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Department of Obstetrics and Gynecology (O.L.B., Z.L., C.D.M., J.J.K.), Northwestern University, Chicago, Illinois 60611; and Departments of Physiology and Biophysics, and Medicine (T.G.U.), University of Illinois at Chicago, College of Medicine and Veterans Affairs Chicago Healthcare System (West Side), Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Dr. J. Julie Kim, Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Northwestern University, 303 East Superior Street, Lurie 4-117, Chicago, Illinois 60611. E-mail: j-kim4{at}northwestern.edu.
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Introduction |
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FOXO1A is a member of the FOXO subfamily of Forkhead/winged helix family of transcription factors that is involved in cell cycle regulation, differentiation, and apoptosis (5). In addition to playing a role in diverse pathways, it has been shown that FOXO1A can function as a master switch to control apoptosis (6). In breast cancer cells, another forkhead transcription factor, FoxA1 is necessary in mediating an estrogen response (7). FOXO1A is expressed in the baboon and human endometrium (8, 9, 10). It is up-regulated during the luteal phase of the menstrual cycle and during pregnancy. FOXO1A can up-regulate both the IGFBP1 and PRL promoter activities in human endometrial stromal cells (HSC) (9) and human term decidual fibroblasts (8). Moreover, we have shown that silencing FOXO1A in human decidual fibroblasts decreases IGFBP1 expression in response to hormones plus dibutyryl cAMP (H+dbcAMP) (11). FOXO1A physically associates with other nuclear transcription factors, such as the progesterone receptor (11), estrogen receptor (7, 12, 13), retinoic acid receptor, and thyroid hormone receptors (13), homeobox A10 (HOXA10) (8) and CCAAT/enhancer-binding protein-ß (C/EBPß) (9). These and other protein-protein interactions can modulate or alter effects of FOXO1A on gene expression (14).
In this study, we further investigate the role of FOXO1A in regulating not only IGFBP1 and PRL but also other genes including decorin (DCN), tissue metalloproteinase inhibitor-3 (TIMP3), cannabinoid receptor 1 (CNR1), and laminin B1 (LAMB1), which have been shown to be up-regulated during decidualization (3, 15). Using primary HSC, FOXO1A was silenced with small interfering RNA (siRNA) technology or overexpressed using an adenoviral construct, and expression levels of the six genes were measured by real-time PCR. The influence of FOXO1A in the presence of other genes induced by H+dbcAMP was also investigated. Here, we demonstrate that all six genes were regulated by FOXO1A and that this regulation varied depending on the gene as well as with H+dbcAMP treatment.
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Materials and Methods |
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siRNA transfection
To silence FOXO1A gene expression, transfection of a siRNA duplex was performed as previously described (11). The FOXO1A siRNA was synthesized by Dharmacon (Lafayette, CO) and corresponded to nucleotides 961–979 of the human FOXO1A coding region (GAGCGTGCCCTACTTCAAG) as described by Potente et al. (17). This sequence also targets the FOXO3A gene. A nonrelated control siRNA that targets the firefly luciferase protein (Dharmacon, catalog no. D-001210-02-05) was used as a control. Cells were grown until 60% confluence at which time they were transfected with FOXO1A siRNA or control siRNA with Lipofectamine 2000. Cells were transfected for 6 h, then treated with H+dbcAMP for 48 h, and then lysed with TriReagent (Molecular Research Center, Cincinnati, OH) for analysis of gene expression. We have previously demonstrated that this siRNA suppresses the expression of FOXO1A protein in human decidual fibroblasts (11). Silencing of the FOXO1A gene was verified by real-time PCR and Western blot in this study.
Adenovirus infection
The cDNA coding for constitutively active human FOXO1A (where Thr24, Ser256, and Ser319 have been replaced by alanines) was subcloned into the pShuttle-IRES-hrGFP vector (Stratagene, La Jolla, CA) in-frame with the carboxyl-terminal Flag epitope tag. The shuttle vector DNA was linearized with Pac1 before transfection of recombination proficient BJ5183-AD-1 cells carrying the pAdEasy-1 plasmid. Transformations were plated and plasmids were screened for recombination by digestion with Pac1. Virus was packaged and amplified in HEK293A cells (Quantum, Montreal, Quebec, Canada), and the expression of Flag-tagged FOXO1A in HEK cells was verified by Western blotting. For studies, adenoviral vectors were reamplified in HEK293 cells, and plaque-forming units were titered by staining for viral hexon protein as a service (Vector Biolabs, Philadelphia, PA).
HSC were grown until 90% confluence at which time they were infected with the adenovirus construct expressing the triple-mutated FOXO1A (constitutively active) protein (AD-FOXO1A) plus green fluorescent protein (GFP) or adenovirus expressing GFP alone (AD-GFP) as a control. Based on previous studies, cells were infected for 24 h with a multiplicity of infection of 100 plaque-forming units per cell. After infection, cells were washed and medium with or without H+dbcAMP was added. Cells were treated for 48 h and then lysed with TriReagent.
Digital photographs were taken before infection and at 2 and 4 d after infection using a Nikon 5.0 MPix CoolPix camera mounted on an inverted microscope (Nikon TS-100F).
Quantitative real-time PCR
Cells were lysed with TriReagent, and total RNA was extracted using the protocol provided by the manufacturer. One microgram of total RNA was reverse transcribed (16) in a total volume of 20 µl, and real-time PCR using SYBR green fluorescence was performed. Each real-time PCR consisted of 1 µl RT product, 10 µl SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA), and 500 nM forward and reverse primers (IDT, Coralville, IA). Reactions were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after an initial 10-min incubation at 95 C. The primers used for real-time PCR are listed in Table 1
. The fold change in expression of each gene was calculated using the 
Ct method (18), with the ribosomal protein 36B4 mRNA as an internal control. Data reported are the mean fold change ± SEM for three independent determinations.
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Reporter gene constructs and expression vectors
The proximal promoter regions of the decidual PRL, corresponding to –1080 to +69 (19) and TIMP3 corresponding to –1002 to +53 from the transcription start site (20), was isolated by PCR from human genomic DNA (Clontech, Mountain View, CA). Additional bases were added to the PRL primers to create a KpnI site on the forward primer and XhoI site on the reverse primer. For TIMP3, an XhoI site on the forward primer and HindII site on the reverse primer were added. The PCR product was subcloned into the corresponding sites of the promoterless pGL4-basic vector (Promega, Madison, WI). The promoter fragments were verified by dideoxy sequencing. The dPRL.Luc and TIMP3.Luc constructs were then used for transient transfections. The human FOXO1A expression vector used in these studies is the mutant form where the three consensus protein kinase B phosphorylation sites, Thr-24, Ser-256, and Ser-319, were mutated to alanines creating a constitutively active form (21).
Cell transfection and reporter gene studies
HSC were grown in 12-well plates. At 80% confluence, cells were transfected with the dPRL.Luc or TIMP3.Luc with FOXO1A expression vector using Lipofectamine 2000 (Invitrogen). Cells were transfected in DMEM with 1 µg/well of the promoter constructs with or without 0.5 µg/well FOXO1A. pcDNA3.1(+) was used as empty vector for the controls. After 4 h, the medium was changed to RPMI 1640 (Invitrogen) with or without H+dbcAMP. Cells were incubated for an additional 48 h. Cell extracts were harvested, and luciferase activity was measured with the luciferase reagent kit (Promega). Fold induction was calculated as relative light units of FOXO1A expression vector divided by relative light units of the control (basal activity with no expression vectors). Data are presented as the mean ± SEM of three independent experiments, each performed in triplicate.
Statistical analysis
A nonparametric t test (Mann-Whitney) was performed on data that were normalized to the controls of each experiment. Data were considered statistically significant when P < 0.05.
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, A–D and F), consistent with previous reports. No change in expression was observed for LAMB1 after 48 h of treatment (Fig. 1E). FOXO1A mRNA and protein expression also increased after H+dbcAMP treatment (Fig. 1, G and H). Thus, with the exception of LAMB1, the other candidate genes increased in expression in response to a 48-h treatment with H+dbcAMP.
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). Interestingly, addition of H+dbcAMP also increased levels of FOXO1A in the AD-FOXO1A-infected cells, similar to the effects on the endogenous FOXO1A (Fig. 1H
) and that of AD-GFP-infected cells (Fig. 2A).
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, B–D), whereas levels of TIMP3 and CNR1 decreased (Fig. 2, E and G). There was no effect of FOXO1A on LAMB1 (Fig. 2F). To evaluate the role of FOXO1A in the presence of other products of decidualization, cells overexpressing AD-FOXO1A were treated with H+dbcAMP for 48 h. For IGFBP1, PRL, and DCN, the addition of H+dbcAMP in AD-GFP-infected cells resulted in an increased expression from that of AD-GFP alone, as expected (Fig. 2, B–D). When H+dbcAMP was added to AD-FOXO1A-overexpressing cells, the levels of IGFBP1, PRL, and DCN were higher than that of AD-FOXO1A or H+dbcAMP alone, suggesting cooperative interactions of FOXO1A with other products of decidualization on the regulation of these three genes. For TIMP3 and CNR1, H+dbcAMP treatment in AD-GFP-infected cells increased mRNA levels compared with AD-GFP alone (Fig. 2, E and G). H+dbcAMP treatment in AD-FOXO1A-overexpressing cells also increased mRNA levels compared with AD-GFP or AD-FOXO1A alone; however, these levels were lower than that of H+dbcAMP alone, suggesting that AD-FOXO1A attenuates the effect of H+dbcAMP on TIMP3 and CNR1. As previously mentioned, FOXO1A overexpression did not affect LAMB1 mRNA levels. However, when H+dbcAMP were added to AD-FOXO1A-infected cells, a decrease in LAMB1 expression was observed (Fig. 2F).
In addition to regulating decidualization-specific genes, overexpression of FOXO1A in the absence of exogenous hormones caused a noticeable change in stromal cell shape. After 2 and 4 d of AD-FOXO1A infection, the cells became round, losing the long fibroblast-like shape (Fig. 3, D–F), whereas cells infected with AD-GFP remained fibroblast-like (Fig. 3, A–C). The change in cell shape induced by FOXO1A is reminiscent of what occurs during in vitro and in vivo decidualization. Nuclear localization of FOXO1A protein after AD-FOXO1A infection was verified by immunofluorescent staining (data not shown), consistent with previous studies (22).
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, levels of FOXO1A mRNA expression decreased with siFOXO1A for both control and H+dbcAMP-treated cells. Similarly, protein levels of FOXO1A diminished with siFOXO1A (Fig. 4B), consistent with previous studies (11). As a result of FOXO1A silencing, mRNA expression of IGFBP1 and DCN decreased (Fig. 4, C and E). However, the mRNA expression of PRL, TIMP3, and CNR1 increased in response to FOXO1A silencing (Fig. 4, D, F, and H). There was no effect on LAMB1 expression (Fig. 4G). These studies demonstrate that FOXO1A contributes to the regulation of IGFBP1, DCN, PRL, TIMP3, and CNR1 in H+dbcAMP-treated cells. The different responses to FOXO1A silencing implicate the involvement of other cofactors and confirm the complexity of gene regulation during decidualization.
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) but not TIMP3.Luc (Fig. 5B). When transfected cells were treated with H+dbcAMP for 48 h, the activity of dPRL.Luc significantly increased, whereas the activity of TIMP3.Luc significantly decreased. This observation is in agreement with the other experiments in this study, in that FOXO1A with H+dbcAMP can increase PRL expression while decreasing TIMP3 expression.
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Discussion |
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After 48 h of H+dbcAMP treatment, mRNA expression of IGFBP1, PRL, TIMP3, CNR1, and DCN increased. However, LAMB1 mRNA levels did not change. Although it has been shown that LAMB1 expression does increase during decidualization (3, 15), it is possible that 48 h of treatment is not sufficient to observe a significant difference.
Reducing expression of FOXO1A with siRNA resulted in a decrease of IGFBP1 and DCN mRNA expression despite treatment with H+dbcAMP. Furthermore, overexpression of FOXO1A in the absence of exogenous hormones was sufficient to increase mRNA levels for IGFBP1 and DCN, demonstrating that FOXO1A is a key molecule responsible for the increased expression of these genes. Although quantity of FOXO1A is important, as shown by the effect of FOXO1A silencing and overexpression, the localization of FOXO1A is also critical in determining function. It has been shown that H+dbcAMP promotes the localization of FOXO1A to the nucleus (9). Thus, although the levels of FOXO1A expression in siFOXO1A and H+dbcAMP-treated cells are similar to that of untreated cells, the localization of FOXO1A is different, and this likely contributes to differences in gene expression in untreated compared with H+dbcAMP-treated cells.
For TIMP3 and CNR1, silencing FOXO1A resulted in an increase of expression in the presence of H+dbcAMP. Moreover, overexpressing FOXO1A reduced expression of these genes from basal levels, suggesting an inhibitory role of FOXO1A on these genes. For PRL, overexpression of FOXO1A caused an increase in mRNA levels, and this is consistent with a previous report that FOXO proteins can interact directly with binding sites in the proximal PRL promoter (9). Furthermore, when H+dbcAMP were added to cells infected with AD-FOXO1A, the increase in PRL was substantial, clearly demonstrating that other factors induced by hormonal treatment act synergistically with FOXO1A to significantly increase PRL expression. This may include C/EBPß, which is induced by cAMP agonists and recruited to the PRL promoter through a direct interaction with FOXO1A (9). Interestingly, when FOXO1A was silenced, the ability of H+dbcAMP to increase PRL mRNA levels was not disrupted, suggesting that FOXO1A-independent mechanisms may also contribute to the induction of PRL. Another possibility is that because the siRNA used in this study is specific for both FOXO1A and FOXO3A, silencing both could regulate PRL expression differently than silencing FOXO1A only. The importance of cofactors in determining the full effect of FOXO1A on gene expression is highlighted by the effects of H+dbcAMP in cells overexpressing FOXO1A on all six genes. The addition of H+dbcAMP increased levels of IGFBP1, DCN, and PRL and decreased TIMP3 and CNR1 beyond that of either AD-FOXO1A or H+dbcAMP alone. LAMB1, which was not responsive to FOXO1A overexpression alone, decreased in expression when H+dbcAMP was added. The increase in AD-FOXO1A protein levels in response to H+dbcAMP (Fig. 2A) could have also contributed to the enhancement of increases or decreases of gene expression.
The effect of FOXO1A overexpression on cell morphology further demonstrates an important role for FOXO1A during decidualization. The transformation of a fibroblast to a rounded epithelioid shape is very similar to that observed during decidualization both in vitro and in vivo. This transformation occurred relatively quickly compared with in vitro decidualization by H+dbcAMP, which takes approximately 10–14 d and could have been the result of the high levels of constitutively active FOXO1A. It is also possible that some of the genes regulated by AD-FOXO1A are involved in the rearrangement of the cytoskeleton. To our knowledge, the role of FOXO1A in cytoskeletal modification has not been studied.
Proximal promoter regions of PRL (19) and TIMP3 (20) were subcloned into a luciferase reporter to more directly examine the effects of FOXO1A. Although dPRL.Luc activity increased significantly with FOXO1A with and without H+dbcAMP, TIMP3.Luc decreased with FOXO1A and H+dbcAMP only. We have previously shown that the transactivation function of FOXO1A on the IGFBP1 promoter decreased when progesterone receptor is coexpressed (11). The decrease in TIMP3.Luc observed here with FOXO1A and H+dcAMP may involve similar mechanisms, in that endogenous progesterone receptor of HSC activated by H+dbcAMP cooperates with FOXO1A to decrease promoter activity. The mechanisms involved in this cooperation are currently under investigation.
In this study, we have shown that FOXO1A is an active and important transcription factor during decidualization. To our knowledge, this is the first report that demonstrates the regulation of DCN, CNR1, LAMB1, and TIMP3 genes by FOXO1A. Although these genes are up-regulated during decidualization, they are differentially regulated by FOXO1A depending on the hormonal milieu. This indicates that FOXO1A functions together with other hormonally regulated factors to contribute to the expression of decidualization genes. Decidualization is a tightly regulated process that takes time to complete. Thus, the induction or amplification of critical genes must be controlled by inhibitors, through cross-talk or by other mechanisms. It has been shown that CNR1, a gene that increases in decidualization, markedly inhibits decidualization (10), and laminin has been reported to decrease PRL and IGFBP1 expression (23). Thus, the aberrant expression or regulation of FOXO1A could offset the critical balance needed in this differentiation process. Furthermore, the different effects of FOXO1A with or without H+dbcAMP demonstrate the versatile nature of FOXO1A and the importance of other cofactors during decidualization.
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Acknowledgments |
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Footnotes |
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Disclosure summary: all authors have nothing to declare.
First Published Online May 11, 2006
Abbreviations: AD, Adenovirus-expressing; C/EBPß, CCAAT/enhancer-binding protein-ß; FOXO1A, forkhead box O1A; GFP, green fluorescent protein; H+dbcAMP, hormones plus dibutyryl cAMP; HOXA10, homeobox A10; HSC, human endometrial stromal cells; IGFBP-1, IGF-binding protein-1; PRL, prolactin; siRNA, small interfering RNA.
Received February 9, 2006.
Accepted for publication May 4, 2006.
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with members of the forkhead transcription factor family. J Biol Chem 276:33554–33560CT method. Methods 25:402–408[CrossRef][Medline]This article has been cited by other articles:
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