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.
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ABSTRACT |
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INTRODUCTION |
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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 [ER
knockout (
ERKO)] indicated that ER
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
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 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
, we hypothesized that they would be lost in the
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
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.
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RESULTS AND DISCUSSION |
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Category 2 genes were ER dependent, as illustrated by their regulation only in the WT samples (yellow boxes, Fig. 1
). These genes were not regulated by a cross-talk mechanism but depended on direct modulation of ER
activity by E2 only. Finally, category 3 included GF-responsive genes. The cluster analysis in Fig. 1
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. 1B), 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 Societys 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-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
-dependent and that
ERKO mice lack uterine growth, demonstrating the requirement for ER
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
.
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 14. In Fig. 2
, dendograms representing the microarray data of the selected genes are presented. Additionally, the RT-PCR data from some of the genes in Tables 14
are also represented graphically in Fig. 2
.
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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. 2A). 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. 2A
). 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 3
). Both EGF and IGF-I also decreased expression of these three genes at 24 h (Table 3
). 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
ERKO samples Txnip mRNA levels were not reduced by E2, but were reduced by GFs (see Table 4
). In the
ERKO, ICI did not alter the GF-induced suppression of Txnip (Fig. 2A
). Western blot analysis showed a similar response pattern of the TXNIP protein in the
ERKO samples (Fig. 2A
). The ER dependence for E response was demonstrated both by the lack of regulation in the absence of ER in the
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 1), whereas the repression after EGF treatment was minimal after 2 h (Table 1
). 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 14), 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
ERKO. GFs also repress these genes to a similar degree in the WT and
ERKO, and the repression by GF is not blocked by ICI.
The mechanism of ER-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
ERKO indicates the ER
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
in the down-regulation might differ from its role in up-regulation. ICI binds to the ER
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
, 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
might remain in a conformation that still allows it to inhibit transcription. Additionally, ICI is known to decrease the level of ER
protein (19); thus, there might not be sufficient ER
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 ERKO, were confirmed by RT-PCR (Fig. 2A
and Tables 14
). 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. 2A
), 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
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 1). ICI decreased or prevented this increase. Cyr61 was minimally increased in the
ERKO by E2 and IGF-I but little by EGF, and much less than the E2 induction of the WT (Table 2
). Additionally, the basal level of Cyr61 in the
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 3). ICI prevented the E2-induced increase, but not the GF-induced increase. In the
ERKO samples, however, Igfbp5 transcript was only increased after IGF-I treatment. The fact that IGF-I increases Igfbp5 mRNA independent of ER
indicates that two different mechanisms of increase may occur: the first is a classic ER
-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
ERKO (using a Mann-Whitney U test to compare
ERKO fold increase to WT fold increase: P = 0.04), suggesting ER
-mediated and IGF receptor pathways may combine in WT samples to increase induction, whereas only the GF pathway is present in the
ERKO. Similarly, the degree of GF regulation of Sox4, (P = 0.04 for both IGF and EGF at 24 h, WT vs.
ERKO) and Baiap (P = 0.04 for EGF at 2 h, WT vs.
ERKO) is blunted in the
ERKO. The regulation of Cyr61 by IGF-I and the regulation of Krt 119 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
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
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. 2B); 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. 2B
and Table 1
). cFos, a subunit of the AP-1 transcription factor (25), was increased after 2 h of E2 in the WT (Table 1
and Fig. 2B
), as has been previously observed (26, 27, 28). ICI decreased or prevented this increase. In the
ERKO samples, cFos was also increased by E2 but less robustly than in the WT (Table 2
and Fig. 2B
). ICI inhibited the E2 increase in the
ERKO sample. cFos has been shown to be rapidly (30 min) increased by EGF, in both WT and
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. 2B
).
Category 2 Genes Respond Exclusively to E2: Mad2, Ramp3, Lactoferrin, Igf-1, and Krt119
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 1 and 2
). Lactoferrin, Mad2, Igf-1, and Krt119 were all elevated 24 h after E2 treatment (Tables 3
and 4
) and were attenuated by ICI. None of these increases were noted with GFs. All required ER
, as demonstrated by the lack of regulation in the
ERKO. Thus, these genes are regulated by a classic ER
-dependent mechanism, but not influenced by ER
cross-talk with GF receptor pathways. The data from the analysis of Mad2 are also shown graphically (Fig. 2B
).
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 14).
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 ERKO (category 3), were examined by RT-PCR (Tables 1
and 2
and Fig. 2C
). The data from Klf-9 are also shown graphically (Fig. 2C
). 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
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 119
The original cluster analysis failed to identify genes showing a profile that would fit a cross-talk model. Interestingly, although 24 h after treatment Krt119 was selectively induced by E2 (Tables 3 and 4
), 2 h after injection, only growth factors induced the transcript and only in the WT samples (Tables 1
and 2
). This seems to indicate a role for ER
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. 2C), but less robustly than with E2. There was no response in the
ERKO samples to E2 or GF. The lack of regulation of Cyclin E, which modulates S phase progression, in the
ERKO by either E2 or GFs, correlates with a lack of epithelial proliferation in response to GFs in the
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 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 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
. This observation suggests that for these genes ER
is upstream of GF receptor signaling, and that E2 increases levels of GFs (see, for example, Igf-1 in Table 3
), 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
; thus, the response could occur in the
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
targets. Indeed, the levels of both EGF and IGF-I are known to increase after E2 treatment, with maximal increase of IGF-I after 46 h (34) and activation of the IGF receptor within 4 h (35), whereas EGF is elevated after 1224 h (36, 37). The transcripts of EGF receptor activators, EGF and TGF
, 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 Splice Variant
Previously, we have reported a residual splice variant of ER, E1-ER
, which is detected in the
ERKO uterine tissue at approximately 510% 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
ERKO, but this possibility is unlikely. First, none of the GF responses was inhibited by ICI, indicating the E1-ER
form is not mediating the response. Second, E2 can activate E1-ER
(38), yet there is minimal genomic response to E2 in the
ERKO. The blunted E response still seen for some of the target genes might indeed be mediated by E1-ER
or possibly by ERß (see p21, cFos Cyr61, and Sox 4 RT-PCR data in Fig. 2
and Table 2
) as ICI does inhibit it. Third, the level and activity of E1-ER
in the uterine tissue are low (38) yet the
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. 3). Additionally, E may interact with the recently described membrane-associated pathway mediated by ER
, 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
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
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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In conclusion, microarray analysis of the global genomic response pattern in uterine tissue after GF treatment of WT or 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
ERKO in response to GF indicates a role for the ER
for a complete biological response. Future studies will lead to a better understanding of the potential gene-specific mechanisms involved in ER
-mediated and ER
-independent responses.
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MATERIALS AND METHODS |
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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 manufacturers protocol. Isolated RNA was then further purified using the QIAGEN Rneasy midi (100500 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 Ambions Northern Max reagents and protocol (Ambion, Austin TX). Membrane was prehybridized and incubated with 32P-labeled riboprobes in Ultrahyb buffer (Ambion) according to the manufacturers 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 manufacturers 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 Societys Journals Online web site at http://mend.endjournals.org) and purchased from Sigma-Genosys. For cDNA amplification, 110 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 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)
Ct(target)/(E18s)
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
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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