of vehicle-treated control immature rats or animals treated with a single EE dose (10 µg/kg/day) at different times (Fig. 2), the Eisen diagram (or heat map) of the genes whose expression is modified at different times after a single EE exposure in the uterus of the prepubertal rat (Fig. 3), and the temporal response of eight selected genes whose expression is significantly regulated by EE exposure (Fig. 4) as supplementary data are available online at http://toxsci.oxfordjournals.org/.
|
ACKNOWLEDGMENTS |
|---|
The authors thank Donald J. Versteeg and Ting Hu for their helpful discussions. Conflicts of interest: None declared.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Anderson JN, Peck EJ Jr, Clark JH. Nuclear receptor estradiol complex: A requirement for uterotrophic responses. Endocrinology (1974) 95:174–178.
Batra S. Increase by oestrogen of calcium entry and calcium channel density in uterine smooth muscle. Br. J. Pharmacol. (1987) 92:389–392.[Web of Science][Medline]
Beissbarth T, Speed TP. GOstat: Find statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics (2004) 12:1464–1465.
Brigstock DR. Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61). Angiogenesis (2002) 5:153–165.[CrossRef][Medline]
Britt KL, Findlay JK. Estrogen actions in the ovary revisited. J. Endocrinol. (2002) 175:269–276.[Abstract]
Buhimschi IA, Yallampalli C, Buhimschi CS, Saade GR, Garfield RE. Distinct regulation of nitric oxide and cyclic guanosine monophosphate production by steroid hormones in the rat uterus. Mol. Hum. Reprod. (2000) 6:404–414.
Butt AJ, McNeil CM, Musgrove EA, Sutherland RL. Downstream targets of growth factor and oestrogen signalling and endocrine resistance: The potential roles of c-Myc, cyclin D1 and cyclin E. Endocr. Relat. Cancer (2005) 12(Suppl. 1):S47–S59.
Catalano RD, Johnson MH, Campbell EA, Charnock-Jones DS, Smith SK, Sharkey AM. Inhibition of Stat3 activation in the endometrium prevents implantation: A nonsteroidal approach to contraception. Proc. Natl. Acad. Sci. U.S.A. (2005) 102:8585–8590.
Catelli MG, Baulieu EE. Estrogen-induced changes of ribonucleic acid in the rat uterus. Mol. Cell. Endocrinol. (1976) 6:129–151.[CrossRef][Web of Science][Medline]
Cheng SV, MacDonald BS, Clark BF, Pollard JW. Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids. Exp. Cell. Res. (1985) 160:459–470.[CrossRef][Web of Science][Medline]
Clark JH, Mani SK. Actions of ovarian steroid hormones. In: The Physiology of Reproduction—Knobil E, Neill J, eds. (1994) Vol. 1, 2nd ed. New York: Raven Press. 1011–1059.
Clemetson CA, Verma UL, De Carlo SJ. Secretion and reabsorption of uterine luminal fluid in rats. J. Reprod. Fertil. (1977) 49:183–187.
Cocchiara R, Albeggiani G, Di Trapani G, Azzolina A, Lampiasi N, Rizzo F, Diotallevi L, Gianaroli L, Geraci D. Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells. Hum. Reprod. (1992) 7:1036–1041.
Couse JF, Dixon D, Yates M, Moore AB, Ma L, Maas R, Korach KS. Estrogen receptor-alpha knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract. Dev. Biol. (2001) 238:224–238.[CrossRef][Web of Science][Medline]
Couse JF, Korach KS. Contrasting phenotypes in reproductive tissues of female estrogen receptor null mice. Ann. NY Acad. Sci. (2001) 948:1–8.[CrossRef][Web of Science][Medline]
Curtis SH, Korach KS. Steroid receptor knockout models: Phenotypes and responses illustrate interactions between receptor signaling pathways in vivo. Adv. Pharmacol. (2000) 47:357–380.[Medline]
Currie RA, Orphanides G, Moggs JG. Mapping molecular responses to xenoestrogens through Gene Ontology and pathway analysis of toxicogenomic data. Reprod. Toxicol. (2005) 20:433–440.[CrossRef][Web of Science][Medline]
Daston GP, Naciff JM. Gene expression changes related to growth and differentiation in the fetal and juvenile reproductive system of the female rat: evaluation of microarray results. Reprod. Toxicol. (2005) 19:381–394.[CrossRef][Web of Science][Medline]
Debidda M, Wang L, Zang H, Poli V, Zheng Y. A role of STAT3 in Rho GTPase-regulated cell migration and proliferation. J. Biol. Chem. (2005) 280:17275–17285.
Fertuck KC, Eckel JE, Gennings C, Zacharewski TR. Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol. Physiol. Genomics (2003) 15:127–141.
Gouon-Evans V, Pollard JW. Eotaxin is required for eosinophil homing into the stroma of the pubertal and cycling uterus. Endocrinology (2001) 142:4515–4521.
Gramantieri L, Chieco P, Giovannini C, Lacchini M, Trere D, Grazi GL, Venturi A, Bolondi L. GADD45-alpha expression in cirrhosis and hepatocellular carcinoma: Relationship with DNA repair and proliferation. Hum. Pathol. (2005) 36:1154–1162.[CrossRef][Web of Science][Medline]
Green AR, Parrott EL, Butterworth M, Jones PS, Greaves P, White IN. Comparisons of the effects of tamoxifen, toremifene and raloxifene on enzyme induction and gene expression in the ovariectomised rat uterus. J. Endocrinol. (2001) 170:555–564.[Abstract]
Hao G, Xie L, Gross SS. Argininosuccinate synthetase is reversibly inactivated by S-nitrosylation in vitro and in vivo. J. Biol. Chem. (2004) 279:36192–36200.
Herve MA, Meduri G, Petit FG, Domet TS, Lazennec G, Mourah S, Perrot-Applanat M. Regulation of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus. J. Endocrinol. (2006) 188:91–99.
Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS. Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol. Endocrinol. (2003) 17:2070–2083.
Hong SH, Nah HY, Lee JY, Gye MC, Kim CH, Kim MK. Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection. J. Endocrinol. (2004) 181:157–167.[Abstract]
Huang SS, Huang JS. TGF-beta control of cell proliferation. J. Cell. Biochem. (2005) 96:447–462.[CrossRef][Web of Science][Medline]
Hung H, Pollak M. Regulation of IGFBP-3 expression in breast cancer cells and uterus by estradiol and antiestrogens: Correlations with effects on proliferation: A review. Prog. Growth Factor Res. (1995) 6:495–501.[CrossRef][Medline]
Inoue A, Takeuchi S, Takahashi S. Insulin-like growth factor-I stimulated DNA replication in mouse endometrial stromal cells. J. Reprod. Dev. (2005) 51:305–313.[CrossRef][Web of Science][Medline]
Joslyn CA, Mniszewski SM, Fulmer A, Heaton G. The gene ontology categorizer. Bioinformatics (2004) 20(Suppl. 1):I169–I177.[CrossRef][Medline]
Kiss R, Lenglet G, Pasteels JL, Danguy A. Hormonal regulation of uterine epithelial cell proliferation. I. Effects of estradiol or progesterone administered separately. In Vivo (1987) 1:281–289.[Medline]
Klinge CM. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res. (2001) 29:2905–2919.
Koshi R, Coutinho-Silva R, Cascabulho CM, Henrique-Pons A, Knight GE, Loesch A, Burnstock G. Presence of the P2X(7) purinergic receptor on immune cells that invade the rat endometrium during oestrus. J. Reprod. Immunol. (2005) 66:127–140.[CrossRef][Web of Science][Medline]
Koos RD, Kazi AA, Roberson MS, Jones JM. New insight into the transcriptional regulation of vascular endothelial growth factor expression in the endometrium by estrogen and relaxin. Ann. NY Acad. Sci. (2005) 1041:233–247.[CrossRef][Web of Science][Medline]
Krumenacker JS, Hyder SM, Murad F. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus. Proc. Natl. Acad. Sci. USA (2001) 98:717–722.
Kwekel JC, Burgoon LD, Burt JW, Harkema JR, Zacharewski TR. A cross-species analysis of the rodent uterotrophic program: Elucidation of conserved responses and targets of estrogen signaling. Physiol. Genomics (2005) 23:327–342.
Lee KW, Cohen P. Nuclear effects: Unexpected intracellular actions of insulin-like growth factor binding protein-3. J. Endocrinol. (2002) 175:33–40.[Abstract]
Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. (1996) 14:1675–1680.[CrossRef][Web of Science][Medline]
Long J, Wang G, Matsuura I, He D, Liu F. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3). Proc. Natl. Acad. Sci. U.S.A. (2004) 101:99–104.
Moggs JG. Molecular responses to xenoestrogens: Mechanistic insights from toxicogenomics. Toxicology (2005) 213:177–193.[CrossRef][Web of Science][Medline]
Moggs JG, Tinwell H, Spurway T, Chang HS, Pate I, Lim FL, Moore DJ, Soames A, Stuckey R, Currie R, et al. Phenotypic anchoring of gene expression changes during estrogen-induced uterine growth. Environ. Health Perspect. (2004) 112:1589–1606.[Web of Science][Medline]
Moyano P, Rotwein P. Mini-review: Estrogen action in the uterus and insulin-like growth factor-I. Growth Horm. IGF Res. (2004) 14:431–435. (Review).[CrossRef][Web of Science][Medline]
Muller RE, Knowler JT. The synthesis of ribosomal RNA and ribosomal protein and their incorporation into ribosomes in the uterus of the oestrogen-stimulated immature rat. FEBS Lett. (1984) 174:253–257.[CrossRef][Web of Science][Medline]
Munoz-Alonso MJ, Acosta JC, Richard C, Delgado MD, Sedivy J, Leon J. p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells. J. Biol. Chem. (2005) 280:18120–18129.
Naciff JM, Jump ML, Torontali SM, Carr GJ, Tiesman JP, Overmann GJ, Daston GP. Gene expression profile induced by 17alpha-ethynyl estradiol, bisphenol A, and genistein in the developing female reproductive system of the rat. Toxicol. Sci. (2002) 68:184–199.
Naciff JM, Overmann GJ, Torontali SM, Carr GJ, Tiesman JP, Richardson BD, Daston GP. Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat. Toxicol. Sci. (2003) 72:314–330.
Naciff JM, Overmann GJ, Torontali SM, Carr GJ, Tiesman JP, Daston GP. Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat. Environ Health Perspect. (2004) Nov;112(15):1519–1526.
Naciff JM, Torontali SM, Overmann GI, Carr GJ, Tiesman JP, Daston GP. Evaluation of the gene expression changes induced by 17-alpha-ethynyl estradiol in the immature uterus/ovaries of the rat using high density oligonucleotide arrays. Birth Defects Res. B Dev. Reprod. Toxicol. (2005) 74:164–184.[CrossRef][Web of Science][Medline]
Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA. Mechanisms of estrogen action. Physiol. Rev. (2001) 81:1535–1565.
Noda S, Sawaki M, Shiraishi K, Yamasaki K, Yamaguchi R. Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation. J. Vet. Med. Sci. (2002) 64:315–319.[CrossRef][Web of Science][Medline]
O'Toole SA, Dunn E, Sheppard BL, Sheils O, O'Leary JJ, Wuttke W, Seidlova-Wuttke D. Oestrogen regulated gene expression in normal and malignant endometrial tissue. Maturitas (2005) 51:187–198.[CrossRef][Web of Science][Medline]
Pines J, Hunter T. Cyclins A and B1 in the human cell cycle. Ciba Found. Symp (1992) 170:187–196. discussion 196–204.[Medline]
Pollard JW, Pacey J, Cheng SV, Jordan EG. Estrogens and cell death in murine uterine luminal epithelium. Cell Tissue Res. (1987) 249:533–540.[CrossRef][Web of Science][Medline]
Schulz S. C-type natriuretic peptide and guanylyl cyclase B receptor. Peptides (2005) 26:1024–1034.[CrossRef][Web of Science][Medline]
Sherr CJ. D-type cyclins. Trends Biochem. Sci. (1995) 20:187–190.[CrossRef][Web of Science][Medline]
Stephanou A, Latchman DS. Opposing actions of STAT-1 and STAT-3. Growth Factors (2005) 23:177–182.[CrossRef][Web of Science][Medline]
Thurston G, Wang Q, Baffert F, Rudge J, Papadopoulos N, Jean-Guillaume D, Wiegand S, Yancopoulos GD, McDonald DM. Angiopoietin 1 causes vessel enlargement, without angiogenic sprouting, during a critical developmental period. Development (2005) 132:3317–3326.
Vairapandi M, Balliet AG, Hoffman B, Liebermann DA. GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J. Cell. Physiol. (2002) 192:327–338.[CrossRef][Web of Science][Medline]
Watanabe H, Suzuki A, Mizutani T, Khono S, Lubahn DB, Handa H, Iguchi T. Genome-wide analysis of changes in early gene expression induced by oestrogen. Genes Cells (2002) 7:497–507.[Abstract]
Waters KM, Safe S, Gaido KW. Differential gene expression in response to methoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductive tissues of female mice. Toxicol. Sci. (2001) 63:47–56.
Wu X, Pang ST, Sahlin L, Blanck A, Norstedt G, Flores-Morales A. Gene expression profiling of the effects of castration and estrogen treatment in the rat uterus. Biol. Reprod. (2003) 69:1308–1317.
Yamada AT, Nagata T. Light and electron microscopic radioautographic studies on the RNA synthesis of peri-implanting pregnant mouse uterus during activation of receptivity for blastocyst implantation. Cell. Mol. Biol. (Noisy-le-grand) (1993) 39:221–233.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Rouquie, C. Friry-Santini, F. Schorsch, H. Tinwell, and R. Bars Standard and Molecular NOAELs for Rat Testicular Toxicity Induced by Flutamide Toxicol. Sci., May 1, 2009; 109(1): 59 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Naciff, Z. S. Khambatta, R. G. Thomason, G. J. Carr, J. P. Tiesman, D. W. Singleton, S. A. Khan, and G. P. Daston The Genomic Response of a Human Uterine Endometrial Adenocarcinoma Cell Line to 17{alpha}-Ethynyl Estradiol Toxicol. Sci., January 1, 2009; 107(1): 40 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. P. Daston Gene Expression, Dose-Response, and Phenotypic Anchoring: Applications for Toxicogenomics in Risk Assessment Toxicol. Sci., October 1, 2008; 105(2): 233 - 234. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||