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Molecular Endocrinology, doi:10.1210/me.2006-0118
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Molecular Endocrinology 20 (10): 2444-2455
Copyright © 2006 by The Endocrine Society

Differential Expression of FOXO1 and FOXO3a Confers Resistance to Oxidative Cell Death upon Endometrial Decidualization

Takeshi Kajihara, Marius Jones, Luca Fusi, Masashi Takano, Fakhera Feroze-Zaidi, Grisha Pirianov, Huseyin Mehmet, Osamu Ishihara, Jenny M. Higham, Eric W.-F. Lam and Jan J. Brosens

Institute of Reproductive and Developmental Biology (T.K., M.J., L.F., M.T., F.F.-Z., G.P., H.M., J.J.B.), and Cancer Research-UK Labs and Section of Cancer Cell Biology (E.W.-F.L.), Department of Oncology, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; Department of Obstetrics and Gynaecology (J.M.H.), Imperial College London, St. Mary’s Hospital, London W2 1PG, United Kingdom; and Department of Obstetrics and Gynecology (O.I.), Saitama Medical School, Moroyama, Saitama 350-0495, Japan

Address all correspondence and requests for reprints to: Jan J. Brosens, Institute of Reproductive and Developmental Biology, Imperial College London, Faculty of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom. E-mail: j.brosens{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The integrity of the feto-maternal interface is critical for survival of the conceptus. This interface, consisting of the maternal decidua and the invading placental trophoblast, is exposed to profound changes in oxygen tension during pregnancy. We demonstrate that human endometrial stromal cells become extraordinarily resistant to oxidative stress-induced apoptosis upon decidualization in response to cAMP and progesterone signaling. This differentiation process is associated with the induction of the forkhead transcription factor FOXO1, which in turn increases the expression of the mitochondrial antioxidant manganese superoxide dismutase. However, silencing of FOXO1 did not increase the susceptibility of decidualized cells to oxidative cell death. Comparative analysis demonstrated that hydrogen peroxide, a source of free radicals, strongly induces FOXO3a mRNA and protein expression in undifferentiated human endometrial stromal cells but not in decidualized cells. Expression of a constitutively active FOXO3a mutant elicited apoptosis in decidualized cells. Furthermore, silencing of endogenous FOXO3a in undifferentiated cells abrogated apoptosis induced by hydrogen peroxide. These results suggest that the induction of FOXO1 may enhance the ability of decidualized cells to prevent oxidative damage while the simultaneous repression of FOXO3a expression disables the signaling pathway responsible for oxidative cell death. The differential regulation of FOXO expression provides the decidua with a robust system capable of coping with prolonged episodes of oxidative stress during pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SURVIVAL AND growth of the developing fetus are critically dependent upon adequate utero-placental blood flow. In early pregnancy, a subpopulation of fetal trophoblast cells, the extravillous trophoblast, invades the decidualized (differentiated) endometrial stromal compartment (interstitial invasion) and its spiral arteries (endovascular invasion) (1, 2). Initially, plugs of endovascular trophoblast effectively occlude the terminal decidual vessels, thereby restricting the exposure of the early placenta and fetus to maternal arterial blood (3, 4). Subsequently, the trophoblast plugs are dislocated, allowing perfusion of the placental intervillous space. The endovascular trophoblast continues to invade the spiral arteries and replaces the endothelial lining and most of the musculo-elastic tissue in the vessel wall. This process, termed physiological transformation of the spiral arteries, ultimately establishes a high-flow, low-resistance utero-placental circulation, capable of accommodating the increasing fetal demand for nutrients and oxygen in later pregnancy (1, 2).

As a consequence of these vascular adaptations, major fluctuations in oxygen concentrations occur at the feto-maternal interface during normal pregnancy. The partial oxygen pressure measured within the human placenta between 7 and 10 wk gestation is less than 20 mmHg (5). This rises sharply to greater than 50 mmHg when the maternal perfusion of the placenta is fully established, a process completed between 11 and 14 wk. The dramatic changes in oxygen tension at the utero-placental interface induce a burst of intracellular reactive oxygen species (ROS) (5). In the absence of effective defense mechanisms, ROS, including superoxide anion, hydrogen peroxide, and hydroxyl radicals, cause indiscriminately damage to proteins, lipids, and nucleic acids. To counter oxidative stress, cells constitutively express enzymes that neutralize ROS and repair and replace the damage caused by ROS (6, 7). In addition, cells also mount an "adaptive response" to elevated levels of oxidative stress, such as increased expression of antioxidant enzymes, including glutathione S-transferases, peroxidases, and superoxide dismutases, and activation of protective and repair genes, such as heat shock proteins and growth arrest- and DNA damage-inducible protein {alpha} of 45 kDa (GADD45{alpha}), respectively (8, 9). Depending on the level of oxidative stress experienced, cells will either undergo transient cell cycle arrest and repair, senescence, apoptosis, or ultimately necrosis (10).

The FOXO subfamily of Forkhead transcription factors in mammalian cells comprises three functionally related members, FOXO1, FOXO3a, and FOXO4, which are orthologs of the Caenorhabditis elegans transcription factor DAF-16 (11, 12). FOXO proteins are evolutionarily conserved transcriptional activators of genes involved in cell cycle inhibition (e.g. p27Kip1) (13, 14), apoptosis [e.g. Bim, TRAIL, and Fas ligand (FasL)] (15, 16, 17, 18), defense against oxidative stress [e.g. manganese superoxide dismutase (MnSOD) and catalase] (19, 20, 21), and DNA repair (e.g. GADD45{alpha}) (9, 22). Posttranslational modification of FOXO proteins is an important mechanism that regulates the ability of these transcription factors to activate distinct gene sets. Phosphorylation in response to growth factor-mediated activation of the phosphatidylinositol 3-kinase/Akt signaling pathway results in the cytoplasmic retention of FOXO proteins and, hence, inhibition of forkhead-dependent transcriptional activity (19, 23). Conversely, targeted phosphorylation of cytoplasmic FOXO factors by Jun N-terminal kinase promotes nuclear import and increases cellular protection against oxidative stress via the transcriptional activation of MnSOD and catalase (21, 24, 25). FOXO proteins activated by oxidative stress signals also recruit SIRT1, a nicotinamide adenine dinucleotide-dependent protein deacetylase (22, 26, 27, 28). SIRT1-dependent deacetylation of FOXO proteins is thought to promote cell cycle inhibition and stress resistance while simultaneously attenuating the apoptotic response to oxidative stress.

FOXO proteins are not constitutively expressed in human endometrium. Previous studies have shown that FOXO1 is induced in decidualizing human endometrial stromal cells (HESCs), both in vitro and in vivo, whereas FOXO3a appears repressed (29, 30, 31). FOXO4 is not expressed in normal endometrium (31). Decidualization of the endometrial stromal compartment is initiated in the midsecretory phase of the cycle and continues in pregnancy. We now demonstrate that FOXO1 induces the expression of MnSOD upon HESC differentiation, indicating that the decidua possesses heightened defense mechanisms against oxidative stress. Conversely, oxidative stress induces FOXO3a and apoptosis in undifferentiated but not in decidualizing HESCs. The data suggest that the distinct regulation of FOXO transcription factors upon endometrial decidualization favors tissue preservation and integrity over apoptotic clearance of defective cells when faced with prolonged oxidative insult during pregnancy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HESCs Become Resistant to Oxidative Cell Death upon Decidualization
We sought to determine whether HESCs become more resistant to oxidative cell death upon differentiation. Confluent primary cultures remained untreated or were decidualized by treating cells with 8-bromoadenosine-cAMP (8-br-cAMP) and a progestin [medroxyprogesterone acetate (MPA)] for 3 d (32, 33, 34). Subsequently, the cultures were exposed to various concentrations of H2O2, a source of oxidative stress, for 24 h. Under light microscopy, cell death was apparent in undifferentiated but not decidualized cultures (data not shown). To confirm these observations, we examined the cell cycle phase distributions in the different cultures using flow cytometry analysis after propidium iodide staining of cells. As shown in Fig. 1Go, A and B, there was a dose-dependent increase in the number of dead or dying cells containing <2 N DNA content in undifferentiated HESCs treated with H2O2. In contrast, no discernible increase in the apoptotic cell fraction was observed in decidualized HESC treated with 100 or 250 µM H2O2 for 24 h. Even in the presence of 1 mM H2O2, cell death was more than 10-fold lower in 8-br-cAMP- plus MPA-treated cultures when compared with control cells. Western blot analysis further demonstrated that undifferentiated but not decidualized cells express cleaved poly(ADP-ribose) polymerase-1 (PARP), an apoptotic marker, upon treatment with H2O2 (Fig. 1CGo). The data supported our assertion that decidualizing cells are more resistant to oxidative cell death, although the level of this resistance was unexpected.


Figure 1
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  		Fig. 1. HESCs Become Resistant to Cell Death Induced by Oxidative Stress upon Decidualization

A, Cell cycle analysis of undifferentiated and decidualizing cultures exposed to various concentrations of H2O2 for 24 h. Decidualizing cultures were pretreated with 8-br-cAMP (cAMP) and MPA for 72 h. The percentage of cells in each phase of the cell cycle (<2 N, G0/G1, S, and G2/M) is indicated. B, Mean apoptotic fraction (<2 N) of three separate experiments. Error bars represent ±SEM. C, Western blot analysis of cleaved PARP expression in undifferentiated and decidualized HESC cultures exposed to various concentrations of H2O2 as indicated. ß-Actin expression was used as loading control.

 
Up-Regulation of MnSOD and GADD45{alpha} Expression in Decidualizing HESCs
FOXO transcription factors regulate the expression of several key enzymes in the antioxidant defense system, including the DNA repair enzyme GADD45{alpha}, Mn SOD, and catalase, the enzyme that degrades hydrogen peroxide (9, 19, 20, 21). Although cytosolic copper/zinc superoxide dismutase (Cu/ZnSOD) accounts for the majority of SOD activity in most cells, MnSOD is the major antioxidant enzyme involved in protecting mitochondria from superoxide anions (35). We speculated that the resistance to oxidative cell death observed in decidualizing cells could reflect FOXO1-dependent up-regulation of these enzymes. To test this, we first examined the expression of Cu/ZnSOD,MnSOD, catalase, and GADD45{alpha} in primary HESCs treated with 8-br-cAMP and MPA for 1, 3, and 10 d. As shown in Fig. 2Go, both MnSOD and GADD45{alpha} mRNA and protein levels increased in decidualizing cells. This was apparent after 3 d of treatment with 8-br-cAMP and MPA, and the levels were considerably higher in cells stimulated for 10 d. Transcript levels for catalase, and to a lesser extent those for Cu/ZnSOD, were modestly higher in 8-br-cAMP- plus MPA-treated cells, although no regulation was apparent at protein level. We also examined whether decidualization is associated with regulated expression of SIRT1, the nicotinamide adenine dinucleotide-dependent deacetylase implicated in promoting FOXO-dependent antioxidant defense responses (22). Upon treatment with 8-br-cAMP plus MPA, SIRT1 levels remained more or less unchanged after 1 and 3 d of stimulation. However, a noticeable rise in SIRT1 expression was seen after prolonged treatment (10 d), which consistently coincided with a noticeable reduction in its expression in undifferentiated control cells (Fig. 2BGo).


Figure 2
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  		Fig. 2. MnSOD Expression Is Up-Regulated upon HESC Differentiation

A, RTQ-PCR analysis of MnSOD, Cu/ZnSOD, catalase, and GADD45{alpha} transcript levels in HESCs treated with 8-br-cAMP (cAMP) and MPA for 1, 3, or 10 d. B, Western blot analysis of MnSOD, Cu/ZnSOD, catalase, GADD45{alpha}, and SIRT1 proteins in whole-cell lysates obtained from primary HESC cultures treated as indicated. {alpha}-Tubulin expression was used as loading control.

 
As reported previously (36, 37), immunohistochemistry demonstrated stronger staining for MnSOD, but not for catalase or Cu/ZnSOD, in the midsecretory endometrium when compared with proliferative endometrium (data not shown). However, the expression profiles of GADD45{alpha} and SIRT1 in human endometrium have not yet been reported. SIRT1 was detectable in both epithelial and stromal compartments of human endometrium, although we found no evidence of cycle-dependent changes in either mRNA or protein expression (Fig. 3Go, A and B). In contrast, analysis of timed endometrial biopsies showed a 10-fold increase in the abundance of GADD45{alpha} transcripts in secretory endometrium when compared with proliferative samples (Fig. 3AGo). GADD45{alpha} immunoreactivity was diffusely cytoplasmic in the epithelial compartment, irrespectively of the phase of the cycle (Fig. 3BGo). In contrast, stromal staining was invariably nuclear and much more pronounced in secretory endometrium (Fig. 3BGo). Together, the results suggest that, upon decidualization in vivo, the endometrium is better equipped to detoxify ROS and to repair damaged DNA.


Figure 3
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  		Fig. 3. Expression of GADD45{alpha} in Cycling Human Endometrium

A, RTQ-PCR analysis of GADD45{alpha} and SIRT1 transcripts in endometrial biopsies obtained from either proliferative (n = 10) or mid-secretory (n = 10) of the cycle. Expression levels were analyzed using Student’s t test. B, GADD45{alpha} and SIRT1 immunostaining in proliferative (d 8) and midsecretory (d 23) endometrium (original magnification, x100).

 
Silencing of FOXO1 Expression Does Not Sensitize HESCs to Oxidative Cell Death
We next investigated whether the induction of MnSOD and GADD45{alpha} upon HESC differentiation in response to 8-br-cAMP plus MPA treatment is dependent upon FOXO1. Parallel cultures were either mock transfected or transfected with small interfering RNA (siRNA) targeting FOXO1 or control (nontargeting) siRNA. The cultures were subsequently treated with 8-br-cAMP and MPA for 72 h, and then harvested for mRNA and protein analysis. Knockdown of FOXO1 expression abrogated the induction of MnSOD in decidualizing cells but had no effect on either Cu/ZnSOD, GADD45{alpha}, or catalase mRNA (data not shown) or protein expression levels (Fig. 4AGo). The supernatants of transfected cultures were assayed for prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP-1), two cardinal decidual markers thought to be under transcriptional control of FOXO1 (29, 30). In agreement with this, FOXO siRNA attenuated the induction of PRL and abolished IGFBP-1 expression in primary cultures treated with 8-br-cAMP and MPA for 3 d (Fig. 4BGo).


Figure 4
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  		Fig. 4. FOXO1 Expression Induces Up-Regulation of MnSOD, GADD45{alpha}, and Decidualization Markers in Differentiating HESCs

A, Mock transfected HESCs or cells transfected with FOXO1 siRNAs or nontargeting (control) siRNAs were treated for 3 d with 8-br-cAMP and MPA (cAMP/MPA). Parallel cultures were harvested for RTQ-PCR analysis (upper panel) or immunoblotting (lower panel). The expression of FOXO1 and MnSOD transcripts in decidualizing cultures treated was calculated relatively to the expression in undifferentiated cells. Total protein lysates were probed for expression of FOXO1, MnSOD, Cu/ZnSOD, catalase, GADD45{alpha}, and {alpha}-tubulin as loading control. B, The secretion of PRL and IGFBP-1 in mock transfected HESCs or cells transfected with FOXO1 siRNAs or nontargeting (control) siRNAs was measured and levels corrected for total protein concentration in each culture. C, Mock transfected HESCs or cells transfected with FOXO1 siRNAs or nontargeting (control) siRNAs were treated for 3 d with 8-br-cAMP and MPA (cAMP/MPA). Subsequently, some cultures were treated with 250 µM H2O2, as indicated. After 24 h, the cultures were harvested and analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle (<2 N, G0/G1, S, and G2/M) is indicated.

 
Because FOXO1 silencing attenuated the expression of differentiation markers and interfered with antioxidant defense mechanisms, we speculated that it would also sensitize decidualizing cultures to stress-induced apoptosis. However, this was not the case. Flow cytometry analysis showed no increase in the apoptotic cell fraction in differentiating cultures transfected with FOXO1 siRNA and treated with 250 µM H2O2 for 24 h (Fig. 4CGo).

Oxidative Stress Induces FOXO3a Expression in Undifferentiated HESCs
The induction of FOXO1 and expression of antioxidant enzymes did not account for the resistance to oxidative cell death in decidualizing cells. To further investigate this apparent paradox, we examined in more detail the stress responses in both undifferentiating and decidualizing cells. Primary cultures untreated or first decidualized with 8-br-cAMP plus MPA for 72 h were exposed to 250 µM H2O2 for 24 h. As reported previously (29), FOXO1 was induced upon decidualization, although occasionally the levels declined somewhat in response to oxidative stress (Fig. 5AGo). In contrast, prolonged exposure to H2O2 induced FOXO3a expression in undifferentiated cells but not in decidualizing cultures. The mRNA expression profiles of FOXO1 and FOXO3a mirrored that of the proteins (Fig. 5BGo), suggesting that oxidative stress signaling differentially regulates FOXO expression at the level of gene transcription. The induction of FOXO3a in H2O2-stressed undifferentiated cells had little or no effect on the expression of MnSOD, Cu/ZnSOD, or catalase (Fig. 5AGo). Cleaved PARP was used to monitor the activation of the proapoptotic machinery.


Figure 5
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  		Fig. 5. The Expression of FOXO1 and FOXO3a Is Differentially Regulated by Oxidative Stress

A, Undifferentiated HESCs or cells pretreated with 8-br-cAMP and MPA (cAMP/MPA) for 72 h were exposed to 250 µM H2O2 for 24 h, after which cells were lysed and total lysates probed for the expression of FOXO1, FOXO3a, MnSOD, Cu/ZnSOD, catalase, and cleaved PARP. {alpha}-Tubulin expression served as loading control. B, Parallel cultures were harvested for RTQ-PCR analysis of FOXO1 and FOXO3a mRNA expression. C, HESCs transfected with FOXO1 siRNAs or nontargeting (control) siRNAs were treated for 3 d with 8-br-cAMP and MPA (cAMP/MPA), and the expression of FOXO1 and FOXO3a was determined by RTQ-PCR.

 
The distinct and opposing pattern of FOXO expression prompted us to examine whether FOXO1 is involved in the repression of FOXO3a upon decidualization. To test this, HESCs were transfected with nontargeting siRNA or FOXO1 siRNA. Subsequently, cells were decidualized for 72 h and harvested for mRNA analysis. As shown in Fig. 5CGo, FOXO3a transcript levels were slightly higher in differentiated HESCs in which FOXO1 expression was silenced but lower when compared with undifferentiated cells.

FOXO3a Silencing in Undifferentiated HESCs Confers Resistant to Oxidative Cell Death
The data suggested that resistance to oxidative cell death in decidualizing cells could be accounted for by the repression of FOXO3a expression. We took two approaches to test this hypothesis. First, we transfected decidualized HESCs with an expression vector that encodes for a mutant FOXO3a protein in which the conserved Akt phosphorylation acceptor sites are changed to alanine (29, 38). Not surprisingly, overexpression of constitutively active FOXO3a elicited activation of the proapoptotic machinery as shown by the appearance of cleaved PARP (Fig. 6AGo). Second, we tested whether FOXO3a silencing would render undifferentiated cells resistant to oxidative cell death. Confluent undifferentiated cultures were either mock transfected or transfected with siRNA targeting FOXO3a or control siRNA. Seventy-two hours later, some cultures were stressed with 250 µM H2O2 for 24 h. Western blot analysis demonstrated that the FOXO3a silencing was very effective and completely abrogated the induction of this transcription factor upon H2O2 treatment (Fig. 6BGo). Furthermore, cellular stress induced the expression of cleaved PARP in control cells but not in cell transfected with FOXO3a siRNA. Flow cytometry analysis further confirmed these findings and demonstrated a total absence of an apoptotic response in undifferentiated cells in which FOXO3a expression was inhibited (Fig. 6CGo).


Figure 6
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  		Fig. 6. HESC Apoptosis in Response to Oxidative Stress Is Mediated by FOXO Transcription Factors

A, Primary HESC cultures pretreated with 8-br-cAMP and MPA (cAMP/MPA) for 72 h were transfected with a control plasmid or an expression vector encoding for a constitutively active FOXO3a mutant (mFOXO3a). Some cultures were subsequently exposed to 250 µM H2O2 as indicated. After 24 h, cells were lysed and the nuclear and cytoplasmic cell fractions probed for FOXO1 and cleaved PARP1 expression, respectively. B, Undifferentiated mock transfected HESCs or cells transfected with FOXO3a siRNAs or nontargeting (control) siRNAs were treated 72 h later with 250 µM H2O2 for 24 h. Whole-cell lysates were harvested and probed for FOXO3a and cleaved PARP expression with {alpha}-tubulin serving as a loading control. C, Parallel cultures were analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle (<2 N, G0/G1, S, and G2/M) is indicated.

 
Expression of Regulators of Apoptosis in Undifferentiated and Decidualizing HESCs
Next, we examined the expression of potential downstream FOXO3a targets in undifferentiated and decidualizing HESCs treated with 250 µM H2O2 for 3, 15, and 30 h. As shown in Fig. 7Go, the induction of FOXO3a in undifferentiated cells was already apparent upon 15-h exposure to H2O2. Notably, the appearance of active cleaved caspase 3, an apoptotic marker, preceded the induction of FOXO3a expression, which could reflect rapid activation of existing FOXO3a, as suggested by the discrete change in protein mobility. SIRT1 levels did not change in response to H2O2 in either undifferentiated or decidualized cells. Although cleaved caspase 3 was strongly induced in undifferentiated HESCs exposed to H2O2, the levels declined upon prolonged exposure to oxidative stress (30 h), indicating possible necrosis. Surprisingly, the expression of proapoptotic factors such as FasL, Bim, and Bax was considerable higher in decidualizing cells, irrespectively of H2O2. The short isoform of cellular Fas-associated death domain-like IL-1ß-converting enzyme-inhibitory protein (c-FLIPS), a potent inhibitor of death-receptor induced apoptosis (39), was also strongly induced in HESCs treated with 8-br-cAMP and MPA for 72 h, although there was little or no change in the expression of other antiapoptotic factors such as Bcl-2 and Bcl-xL. Notably, only Bim expression correlated with the induction of FOXO3a in undifferentiated HESCs exposed to oxidative stress.


Figure 7
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  		Fig. 7. Expression of Putative FOXO Target Genes in Undifferentiated and Decidualized HESCs Exposed to Oxidative Stress

Undifferentiated HESCs or cells pretreated with 8-br-cAMP and MPA (cAMP/MPA) for 72 h were exposed to 250 µM H2O2 for 3, 15, or 30 h. The cultures were harvested and total lysates probed for the expression of cleaved caspase 3 and various proapoptotic and antiapoptotic factors as indicated. ß-Actin expression served as loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated for the first time that HESC become refractory to cell death induced by oxidative stress upon decidualization. There are multiple components to the mechanism underlying this resistance. First, the induction of MnSOD upon HESC differentiation suggests that the decidua has heightened ROS scavenging capacity (37). In agreement with previous observations (40), differentiation of HESCs with 8-br-cAMP and MPA did not increase Cu/ZnSOD or catalase expression. The increase in MnSOD was abrogated upon silencing of FOXO1 expression in decidualizing cells, confirming the importance of this family of transcription factors as sensors of oxidative stress and their role in regulating cellular antioxidant defenses (41). Second, we demonstrate that GADD45{alpha} is induced upon HESC differentiation in vivo as well as in culture, although we found no evidence that its expression is under the control of FOXO1. GADD45 proteins are multifaceted factors implicated in the regulation of diverse stress responses including cell cycle arrest at G2/M, chromatin remodeling, and nucleotide excision repair. GADD45 proteins can also trigger apoptosis, which has led to the suggestion that they serve as gatekeepers capable of eliminating cells unable to repair DNA damage caused by cellular stress (42, 43, 44). The GADD45 family consists of three structurally related DNA damage- and stress-inducible proteins, GADD45{alpha}, -ß, and -{gamma}. GADD45{alpha} is the only member of this family that is regulated by the tumor suppressor p53 (45). Like FOXO1, p53 protein levels increase upon treatment of primary HESC cultures with 8-br-cAMP (46). Furthermore, the expression of this tumor suppressor in normal endometrium is confined to the secretory phase of the cycle, suggesting that it may be involved in regulating GADD45{alpha} expression. Finally, and most importantly, we demonstrate that H2O2 induces FOXO3a expression in undifferentiated cells but not in decidualizing cultures. In fact, basal FOXO3a levels were approximately 40–60% lower in HESCs treated with 8-br-cAMP plus MPA when compared with untreated cells, suggesting that its expression is actively repressed upon decidualization. Silencing of FOXO1 in differentiated cells did increase but not restore FOXO3a expression and was insufficient to sensitize the cells to oxidative cell death. In contrast, overexpression of a constitutive active FOXO3a mutant in decidualizing cells caused apoptosis, and silencing of endogenous FOXO3a conferred resistance to oxidative apoptosis in undifferentiated cells. Together, the data demonstrate that different FOXO family members have distinct and nonredundant roles human endometrium: induction and activation of FOXO3a upon oxidative stress elicits apoptosis, whereas FOXO1 activates gene sets involved in differentiation and stress resistance.

The role of FOXO transcription factors in cell fate decisions appear to be highly dependent upon hormonal and environmental cues. For instance, although FOXO1 promotes differentiation of HESCs, characterized by expression of PRL and IGFBP-1, it also enhances Bim expression and induces apoptosis upon progesterone withdrawal from decidualized cultures (31). This strongly suggests that the ability of FOXO transcription factors to transactivate different gene sets in HESCs is tightly controlled by additional regulatory mechanisms. Recent studies have shown that deacetylation of FOXO transcription factors by SIRT1 promotes cell cycle arrest and quiescence over programmed cell death (21, 22, 27). Although the levels of SIRT1 increase in primary HESC cultures upon prolonged stimulation with 8-br-cAMP and MPA, we found no evidence that its expression in vivo changes during the cycle. Whether SIRT1 regulates the activity of FOXO1 or FOXO3a in endometrial cells through binding and deacetylation of these transcription factors is currently under investigation. Physical interaction with other decidua-specific transcription factors, such as C/EBPß, HOXA10, and the liganded PR, could constitute another mechanism that promotes preferential transcriptional activation of differentiation and antioxidative defense genes by FOXO1 (29, 30, 47). FOXO3a has also been implicated in seemingly opposing cell fate decisions. For instance, there is compelling evidence to suggest that FOXO3a promotes cell survival in response to a low level of oxidative stress (21, 41, 48), yet it is also a key mediator of cell death in response to a variety of proapoptotic signals (16, 18, 49, 50).

The mechanism whereby FOXO proteins promote apoptosis in different cell types appears also surprisingly diverse and includes induction of death-receptor ligands (e.g. FasL) and proapoptotic factors (e.g. Bim) as well as repression of cytoplasmic (e.g. c-FLIP) and mitochondrial antiapoptotic proteins (e.g. Bcl-2 and Bcl-xL) (16, 18, 49, 50). We examined the expression of these factors in H2O2-treated HESCs in a time course experiment and found that only Bim is regulated in a manner that paralleled the induction of FOXO3a. However, the levels of Bim are much higher in decidualizing cells when compared with H2O2-treated undifferentiated HESCs. Furthermore, the induction of cleaved caspase 3 preceded the increase in Bim expression in undifferentiated cells, suggesting that other mechanisms are involved in FOXO3a-mediated cell death. Using a gene microarray approach, Alikhani et al. (51) recently identified 26 FOXO1-regulated genes involved in apoptosis of TNF-{alpha}-treated dermal fibroblasts. A similar approach will be useful to identify the array of genes regulated by FOXO3a in HESCs exposed to oxidative stress. Somewhat fortuitously, we also observed that decidualizing cells highly express FasL as well as c-FLIP. Local expression of FasL in the decidua during pregnancy is thought to confer maternal immunotolerance to fetal alloantigens by eliminating activated T cells (52). However, HESCs also express Fas, also termed CD95 or APO-1, the cognate cell surface receptor for FasL and member of the TNF/nerve growth factor receptor family. Fas-induced cell death is tightly controlled by various cytoplasmic regulators, among which is c-FLIP, a procaspase-8 like protease-deficient protein (53). The various c-FLIP mRNA splice variants encode either for a long (c-FLIPL) or two short forms (c-FLIPS and the recently identified c-FLIPR) (54). Although proapoptotic as well as antiapoptotic functions have been ascribed to c-FLIPL, the short isoforms are potent inhibitors of caspase-8 activation and death receptor-mediated apoptosis (53). It is tempting to speculate that the high expression of c-FLIP in decidualizing cells serves to prevent autoactivation of the death receptor signaling pathway.

Excessive oxidative stress and increased cell death are thought to represent a common pathological pathway in a spectrum of pregnancy disorders, from recurrent first trimester pregnancy loss to preeclampsia and fetal growth restriction (4, 55, 56, 57). Consequently, there is considerable interest in using antioxidant supplements during pregnancy for the prevention of obstetrical disorders associated with impaired placental perfusion. This approach is supported by the findings of a recent randomized-controlled clinical trial demonstrating a significant reduction in the incidence of preeclampsia in at-risk women taking vitamin C and E supplements in pregnancy (58). Our data indicate that impaired decidualization will render the feto-maternal interface vulnerable to tissue destruction in response to oxidative stress. Conversely, treatments aimed capable of supporting the decidual process, such as human chorionic gonadotrophin, phosphodiesterase inhibitors, or progesterone (34, 59), could act complementary to antioxidant supplements in women with a poor obstetrical history.

In summary, we have shown that human endometrium becomes resistant to oxidative apoptosis upon differentiation. This resistance is not accounted for by an increase in defense or repair capacity. Instead, we demonstrate that repression of FOXO3a expression disables the activation of the apoptotic machinery in decidualizing cells upon prolonged oxidative stress. Moreover, our results also highlight, for the first time, the distinct roles of different FOXO family members in human endometrial function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tissue Collection and Primary Culture
The Local Research and Ethics Committee approved this study and patient consent was obtained before tissue collection. Endometrial samples from cycling women without uterine pathology were divided for histological dating, using standard criteria, and for storage in RNAlater (Ambion, Austin, TX). HESCs were isolated from fresh biopsies and primary cultures established, maintained, and decidualized with 0.5 mM 8-br-cAMP (Sigma, St. Louis, MO) and 10–6 M MPA (Sigma) as described previously (33, 60). All experiments were carried out before the third cell passage.

Real-Time Quantitative (RTQ)-PCR
Total RNA extracted from untreated or decidualizing HESC cultures and from tissue samples stored in RNAlater (Ambion), was reversed transcribed, and the resulting cDNA amplified using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) (33) and the following gene-specific primer pairs: L19-sense (5'-GCG GAA GGG TAC AGC CAA T-3') and L19-antisense (5'-GCA GCC GGC GCA AA-3'); FOXO1-sense (5'-TGG ACA TGC TCA GCA GAC ATC-3') and FOXO1-antisense (5'-TTG GGT CAG GCG GTT CA-3'); FOXO3a-sense (5'-CCC AGC CTA ACC AGG GAA GT-3') and FOXO3a-antisense (5'-AGC GCC CTG GGT TTG G-3'); MnSOD-sense (5'-AAT TGC TGC TTG TCC AAA TCA G-3') and MnSOD-antisense (5'-TCC CCA GCA GTG GAA TAA GG-3'); Cu/ZnSOD-sense (5'-AAA GGA TGA AGA GAG GCA TGT TG-3') and Cu/ZnSOD-antisense (5'-TCG GCC ACA CCA TCT TTGT-3'); catalase-sense (5'-TGC TGG AGA ATC GGG TTC A-3') and catalase-antisense (5'-ATT TCA CTG CAA ACC CAC GAG-3'); SIRT1-sense (5'-TAC GAC GAA GAC GAC GAC GA-3') and SIRT1-antisense (5'-CGC CGC CGC CGC CTC TTC C-3'); GADD45{alpha}-sense (5'-TCT CGG CTG GAG AGC A-3') and GADD45{alpha}-antisense (5'-GGC TTT GCT GAG CAC T-3'). Specificity of each primer was determined using NCBI BLAST module. L19, a nonregulated ribosomal housekeeping gene, served as an internal control and was used to normalize for differences in input RNA. All measurements were performed in triplicate.

Western Blot Analysis
Whole-cell extracts or nuclear and cytoplasmic protein fractions were immunoblotted as described (32, 33). Primary antibodies used were as follows: rabbit polyclonal anti-FOXO1 (Cell Signaling Technology, Beverly, MA); rabbit polyclonal anti-FOXO3a (Upstate Biotechnology, Lake Placid, NY); goat polyclonal anti-MnSOD (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-Bax (Santa Cruz Biotechnology); mouse monoclonal anti-Bcl-2 (Santa Cruz Biotechnology); rabbit polyclonal anti Bcl-xL (Santa Cruz Biotechnology); rat monoclonal anti-Bim (Calbiochem, San Diego, CA), rabbit polyclonal anti-Cu/ZnSOD (Santa Cruz Biotechnology); mouse monoclonal anticatalase (Sigma); rabbit polyclonal anti-Fas (Santa Cruz Biotechnology); rabbit polyclonal anti-FasL (Santa Cruz Biotechnology); rabbit polyclonal anti-FLIP (ProSci, Poway, CA); mouse monoclonal anti-{alpha}-tubulin (Santa Cruz Biotechnology); rabbit polyclonal anti-PARP cleavage site (214/215) (BioSource International, Camarillo, CA); and rabbit polyclonal anti-GADD45{alpha} (Santa Cruz Biotechnology); and mouse monoclonal anti-SIRT1 (Upstate Biotechnology). The primary antibodies were used at 1:1000 except for the antibodies to ß-actin (diluted 1:100,000, Abcam), Bax (diluted 1:500), Bcl-xL (diluted 1:500), and FLIP (diluted 1:250).

Immunohistochemistry
Paraffin-embedded, formalin-fixed endometrial specimens were examined for in vivo MnSOD, Cu/ZnSOD, catalase, and GADD45{alpha} immunoreactivity. All specimens were obtained for cycling premenopausal women and were free of intrauterine disease such as endometrial hyperplasia or polyps. Using standard criteria, endometria were allocated to proliferative phase (n = 7) and mid- to late-secretory phase (n = 8). Five-micrometer sections, placed on 1% wt/vol poly-lysine slides, were deparaffinized, dehydrated, exposed to 0.3% vol/vol H2O2 for 15 min, and, if indicated, microwaved in 0.01 M citrate buffer (pH 6.0). Immunostaining was performed using the Universal LSAB Plus Kits (DakoCytomation, Carpinteria, CA). The sections were incubated for 1 h with the dilutions specified; goat polyclonal anti-MnSOD (Santa Cruz Biotechnology) diluted 1:100; rabbit polyclonal anti-Cu/ZnSOD (Santa Cruz Biotechnology) diluted 1:200; mouse monoclonal anticatalase (Sigma) diluted 1:200; mouse monoclonal anti-SIRT1 (Upstate Biotechnology) diluted 1:200; or rabbit polyclonal anti-GADD45{alpha} (Santa Cruz Biotechnology) diluted 1:100. For negative controls, sections were incubated with 1% BSA instead of primary antibody. Immunoreactivity was not observed in the absence of primary antibody.

Transient Transfection, siRNA, and Flow Cytometry
Decidualizing HESCs cultured in six-well plates were transiently transfected, using calcium phosphate precipitation as described (32, 33), with an expression vector (1 µg per well) encoding for a constitutively active FOXO3a mutant. Cells were harvested after 24 h later for Western blot analysis or flow cytometry. Transfections studies were repeated at least three times. For gene silencing, undifferentiated or decidualized HESCs were transiently transfected with 50 nM of the following siRNA reagents purchased from Dharmacon (Lafayette, CO): FOXO1 siGENOME SMARTpool, FOXO3a siGENOME SMARTpool, or siCONTROL Non-Targeting siRNA Pool. Flow cytometry analysis was used to quantify apoptosis in primary cultures by evaluating the sub-G1 fraction (<2 N) after propidium iodide staining of ethanol-fixed cells.

PRL and IGFBP-1 Measurements
PRL levels in supernatant were measured by microparticle enzyme immunoassay (AxSYM System; Abbott Laboratories, Abbott Park, IL) and IGFBP-1 levels by ELISA (Diagnostic Systems Laboratories, Webster, TX). Measurements were performed in triplicate and normalized to the total protein content of cultures.


   FOOTNOTES
 
This work was supported by the Great Britain Sasakawa Foundation (to J.J.B.) and the Daiwa Foundation (to T.K.).

The authors have nothing to disclose.

First Published Online May 18, 2006

Abbreviations: 8-br-cAMP, 8-Bromoadenosine-cAMP; c-FLIP, cellular Fas-associated death domain-like IL-1ß-converting enzyme-inhibitory protein; Cu/ZnSOD, copper/zinc superoxide dismutase; FasL, Fas ligand; GADD45{alpha}, growth arrest- and DNA damage-inducible protein {alpha} of 45 kDa; HESC, human endometrial stromal cell; IGFBP-1, IGF binding protein 1; MnSOD, manganese superoxide dismutase; MPA, medroxyprogesterone acetate; PARP, poly(ADP-ribose) polymerase-1; PRL, prolactin; ROS, reactive oxygen species; RTQ-PCR, real-time quantitative-PCR; siRNA, small interfering RNA.

Received for publication March 9, 2006. Accepted for publication May 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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