Global Uterine Genomics in Vivo: Microarray Evaluation of the Estrogen Receptor {alpha}-Growth Factor Cross-Talk Mechanism

Sylvia Curtis Hewitt, Jennifer Collins, Sherry Grissom, Bonnie Deroo and Kenneth S. Korach

Receptor Biology Section (S.C.H., B.D., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, and Microarray Group (J.C., S.G.), National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Sylvia C. Hewitt, NIEHS, PO Box 12233, Research Triangle Park, North Carolina 27709. E-mail: curtiss{at}niehs.nih.gov.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cross-talk between growth factor receptors and the estrogen receptor (ER) has been proposed as a signaling mechanism in estrogen target tissues, with ER{alpha} as a direct target of growth factor receptor-activated signals, leading to regulation of estrogen target genes and estrogen-like biological responses to growth factors. We evaluated whether global genomic changes in the mouse uterus in response to epidermal growth factor or IGF-I mimic those of estradiol (E2), reflecting the cross-talk mechanism. Overlapping responses to growth factors and E2 were expected in the wild type (WT) whereas no response was expected in mice lacking ER{alpha} (ER{alpha} knockout). Surprisingly, although most of the E2 response in the WT also occurred after growth factor treatment, some genes were induced only by E2. Second, although E2 did not induce gene changes in the {alpha}ER knockout, the growth factor response was almost indistinguishable from that of the WT. Differences in response of some genes to IGF-I or epidermal growth factor indicated selective regulation mechanisms, such as phosphatidylinositol 3-kinase or MAPK-dependent responses. The robust ER{alpha}-independent genomic response to growth factor observed here is surprising considering that the biological growth response is ER{alpha} dependent. We propose two mechanisms as alternatives to the cross-talk mechanism for uterine gene regulation. First, E2 increases uterine growth factors, which activate downstream signaling cascades, resulting in gene regulation. Second, growth factors and estrogen regulate similar genes. Our results suggest that the estrogen response in the uterus involves E2-specific ER{alpha}-mediated responses as well as responses resulting from convergence of growth factor and ER-initiated activities.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN (E) HAS profound roles in the development and function of numerous tissues. Although E circulates in the serum of cycling females at significant levels and can reach all tissues, its effects are selective and distinct. E target tissues contain estrogen receptors (ER), transcription factors with high-affinity binding sites for E, which interact with and regulate E-responsive genes (1, 2). In the simplest model, E binds to the ER, which interacts via high-affinity binding of specific DNA sequences in target genes, causing recruitment of transcriptional modulators and consequent regulation of transcription. However, numerous variations in this mechanism have been described. Many E-responsive genes lack the canonical E-responsive enhancer sequence, but are regulated by ER complexes tethered to SP1 transcription factor or activator protein 1 (AP-1) transcription factors (3, 4, 5). In addition, several mechanisms to account for very rapid nongenomic or nongenotropic E responses indicate that E receptors, either distinct or identical to the nuclear ERs, interact with and activate signal cascades at the membrane (6, 7). Finally, several activators of growth factor (GF) receptor pathways, including IGF-I and epidermal growth factor (EGF), can activate ER-mediated transcription (6).

We have previously characterized these ER-GF receptor cross-talk mechanisms in vivo, by showing the estrogen-like effects of an increase in uterine weight and proliferation of uterine epithelial cells in ovariectomized mice after EGF or IGF-I treatment (8, 9, 10). The lack of these responses in similarly tested mice lacking ER{alpha} [ER{alpha} knockout ({alpha}ERKO)] indicated that ER{alpha} is downstream of the GF receptor signaling in this response (9, 10, 11). This mechanism has been studied in vitro as well, using reporter gene assays, which also show that ER{alpha} is required for GF receptor activators to modulate E-regulated reporter genes (12, 13). The approaches have been combined, and we showed that IGF-I treatment increases expression of an E-responsive luciferase reporter gene in transgenic mice (10), providing support for the cross-talk hypothesis in vivo.

Here, we use microarray technology to investigate the endogenous uterine genomic profile of ovariectomized WT and {alpha}ERKO mice that were treated with EGF or IGF-I. The responses were compared with those resulting from estradiol (E2) treatment, which were characterized previously (14). Considering the model of GF receptor-ER cross-talk, we hypothesized that this approach would yield two patterns or clusters of genes with similar response profiles. The first would include genes regulated in the wild type (WT) in response to E2 or GF receptor signaling, representing the cross-talk response; because these responses require ER{alpha}, we hypothesized that they would be lost in the {alpha}ERKO. The second pattern would include genes that were directly regulated by GF receptor pathways. These genes would not overlap with ER-mediated responses and therefore would be similarly regulated in the {alpha}ERKO, because they depend only on GF receptor pathways. Surprisingly, the global genomic response in the uterus did not fit these expected patterns and has uncovered alternate models of GF-ER pathway interaction.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Microarray Analysis: Genomic Response of the Uterus to Growth Factors
Previously, we have used microarrays to characterize the genomic response of the ovariectomized mouse uterus to acute E2 treatment (14) and demonstrated distinct early (2 h after treatment) and late (24 h) gene responses. To compare the GF and E2 responses, we examined the uterine profile after treatment for 2 or 24 h with long R3 IGF-I (an IGF-I analog with low affinity for IGF-binding proteins) or EGF. The cluster analysis of the global genomic responses of the uterus is shown in Fig. 1Go. Surprisingly, the patterns of gene regulation were not as anticipated. Instead, the gene changes generally fell into three categories, which are indicated on Fig. 1Go.



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Fig. 1. Cluster Analysis of Significant Gene Changes

A, Data obtained from microarray analysis (as described in Materials and Methods) were used to generate a cluster diagram of significant gene changes (99% confidence). Each vertical line represents a single gene. Genes that were increased by E2 or GF treatment relative to vehicle are indicated by red, with relative intensity representing degree of regulation. Down-regulated genes are similarly shown in green. Each horizontal line shows gene changes at 2 h (top panel) or 24 h (bottom panel) of treatment compared with vehicle, respectively. Response pattern categories are indicated above each chart, including category 1, genes regulated by E2 or GFs in the WT but only by GFs in the {alpha}ERKO; category 2, genes that are regulated only by E2 and only in the WT (yellow boxes); category 3, genes that are regulated only by GF. B, Lists of genes significantly regulated by E, IGF-I, or EGF (99% confidence) in 75% of hybridizations were created by querying the data sets in the NIEHS MAPS (MicroArray Project System) database and copied into GeneSpring for display as Venn diagrams for each genotype (WT or {alpha}ERKO) and time point (2 or 24 h). Number of genes in each group are indicated.

 
Category 1 included ER{alpha} or GF-dependent genes that were regulated similarly by either E2 or GFs in the WT samples. These genes might seem to be regulated as described by the cross-talk mechanism in which GF receptor signaling results in ER-mediated transcription. However, in this model, no GF gene regulation would be expected in the {alpha}ERKO, yet the category 1 genes retained GF responsiveness in the absence of ER{alpha}. The ER{alpha} dependence of category 1 genes for E2 regulation was demonstrated by lack of E regulation in the absence of ER{alpha} (Fig. 1Go).

Category 2 genes were ER{alpha} dependent, as illustrated by their regulation only in the WT samples (yellow boxes, Fig. 1Go). These genes were not regulated by a cross-talk mechanism but depended on direct modulation of ER{alpha} activity by E2 only. Finally, category 3 included GF-responsive genes. The cluster analysis in Fig. 1Go did not reveal any genes that followed the pattern of expression predicted by a cross-talk mechanism.

Significant gene changes (99% confidence) in each condition are displayed as Venn diagrams (Fig. 1BGo), allowing visualization of relative numbers of genes in each response category.

Overall, genes in the WT samples followed a biphasic pattern (distinct clusters regulated primarily at the early 2-h time point or the later 24-h time point) as reported previously (14), both with E2 and with GFs (see Supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, in which the clusters for 2 and 24 h are compared). In addition, the three response categories were represented at both time points, although the GF-specific responses (category 3) were more apparent at 2 h. Both up- and down-regulated genes were evident at both time points and all treatments; however, the ER{alpha}-specific response (category 2) included only up-regulated genes at both time points. Previous GF studies had shown that transcription of model reporter genes was ER{alpha}-dependent and that {alpha}ERKO mice lack uterine growth, demonstrating the requirement for ER{alpha} in GF responses in support of a cross-talk mechanism (9, 10, 11, 12, 15). Thus, it was surprising that the response of numerous endogenous uterine genes to GF is independent of ER{alpha}.

Verification of Response Patterns
To verify the gene regulation patterns observed in the microarray analysis, examples of genes that followed these patterns were chosen and confirmed by real-time RT-PCR. The data from all analyses are shown in Tables 1–4GoGoGoGo. In Fig. 2Go, dendograms representing the microarray data of the selected genes are presented. Additionally, the RT-PCR data from some of the genes in Tables 1–4GoGoGoGo are also represented graphically in Fig. 2Go.


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Table 1. Real-Time PCR Values from WT 2-h Samples

 

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Table 2. Real-Time PCR Values from {alpha}ERKO 2-h Samples

 

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Table 3. Real-Time PCR Values from 24-h WT Samples

 

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Table 4. Real-Time PCR Values from {alpha}ERKO 24-h Samples

 



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Fig. 2. Verification of Responses Seen in the Microarray Analysis by Real-Time RT-PCR and Western Blot

Genes that were in the categories indicated in Fig. 1Go were identified, and their regulation by E2 or GFs in WT or {alpha}ERKO samples was determined by real-time RT-PCR. Each category panel includes a dendogram showing the microarray data of the genes further analyzed. The number next to the gene names on the dendogram is the clone number from the microarray chip. For some genes, the effects of the E antagonist ICI 182,780 on E2 or GF regulation were also determined. Some were also analyzed for protein expression levels by Western blot. All analyses consisted of two to three replicate samples, some of which were pools of two to three uteri. Maroon bars represent analysis of samples treated with ICI 182,780, together with the adjacent ligand. A, Category 1: ER{alpha}- or GF-regulated genes. Txnip and p21 transcripts were assayed by real-time RT-PCR in WT and {alpha}ERKO samples. Values are expressed relative to the vehicle-treated sample from the respective ER{alpha} genotype and normalized to 18s transcript. V, Vehicle treated; E, E2, IGF, (long R3 IGF-I). TXNIP protein was also analyzed by Western blot. Values from a densitometry scan are indicated below the Western blot. B, Category 2: ER{alpha}-regulated genes. cFos and Mitotic arrest deficient 2 (Mad2) cDNA was assessed in both WT and {alpha}ERKO samples at 2 and 24 h, respectively. cFOS protein was also examined by Western blot. C, Category 3: genes regulated only by GFs and cross-talk genes. Kruppel-like factor 9 (Klf-9) [also called basic transcription element binding protein 1 (Bteb1)] transcripts were measured in WT and {alpha}ERKO samples from the 2-h treatment time point. Cyclin E cDNA was assessed 24 h after treatment.

 
Category 1 Down-Regulated Genes: Txnip (Thioredoxin-Interacting Protein), Igfbp3 (IGF-Binding Protein 3), and Sox 4 (SRY Box Containing Gene 4)
The expression patterns of genes that were repressed in an ER- or GF-dependent manner (category 1) were confirmed (see Fig. 2AGo and Tables 1–4GoGoGoGo). These genes included integral membrane 2b (Itmp2b), Igfbp3, the predominant serum IGF-binding protein, which binds and increases the half-life of serum IGFs and modulates their activity on target cells (16), Sox 4, and Txnip, which inhibits thioredoxin activity (17).

As previously reported, Txnip is repressed after E treatment of the uterus (18), and our microarray analysis shows a GF-dependent regulation as well. The data from the RT-PCR analysis of Txnip at 24 h (17) are shown graphically (Fig. 2AGo). In addition, Western blot analysis revealed the pattern of regulation of the TXNIP protein at 24 h mirrored that of the mRNA in the WT samples (Fig. 2AGo). The repression of Txnip (and Igfbp3) transcript at 24 h by E was partially relieved by antiestrogen ICI 182,780 (ICI), whereas that of Sox4 was completely inhibited (see Table 3Go). Both EGF and IGF-I also decreased expression of these three genes at 24 h (Table 3Go). Unlike E2 treatment, ICI did not prevent the decrease of Txnip mRNA after GF treatment, indicating that the mechanism of repression by GFs differs from that of E2-mediated repression and does not involve the ER. In similarly treated {alpha}ERKO samples Txnip mRNA levels were not reduced by E2, but were reduced by GFs (see Table 4Go). In the {alpha}ERKO, ICI did not alter the GF-induced suppression of Txnip (Fig. 2AGo). Western blot analysis showed a similar response pattern of the TXNIP protein in the {alpha}ERKO samples (Fig. 2AGo). The ER dependence for E response was demonstrated both by the lack of regulation in the absence of ER in the {alpha}ERKO as well as by the inhibition of response by ICI in the WT. The GF dependence was demonstrated by their regulation independent of the presence of ER and the lack of ICI effect.

Interestingly, in the cases of Txnip or Sox4, repression was apparent by 2 h after estradiol or IGF-I treatment in the WT (Table 1Go), whereas the repression after EGF treatment was minimal after 2 h (Table 1Go). This difference in the timing of responses of these genes to E2 or EGF indicates differences in the signaling mechanisms involved in their regulation.

The general pattern of expression of these category 1 down-regulated genes is strikingly similar (see Tables 1–4GoGoGoGo), with E2 causing profound repression in the WT, which is completely or partially blocked by ICI, combined with diminishment or loss of response to E2 in the {alpha}ERKO. GFs also repress these genes to a similar degree in the WT and {alpha}ERKO, and the repression by GF is not blocked by ICI.

The mechanism of ER{alpha}-mediated gene down-regulation might be quite different than the canonical ER-mediated gene up-regulation responses that have been characterized. The observation that the repression in response to E2 is diminished or lost in the {alpha}ERKO indicates the ER{alpha} has a significant role in the mechanism. In addition, ICI did not completely inhibit the E-mediated repression in all cases, indicating that the role of ER{alpha} in the down-regulation might differ from its role in up-regulation. ICI binds to the ER{alpha} and induces a conformation that does not interact effectively with transcriptional coregulators (2), thus resulting in loss of induction of up-regulated genes. In the case of down-regulated genes, the role of the ER{alpha}, rather than recruiting activating factors to the transcriptional apparatus, might be to interfere with basal transcription of target genes or to recruit repressors to the transcriptional apparatus. When bound to ICI, the ER{alpha} might remain in a conformation that still allows it to inhibit transcription. Additionally, ICI is known to decrease the level of ER{alpha} protein (19); thus, there might not be sufficient ER{alpha} present to completely repress transcription.

Category 1 Up-Regulated Genes: Igfbp5, p21, and Cyr 61
The expression patterns of Igfbp5, Cyr61, and p21, genes that were increased by E2 or GF in the WT but only by GFs in the {alpha}ERKO, were confirmed by RT-PCR (Fig. 2AGo and Tables 1–4GoGoGoGo). The data from analysis of p21 are shown graphically. Two hours after E2 treatment, as previously observed (14), mRNA levels of the cyclin-dependent kinase inhibitor p21 increased in the WT samples (Fig. 2AGo), and ICI prevented this increase. p21 levels also increased 2 h after GF treatment, but not to the extent seen with E2. p21 mRNA levels were also increased by GF in the {alpha}ERKO, but not by E2.

Cysteine-rich protein 61 (Cyr61), a secreted cell surface protein (20, 21), was also increased in the WT samples (Table 1Go). ICI decreased or prevented this increase. Cyr61 was minimally increased in the {alpha}ERKO by E2 and IGF-I but little by EGF, and much less than the E2 induction of the WT (Table 2Go). Additionally, the basal level of Cyr61 in the {alpha}ERKO was 5-fold higher than the WT basal level. The differential response to the two growth factors indicates Cyr 61 is selectively regulated by signaling initiated by IGF-I but not EGF, such as phosphatidylinositol 3-kinase (PI3 kinase) signaling, rather than a MAPK-dependent response.

The level of Igfbp5 mRNA, another serum IGFBP, was increased by 24 h of E2 as previously observed (14) and also by IGF-I treatment in the WT samples, but little by EGF (Table 3Go). ICI prevented the E2-induced increase, but not the GF-induced increase. In the {alpha}ERKO samples, however, Igfbp5 transcript was only increased after IGF-I treatment. The fact that IGF-I increases Igfbp5 mRNA independent of ER{alpha} indicates that two different mechanisms of increase may occur: the first is a classic ER{alpha}-dependent mechanism; the second is through the IGF-I induced pathway. Igfbp5 has been shown to be elevated in vascular smooth muscle cells via a PI3 kinase-mediated pathway (22, 23). We noted that Igfbp3 is repressed and Igfbp5 is increased by E2 or IGF-I, suggesting different roles for these IGFBPs in uterine biology. In support of divergent roles for these two IGFBPs, IGFBP5 protein has been reported to be predominantly expressed in the myometrial region of the uterus, whereas IGFBP3 has been detected in the stroma (24). The IGF-I used for our studies was long R3 IGF-I, an analog designed to have decreased affinity for IGFBPs. It is not known whether this may affect the regulation of Igfbp transcripts. It is interesting that in the WT sample the IGF-I increased Igfbp5 more than E2 did, and this increase by IGF-I is significantly lower in the {alpha}ERKO (using a Mann-Whitney U test to compare {alpha}ERKO fold increase to WT fold increase: P = 0.04), suggesting ER{alpha}-mediated and IGF receptor pathways may combine in WT samples to increase induction, whereas only the GF pathway is present in the {alpha}ERKO. Similarly, the degree of GF regulation of Sox4, (P = 0.04 for both IGF and EGF at 24 h, WT vs. {alpha}ERKO) and Baiap (P = 0.04 for EGF at 2 h, WT vs. {alpha}ERKO) is blunted in the {alpha}ERKO. The regulation of Cyr61 by IGF-I and the regulation of Krt 1–19 by EGF at 2 h seems greater in the WT; however, the difference is not statistically significant (P = 0.095) but further analysis might indicate whether ER{alpha} is involved in the GF-mediated regulation of these genes. Conversely, ICI does not decrease the GF regulation of these transcripts in the WT, suggesting a lack of a contribution from ER{alpha} for GF regulation of these genes. More extensive future study might indicate the validity of this preliminary suggestion.

cFos: RT-PCR Moves cFos from Category 2 to Category 1
Although cFos was in the E2-specific category 2 cluster on the microarray analysis, it was also regulated by GF and follows the regulation pattern of the category 1 group. The GF regulation is also apparent in the cluster analysis (see dendogram in Fig. 2BGo); however, the robust increase with E probably caused the data to cluster with the E2-only set. RT-PCR analysis indicated an increase in cFos 2 h after GF treatment as well (Fig. 2BGo and Table 1Go). cFos, a subunit of the AP-1 transcription factor (25), was increased after 2 h of E2 in the WT (Table 1Go and Fig. 2BGo), as has been previously observed (26, 27, 28). ICI decreased or prevented this increase. In the {alpha}ERKO samples, cFos was also increased by E2 but less robustly than in the WT (Table 2Go and Fig. 2BGo). ICI inhibited the E2 increase in the {alpha}ERKO sample. cFos has been shown to be rapidly (30 min) increased by EGF, in both WT and {alpha}ERKO uteri (9), and the degree of regulation by GF observed here after 2 h may be maintained from the earlier response. Western blot analysis of protein extracts from identical treatments showed the pattern of cFOS protein expression mirrored that of the mRNA (Fig. 2BGo).

Category 2 Genes Respond Exclusively to E2: Mad2, Ramp3, Lactoferrin, Igf-1, and Krt1–19
The observation of exclusively E2-regulated genes (category 2) was somewhat unexpected. We verified the expression patterns of several genes that followed this pattern. As we previously observed (14), RNA levels of receptor (calcitonin) activity-modifying protein 3 (Ramp3), involved in transport and activation of calcitonin-like receptors (29), and mitotic arrest-deficient, homolog-like 1 (Mad2), which aligns the mitotic spindle (30), were robustly increased 2 h after E2 treatment (Tables 1Go and 2Go). Lactoferrin, Mad2, Igf-1, and Krt1–19 were all elevated 24 h after E2 treatment (Tables 3Go and 4Go) and were attenuated by ICI. None of these increases were noted with GFs. All required ER{alpha}, as demonstrated by the lack of regulation in the {alpha}ERKO. Thus, these genes are regulated by a classic ER{alpha}-dependent mechanism, but not influenced by ER{alpha} cross-talk with GF receptor pathways. The data from the analysis of Mad2 are also shown graphically (Fig. 2BGo).

Interestingly, Igf-1 regulation by E involves tethering of the ER to the promoter sequences via an AP-1 site (3, 31); thus, our observation indicates that the E-exclusive mode of response can occur in genes regulated by this tethering mechanism. Note the lack of response of this AP-1-mediated gene to GFs despite the increase in the AP-1 component cFOS by GF (Tables 1–4GoGoGoGo).

Category 3: Genes Responding Exclusively to GFs: Baiap2 and Kruppel-Like Factor 9 (Klf 9)
The expression patterns of brain-specific angiogenesis inhibitor 1-associated protein 2, (Baiap2), an IRS p53 isoform (32, 33), and Klf-9 (also called basic transcription element binding protein or Bteb1), genes that were regulated only by GF in the WT and the {alpha}ERKO (category 3), were examined by RT-PCR (Tables 1Go and 2Go and Fig. 2CGo). The data from Klf-9 are also shown graphically (Fig. 2CGo). RT-PCR analysis confirmed the GF-specific regulation of Klf-9 and Baiap2. Caveolin (Cav-1), which encodes CAV protein found in caveoli and has been shown to sequester ER{alpha} in these membrane structures, appeared to be EGF regulated in the microarray dataset, but the regulation was not confirmed by RT-PCR (data not shown).

Category 4 Cross-Talk Genes: Cyclin E and Krt 1–19
The original cluster analysis failed to identify genes showing a profile that would fit a cross-talk model. Interestingly, although 24 h after treatment Krt1–19 was selectively induced by E2 (Tables 3Go and 4Go), 2 h after injection, only growth factors induced the transcript and only in the WT samples (Tables 1Go and 2Go). This seems to indicate a role for ER{alpha} and a cross-talk-type mechanism, which perhaps has an altered time course in response to E2 or GF and is therefore included in the cross-talk section.

A second cross-talk gene, Cyclin E, was also identified. Cyclin E was not present on the microarray chip, but was previously shown to be increased by E2 after 24 h (14). Real-time RT-PCR analysis revealed that it was also increased by GF in the WT samples (Fig. 2CGo), but less robustly than with E2. There was no response in the {alpha}ERKO samples to E2 or GF. The lack of regulation of Cyclin E, which modulates S phase progression, in the {alpha}ERKO by either E2 or GFs, correlates with a lack of epithelial proliferation in response to GFs in the {alpha}ERKO (9, 10).

The microarray dataset was further analyzed using Genespring software to identify genes that were regulated by E2 or GF but only in the WT samples; however, no additional genes fitting the cross-talk pattern of expression were identified.

Mechanisms Accounting for Gene Responses to GF in the {alpha}ERKO
Direct E-Mediated Regulation of GFs.
The global genomic response of the uterus to acute E2 or GF treatment observed in this study differed from our original hypothesis based on a cross-talk mechanism. Clearly, ER{alpha} is required for E2 to regulate levels of responsive genes. For a subset of these E2-responsive genes, however, GF can similarly regulate responses independently of ER{alpha}. This observation suggests that for these genes ER{alpha} is upstream of GF receptor signaling, and that E2 increases levels of GFs (see, for example, Igf-1 in Table 3Go), thus activating GF receptor pathways and the genomic response resulting from regulation of its targets. These responses, then, could also be elicited directly by GFs, bypassing the need for ER{alpha}; thus, the response could occur in the {alpha}ERKO. This mechanism suggests that for many endogenous uterine genes, regulation is not occurring via a cross-talk mechanism, and that the genes regulated exclusively by E2 are the only actual ER{alpha} targets. Indeed, the levels of both EGF and IGF-I are known to increase after E2 treatment, with maximal increase of IGF-I after 4–6 h (34) and activation of the IGF receptor within 4 h (35), whereas EGF is elevated after 12–24 h (36, 37). The transcripts of EGF receptor activators, EGF and TGF{alpha}, were not increased by E2 in the microarray dataset (data not presented). We did observe a 2-fold increase in Igf-1 mRNA by ribonuclease protection assay analysis in our 2-h E2-treated sample but were unable to detect EGF transcript by RT-PCR at 2 or 24 h after E2 treatment (data not shown). Whereas the Igf-1 transcript increases rapidly, whether the IGF-I protein is increased rapidly enough to account for the genomic responses observed as early as 2 h is not known.

Residual ER{alpha} Splice Variant
Previously, we have reported a residual splice variant of ER{alpha}, E1-ER{alpha}, which is detected in the {alpha}ERKO uterine tissue at approximately 5–10% of the WT level and contains a deletion in the AF-1 region and retains the entire AF-2 and ligand-binding domains (38). We considered whether it may account for the remaining E-like responsiveness to GF observed in the {alpha}ERKO, but this possibility is unlikely. First, none of the GF responses was inhibited by ICI, indicating the E1-ER{alpha} form is not mediating the response. Second, E2 can activate E1-ER{alpha} (38), yet there is minimal genomic response to E2 in the {alpha}ERKO. The blunted E response still seen for some of the target genes might indeed be mediated by E1-ER{alpha} or possibly by ERß (see p21, cFos Cyr61, and Sox 4 RT-PCR data in Fig. 2Go and Table 2Go) as ICI does inhibit it. Third, the level and activity of E1-ER{alpha} in the uterine tissue are low (38) yet the {alpha}ERKO uterine gene response to GFs is as robust as in the WT in most cases.

Proposed Model: Converging Responses
We propose that rather than a strict cross-talk pathway, uterine responses result from diverse signals (E, GF) that converge to regulate overlapping genes (see model, Fig. 3Go). Additionally, E may interact with the recently described membrane-associated pathway mediated by ER{alpha}, androgen receptor (AR), or a hypothesized membrane E receptor that initiates a signaling cascade (6, 7). Several studies using cultured cells have shown E2-dependent ER{alpha} or androgen receptor interaction with, and activation of, membrane-associated components (39, 40, 41, 42, 43, 44, 45, 46). Studies using cultured rat pituitary cells indicated that inhibition of the MAPK-signaling cascade prevented E-mediated induction of endogenous prolactin transcription, illustrating the essential role of this mechanism in some E-responsive gene expression (47). This novel mechanism has been termed the "rapid," "nongenomic," or "nongenotropic" mechanism of E action. The ability of ICI to inhibit nongenotropic signaling has been suggested to indicate the involvement of the ER{alpha} in the mechanism (39, 40, 43). We propose that the activation of the same intracellular signaling pathways by GF receptors or by the nongenomic E mechanism might result in regulation of common genes by these divergent ligands.



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Fig. 3. Proposed Model: Diverse Signal Initiators Converge to Regulate Uterine Genes

E binds nuclear ER{alpha}, recruiting coregulators (CoAc), modulating gene transcription by directly interacting with estrogen response element (ERE) DNA sequences or through tethered interaction with AP-1 or SP1 transcription factors. Initiation of nongenotropic signals at the cell membrane is also depicted where E activates signaling pathways such as the MAPK pathway or the PI3 kinase pathway. ER{alpha}, AR, or another E-binding molecule may be involved in mediating this response (represented by ?). GFs also activate MAPK or PI3 kinase pathways by interacting with and activating their membrane receptors (GFR). These signals converge at the level of genomic modulation, resulting in similar genomic responses to E and GFs, either by direct activation of nuclear ER-mediated transcription, or other transcriptional mediators. Igf-1 is increased by E and further activates the GF receptor-mediated pathways. txn, Transcription.

 
A significant number of endogenous uterine genes were found to be regulated exclusively by E and to require the presence of ER{alpha} (category 2). Because these genes are not regulated after activation of GF receptor signaling, yet GF treatment leads to epithelial proliferation, these ER{alpha}-dependent responses seem not to be obligatory for proliferation.

In conclusion, microarray analysis of the global genomic response pattern in uterine tissue after GF treatment of WT or {alpha}ERKO mice has led us to reevaluate the role of cross-talk mechanisms in the uterine genomic responses. Clearly, in a biological system, the interaction between E and GF signaling is more complex than originally envisioned. Despite a robust genomic response, the lack of epithelial proliferation in the {alpha}ERKO in response to GF indicates a role for the ER{alpha} for a complete biological response. Future studies will lead to a better understanding of the potential gene-specific mechanisms involved in ER{alpha}-mediated and ER{alpha}-independent responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals and Treatments
All animals were handled according to National Institutes of Health guidelines and in compliance with a National Institute of Environmental Health Sciences approved animal protocol. All mice used were ovariectomized and housed for at least 10 d before studies to allow endogenous ovarian steroids to decrease. Mice are maintained on standard NIH chow. WT C57 Bl/6 mice were purchased from Charles River Laboratories, Inc. (Raleigh, NC) or obtained from Taconic Farms, Inc. (Germantown, NY). {alpha}ERKO mice were obtained from Taconic Farms. Control animals were treated with sesame oil vehicle (Sigma Chemical Co., St. Louis, MO). For E2 treatments, 1 µg E2 (Steraloids, Newport, RI) was either dissolved in 100 µl sesame oil and injected sc (24-h group) or 100 µl saline and injected ip (2-h group). EGF (Biosource Technologies, Inc., Camarillo, CA) or long R3 IGF-I (Diagnostic Systems Laboratories, Inc., Webster, TX), a synthetic IGF-I analog with low affinity for IGF binding proteins (DSL), were either dissolved in sesame oil at 1 µg/µl and 200 µl was injected ip (2-h treatment), or dissolved at 2.5 µg/µl in 0.1 N acetic acid and administered in osmotic pumps (83 µg delivered in 24 h) (Alza, Durect Corp., Cupertino, CA) as previously described (10). Some animals were treated with 45 µg ICI 182,780 (kindly provided by Zeneca Pharmaceuticals, Cheshire UK; dissolved in 50 µl dimethylsulfoxide injected ip) 30 min before E2 or GF treatments. Animals were killed at the indicated times using CO2 and the uteri were collected, weighed, and snap frozen in liquid nitrogen for RNA or protein isolation.

Microarray Analysis
Frozen uterine tissue was pooled (at least five uteri per group), pulverized, and then homogenized in Trizol (Invitrogen, Carlsbad, CA), and RNA was prepared according to the manufacturer’s protocol. Isolated RNA was then further purified using the QIAGEN Rneasy midi (100–500 µg RNA) or mini prep kit (<100 µg RNA) clean up protocol (QIAGEN, Valencia, CA).

A cDNA Mouse Chip (NIEHS Mouse chip version 1.0), developed in house at NIEHS, was used for gene expression profiling experiments as previously described. A complete listing of the genes on this chip is available at the following website: https://dir-apps.niehs.nih.gov/maps/guest/clonesrch.cfm. cDNA microarray chips were prepared according to the method of DeRisi et al (48). Microarray hybridizations and analyses were performed as previously described (14). The entire dataset is available for journal editors at http://dir.niehs.nih.gov/microarray/hewitt.

The microarray data were obtained from a single experiment, with each sample pair hybridized on at least four replicate chips. The data from E treatments were compiled with results from identical treatments used in two additional experiments.

Northern Blot
Total RNA (2 µg) was run on a 1% agarose formaldehyde gel and transferred to Bright Star membrane using Ambion’s Northern Max reagents and protocol (Ambion, Austin TX). Membrane was prehybridized and incubated with 32P-labeled riboprobes in Ultrahyb buffer (Ambion) according to the manufacturer’s protocol. Riboprobes were labeled using the Maxiscript kit (Ambion) and 32P CTP (Amersham Pharmacia Biotech, Arlington Heights, IL). The IGF-I cDNA template was as previously reported (11). The cFos cDNA template was purchased from Ambion. Signal was detected and quantified using the Storm phosphor imager and ImageQuant software (GE Healthcare, Piscataway, NJ) and was normalized to the 18s ethidium bromide band using the UVP EC3 Bioimaging system and Labworks software (Ultraviolet Products, Upland, CA).

Verification of Microarray Results by Real-Time RT-PCR
Ovariectomized mice were treated as described for the microarray analysis, and RNA was prepared from mice treated with vehicle, E2, EGF, or long R3 IGF-I with or without ICI pretreatment as described above, and then collected 2 h or 24 after treatment. In some cases, uteri were pooled (two to three uteri per pool), whereas others were used individually. Frozen tissue was pulverized, and RNA was prepared using Trizol reagent according to the manufacturer’s protocol.

cDNA was synthesized as previously described (14). Ct values were obtained by real-time PCR with SYBR Green I dye using the ABI PRISM 7900 Sequence Detection System and analysis software (Applied Biosystems, Foster City, CA). Primers were created using Applied Biosystems’ Primer Express Software version 2.0 (see supplemental Table 1 published as supplemental data on the Endocrine Society’s Journals Online web site at http://mend.endjournals.org) and purchased from Sigma-Genosys. For cDNA amplification, 1–10 ng of cDNA were combined with 40 µl of a mixture containing SYBR Green PCR Master Mix (Applied Biosystems), and 200 nM reverse and forward primers. Samples were analyzed in duplicate or triplicate. Amplification was carried out as follows: a, 50 C, 2 min; b, 95 C, 10 min (denaturation); c, 95 C, 15 sec; 60 C, 30 sec (denaturation/amplification); for 40 cycles. Dissociation curves were also created by adding the following steps to the end of the amplification reaction: 95 C, 15 sec (denaturation); 60 C, 20 sec; then gradually increasing to 95 C over 20 min, finally holding at 95 C for 15 sec. Expression ratios relative to a calibrator [vehicle control from the appropriate (WT or {alpha}ERKO) tissue] and normalized to a 18s reference were determined by quantification of cDNA according to the mathematical model described by Pfaffl (49), in which ratio = (Etarget){Delta}Ct(target)/(E18s){Delta}Ct(18s). Where E = efficiency of the primer set, calculated from the slope of a standard curve of log(ng of cDNA) vs. Ct value for a sample that contains the target according to the formula E = 10 – (1/slope) and {Delta}Ct = Ct(vehicle) – Ct(treated sample).

Western Blot Analysis
Protein extracts were prepared and analyzed by Western blots as previously described (14). The antibody for cFOS was purchased from Santa Cruz Biotechnology, Inc. (sc-52, Santa Cruz, CA), and diluted 1:1000. The antibody for TXNIP was kindly provided by Dr. Richard T. Lee at Harvard University and used at 1:1000. Horseradish peroxidase-linked antirabbit antibody was purchased from Cell Signaling Technologies (Beverly, MA) and diluted 1:5000. ECL-Plus (Amersham) was used for detection. The TXNIP blot was quantified with the Molecular Dynamics densitometer and ImageQuant software.


    ACKNOWLEDGMENTS
 
We thank Dr. Richard T. Lee at Harvard University for the antibody for TXNIP and Dr. Grace Kissling (NIEHS Biostatistics Branch, Statistical Consulting) for statistical analysis. We also thank James Clark, Page Myers, and Tracy Demianenko for their surgical expertise; Vickie Walker and Linwood Koonce for management and oversight of the transgenic animal colonies; and Katherine Hansen for technical expertise. We thank Derek Henley, John Couse, and all the other members of the Receptor Biology Group for helpful suggestions and discussions. We are indebted to Drs. Diane Klotz and Perry Blackshear for critically reading the manuscript.


    FOOTNOTES
 
First Published Online November 4, 2004

Abbreviations: AP-1, Activator protein 1; E, estrogen; E2, estradiol; EGF, epidermal growth factor; ER, estrogen receptor; ERKO, ER knockout; GF, growth factor; ICI, ICI 182,780; Klf-9, Kruppel-like factor 9; PI3 kinase, phosphatidylinositol 3-kinase; TXNIP, thioredoxin-interacting protein; WT, wild- type.

Received for publication April 6, 2004. Accepted for publication October 26, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Shao W, Brown M 2004 Advances in estrogen receptor biology: prospects for improvements in targeted breast cancer therapy. Breast Cancer Res 6:39–52[CrossRef][Medline]
  2. Hall JM, Couse JF, Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869–36872[Free Full Text]
  3. Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P 2000 Estrogen receptor pathways to AP-1. J Steroid Biochem Mol Biol 74:311–317[CrossRef][Medline]
  4. Safe S 2001 Transcriptional activation of genes by 17ß-estradiol through estrogen receptor-Sp1 interactions. In: Vitamins and hormones—advances in research and applications. Litwack G, Begley T, eds. San Diego: Academic Press, vol 62:231–252
  5. Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL 2001 Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway. J Biol Chem 276:13615–13621[Abstract/Free Full Text]
  6. Coleman KM, Smith CL 2001 Intracellular signaling pathways: nongenomic actions of estrogens and ligand-independent activation of estrogen receptors. Front Biosci 6:D1379–D1391
  7. Cato AC, Nestl A, Mink S 2002 Rapid actions of steroid receptors in cellular signaling pathways. Sci STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2002/138/re9
  8. Nelson KG, Takahashi T, Bossert NL, Walmer DK, McLachlan JA 1991 Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation. Proc Natl Acad Sci USA 88:21–25[Abstract/Free Full Text]
  9. Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF, Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci USA 93:12626–12630[Abstract/Free Full Text]
  10. Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor-{alpha} in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537[Abstract/Free Full Text]
  11. Klotz DM, Hewitt SC, Korach KS, Diaugustine RP 2000 Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor-{alpha}. Endocrinology 141:3430–3439[Abstract/Free Full Text]
  12. Ignar Trowbridge DM, Teng CT, Ross KA, Parker MG, Korach KS, McLachlan JA 1993 Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element. Mol Endocrinol 7:992–998[Abstract]
  13. Ignar-Trowbridge DM, Pimentel M, Parker MG, McLachlan JA, Korach KS 1996 Peptide growth factor cross-talk with the estrogen receptor requires the A/B domain and occurs independently of protein kinase C or estradiol. Endocrinology 137:1735–1744[Abstract]
  14. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS 2003 Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 17:2070–2083[Abstract/Free Full Text]
  15. Ignar Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662[Abstract/Free Full Text]
  16. Clemmons DR, Busby W, Clarke JB, Parker A, Duan C, Nam TJ 1998 Modifications of insulin-like growth factor binding proteins and their role in controlling IGF actions. Endocr J 45(Suppl):S1–S8
  17. Ludwig DL, Kotanides H, Le T, Chavkin D, Bohlen P, Witte L 2001 Cloning, genetic characterization, and chromosomal mapping of the mouse VDUP1 gene. Gene 269:103–112[CrossRef][Medline]
  18. Deroo BJ, Hewitt SC, Peddada SD, Korach KS 2004 Estradiol regulates the thioredoxin antioxidant system in the mouse uterus. Endocrinology 145:5485–5492[Abstract/Free Full Text]
  19. Gibson MK, Nemmers LA, Beckman WC, Jr., Davis VL, Curtis SW, Korach KS 1991 The mechanism of ICI 164,384 antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. Endocrinology 129:2000–2010[Abstract]
  20. Xie D, Miller CW, O’Kelly J, Nakachi K, Sakashita A, Said JW, Gornbein J, Koeffler HP 2001 Breast cancer-Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease. J Biol Chem 276:14187–14194[Abstract/Free Full Text]
  21. Sampath D, Winneker RC, Zhang ZM 2001 Cyr61, a member of the CCN family, is required for MCF-7 cell proliferation: regulation by 17ß-estradiol and overexpression in human breast cancer. Endocrinology 142:2540–2548[Abstract/Free Full Text]
  22. Duan C, Clemmons DR 1998 Differential expression and biological effects of insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells. J Biol Chem 273:16836–16842[Abstract/Free Full Text]
  23. Duan C, Liimatta MB, Bottum OL 1999 Insulin-like growth factor (IGF)-I regulates IGF-binding protein-5 gene expression through the phosphatidylinositol 3-kinase, protein kinase B/Akt, and p70 S6 kinase signaling pathway. J Biol Chem 274:37147–37153[Abstract/Free Full Text]
  24. Girvigian MR, Nakatani A, Ling N, Shimasaki S, Erickson GF 1994 Insulin-like growth factor binding proteins show distinct patterns of expression in the rat uterus. Biol Reprod 51:296–302[Abstract]
  25. Franza Jr BR, Rauscher III FJ, Josephs SF, Curran T 1988 The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites. Science 239:1150–1153[Medline]
  26. Loose Mitchell DS, Chiappetta C, Stancel GM 1988 Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2:946–951[Abstract]
  27. Weisz A, Bresciani F 1988 Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus. Mol Endocrinol 2:816–824[Abstract]
  28. Boettger-Tong HL, Murthy L, Stancel GM 1995 Cellular pattern of c-fos induction by estradiol in the immature rat uterus. Biol Reprod 53:1398–1406[Abstract]
  29. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339[CrossRef][Medline]
  30. Shah JV, Cleveland DW 2000 Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell 103:997–1000[Medline]
  31. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269:16433–16442[Abstract/Free Full Text]
  32. Okamura-Oho Y, Miyashita T, Yamada M 2001 Distinctive tissue distribution and phosphorylation of IRSp53 isoforms. Biochem Biophys Res Commun 289:957–960[CrossRef][Medline]
  33. Alvarez CE, Sutcliffe JG, Thomas EA 2002 Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region. J Biol Chem 277:24728–24734[Abstract/Free Full Text]
  34. Murphy LJ, Murphy LC, Friesen HG 1987 Estrogen induces insulin-like growth factor-I expression in the rat uterus. Mol Endocrinol 1:445–450[Abstract]
  35. Richards RG, DiAugustine RP, Petrusz P, Clark GC, Sebastian J 1996 Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus. Proc Natl Acad Sci USA 93:12002–12007[Abstract/Free Full Text]
  36. DiAugustine RP, Petrusz P, Bell GI, Brown CF, Korach KS, McLachlan JA, Teng CT 1988 Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinology 122:2355–2363[Abstract]
  37. Huet Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK 1990 Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 4:510–523[Abstract]
  38. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS 1995 Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–1454[Abstract]
  39. Kousteni S, Bellido T, Plotkin LI, O’Brien CA, Bodenner DL, Han L, Han K, DiGregorio GB, Katzenellenbogen JA, Katzenellenbogen BS, Roberson PK, Weinstein RS, Jilka RL, Manolagas SC 2001 Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104:719–730[Medline]
  40. Migliaccio A, Castoria G, Di DM, De FA, Bilancio A, Auricchio F 2002 Src is an initial target of sex steroid hormone action. Ann NY Acad Sci 963:185–190[Abstract/Free Full Text]
  41. Migliaccio A, Castoria G, Di DA, de FA, Bilancio A, Lombardi M, Bottero D, Varricchio L, Nanayakkara A, Rotondi A, Auricchio F 2002 Sex steroid hormones act as growth factors. J Steroid Biochem Mol Biol 83:31–35[CrossRef][Medline]
  42. Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, Auricchio F 2000 Steroid-induced androgen receptor-oestradiol receptor ß-Src complex triggers prostate cancer cell proliferation. EMBO J 19:5406–5417[Abstract/Free Full Text]
  43. Song RX, McPherson RA, Adam L, Bao Y, Shupnik M, Kumar R, Santen RJ 2002 Linkage of rapid estrogen action to MAPK activation by ER{alpha}-Shc association and Shc pathway activation. Mol Endocrinol 16:116–127[Abstract/Free Full Text]
  44. Zhang ZG, Maier B, Santen RJ, Song RXD 2002 Membrane association of estrogen receptor {alpha} mediates estrogen effect on MAPK activation. Biochem Biophys Res Commun 294:926–933[CrossRef][Medline]
  45. Song RX, Barnes CJ, Zhang Z, Bao Y, Kumar R, Santen RJ 2004 The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor {alpha} to the plasma membrane. Proc Natl Acad Sci USA 101:2076–2081[Abstract/Free Full Text]
  46. Stoica GE, Franke TF, Moroni M, Mueller S, Morgan E, Iann MC, Winder AD, Reiter R, Wellstein A, Martin MB, Stoica A 2003 Effect of estradiol on estrogen receptor-{alpha} gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway. Oncogene 22:6054–6067[CrossRef][Medline]
  47. Watters JJ, Chun TY, Kim YN, Bertics PJ, Gorski J 2000 Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Mol Endocrinol 14:1872–1881[Abstract/Free Full Text]
  48. DeRisi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM 1996 Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet 14:457–460[Medline]
  49. Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007