Gene Expression Profiling of 17ß-Estradiol and Genistein Effects on Mouse Thymus

Vimal Selvaraj*,1, David Bunick*, Carrol Finnigan-Bunick*, Rodney W. Johnson{dagger}, Huixia Wang{ddagger}, Lei Liu{dagger},§ and Paul S. Cooke*,2

Departments of * Veterinary Biosciences, {dagger} Animal Sciences and {ddagger} Statistics, § W.M. Keck Center for Comparative and Functional Genomics, and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

2 To whom correspondence should be addressed at Department of Veterinary Biosciences, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802. E-mail: p-cooke{at}uiuc.edu.

Received February 3, 2005; accepted June 1, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen regulates thymic development and involution and modulates immune function. Despite its critical role in thymus, as well as in autoimmune disorders, the mechanism by which estrogen affects the thymus is not well understood. We previously reported that the estrogenic soy isoflavone genistein, as well as 17ß-estradiol (E2), could induce thymic involution, but genistein effects were only partially mediated through estrogen receptors. To provide insights into mechanisms of estrogenic effects in the thymus, we investigated thymic gene expression changes induced by E2 (125 ng/day) and genistein (1500 ppm in feed) in weanling mice using high-density DNA arrays. We identified several E2-responsive genes involved in thymic development and thymocyte signaling during selection and maturation. Functional characterization indicated effects on genes involved in transcription, apoptosis, and the cell cycle. This study also identified changes in several E2-regulated transcripts essential to maintain immune self-tolerance. E2 upregulated more genes than genistein, while genistein downregulated more genes than E2. Though each treatment regulated several genes not altered by the other, there was considerable overlap in the genes regulated by E2 and genistein. Changes in transcription factors and cell cycle factors were consistent with decreases in cell proliferation induced by both genistein and E2. As indicated by the regulation of non-E2-responsive genes, genistein also induced unique effects through non-estrogenic mechanisms. The specific downregulation of the CD4 coreceptor transcript by genistein was consistent with the decline of CD4+ thymocytes in genistein-treated mice in our previous study. This is the first study identifying E2 and genistein target genes in the thymus. These findings provide new mechanistic insights toward explaining estrogen action on thymocyte development, selection, and maturation, as well as the effects of genistein on prenatal and neonatal thymic development and function.

Key Words: immune system; estrogen; T cells; soy; phytoestrogen; autoimmunity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen plays a critical role in immune system development and adult function, and females typically have a more robust immune responsiveness than males (Martin, 2000Go; Olsen and Kovacs, 1996Go). Estrogen receptor (ER){alpha} is expressed in both thymocytes and thymic epithelial cells (TECs) (Kawashima et al., 1992Go), and ER{alpha} mediated signaling is critical for normal thymic organogenesis, as shown by decreased thymic weights in ER{alpha} knockout mice (Erlandsson et al., 2001Go; Staples et al., 1999Go; Yellayi et al., 2000Go). The impaired thymic development in the absence of ER{alpha} is consistent with earlier reports that E2 produces increases in mature T-cell phenotypes (Screpanti et al., 1991Go), formation of extra-thymic loci of T-cell maturation (Okuyama et al., 1992Go) and humoral immunity (Cutolo et al., 2002Go; Erbach and Bahr, 1991Go; Trawick and Bahr, 1986Go). Estrogen also increases cytokine activity (Olsen and Kovacs, 1996Go) and macrophage function in some species (Miller and Hunt, 1996Go).

Despite the numerous studies showing estrogen positively regulates thymic development and certain aspects of its function, an equally large literature indicates that high doses of exogenous estrogen can induce thymic involution in mature/immature, castrated/noncastrated and adrenalectomized animals in both sexes (Ahlqvist, 1976Go; Dougherty, 1952Go; Forsberg, 1984Go; Luz et al., 1969Go; Sobhon and Jirasattham, 1974Go; Thompson, 1981Go). In addition, humoral and some cell-mediated immune responses are suppressed by estrogen (Luster et al., 1984Go; Trawick and Bahr, 1986Go). Furthermore, estrogen effects have been reported to differ based on age, and estrogen exposure in neonatal mice can result in an increased adult thymic size (Forsberg, 1995Go), emphasizing the complexity of estrogen's thymic actions.

Exposure to the synthetic estrogen diethylstilbestrol (DES) produces thymic atrophy similar to that seen with E2 (Forsberg, 1996Go). Our laboratory made the observation that genistein, a phytoestrogen present in high quantities in soy, could also induce thymic atrophy in mice (Yellayi et al., 2002Go). Genistein administration caused decreases in both humoral (Yellayi et al., 2002Go) and cell-mediated immunity (Yellayi et al., 2002Go, 2003Go). The decrease in thymic size induced by genistein was only partially inhibited by the anti-estrogen ICI 182,780 (which blocks both ER{alpha} and ß), suggesting that genistein effects occurred via ER-mediated and non-ER-mediated pathways (Yellayi et al., 2002Go). Furthermore, estrogen increases the percentage of CD4+CD8 T cells relative to CD4CD8+ (Screpanti et al., 1989Go), in contrast to the selective decreases in the CD4+CD8 T-cell fraction in the thymus in response to genistein (Yellayi et al., 2002Go). Effects on thymic size and immune function were seen at serum concentrations of genistein comparable to those in humans consuming high amounts of soy, such as infants fed soy formula (King and Bursill, 1998Go; Setchell et al., 1997Go; Watanabe et al., 1998Go). Though these findings show that genistein can suppress immune function and have been corroborated by other data in humans and laboratory animals showing immune inhibition with genistein (Calemine et al., 2003Go; Huang et al., 2005Go; O'Connor et al., 2002Go), other studies using rodent model systems have reported that genistein or other phytoestrogens induce stimulatory effects on various aspects of immune function (Guo et al., 2001Go, 2002Go; Klein et al., 2002Go; Wang et al., 1997Go; Zhang et al., 1999Go). The overall effect of genistein on the thymus and immune system is therefore not clear, and may vary with the age and type of animal used and the specific endpoint measured. Likewise, older literature suggests that consumption of soy formula by human infants may impair their immune responses and increase morbidity (Zoppi, 1983Go; Zoppi et al., 1979Go, 1982Go, 1983Go), but more extensive recent studies have disputed these findings and indicate that a wide variety of immune parameters are normal in human infants fed soy formula (Cordle et al., 2002Go; Ostrom et al., 2002Go).

Estrogenic responses in the thymus have not been studied extensively at the molecular level, although some genes regulated by estrogens have been identified (Mor et al., 2001Go; Morris et al., 2003Go; Screpanti et al., 1991Go). In addition, genistein has other effects, such as inhibiting protein tyrosine kinases and topoisomerase II (Markovits et al., 1989Go; McCabe and Orrenius, 1993Go) not involving ER.

One useful tool for gaining insight into the transcriptional changes induced in the thymus by E2 and genistein is microarray analysis. The objectives of this study were to use high-density DNA arrays to identify E2-regulated genes and contrast genistein-regulated gene expression over time, and to gain insight into how estrogen-mediated gene expression changes might affect signaling pathways that regulate various stages of thymocyte maturation. Our results indicate that the effects of genistein and E2 on thymic gene expression were similar, but not identical, with both E2 and genistein having effects on genes involved in a wide variety of signaling and physiological processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment.
Juvenile age-matched (21-day-old) weanling female C57BL/6 mice were purchased from Harlan (Indianapolis, IN) and ovariectomized on day 26 to minimize endogenous estrogen production. Mice were housed individually in polyethylene cages and placed on a phytoestrogen-free diet (AIN-93G purified rodent diet, Dyets, Inc., Bethlehem, PA) two days later. Experimental treatments began on day 30. All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all experiments were approved by the Institutional Animal Care and Use Committee of the University of Illinois.

Animals were fed for 0 (control), 2, 3, and 6 days (n = 6/time point/group) with a diet containing 0 (control) or 1500 ppm genistein (Indofine Chemicals, Somerville, NJ) ad libitum by adding genistein to the AIN-93G diet. The genistein dose was chosen based on previous studies showing that serum genistein concentrations at these doses were comparable to those in human infants fed soy formula (Naaz et al., 2003Go). Some mice given control diet received daily subcutaneous injections of 125 ng of E2 (Sigma-Aldrich, St. Louis, MO) in 0.02 ml corn oil, with the injection sites changed each time. Individual feed intake was measured during treatment to assess effects on feed consumption by genistein or E2. Following treatment, animals were killed, and thymuses removed, weighed, and snap-frozen in liquid nitrogen, then stored at –80°C until RNA extraction. Uteri were weighed and fixed in neutral buffered formalin for histology.

RNA extraction.
Total RNA was extracted from the frozen samples using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA) following a standard protocol. RNA was quantified spectrophotometrically. For quality control, RNA purity was estimated using the OD260/280 ratio. RNA integrity was tested by loading 10 µg of total RNA in a denaturing gel and visualizing the 2:1 ratio of 28S and 18S band intensities with ethidium bromide and checking for RNA degradation. Only samples with OD260/280 ratios from 1.8 to 2.1 and with tight bands with approximate 2:1 ratios of 28S/18S band intensities were used.

Selecting time points.
Differential display RT-PCR was initially used for detecting changes in gene expression and to determine the time course of thymic changes with E2 or genistein. Changes were observed as early as day 1 and 2 following E2 or genistein, respectively (not shown), and therefore treatments of 2, 3, and 6 days were chosen for microarray experiments.

Oligonucleotide array.
GeneChip Mouse Expression Set 430A (Affymetrix, Santa Clara, CA), an oligonucleotide array for 22,680 genes containing 11 probe pairs per gene, was used. Each probe pair consisted of a Perfect Match (PM) and a Mismatch (MM), which were identical except for a single nucleotide change to the complement in the middle of the MM probe sequence that allowed for determination of nonspecific binding. Analyses were carried out at the W.M. Keck Center for Comparative and Functional Genomics, UIUC. The experiment was performed with six animals per time point (n = 6) for each treatment group. Total RNA samples were pooled from three animals for each time point forming two subgroups, and one array was hybridized from each subgroup, making it two independent biological samples for each time point.

Target labeling was performed using total RNA for cDNA synthesis and preparing the biotinylated antisense cRNA using SuperScript II (Invitrogen Life Technologies, Rockville, MD) and EnzoBioArray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Quality control, hybridization, washing, and scanning were performed as described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA; Rev. 4).

Probe set model-based statistics.
To validate expression results obtained from the two subgroups per time point, we used the probe set model-based expression indexes (MBEI) from dChip (Li and Wong, 2001Go) and calculated the Pearson correlation coefficients using the PM probe set expression intensities for each gene. We also performed another comparison between the PM probe set intensities of two subgroups per time point by fitting an Anova model using SAS (SAS/STAT software version 8, SAS Institute, Cary, NC) for each gene at each time point:

Here PMkl is the Perfect Match intensity after quantile normalization using Microarray Suite 5.1 from Affymetrix (Santa Clara, CA) of the kth probe and lth replicate, P and A represent probe and replicate effects, and ekl is random error assumed to be normally distributed with mean 0 and variance {sigma}2.

Data management and analysis.
Expression intensities were prepared with default smoothing adjustments, noise correction, and quantile normalization in Microarray Suite. Gene expression value calculations and comparison analysis between treatments (E2 and genistein) and baseline (control) were performed according to guidelines in the Affymetrix Statistical Algorithms Description Document (Affymetrix, Santa Clara, CA). Detection calls were made with {gamma}1 = 0.05 and {gamma}2 = 0.065 and change calls were made using thresholds {gamma}1L = {gamma}1H = 0.0045 and {gamma}2L = {gamma}2H = 0.006. If the PM or MM cell was saturated (PM or MM > 46,000), the corresponding probe pair was not used for further computations. Data outputs and gene lists were imported into GeneSpring 5.0 (Redwood City, CA) for filtering and clustering. Genes up- or downregulated two-fold or more were used for further analysis.

Hierarchical clustering.
Hierarchical clustering of the filtered genes showing two-fold or greater changes at one or more time points with either E2 or genistein treatment was done in GeneSpring using standard correlation as a similarity measure across time points in both E2 and genistein groups. A minimum distance of 0.001 and the correlation difference between the groups specified by separation ratio 0.5 (default) were used in the hierarchical clustering. This analysis was used to visualize gene expression clusters and how their expression patterns correlated across treatment and time.

Quantitative RT-PCR.
In order to confirm the relative changes in gene expression induced by E2 and genistein that led to the major conclusions in this paper, we used a real-time quantitative reverse transcriptase-polymerase chain reaction (QPCR) approach for selected transcripts. All analyses were carried out using the same total RNA preparations from the microarray analysis. Total RNA (1.0 µg) was reverse-transcribed to cDNA using the Invitrogen Superscript III first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad CA.) The analyses were performed on an ABI Prism 7000 Sequence Detection System using validated ABI Taqman Gene Expression assays (Applied Biosystems, Foster City, CA). All assays were carried out in triplicate prepared for each target mRNA and an internal control gene (18S) for each experimental group. Assays were performed using standard ABI assay conditions and 35 cycles of PCR. Quantitation was performed using the averaged experimental Ct values normalized to the averaged 18S Ct values to generate a {Delta}Ct value. The {Delta}Ct values were then used to calculate a relative quantitative value of fold change using the {Delta}{Delta}Ct method (ABI Chemistry guide # 4330019). ABI Taqman Gene Expression assays used for specific transcripts were: Mm 01340213 m1 (IL-2R{alpha}), Mm00434256 m1 (IL-2), Mm01216171 m1 (CCR5), Mm00726417 s1 (GILZ), Mm00480629 m1 (Pax9), Mm00435245 m1 (Notch1), Mm00432322 m1 (Caspase 7), and Ms99999901 s1 (18S rRNA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uterotropic Response and Thymic Weight Changes
Thymic and uterine (positive control) weights were measured at various time points during E2 or genistein treatment. Both E2 and genistein decreased thymic weights (Fig. 1A), although effects of E2 were more pronounced. Uterine weights showed similar increases with both E2 and genistein, although there was a trend toward a larger increase with E2 (Fig. 1B).



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FIG. 1. Effect of E2 and genistein treatment on thymic and uterine weights. Weights of (A) thymuses and (B) uteri of ovariectomized 30-day-old mice treated with E2 or genistein for 2, 3, and 6 days. Uterine and thymic weights are compared to ovariectomized untreated mice on a phytoestrogen-free diet at 0 and 6 days.

 
Global Gene Expression Categorizations
Of the ~22,600 genes investigated, 807 genes were significantly regulated by genistein or E2 at one or more time points (E2 and genistein microarray data are available from GEO database http://www.ncbi.nlm.nih.gov/geo/ with the accession number GSE2889.). Of these, 538 genes were regulated by E2, and 456 genes were regulated by genistein, and 187 genes (23%) were similarly regulated by both. Besides commonly regulated genes, E2 and genistein exclusively regulated a number of genes; 351 genes (44%) were regulated by E2, and 269 (33%) were regulated by genistein (Fig. 2A). In total, 372 genes were upregulated and 458 downregulated by either or both treatments. Of the upregulated genes, 84 (23%) were upregulated by both treatments; 212 genes (57%) and 76 genes (20%) were exclusively upregulated by E2 or genistein, respectively (Fig. 2B). Of 458 downregulated genes, 103 (22%) were downregulated by both E2 and genistein; 140 genes (31%) and 215 genes (47%) were exclusively regulated by E2 or genistein, respectively (Fig. 2C). More genes were upregulated by E2 (296) than genistein (160), and more genes were downregulated by genistein (318) compared to E2 (243).



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FIG. 2. Global gene expression categorizations. (A) Genes significantly regulated by E2 and genistein. Thymus microarray data for significantly regulated genes, filtered for at least one time point in E2 and genistein treatment showing similar or unique effects. Percentages were calculated based on a total of 807 significantly regulated genes. (B and C) Genes upregulated or downregulated by E2 and genistein. Thymus microarray data showing (B) upregulated genes, percentages based on total 372 genes, and (C) downregulated genes, percentages based on total of 458 genes. (D and E) Selective modulation of E2 regulated genes by genistein. Genistein modulation of (D) E2 upregulated genes, with percentages based on total 296 genes, and (E) E2 downregulated genes, with percentages based on total 243 genes.

 
Of the 296 genes upregulated by E2, 212 (72%) were unaffected by genistein, 69 (23%) were upregulated by genistein, 12 (4%) showed opposite responses to genistein, and 3 (1%) were biphasic (both similar and opposite effects relative to E2 at different time points; Fig. 2D). Of the 243 genes downregulated by E2, 141 (58%) were unaffected by genistein, 97 (40%) showed similar changes that did not reach significance. and 5 (2%) showed opposite changes (Fig. 2E).

Functional Characterization
The Gene Ontology (GO) Consortium database was used to assign molecular function for the annotated transcripts. Significantly upregulated and downregulated genes with either E2 or genistein treatment were classified under nine functional categories (immunity proteins, immune response, cell adhesion/ECM, chaperones, transport, transcription factors, signal transducers, cell cycle, and apoptosis). In all categories, more genes were upregulated by E2 than genistein (Fig. 3). E2 and genistein regulated several transcription factors (E2, 14 genes upregulated, 12 downregulated; genistein, 10 upregulated, 12 downregulated; Table 1). Numerous genes involved in cell proliferation and death were affected. Eight apoptotic genes (Table 2) were upregulated, and only one gene was downregulated by E2 treatment. Several cell cycle genes (Table 3) were differentially regulated by E2 (3 genes upregulated, 3 downregulated) and genistein (1 gene upregulated, 4 downregulated). Genes involved in signal transduction pathways (Table 4) were also regulated (E2, 17 genes upregulated, 14 downregulated; genistein, 7 upregulated, 11 downregulated). A larger number of genes related to immunity (Table 5) were regulated by E2 (16 genes upregulated, 5 downregulated) compared to genistein (3 upregulated, 3 downregulated). Factors involved in the immune response were also affected by E2 (8 genes upregulated, 3 downregulated) and genistein (3 genes upregulated, 3 downregulated). Both E2 and genistein induced changes in cell adhesion and extracellular matrix genes (E2, 13 genes upregulated, 4 downregulated; genistein, 9 upregulated, 7 downregulated). Besides these, there was also differential expression of genes involved in transport (E2, 24 genes upregulated, 16 downregulated; genistein, 9 upregulated, 21 downregulated) and chaperone molecules (E2, 9 genes upregulated, 9 downregulated; genistein, 9 upregulated and 7 downregulated). Several unknown genes were also identified that were expressed sequence tags (ESTs) and were significantly regulated by E2 and genistein.



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FIG. 3. Functional characterization of genes regulated by E2 and genistein. The Gene Ontology database was used to assign molecular function for annotated transcripts of regulated genes. Nine selected functional groups with the number of genes upregulated and downregulated in the thymus by E2 and genistein are shown.

 

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TABLE 1 Transcription Factors Regulated by E2 and Genistein

 

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TABLE 2 Apoptotic Genes Regulated by E2 and Genistein

 

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TABLE 3 Cell Cycle Genes Regulated by E2 and Genistein

 

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TABLE 4 Enzymes and Signal Transducers Regulated by E2 and Genistein

 

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TABLE 5 Genes of Immune Significance Regulated by E2 and Genistein

 
Time Course Patterns
The dendrogram obtained with hierarchical clustering was used to visualize and select clusters that varied temporally or with treatment (Fig. 4). Large numbers of upregulated genes in both E2 and genistein treatment were clustered together at all time points (Fig. 4A). A number of E2-upregulated genes in a cluster were not affected by genistein (Fig. 4B). Genistein caused early downregulation of a group of genes not affected at later time points or with E2 (Fig. 4E). Another cluster was downregulated early by E2, but less affected at later time points or with genistein (Fig. 4D). Similar patterns of downregulation were observed in two clusters with both treatments, one with a relatively higher downregulation with E2 (Fig. 4C) and another with a relatively higher downregulation with genistein (Fig. 4F). A group of genes was downregulated at day 6 by genistein, not affected at other time points, and affected to a lesser degree by E2 (Fig. 4G). In all cases, the clusters of genes whose expression was altered in the various treatments could not be directly attributed to any one functional group.



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FIG. 4. Hierarchical clustering of E2- and genistein-regulated genes with time. Genes significantly regulated by E2 and genistein (807 genes) were clustered hierarchically using standard correlation. Clusters were identified that were upregulated (A) or downregulated (C, F) by both E2 and genistein. Some clusters were upregulated by E2 but unaffected by genistein (B), while other clusters of genes were unaffected by E2 but downregulated early by genistein (E). Finally, some clusters were downregulated early by E2 treatment but less affected by genistein (D), or downregulated late by genistein (G).

 
T-cell development is regulated at multiple levels by a network of signaling events. To more clearly show the critical developmental events in T-cell development that are altered by E2 and genistein, the transcripts modulated in various stages of thymocyte development by either or both treatments are shown in Figure 5.



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FIG. 5. Schematic of E2-regulated genes in thymocyte development. Intrathymic T cell development takes place through several stages of maturation and final selection to form the SP CD4 or CD8 T cell. Genes regulated by E2 that influence various stages of thymocyte development are shown. Genes also regulated by genistein are bolded.

 
Validation
The reliability of the gene expression changes identified here was independently corroborated by two approaches. First, by identification of the same gene expression changes using two independent samples for each time point, and second, by QPCR validation of selected genes. From the array comparisons, all of the six Pearson correlation coefficients were higher than 0.984, indicating high consistency between the two independent subgroups per time point. The gene-by-gene comparison of the subgroups identified 16 genes in the control, 6 genes in the E2 group at day 2, and 9 genes in the genistein group at day 6 that were differentially expressed between the two subgroups at a time point; all other gene expression changes between the subgroup chips for all time points were insignificant. These 31 genes that differed between subgroups were not included in further analysis. Fold-change in gene expression with QPCR largely agreed with the microarray data (Table 6). However, there was a discrepancy with IL-2 expression between the array and QPCR. IL-2 transcript was found to be upregulated with QPCR but downregulated in the microarray. This finding highlights that global gene expression results are statistical selections carrying a significant degree of uncertainty. While global patterns are revealed, absolute expression values for critical molecules should be further validated by a secondary method such as QPCR.


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TABLE 6 Quantitative PCR Validation for Genes Regulated by E2 and Genistein

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite important effects of estrogens and ER{alpha} on thymic development and immune function (Ahmed, 2000Go; Ahmed et al., 1999Go; Erlandsson et al., 2001Go; Li et al., 2002Go; Rao and Richardson, 1999Go; Yellayi et al., 2000Go), mechanisms by which estrogens affect thymus are unknown. We previously showed that genistein, an estrogenic soy isoflavone, causes thymic atrophy and inhibits both humoral (Yellayi et al., 2002Go) and cell-mediated immunity (Yellayi et al., 2003Go) in mice; these effects are only partially inhibited by anti-estrogens (Yellayi et al., 2002Go). Furthermore, genistein effects on humoral immunity differ from E2 effects (Trawick and Bahr, 1986Go), and selective suppression of CD4+CD8 thymocytes by genistein was opposite to that seen with E2 (Screpanti et al., 1989Go). This study investigated effects of E2 and genistein on gene expression in mouse thymus, using oligonucleotide arrays to provide insight into their molecular mechanisms of action.

Global Gene Expression Patterns
Microarray experiments give global patterns of gene expression. Only 3.5% of the genes analyzed were significantly regulated by E2 and genistein, indicating that these treatments alter only a small subset of genes despite their phenotypic effects. There was considerable overlap in the genes regulated by both E2 and genistein, but there were clear differences as well, indicating that genistein has significant actions in addition to its estrogenic effects.

Functional categorization of gene expression showed that E2 and genistein had effects on a wide variety of genes involved in many critical cellular processes. E2 and genistein affected cell proliferation and apoptosis genes, processes occurring extensively during thymocyte maturation. They also modulated transcription factor expression and genes involved in signaling, immune response, and immunoglobulin synthesis.

Specific gene expression changes observed in thymus due to E2 and genistein treatment could be due to (a) direct influence of either E2 or genistein, (b) compensatory homeostatic mechanisms, or (c) downstream effects brought about by (a) and/or (b). There may also be compositional changes in the thymus due to different effects of genistein or E2 on one or more cell types that could alter relative cell proportions and be responsible for some of the changes detected in the arrays.

E2 and Genistein Modulate Thymocyte Signaling
TECs produce cytokines essential for expansion and development of DN thymocytes (Di Santo and Rodewald, 1998Go; Malek et al., 1999Go). The IL-2/IL-2 receptor pathway is involved in thymic selection (Bassiri and Carding, 2001Go; Zuniga-Pflucker and Kruisbeek, 1990Go) and CD25, the interleukin 2 receptor {alpha} (IL-2R{alpha}) chain, plays a crucial role in development of {alpha}ßT cells (Fehling et al., 1995Go). Signaling through pre-TCR (T-cell receptor) mediates transcriptional repression of IL-2R{alpha} (Malissen et al., 1999Go), and after the transition to the DP stage, thymocytes do not express IL-2R{alpha} (Yeh et al., 2002Go). Binding of IL-2 to IL-2R{alpha} mediates clonal expansion and induces IFN{gamma} and TNF production (Reddy et al., 2001Go). IL-2R{alpha} was upregulated by E2 and genistein (also determined using QPCR). Array data showed that IL-2 was downregulated by both E2 and genistein; however, QPCR results showed an increase in both E2 and genistein groups (Table 6). IL-2R{alpha} induction usually occurs to increase affinity for IL-2, allowing responsiveness when IL-2 levels are low (Yeh et al., 2002Go). This suggests the possibility that downstream signaling events in the IL-2/IL-2R pathway are dampened due to glucocorticoid-induced leucine zipper (GILZ) overexpression and suppression of activation-induced responses in E2-treated thymus (discussed below). IL-2 signaling is essential for self-tolerance, and IL-2/IL-2R deficient mice exhibit autoimmunity due to lack of activation-induced cell death (AICD) (Malek, 2003Go).

IL-6 is another important cytokine for proliferation and survival of thymocytes and their committed progenitors (Bernad et al., 1994Go). IL-6 has anti-apoptotic and proliferative effects mediated through STAT3 activation (Takeda et al., 1998Go), and mice lacking IL-6 have decreased peripheral T cells (Kopf et al., 1994Go). In this study, E2 reduced IL-6 mRNA expression at days 3 and 6, which may reduce thymocyte survival and proliferation.

IL-12b, a cytokine produced mainly by dendritic cells, induces Th1 polarization of the immune response (Rizzitelli et al., 2002Go). IL-12b signaling plays an important role in Th1/Th2 differentiation of SP CD4 thymocytes (Kikkawa et al., 2002Go). E2 downregulated IL-12b, facilitating the formation of more Th2 cells and enhancing humoral immunity in E2-treated mice. However, TCR-mediated signaling events also influence the generation of Th1/Th2 cells (Abbas et al., 1996Go). Further, the inducible costimulator (ICOS)/ICOS ligand (ICOS-L) plays an important role in Th1/Th2 isotype class switching and germinal center formation (Tafuri et al., 2001Go). ICOS-L was downregulated early during genistein treatment. This could affect Th1/Th2 cytokine secretion by CD4 memory cells and severely affect T cell–dependent B-cell responses (Khayyamian et al., 2002Go; Tafuri et al., 2001Go).

Thymocyte selection is mediated by cortical TECs expressing MHC and peptide ligand for TCR (Anderson et al., 1993Go). Loading this peptide ligand on MHC is governed by histocompatibility 2-M (H2-M) molecules, which help the replacement of invariant chain (Ii)–derived MHC class II associated Ii peptides (CLIP) from MHC class II with the antigenic peptide (Wolf and Ploegh, 1995Go). Three H2-M genes (M{alpha}, Mb1, and Mb2) control the nature of the peptide repertoire loaded on the MHC (Cho et al., 1991Go); if the affinity of MHC class II-H2-M is higher than the peptide ligand it would not be loaded. Mb2, predominantly expressed in lymph nodes and spleen (Walter et al., 2001Go), was upregulated by both E2 and genistein. This differential pattern of M{alpha}/Mb1/Mb2 reflects changes in the qualitative composition of immune cells and may be associated with development of autoimmune diseases (Walter et al., 2001Go).

During MHC-TCR interaction in DP thymocytes, CD4/CD8 coreceptors bind. Based on MHC (class I or II), there is irreversible repression or maintenance of either CD4 or CD8 expression, forming the single positive (SP) T cell. Genistein treatment downregulated CD4 mRNA. The transcriptional regulation of CD4 gene is believed to be linked to the signals from TCR interaction (Siu, 2002Go). Genistein affects tyrosine phosphorylation of TCR signaling events in vitro by inhibiting protein tyrosine kinases (PTKs) (Mustelin et al., 1990Go; Trevillyan et al., 1990Go). CD4 is associated with the PTK lck more tightly than CD8 and delivers a stronger signal (Werlen et al., 2003Go), which when inhibited by genistein can cause thymocytes expressing MHC class II restricted TCRs to develop into SP CD8 T cells. This explains our findings in previous experiments where SP thymic CD4 T cells were selectively depleted by genistein in vivo (Yellayi et al., 2002Go), and may reflect PTK inhibitory effects of genistein (Akiyama et al., 1987Go).

CD84, a member of the CD2 subfamily of cell surface molecules, is a costimulator for thymocyte and T-cell activation (Martin et al., 2001Go). CD84 is expressed on CD4CD8 thymocytes and decreases with thymocyte maturation (Martin et al., 2001Go). In T-cell activation studies, the level of proliferation induced by anti-CD84 mAb exceeded that induced by anti-CD3 mAb and IL-2 (Tangye et al., 2003Go). CD84 mRNA was downregulated by both E2 and genistein, which may impair the costimulatory signal for thymocyte activation and cytokine secretion required for thymocyte expansion.

Signaling events in the thymus involve several pathways with distinct and common signaling molecules. The MAP kinase pathway is a common mitogenic signal transduction pathway triggered by many growth and survival signals, and inhibiting this pathway reduces signaling associated with MAP kinases (Schultz, 2003Go). Genistein inhibited the MAP kinase pathway by downregulating MAPKKK 7 mRNA, which may disrupt thymocyte response to signals vital for selection and final T-cell repertoire.

Changes in chemokines induced by either genistein or E2 can regulate specific processes involved in thymic and immune function and affect movement of cells and the thymic microenvironment (Bleul and Boehm, 2000Go). Chemokine receptor 5 (CCR5) expressed on thymocytes binds RANTES and MIP1({alpha} and ß) and acts in stimulating IL-2 production and CD25 expression and costimulates T-cell activation (Ward and Westwick, 1998Go). CCR5 was upregulated by E2 and genistein (also determined using QPCR). This allows increased affinity for ligands RANTES and MIP1 and could explain the increased IL-2 transcripts observed. Chemokine ligands 6, 8, and 11 were significantly upregulated by E2 at various time points, suggesting alterations in thymocyte migration in response to receptor expression.

E2- and Genistein-Regulated Transcription Factors Affect Thymocyte Development
Several transcription factors were regulated by E2 and genistein, which may influence thymic cell growth and proliferation. Myc has a highly conserved function in cell growth and proliferation. Mad1 is a transcription repressor that antagonizes Myc function and inhibits thymocyte growth, expansion, and maturation following TCR stimulation (Iritani et al., 2002Go). Myc binds E box sequences as a heterodimer with Max and promotes transcription (Grandori et al., 2000Go). Mad1 also heterodimerizes with Max and represses transcription at E box sites (Knoepfler and Eisenman, 1999Go). The balance between Myc-Max heterodimers and Mad1-Max heterodimers provides a mechanism to control lymphocyte proliferation in lymphomas, autoimmune disease, or clonal expansion of activated T cells (Iritani et al., 2002Go). Transgenic mice overexpressing Mad1 had a drastically reduced thymus, but thymocyte subpopulations were unaffected (Rudolph et al., 2001Go). Mad1 was significantly upregulated by E2 and unaffected by genistein, while both E2 and genistein downregulated Max. This results in a repression of proliferation in the thymus with relatively high levels of Mad1 and decreases in Max, further competing out Myc heterodimerization, severely impairing proliferation.

T-cell signaling, as a result of MHC loaded antigen interaction with TCR/CD3 complex, controls negative selection (Jenkinson et al., 1989Go). Glucocorticoid-induced leucine zipper (GILZ) expression protects T cells from apoptosis induced by TCR/CD3 signaling (D'Adamio et al., 1997Go). GILZ overexpression inhibits TCR activation-induced responses such as NF{kappa}-B nuclear translocation, FasL expression, and subsequent activation-induced cell death (AICD) (Ayroldi et al., 2002Go). GILZ was upregulated by both E2 and genistein (also determined using QPCR). This decreases AICD in maturing thymocytes and could modulate IL-2 production, a key growth factor for antigen-activated thymocytes. This agrees with our finding of significant changes in IL-2/IL-2R{alpha} transcripts in both E2 and genistein treatment.

PU.1 is an ets-family transcription factor required for myeloid and lymphoid development. Inactivation of the PU.1 gene causes developmental defects in B and T lymphocytes, monocytes, and granulocytes (McKercher et al., 1996Go; Scott et al., 1994Go). C/EBP{alpha}, upregulated by E2 and genistein, can block PU.1 function (Reddy et al., 2002Go). This could affect thymic dendritic cell activity and decrease SP CD4 thymocytes, as PU.I null mice do not produce MHC class IIhigh dendritic cells in vitro (Anderson et al., 2000Go). C/EBPß also controls lymphocyte proliferation, possibly by downregulating IL-6 (Alonzi et al., 1997Go). C/EBPß was upregulated late in E2 treatment and downregulated early in genistein treatment. IL-6 mRNA was inversely regulated with C/EBPß expression and was downregulated by E2.

Notch signaling through the Notch1, 2, and 3 receptors plays an important role in lymphoid development (Felli et al., 1999Go). Notch1, expressed only on early DN thymocytes, is critical for regulating T lineage commitment during differentiation and the DN to DP stage of thymocyte maturation (Allman et al., 2002Go). A relatively higher percentage of thymocytes express Notch3 compared to Notch1 (Felli et al., 1999Go). Injecting anti-CD3 antibodies in mice with an active Notch1 transgene and disrupted recombinase activating gene 2 (RAG2–/–) increased progression from DN to DP stage with a three-fold increase in the SP CD8 compartment (Huang et al., 2003Go). An active Notch3 transgene in mice caused expansion of DN cells, retention of IL-2R{alpha} in post-DN cells, constitutive activation of NF{kappa}-B, and induction of T-cell lymphomas in spleen and lymph nodes (Bellavia et al., 2000Go). Notch1 was upregulated by both E2 and genistein (also determined using QPCR); Notch3 was upregulated only by E2. Though the function of Notch1 and Notch3 is unclear, this could be compensatory, causing lymphoproliferative alterations driving DN to DP stage maturation and causing a bias in phenotype toward SP CD8 versus CD4. IL-2R{alpha}, which is retained past the DN stage in Notch3 overexpression, was significantly upregulated with E2 treatment but less affected by genistein.

Paired box (Pax) proteins are important regulators of thymic embryogenesis. Pax1 is expressed in TECs throughout development and adulthood; it promotes differentiation of cortical TECs, and Pax1 mutants show decreased thymic size and accumulation of CD4CD8 thymocytes due to reduced functional capacity of cortical TECs (Wallin et al., 1996Go). Pax1 was upregulated at the late time point, with E2 treatment probably promoting cortical TEC differentiation. Pax9 is another gene homologous to Pax1. Pax9 deletion causes agenesis of thymus, parathyroid glands, and ultimobranchial bodies (Peters et al., 1998Go). The thymic rudiment which initially develops atopically has significantly small numbers of TCR{gamma}{delta} thymocytes (Hetzer-Egger et al., 2002Go). Pax9 was upregulated by both E2 and genistein (also determined using QPCR). Besides its role in organogenesis, Pax9 function in thymus is unknown.

E2 and Genistein Induce Thymocyte Apoptosis
Thymocyte apoptosis is a continuous process in the thymus undergoing active thymocyte selection. E2 and genistein increases thymic apoptosis (Do et al., 2002Go; Okasha et al., 2001Go; Yellayi et al., 2002Go). Thymic apoptosis occurs through two distinct pathways in the thymus. The first is death by neglect due to lack of critical survival signals. Here, absence of cytokine signaling induces the caspase cascade with cytochrome C release from the mitochondria, which activates the caspases 7 and 9, and causes cell death. E2 treatment induced overexpression of caspase 7 and 9 mRNAs. Genistein upregulated only caspase 7. Thus, E2 and genistein induce passive cell death in the thymus due to lack of crucial pro-survival signals.

The second type of apoptosis is AICD. The IL-2/IL-2R{alpha} pathway has essential functions in promoting Fas-dependent AICD (O'Shea et al., 2002Go), which is important in terms of autoimmunity. In addition, GILZ is also critical in preventing AICD and is upregulated by both E2 and genistein. Apoptotic sensitivity of lymphocytes undergoing passive cell death due to lack of survival signals has been correlated with the presence of cell surface receptors of the lipocalin family (Devireddy et al., 2001Go; Orabona et al., 2001Go). Lipocalin-7 was significantly upregulated by E2, possibly indicating increased sensitivity to death by neglect in these thymocytes. DNA repair enzymes topoisomerase IIß and topoisomerase IIIß, which help prevent apoptosis initiated by DNA damage, were downregulated by E2 and genistein, and this is another possible cause for the increased apoptosis (McCabe and Orrenius, 1993Go; Markovits et al., 1989Go). Cell-death-inducing DNA fragmentation factor, alpha subunit-like effector A (CIDE-A) functions as a positive effector of apoptotis (Inohara et al., 1998Go). CIDE-A was upregulated by both E2 and genistein and, potentially, may be involved in increased thymocyte apoptosis.

E2-Mediated Thymic Changes and Autoimmunity
During T-cell development, autoreactive thymocytes recognizing self-antigens are negatively selected and undergo apoptosis by AICD. Any alteration in the selection and maturation of thymocytes can allow release of self-reactive autoimmune cells. Females have higher incidences of autoimmune diseases than males (Whitacre, 2001Go), and estrogen may contribute to this, but the molecular basis for estrogen effects on autoimmunity is unknown. Here we show that thymic mRNA expression for critical factors involved in thymocyte selection and maturation are modulated with E2 treatment. Several articles have reviewed autoimmunity associated with IL-2/IL-2R{alpha} pathway (Nelson, 2002Go; O'Shea et al., 2002Go). IL-2 or IL-2R deficient mice show severe autoimmune pathology (Horak et al., 1995Go). Further, blocking IL-2/IL-2R in vivo rescues thymocytes from AICD, showing its nonredundant role in maintaining central tolerance (Bassiri and Carding, 2001Go). We have shown that IL-2 and IL-2R{alpha} mRNA are significantly increased by E2 and genistein. This suggests that there is immune modulation, possibly due to reduced signaling through downstream effectors in this pathway. One mechanism that has been used to explain this is the ability of IL-2 to promote Fas-dependent AICD (O'Shea et al., 2002Go). Negative selection by AICD of high-avidity TCR-activated thymocytes is rescued by IL-2 deficiency, leading to release of self-reactive T cells. IL-2R{alpha} (CD25) was significantly upregulated by E2 but not genistein. Upregulation of IL-2 and IL-2R{alpha} is probably a homeostatic mechanism to compensate for reduced IL-2 signaling in thymocytes. This was also seen with genistein treatment, where IL-2 and IL-2R{alpha} mRNA levels were again upregulated. In another model, IL-2 controls autoimmunity by promoting the development of T regulatory cells (Malek, 2003Go). Regulatory T cells suppress a self-reactive response by autoimmune cells. Modulation of IL-2 and IL-2R{alpha} signaling potentially indicated in this study can affect maturation of different T-cell populations and immune function. In addition, E2 regulates the qualitative characteristics of the T-cell repertoire by upregulating Mb2, which controls loading of processed antigen on MHC. Central tolerance by thymic selection is not the principal regulator of all autoimmune responses, and to some degree autoreactivity is normal (O'Shea et al., 2002Go). However, the nonredundancy of these pathways indicates a mechanism by which E2 could facilitate thymic production of autoimmune cells.

Conclusions
This microarray experiment utilized whole thymus consisting of several specialized cells, including thymocytes at various maturational stages and TECs, all expressing ER. Specific transcriptional responses based on thymocyte subsets cannot be delineated from these results; however, expression patterns of specific known transcripts in either thymocytes or TECs can be interpreted accurately. This study describes several mechanisms of E2 and genistein action on thymocyte development and maturation that may be of potential importance to the overall effects of E2 and genistein on thymopoiesis, which appears to be a complex process involving a number of different pathways.


    NOTES
 
1 Current address: James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Road, Ithaca, NY 14850. Back


    ACKNOWLEDGMENTS
 
This work was supported by NIH grant ES 011590 (to P.S.C.) and the Thanis A. Field Endowment. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR16515 from the National Center for Research Resources, National Institutes of Health. Conflict of interest: none declared.


    REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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