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Molecular Endocrinology, doi:10.1210/me.2006-0131
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Molecular Endocrinology 20 (10): 2304-2314
Copyright © 2006 by The Endocrine Society

Glucocorticoid Receptor Activation Signals through Forkhead Transcription Factor 3a in Breast Cancer Cells

Wei Wu, Min Zou, Deanna R. Brickley, Travis Pew and Suzanne D. Conzen

Department of Medicine and Committee on Cancer Biology, University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Suzanne D. Conzen, Department of Medicine and Committee on Cancer Biology, MC 2115, University of Chicago, Chicago, Illinois 60637. E-mail: sdconzen{at}uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the glucocorticoid receptor (GR) plays a critical role in the stress response of virtually all cell types. Despite recent advances in large-scale genomic and proteomic data acquisition, identification of physiologically relevant molecular events downstream of nuclear hormone receptor activation remains challenging. By analyzing gene expression changes 30 min after dexamethasone (Dex) treatment, we previously found that immediate induction of serum and glucocorticoid-regulated kinase-1 (SGK-1) expression is required for GR-mediated mammary epithelial cell survival signaling. We now report that activation of the GR mediates Forkhead transcription factor 3a (FOXO3a) phosphorylation and inactivation in mammary epithelial cells. GR-mediated induction of SGK-1 expression is required for FOXO3a inactivation; additional growth factor stimulation is not required. To further explore the gene expression changes that occur downstream of GR-mediated FOXO3a inactivation, we analyzed temporal gene expression data and selected GR-down-regulated genes containing core FOXO3a binding motifs in their proximal promoters. This approach revealed several previously unrecognized transcriptional target genes of FOXO3a, including IGF binding protein-3 (IGFBP-3). Endogenous IGFBP-3 expression was confirmed to be dependent on the GR-SGK-1-FOXO3a signaling pathway. Moreover, GR activation decreased FOXO3a-induced apoptosis in SK-BR-3 breast cancer cells. Collectively, our data suggest that GR-mediated FOXO3a inactivation is an important mechanism contributing to glucocorticoid-mediated mammary epithelial cell survival.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOID RECEPTOR (GR) activation has been linked to both proapoptotic (reviewed in Ref. 1) and antiapoptotic (2, 3, 4, 5, 6) signaling events in a variety of cell types. The GR is a classic nuclear hormone receptor that dimerizes after ligand binding, translocates to the nucleus, and modulates the expression of many target genes (reviewed in Refs. 7 and 8). As with other nuclear hormone receptors, the GR also appears to have important nongenomic effects (9). These may act in concert with the genomic functions, although in a much more rapid time frame. Several reports have demonstrated that direct target genes of GR activation encode proteins that include kinases, phosphatases, and transcription factors that, in turn, are likely to mediate downstream transcriptional events (10). However, relatively little is known about how immediate GR-mediated gene regulation is connected with later gene expression changes to form molecular pathways that underlie GR-mediated cell signaling.

The serum and glucocorticoid-regulated kinase-1 (SGK-1) gene was originally identified as an immediate early response gene induced following serum or glucocorticoid treatment in rat mammary cancer cells (11). More recently, we (3) and others (12) have demonstrated that GR activation rapidly induces the transcription of SGK-1 in human mammary epithelial cells and that SGK-1, a downstream effector of the phosphoinositide-3 kinase signaling pathway (13), plays an important role in GR-mediated cell survival signaling (3, 12, 14). In turn, SGK-1, in coordination with another protein kinase AGC family member, Akt-1, has been reported to preferentially phosphorylate Forkhead transcription factor 3a (FOXO3a) at Thr32 and Ser315 (15). In addition, in Caenorhabditis elegans, SGK-1 activity is required for the inactivating phosphorylation of the FOXO3a homolog Daf 16 (16), suggesting a highly conserved pathway involving SGK-1 and FOXO3a.

Studies in mammalian cells have shown that the family of forkhead transcription factors, including FOXO1, FOXO3a, and FOXO4, has an important role in diverse biological processes such as metabolism, apoptosis, and aging (reviewed in Ref. 17). In particular, FOXO3a functions to regulate genes that mediate potent proapoptotic signals under different stimuli (reviewed in Ref. 18). Inactivation of FOXO3a after phosphorylation by Akt-1 and SGK-1 can subsequently reduce proapoptotic target gene expression and is postulated to contribute to cell survival mediated by serum and IGF-I (15, 19). However, in addition to cell survival mediated by serum and individual growth factors such as insulin, we have observed that GR activation alone is sufficient to provide a potent signal for cell survival in mammary epithelial cells (2).

Although GR activation induces SGK-1 expression (3, 5, 12), it is not associated with Akt-1 activation (2). However, the role of GR activation in regulating FOXO3a function, previously studied in the context of serum or insulin stimulation and subsequent Akt-1 activation, had not been explored in detail. Here, we hypothesized that GR activation alone might be sufficient for FOXO3a inactivation because of the rapid and significant induction of SGK-1 expression by glucocorticoids, even in the absence of serum. We demonstrate that early SGK-1 induction is required for GR-mediated FOXO3a phosphorylation and subsequent inactivation. To identify downstream target genes of FOXO3a inactivation, we used a bioinformatic approach combining temporal gene expression data and FOXO3a binding motif analyses. This approach identified a group of known and novel FOXO3a target genes. Included among these genes are TRAIL, IGFBP-3, and STK11, all of which encode proapoptotic proteins. Although TRAIL is a known target of FOXO3a, IGFBP-3 has not been previously identified as a FOXO3a target gene. To validate this relationship, we used chromatin immunoprecipitation (ChIP) to show that dexamethasone (Dex) completely inhibits TRAIL and IGFBP-3 promoter occupancy by FOXO3a, whereas the GR antagonist RU486 restores occupancy. In addition, expressing a constitutively active FOXO3a abrogated the cell survival effect of Dex and was associated with induction of IGFBP-3 expression. Furthermore, blocking GR-mediated SGK-1 induction through expression of SGK-1 short interfering RNA (siRNA) reversed IGFBP-3 down-regulation. Finally, GR activation decreased FOXO3a-induced apoptosis in SK-BR-3 cells in association with a reduction in IGFBP-3 expression. These results suggest that GR-mediated mammary epithelial cell survival requires FOXO3a inactivation and provide an example of using temporal gene expression data and transcription factor binding motif analysis to identify a novel downstream pathway after nuclear hormone receptor activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GR Activation Is Associated with FOXO3a (Thr32) Phosphorylation
Glucocorticoids increase epithelial cell survival in response to diverse apoptotic stimuli (reviewed in Ref. 20). SGK-1 is a direct GR target (11) and encodes a serine-threonine kinase with significant homology to Akt-1 (3, 12). However, the precise mechanism through which SGK-1 mediates GR-induced cell survival is not fully understood. We therefore hypothesized that GR-induced SGK-1 expression might be sufficient to phosphorylate and inactivate FOXO3a independently of serum stimulation.

To ascertain the consequence of GR activation on FOXO3a phosphorylation, we performed time course experiments after GR activation and examined SGK-1 induction, Akt-1 phosphorylation (Ser473), and FOXO3a phosphorylation (Thr32) in MCF10A-Myc breast epithelial cells. As shown in Fig. 1AGo, slight induction of SGK-1 protein after Dex treatment (in the absence of serum) was seen at 2 h, with a maximum induction of SGK-1 expression at 8 h, and then a return to baseline by 24 h. Consistent with previous observations (3), GR-mediated induction of SGK-1 was inhibited by the progesterone receptor (PR)/GR antagonist RU486. (MCF10A-Myc cells do not express detectable PR-A or PR-B, and therefore RU486 specifically inhibits GR activation.) As expected, both pan and p-Akt-1 (Ser473) expression did not change significantly after GR activation with either Dex alone or Dex/RU486 treatment. However, FOXO3a (Thr32) phosphorylation increased dramatically beginning at 8 h after GR activation and was consistently blocked by concomitant RU486 treatment. The ratio of p-FOXO3a/FOXO3a (as measured by densitometry) increased more than 10-fold at 8, 12, and 24 h in comparison to 0 h. Interestingly, total FOXO3a protein also increased slightly after Dex treatment, and this effect was reversed by RU486 (Fig. 1AGo). Gene array and quantitative real-time PCR analysis showed an approximately 2-fold increase in FOXO3a mRNA levels at 2 and 4 h after Dex treatment (data not shown), suggesting that FOXO3a may also be transcriptionally induced by GR activation. However, given the high ratio of Thr32 FOXO3a phosphorylation to total FOXO3a protein (densitometry ratios are shown below the FOXO3a panel in Fig. 1AGo), there appears to be a much more dramatic GR-mediated induction of FOXO3a phosphorylation than induction of total FOXO3a protein.


Figure 1
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  		Fig. 1. GR Activation Is Associated with FOXO3a Phosphorylation (Thr32)

A, MCF10A-Myc cells were treated with Dex (10–6 M) or vehicle (EtOH) for the indicated time. Protein lysates were collected and subjected to SDS-PAGE, and SGK-1, p-Akt-1 (Ser473), p-FOXO3a (Thr32), pan FOXO3a, and Akt-1 were examined. The ratios of p-FOXO3a/FOXO3a for each experimental condition are indicated. The blot is representative of three independent experiments. B, MCF10A-Myc cells stably expressing scrambled sequence (–) or SGK-1 siRNA (+) were treated with vehicle or Dex as described above. Western analysis shows the SGK-1 and p-FOXO3a (Thr32) expression in these samples using specific antibodies. The ratios of p-FOXO3a/FOXO3a for each experimental condition are indicated. ß-Actin was probed as a loading control. The blot shown is representative of three independent experiments. C, MCF10A-Myc cells were starved overnight and treated with Dex (10–6 M) for the indicated time periods. Cells were incubated with an anti-FOXO3a antibody followed by secondary Alexa Fluor 568 and examined for FOXO3a localization at x400. Similar results were obtained in four independent experiments.

 
The timing of FOXO3a phosphorylation after SGK-1 protein accumulation implied that FOXO3a might be a direct or indirect target of SGK-1 activity. We therefore predicted that inhibiting SGK-1 induction using siRNA would also inhibit Dex-mediated FOXO3a phosphorylation. To test this, we treated MCF10A-Myc cells stably expressing either SGK-1 siRNA (+) or a scrambled sequence (SS) siRNA (–) with Dex and examined SGK-1 expression (Fig. 1BGo, top panel). As expected, cells expressing SGK-1 siRNA (+) exhibited dramatically less SGK-1 after GR activation (lanes 4, 6, 8, and 10) compared with cells expressing SS siRNA (–) (lanes 3, 5, 7, and 9). As seen in Fig. 1BGo, middle panel, when SGK-1 expression was blocked by siRNA, detection of p-FOXO3a was also significantly diminished (lanes 8, 10, and 12 vs. lanes 7, 9, and 11). Therefore, phosphorylation of FOXO3a after GR activation appears to be dependent, at least in part, upon SGK-1 induction.

Phosphorylation of FOXO3a at Thr32, Ser253, and Ser315 has previously been associated with its nuclear exclusion and consequent functional inactivation (15, 19). Subcellular localization of FOXO3a after phosphorylation was therefore evaluated by immunofluorescent detection of endogenous FOXO3a in MCF10A-Myc cells. As shown in Fig. 1CGo, endogenous FOXO3a was found to be both nuclear and cytoplasmic after overnight serum starvation (0 h). However, beginning 8 h after Dex treatment, FOXO3a exhibited progressive nuclear exclusion, correlating with the time of initial FOXO3a phosphorylation. Taken together, these data suggest that GR activation alone, independently of serum stimulation, results in FOXO3a phosphorylation and subsequent nuclear exclusion.

GR Activation Results in Decreased FOXO3a Transcriptional Activity
Because glucocorticoid treatment resulted in FOXO3a phosphorylation and its exclusion from the nucleus, we predicted that GR activation might lead to a reduction in FOXO3a-dependent transcription. To examine this, we cotransfected SK-BR-3 breast cancer cells with a plasmid containing three FOXO3a response elements fused to a gene encoding luciferase (FRE-Luc) (19). These cells were cotransfected with a plasmid encoding either wild-type FOXO3a-FLAG (WT-FOXO3a) or a nonphosphorylatable, constitutively active FOXO3a-FLAG [triple mutant (TM)-FOXO3a, T32A/S253A/S315A]. As shown in Fig. 2AGo, ectopic expression of either WT-FOXO3a or TM-FOXO3a was detected with an anti-FLAG antibody. After transfection (48 h), cells were then split into duplicate dishes, and treated with either vehicle (EtOH) or Dex for 24 h. To determine whether or not FOXO3a activity was decreased by Dex treatment, relative luciferase activity was measured. As shown in Fig. 2BGo, the transcriptional activity of WT-FOXO3a was significantly decreased after Dex treatment, whereas in TM-FOXO3a-expressing cells, luciferase activity was not affected by Dex. These observations suggest that GR-induced FOXO3a phosphorylation inhibits FOXO3a transcriptional activity and are consistent with a novel signaling pathway connecting GR activation to FOXO3a inactivation.


Figure 2
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  		Fig. 2. GR Activation Decreases FOXO3a Transcriptional Activity

SK-BR-3 breast cancer cells were transiently transfected with either pLPCX, WT-FOXO3a-FLAG, or TM-FOXO3a-FLAG, together with FRE-Luciferase reporter plasmid, and pCMV-ß-galactosidase encoding vectors. A, Forty-eight hours after transfection, Western analysis shows FOXO3a protein expression with anti-FLAG-HRP antibody. B, Cells were split into duplicates (48 h after transfection), and transfected cells were treated with either vehicle (EtOH) or Dex (10–6 M) for 24 h. Luciferase activity was examined following manufacturer’s instructions. The relative luciferase activity in each condition was normalized to ß-galactosidase activity to account for transfection efficiency. Fold change was calculated after normalization to pLPCX control and reported as an average ± SE of three independent experiments. **, Significantly less luciferase activity (P < 0.05) in WT-FOXO3a-expressing cells treated with Dex compared with vehicle. N.D., No significant difference.

 
Identification of Putative FOXO3a Target Genes Using a Bioinformatic Strategy Combining GR-Mediated Time Course Microarray Data and Promoter Analysis for FOXO3a Binding Motifs
FOXO3a activity regulates the expression of a number of genes with diverse cellular roles (reviewed in Ref. 18). We approached the discovery of unknown FOXO3a target genes the expression of which is down-regulated by FOXO3a nuclear exclusion using a combination of temporal gene expression data (looking for significantly down-regulated genes at a time point after FOXO3a phosphorylation), and promoter analysis for FOXO3a binding motifs. Figure 3AGo depicts the predicted relationship between SGK-1 expression, FOXO3a phosphorylation, and FOXO3a target gene expression in response to GR activation. Initially, SGK-1 mRNA expression levels increase, followed by phosphorylation, nuclear exclusion, and inactivation of FOXO3a. By 24 h we predicted that FOXO3a target gene expression would be significantly down-regulated. Of course, the decrease in steady-state mRNA levels of any gene is also a function of the individual mRNA half-life, because mRNAs with particularly long half-lives will exhibit a much slower rate of decrease than mRNAs with rapid turnover. The average half-life of a mammalian mRNA is about 5 h (21), and therefore we predicted a significant time lag between FOXO3a phosphorylation and decreased mRNA expression of FOXO3a-dependent genes. Although SGK-1 mRNA is induced as early as 30 min after Dex treatment (3, 5), 24 h was chosen as a reasonable time point to identify putative FOXO3a target genes. The patterns of gene expression changes predicted by this model underscore the importance of using a time course gene expression approach (rather than a single time point) for studying dynamic cell signaling pathways.


Figure 3
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  		Fig. 3. Prediction of FOXO3a Targets after GR Activation Using a Combination of Time Course Gene Expression Data and Promoter Analysis

A, A cartoon depicting SGK-1 induction after Dex treatment, FOXO3a phosphorylation, and predicted subsequent down-regulation of FOXO3a target genes. B, Bioinformatic analysis: putative FOXO3a target genes were selected as those that both were down-regulated 30% at 24 h after Dex treatment and contained FOXO3a core binding motif(s) in their promoter regions. C, Quantitative real-time PCR (Q-RT-PCR) evaluation of three FOXO3a target genes IGFBP-3, uPA, LKB-1 24 h after Dex treatment. The fold change of gene expression is shown relative to vehicle, and data are reported as a mean ± SE of triplicate experiments.

 
We used the Affymetrix human genechip HG-U133A containing 22,215 probe sets (representing ~16,000 human genes) to examine gene expression in MCF10A-Myc cells treated with Dex or vehicle for 0.5, 2, 4, and 24 h (all data are available on http://www.ncbi.nlm.nih.gov/geo/). We averaged the gene expression ratios (Dex/vehicle) from three independent experiments by comparing Dex and vehicle treatment and identified 509 genes that had at least a 30% average reduction in gene expression 24 h after Dex treatment. We chose this relatively modest 30% down-regulation as a cutoff because in our experience, a 30% reduction in gene expression by array technology correlates with at least a 50% reduction in gene expression as determined by quantitative real-time PCR or Northern analysis (Zou, M., and S. D. Conzen, unpublished data). At this point, we did not further restrict these 509 genes based on a P value cutoff because of the relatively small number of replicate experiments (n = 3) and the moderate degree of down-regulation that we considered to be biologically relevant. Instead, using a human genome promoter database (http://dbtss.hgc.jp) and a transcription factor binding site search program (see Materials and Methods for details), we next identified 2860 genes in the human genome that have one or more FOXO3a core binding motifs [AAA(C/T)A] (18) between –1000 nucleotides (nt) and +200 nt. Genes that were both down-regulated at 24 h after Dex treatment and contained at least one FOXO3a core binding motif (n =41) were considered putative target genes of GR-induced FOXO3a inactivation (Fig. 3BGo). These genes included TRAIL, a known target gene of FOXO3a (22). To our knowledge, the other 40 genes (supplemental Table 1, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) have not been previously reported as FOXO3a targets. Of the 41 genes, 11 have roles in apoptotic physiology based on a PubMed search using putative FOXO3a target gene names and the keywords "apoptosis" or "cell survival." Because of their known roles in breast cancer biology, expression of three of these 11 genes, insulin-like growth factor binding protein-3 (IGFBP-3), urokinase plasminogen activator (uPA/PLAU), and serine/threonine kinase 11 (LKB1/STK11), were evaluated by quantitative real-time PCR, and all three were confirmed to be down-regulated after glucocorticoid treatment (Fig. 3CGo). Although, IGFBP-3 protein secretion has been previously shown to be decreased in hepatocytes after glucocorticoid treatment (23), the exact mechanism of IGFBP-3 down-regulation is not known. Because of the evidence in the literature implicating up-regulation of IGFBP-3 in breast cancer cell apoptosis (reviewed in Ref. 24), IGFBP-3 was selected for further validation as a bona fide FOXO3a target that is regulated indirectly by GR activation.

GR Activation Inhibits Binding of FOXO3a to the IGFBP-3 Promoter
Based on this analysis, the IGFBP-3 proximal promoter contains two canonical FOXO3a core binding elements [AAA(C/T)A] at –560 nt and –784 nt, which we next examined for FOXO3a occupancy using ChIP analysis (Fig. 4AGo). As a positive control, the sequence of the TRAIL promoter containing a known FOXO3a binding site (22) was examined. As seen in Fig. 4BGo, Dex treatment resulted in the loss of PCR amplification of the known FOXO3a binding site in the TRAIL promoter (–1005/–1001); sequence amplification was restored after concomitant treatment with RU486. These observations are consistent with a requirement for GR activation in causing FOXO3a nuclear exclusion, thereby preventing TRAIL promoter occupancy. In a parallel experiment, we found that the putative FOXO3a binding sequence (–784/–780) within the IGFBP-3 promoter was also amplified from the FOXO3a immunoprecipitate in the presence of vehicle but not after Dex treatment. Furthermore, concomitant treatment with RU486 also restored IGFBP-3 promoter occupancy by FOXO3a. Of note, the other predicted core sequence (–560/–556) could not be amplified after FOXO3a immunoprecipitation. This is most likely because the core sequence [AAA(C/T)A] we used as our search criteria is essential, but not sufficient, for FOXO3a promoter occupancy. As an additional negative control, we carried out the amplification of an irrelevant upstream IGFBP-3 promoter sequence (–2000/–1402), which does not contain a FOXO3a core binding motif. The anti-FOXO3a antibody also failed to immunoprecipitate this irrelevant sequence. These results support our hypothesis that 1) the IGFBP-3 promoter contains a bona fide FOXO3a binding site, and 2) IGFBP-3 promoter-FOXO3a interaction is disrupted after GR activation as a result of phosphorylation and nuclear exclusion of FOXO3a.


Figure 4
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  		Fig. 4. GR Activation Disrupts IGFBP-3 Promoter Occupancy by FOXO3a

A, Diagram demonstrating the FOXO3a core-binding sites [AAA(C/T)A] in the TRAIL and IGFBP-3 promoters. The known FOXO3a binding site in the TRAIL promoter served as a positive control whereas an irrelevant sequence in the IGFBP-3 upstream promoter was used as negative control. Two core binding sequences are illustrated in the IGFBP-3 promoter. Primers used to amplify the corresponding DNA fragments are indicated with arrows. B, MCF10A-Myc cells were treated with either vehicle (EtOH), Dex (10–6 M), or Dex/RU486 (10–7 M) after 72 h of serum starvation. The representative image shows input DNA as a loading control and DNA pulled down in a ChIP assay with either anti-FOXO3a antibody or without antibody. The experiment was performed three independent times with similar results. Ab, Antibody.

 
Down-Regulation of IGFBP-3 Expression Is Mediated by GR-Induced FOXO3a Inactivation
IGFBP-3 expression has been reported to be altered after glucocorticoid treatment (reviewed in Ref. 25), but the mechanism has not been elucidated. To determine whether expression of IGFBP-3 is dependent on the GR/SGK-1/FOXO3a pathway, we examined endogenous IGFBP-3 expression in the presence of the GR agonist Dex or partial antagonist RU486 in MCF10A-Myc cells. As Fig. 5AGo shows, Dex treatment significantly decreased IGFBP-3 mRNA levels when compared with cells treated with vehicle (P < 0.05). In contrast, concomitant RU486 reversed the inhibition of IGFBP-3 gene expression by Dex. Because IGFBP-3 is a predominantly secreted protein, we next examined IGFBP-3 levels in media from cells treated with Dex or vehicle for 24 h. Figure 5BGo shows that secreted IGFBP-3 protein levels decreased after Dex treatment of MCF10A-Myc cells.


Figure 5
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  		Fig. 5. Down-Regulation of IGFBP-3 Expression Is Mediated by GR Activation

A, After 72 h of growth factor withdrawal, MCF10A-Myc cells were treated with either vehicle (EtOH), Dex (10–6 M), or Dex/RU486 (10–7 M) for 24 h. Total RNA was extracted from each condition and quantitative real-time PCR (Q-RT-PCR) was performed using an IGFBP-3-specific primer pair. Gene expression is shown relative to GAPDH and data are reported as mean ± SE of three independent experiments. **, Significantly decreased IGFBP-3 gene expression (P < 0.05) in Dex vs. vehicle treatment. B, After 72 h of growth factor withdrawal, MCF10A-Myc cells were treated with either vehicle or Dex (10–6 M) for 24 h. Medium from either vehicle- or Dex-treated cells were collected and proteins were concentrated and run on SDS-PAGE followed by Western analysis with anti-IGFBP-3 antibody. The blot shown is a representative of three experiments. C, MCF10A-Myc cells infected with either SS or SGK-1 siRNA; or D, MCF10A-Myc cells stably expressing either pLPCX, WT-FOXO3a-FLAG, or TM-FOXO3a-FLAG were treated with either vehicle or Dex (10–6 M) for 24 h, after growth factor withdrawal. Total RNAs were extracted from each condition, and quantitative real-time PCR was performed using an IGFBP-3-specific primer pair. Gene expression is shown relative to GAPDH and data are reported as a mean ± SE of triplicate samples. **, Significantly decreased IGFBP-3 gene expression (P < 0.05) in Dex vs. vehicle treatment. N.D., No significant difference.

 
Because SGK-1 is induced after GR activation, we also asked whether knockdown of SGK-1 expression (Fig. 1BGo) would affect the down-regulation of IGFBP-3 mRNA observed in Dex-treated cells. To examine this, stable MCF10A-Myc cell lines expressing either an SS siRNA or SGK-1 siRNA were treated with vehicle or Dex for 24 h. Figure 5CGo demonstrates that knocking down SGK-1 expression reverses the GR-mediated decrease in IGFBP-3 expression (P < 0.05). Finally, we established MCF10A-Myc cells stably expressing either the empty pLPCX vector, WT-FOXO3a or TM-FOXO3a; individual clones were selected, cells were then treated with either vehicle or Dex for 24 h, and IGFBP-3 mRNA expression was again evaluated using quantitative real-time PCR. As Fig. 5DGo shows, IGFBP-3 expression was significantly decreased in WT-FOXO3a-expressing cells after Dex treatment (P < 0.05), whereas IGFBP-3 mRNA expression levels were relatively high in TM-FOXO3a-expressing cells and were not affected by Dex treatment. These data suggest that FOXO3a activity is associated with GR-mediated down-regulation of IGFBP-3 expression. In addition, SGK-1 expression appears to be required for GR-mediated IGFBP-3 down-regulation.

GR Activation Inhibits FOXO3a-Mediated Apoptosis
FOXO3a activation has been reported to induce apoptosis in leukemia (26) and breast (27) cancer cell lines. To determine whether GR activation can reverse FOXO3a-mediated cell death, we examined apoptosis in either empty vector, WT-FOXO3a or TM-FOXO3a (T32A/S253A/S315A)-expressing cells treated with or without Dex. Figure 6AGo shows the relative percentages of apoptotic cells measured by flow cytometric analysis of the sub-G1 population in transiently transfected SK-BR-3 cells treated with Dex or vehicle. In the presence of Dex, cells expressing WT-FOXO3a showed significantly decreased apoptosis compared with vehicle treatment, whereas TM-FOXO3a-expressing cells showed no significant difference in apoptosis (P < 0.01). These results suggest that GR protection from apoptosis requires WT-FOXO3a phosphorylation of one or more sites on FOXO3a (T32/S253/S315). We next examined IGFBP-3 expression under identical experimental conditions as in Fig. 6AGo. As previously seen in MCF10A-Myc cells (Fig. 5Go), Dex treatment significantly decreased IGFBP-3 mRNA levels in WT-FOXO3a-transfected SK-BR-3 cells but not in TM-FOXO3a-transfected cells (Fig. 6BGo). Taken together, these results suggest that GR activation inhibits FOXO3a-induced apoptosis in association with down-regulating IGFBP-3 gene expression.


Figure 6
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  		Fig. 6. GR Activation Inhibits FOXO3a-Induced Apoptosis

SK-BR-3 cells were transiently transfected with either pLPCX, WT-FOXO3a, or TM-FOXO3a for 48 h. Transfected cells were split into duplicate plates and treated with either vehicle (EtOH) or Dex (10–6 M) for 24 h in serum-free media. A, Cells were fixed and stained with propidium iodide to measure apoptosis by flow cytometry from three experiments. A bar graph depicts the fold change of apoptotic cells determined by analysis of the sub-G1 population relative to pLPCX vector alone. Data represent the mean and SE of three independent experiments. **, Significantly decreased apoptosis (P < 0.01) in Dex vs. vehicle treatment in WT-FOXO3a cells. B, Total RNAs were extracted from each condition, and quantitative real-time PCR (Q-RT-PCR) was performed using an IGFBP-3-specific primer pair. The fold change was normalized to pLPCX vector alone and reported as an average ± SE of three independent experiments. **, Significantly decreased IGFBP-3 gene expression (P < 0.05) in Dex- vs. vehicle-treated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids regulate a variety of essential biological functions, and the ensuing complexity of their actions has made the study of downstream signaling mechanisms a challenge. Using time course microarray technology to explore both early and late gene expression changes after Dex treatment in mammary epithelial cells, we have begun to model the molecular mechanisms of glucocorticoid action beyond its immediate target genes. Our strategy couples analyzing GR-mediated gene expression with promoter analysis to identify downstream transcriptional events indirectly regulated by GR activation. As in the example presented here, these later transcriptional events are likely the result of indirect effects of the GR on transcription factors (e.g. FOXO3a).

Although it is well known that ligand-bound GR interferes directly with activator protein-1 (AP-1) and the nuclear factor {kappa}B (NF-{kappa}B) transcription factors, the indirect effects of the GR on other transcription factors are not well understood. In this study, we demonstrate that GR-mediated mammary epithelial cell survival requires FOXO3a inactivation. To further identify potential downstream target genes affected by FOXO3a inactivation, we used a combination of temporal gene expression data and promoter analysis for FOXO3a core binding motifs. This analysis identified 41 putative FOXO3a target genes that both were down-regulated after GR activation and contained FOXO3a core binding sequences. Among these 41 putative FOXO3a target genes, we identified TRAIL, a well-established target of FOXO3a, whereas the other 40 genes have not been previously reported as FOXO3a targets.

In breast cancer, increased IGFBP-3 mRNA expression has been previously associated with susceptibility to breast cancer cell apoptosis (reviewed in Refs. 24 , 28 , and 29). For example, IGFBP-3 induction potentiates paclitaxel-induced apoptosis in human breast cancer cells (30) and is also observed after antiestrogen ICI 182,780-mediated apoptosis (31). Furthermore, high serum IGFBP-3 levels may be associated with a lower risk of breast cancer (21), suggesting the potential importance of this secreted protein in inhibiting breast cancer cell survival. Our results demonstrate that down-regulation of IGFBP-3 expression after GR activation is at least partially mediated by FOXO3a transcriptional inactivation and is also associated with GR-mediated cell survival.

Recently, paclitaxel-induced apoptosis was shown to correlate with FOXO3a’s nuclear sequestration and subsequent transcriptional regulation of the proapoptotic gene, Bim (27, 32). Interestingly, we have shown previously that Dex pretreatment of breast cancer cells inhibits paclitaxel-induced apoptosis in vitro (5, 10). It will therefore be of interest to determine whether or not GR inhibition of FOXO3a activity plays a more general role in chemotherapy resistance. Furthermore, expression of novel target genes of FOXO3a, such as IGFBP-3, may provide predictive biomarkers for sensitivity to paclitaxel and other chemotherapeutic drugs.

In addition to inhibiting chemotherapy-induced apoptosis, glucocorticoid treatment may inhibit FOXO3a activity in other clinically relevant situations. For example, Dex treatment induces cardiac hypertrophy (33), whereas FOXO3a is a negative regulator of cardiac hypertrophy (34). This suggests that the negative regulation of FOXO3a activity by GR activation might contribute to glucocorticoid-induced hypertrophy of cardiac muscle.

Conversely, GR and FOXO3a may exhibit cooperative effects in coordinating transcription in certain cell types. For example, the GR and FOXO proteins can both increase gluconeogenesis by up-regulating the expression of the glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase 4 genes (35). Prolonged Dex treatment causes skeletal muscle atrophy in patients (36), and expression of a constitutively active form of FOXO3a can also result in atrophy of fully differentiated skeletal muscle cells (37). Therefore, there appears to be a cell type-specific context with respect to GR and FOXO3a activity in regulating specific physiological outcomes. Whether this difference is due to a differential ability of GR activation to induce SGK-1 expression remains to be determined.

Our experiments also suggest that glucocorticoid-mediated FOXO3a (Thr32) phosphorylation is independent of Akt-1 activation. There are other examples of SGK-1-mediated phosphorylation events that do not require Akt-1 activation. For example, Zhang et al. (14) demonstrated that SGK-1 phosphorylates and activates I{kappa}B kinase ß (IKKb) independently of Akt-1 activation. In another study, Hu et al. (38) found that activated IKKb phosphorylates Ser644 on FOXO3a, thereby inhibiting its activity through protein degradation independently of Akt-1. Taken together, one might postulate that SGK-1 can phosphorylate IKKb, in turn causing FOXO3a phosphorylation and degradation. However, we did not find any evidence of IKKb phosphorylation after GR activation at any of the time points studied (data not shown). This suggests that either GR-mediated SGK-1 induction results in direct FOXO3a Thr32 phosphorylation or that SGK-1 mediates Thr32 phosphorylation through a yet-to-be-defined intermediary kinase.

In summary, we demonstrate here, for the first time, that GR activation results in FOXO3a phosphorylation, nuclear exclusion, and inactivation. Using a bioinformatic approach, we also identify known and novel FOXO3a target genes, such as TRAIL and IGFBP-3, which are down-regulated indirectly by GR activation. This approach is likely to be useful for identifying novel signaling pathways from complex time course microarray data after nuclear hormone receptor activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Lines and Transfection
MCF10A-Myc cells were cultured in a 1:1 mixture of DMEM and Ham’s F12 (BioWhittaker, Inc., Walkersville, MD) supplemented with hydrocortisone (0.5µg /ml), human recombinant epidermal growth factor (10 ng/ml), insulin (5 ng/ml; Sigma), 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. SK-BR-3 breast cancer cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Transfection of cells was performed using Polyfect reagent (QIAGEN) per manufacturer’s instructions.

Plasmids, Small Interfering RNA, and Retroviral Infection
The WT-FOXO3a-HA, TM-FOXO3a-HA, and FRE-Luciferase reporter constructs were obtained from Dr. Michael E. Greenberg (Harvard Medical School, Boston, MA). WT-FOXO3a-FLAG (WT-FOXO3a) and TM-FOXO3a-FLAG (TM-FOXO3a) were amplified by PCR and ligated into the XhoI and HindIII restriction enzyme sites of the pLPCX retroviral vector. Short hairpin RNA interference (siRNA) encoding for either SGK-1 or SS (5) were cloned into the BamHI and EcoRI restriction enzymes sites of the retroviral expression vector, RNAi-Ready pSIREN-RetroQ (BD Biosciences, Palo Alto, CA). The siRNA constructs were transiently transfected into Ampho Phoenix cells and 48 h later, supernatant containing virus particles were collected and MCF10A-Myc cells were infected as described previously (3). Clones stably expressing SGK-1 siRNA or SS siRNA were made by selecting cells with puromycin (400 ng/ml).

Western Blot Analysis
Cell lysates were harvested with 2x Laemmli lysis buffer. Equal amounts of protein were resolved by SDS-PAGE, transferred to nitrocellulose membrane, blocked with 5% skim milk in TBS-T (0.1% Tween-20 in TBS), and probed with a 1:1000 dilution of one of the following antibodies: anti-SGK-1 antibody (5), anti-p-FOXO3a (T32) (Cell Signaling Technology, Beverly, MA), anti-FOXO3a (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-p-Akt-1 (S473) (Cell Signaling), or anti-Akt-1 (Cell Signaling). Goat antirabbit or goat antimouse-horseradish peroxidase (HRP) (Santa Cruz, 1:5000) was used as the secondary antibody. Anti-FLAG-HRP-conjugated M2 monoclonal antibody (Sigma Chemical Co., St. Louis, MO) was used to detect the FOXO3a expression in some experiments. Blots were subsequently probed with a mouse antiactin antibody (Sigma) to detect actin expression as a loading control. Proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Chemical Co., Rockford, IL). To measure p-FOXO3a/FOXO3a protein expression ratios, films were scanned on a Bio-Rad GS-710 densitometer, and data were analyzed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA). For secreted IGFBP-3 protein, medium was collected and concentrated using Centricon YM-10 (Millipore, Billerica, MA) filters following manufacturer’s instructions.

Immunofluorescence
MCF10A-Myc cells (2) were plated on coverslips, allowed to adhere overnight, starved of all growth factors for 24 h, and treated with Dex for 0, 2, 4, 8, 12, or 24 h. Cells were fixed for 20 min at room temperature in freshly prepared 4% paraformaldehyde and 5 mM HEPES in Hank’s balanced salt solution (Life Technologies, Gaithersburg, MD) and then blocked and permeabilized simultaneously for 30 min at room temperature using a 1% fetal calf serum/0.01% saponin/Hanks’ balanced salt solution. Fixed cells were incubated for 1 h at 37 C in a humidified chamber with either anti-FOXO3a (Santa Cruz, 1:150) diluted in blocking/permeabilization buffer, washed at room temperature three times for 2min in blocking/permeabilization buffer, and then incubated in the dark for 1 h at 37 C in a humidified chamber with an antirabbit antibody (1:1500) conjugated to a red spectrum Alexa Fluor 568 (Molecular Probes Inc., Eugene, OR). Cells were then washed two times for 3 min using the blocking/permeabilization buffer, mounted onto slides using Gel Mount (Biomeda, Foster City, CA), and examined at x40 with a Zeiss Axiovert 200 inverted fluorescence microscope (Carl Zeiss, Thornwood, NY), using a cooled Orca ER camera. Images were captured and analyzed using the Openlab program.

Apoptosis Assays
SK-BR-3 cells were transiently transfected with either pLPCX, WT-FOXO3a, or TM-FOXO3a and 48 h post transfection, cells were split into duplicate plates and allowed to adhere for 16 h. Either vehicle (EtOH) or Dex (10–6 M) was added to each plate of transfected cells for 24 h, after which cells were collected, fixed with 95% EtOH and stored at 4 C, spun down to remove EtOH, washed twice with 1x PBS, and stained with a solution of RNase (10 µg/ml) and propidium iodide (50 µg/ml) at 4 C for 30 min in the dark. Samples were analyzed on a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ), and data were processed using the Cell Quest program (Becton Dickinson and Co.). Cellular debris was excluded from analysis, and the percentage of apoptosis was calculated by analysis of the sub-G1 cell population.

Luciferase Assays
SK-BR-3 breast cancer cells were transiently transfected with 2.5 µg of pLPCX, WT-FOXO3a-FLAG, or TM-FOXO3a-FLAG, together with FRE-Luciferase reporter DNA and 0.5 µg of a pCMV-ß-galactosidase encoding vector (gift of Dr. Geoffrey Greene, University of Chicago). Cells were treated with EtOH or Dex 48 h after transfection, as described above in Apoptosis Assays. After the culture media was removed and rinsed twice with 1x PBS, 500 µl of 1x passive lysis buffer (Promega Corp., Madison, WI) was added to the plates and shaken at room temperature for 15 min. Luminescence was measured using a Wallace luminescence counter (Beckman), and ß-galactosidase activity was measured at 420 nm. Experiments were done three times, and relative luciferase activity in each condition was normalized to ß-galactosidase activity and reported as an average ± SE.

Time Course Microarray Data and Bioinformatic Analysis
The procedure for cRNA preparation and hybridization to high-density oligonucleotide arrays was described previously (10). Briefly, MCF10A-Myc cells were subjected to 72 h of growth factor withdrawal followed by treatment with either vehicle (ethanol), Dex (10–6 M), or concomitant Dex/RU486 (10–7 M) for 0.5, 2, 4, or 24 h. Total RNA from each sample was extracted using QIAGEN's RNeasy Kit. Preparation of biotinylated cRNA and hybridization to oligonucleotide arrays (Affymetrix human genechip HG-U133A) were performed at the University of Chicago Microarray Core Facility. The HG-U133A genechip contains 22,215 probe sets that represent approximately 16,000 human genes. The complete procedure was repeated independently three times. Genechips were scanned and analyzed using Robust Multiarray Average algorithm (39).

For each time point, genes with an averaged fold change (Dex-treated vs. vehicle-treated) greater than or equal to 1.5-fold or less than or equal to 1/1.5-fold (0.7-fold) were selected as being up-regulated and down-regulated, respectively, at this specific time point. Our main goal was to identify the transcriptional targets of FOXO3a. A time lag is predicted to exist between the initial induction of SGK-1 gene expression and the subsequent transcriptional down-regulation of FOXO3a target genes (due to the time that it takes for SGK-1 protein synthesis, and subsequent FOXO3a phosphorylation and nuclear exclusion). Therefore, we first obtained promoter sequences (–1000 nt/+200 nt) of genes in the human genome from Database of Transcription Start Site (DBTSS at http://dbtss.hgc.jp). A set of genes in the human genome that have a least one consensus FOXO3a core binding motif [AAA(C/T)A] (18) in their promoter region was then identified using a JAVA-based Transcription Binding Site Search program we developed. This program searches for promoter sequences with a specific transcription factor-binding motif and is available upon request. We subsequently identified putative FOXO3a targets by searching for a set of genes that contained both a FOXO3a core-binding motif and were down-regulated, on average, by at least 30% at 24 h after GR activation.

Quantitative Real-Time PCR
Quantitative real-time PCR were performed as previously described (10). The following primers were used: uPA (PLAU), 5'-ACGCAAGGGGAGATGAAG-3' (forward) and 5'-TCAGCAAGGCAATGTCGT-3' (reverse); IGFBP-3, 5'-CAGAGACTCGAGCACAGCAC-3' (forward) and 5'-G ATGACCGGGGTTTAAAGGT-3' (reverse); LKB-1, 5'-CCTGCTGAA AGGGATGCT-3' (forward) and 5'-TTCAGCCGGAGGATGTTT-3' (reverse); TRAIL, 5'-CCAGAGGAAGAAGCAACACA-3' (forward) and 5'-GGAATGAATGCCCACTCC-3' (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GAGTCAACGGATTTGGTCGT-3' (forward) and 5'-TTGATTTTGGA GGGATCTCG-3' (reverse). GAPDH was amplified as an internal control. The samples were loaded in triplicate, and the results of each sample were normalized to GAPDH to obtain an average ± SE. Fold change was calculated as a ratio of treated (Dex) over control (vehicle).

ChIP Analysis
MCF10A-Myc cells were starved of all growth factors for 72 h and then treated with either vehicle (EtOH), Dex (10–6 M) or Dex/RU486 (10–7 M). Cells were fixed with formaldehyde, and lysates were then sonicated and incubated with or without anti-FOXO3a antibody according to manufacturer’s directions in the Upstate Chromatin Immunoprecipitation Assay Kit. Quantitative real-time PCR was performed on the purified DNA using the following primers: IGFBP-3 no. 1, forward, 5'-GTTTCAGCAGTGCCCAGTT-3' (–977/–950); reverse, 5'-TGTTGCTACACCGCAAGTCT-3' (-691/-711). IGFBP-3 no. 2, forward, 5'-TTGCGTTGAGAAGTAAGCCTG-3' (–643/–623); reverse, 5'-GCATTCGTGTGTACCTCGTG-3' (–423/–443). Negative control primers, forward, 5'-GGGCACTCCATTGTTCTTGT-3' (–2000/–1880); reverse, 5'-GATCTCTCGCCCAGTGTCTC-3' (–1402/–1418). TRAIL primers, forward, 5'-CCTGGGCGATAAAGTGAGAT-3' (–1120/–1100); reverse, 5'-GGCCCAGCTGTATGTTGTCT-3' (–790/–810). Analysis of the PCR products was performed on a standard 2% (wt/vol) agarose gel.


   ACKNOWLEDGMENTS
 
We thank Dr. Harinder Singh for advice with ChIP assays and Dr. Michael Greenberg for plasmids. We also thank the University of Chicago Functional Genomics Core Facility for assistance in performing Affymetrix hydridizations and data analysis.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants CA90459 and CA89208 (to S.D.C.) and by the University of Chicago Cancer Research Foundation. M.Z. was supported in part by a postdoctoral fellowship from the Illinois Department of Public Health Penny Severns Breast and Cervical Cancer Research Program, and T.P. was supported in part by a summer undergraduate research fellowship from The Endocrine Society.

Disclosure statement: The authors have nothing to disclose.

First Published Online May 11, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; Dex, dexamethasone; FOXO3a, Forkhead transcription factor 3a; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; HRP, horseradish peroxidase; IGFBP-3, IGF-binding protein 3; IKKb, I{kappa}B kinase; nt, nucleotide; RNAi, RNA interference; SGK-1, serum and glucocorticoid-regulated kinase-1; siRNA, short interfering RNA; SS, scrambled sequence; WT-FOXO3a, wild-type FOXO3a.

Received for publication March 24, 2006. Accepted for publication May 5, 2006.


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 RESULTS
 DISCUSSION
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NURSA Molecule Pages Link:

Nuclear Receptors:   GR
Ligands:   Dexamethasone  |  RU486



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