Expression and Localisation of FoxO3 and FoxO4 in Human Placenta and Fetal Membranes

Placenta 31 (2010) 1043e1050

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Expression and Localisation of FoxO3 and FoxO4 in Human Placenta and Fetal Membranes M. Lappas a, b, *, R. Lim a, b, C. Riley a, R. Menon c, M. Permezel a, b a

Department of Obstetrics and Gynaecology, University of Melbourne, Victoria, Australia Mercy Perinatal Research Centre, Mercy Hospital for Women, Heidelberg, Victoria, Australia c Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 15 September 2010

Forkhead box O (FoxO) proteins regulate inflammation, extracellular matrix (ECM) remodelling and apoptosis. We have previously identified FoxO1 proteins in human gestational tissues, and demonstrated a link between FoxO1 and rupture of fetal membranes. There is, however, no data available on the expression and localisation of FoxO3 and FoxO4 in human intrauterine tissues. Thus the aim of this study was to characterise the localisation and expression of FoxO3 and FoxO4 in (i) human placenta and fetal membranes before term spontaneous labour onset, and (ii) supracervical site (SCS) and distal site (DS) fetal membranes from non-labouring women. Immunohistochemistry, Western blotting and quantitative RT-PCR (qRT-PCR) was used to localise and quantitate FoxO3 and FoxO4 protein and mRNA expressions. Cytoplasmic and nuclear FoxO3 was localised in the syncytiotrophoblast layer, chorionic trophoblasts, amnion epithelium and decidua. Cytoplasmic FoxO4 was localised in the syncytiotrophoblasts and chorionic trophoblasts. No or very little FoxO4 protein and mRNA was present in amnion epithelium. The intensity and extent of staining of FoxO3 and FoxO4 was greater in fetal membranes obtained from the SCS compared to DS. Presence of FoxO3 and FoxO4 are expected to contribute to apoptosis and/or cell cycle regulation associated with fetal membrane rupture. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Placenta Fetal membranes FoxO3 FoxO4 Fetal membrane rupture

1. Introduction In humans, the FoxO subfamily of Forkhead transcription factors consists of FoxO1a (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX) and FoxO6. FoxO1, FoxO3, and FoxO4 are three functionally related members, which are orthologs of the Caenorhabditis elegans transcription factor DAF-16 [1]. These transcription factors are important physiological targets of phosphatidylinositol-3 kinase (PI3K)/ protein kinase B (PKB) signalling. Activation of PI3K/PKB phosphorylates FoxO4 at three different Ser/Thr residues [2e5], which results in nuclear export and cytoplasmic sequestration effectively inhibiting its transcriptional function. Nuclear localisation of FoxO proteins suspends cell cycle progression [6], promotes apoptosis [7], and negatively regulates angiogenesis [8]. FoxO3 has been causally linked to multiple cellular processes, which are activated during human parturition [9e11]. FoxO3

* Corresponding author. Department of Obstetrics and Gynaecology, University of Melbourne, Mercy Hospital for Women, Level 4/163 Studley Road, Heidelberg 3084, Victoria, Australia. Tel.: þ61 3 8458 4370; fax: þ61 3 8458 4380. E-mail address: [email protected] (M. Lappas). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.09.009

induces matrix metalloproteinase (MMP)- 3, 9 and 13 expression and activity in cancer cells [12] and decreases tissue inhibitor of MMP (TIMP)-1 in human umbilical vein endothelial cells [13]. It has also been shown to increase resistance to oxidative stress by upregulation of antioxidants including mitochondrial superoxide dismutase (MnSOD) [14], peroxisomal catalase [15], and peroxiredoxins [16]. Although there is no data available on FoxO4 in human parturition, in non-gestational tissues, FoxO4 is regulated by and regulates pro-labour mediators. Pro-inflammatory tumour necrosis factor (TNF)a has been shown to activate the c-Jun N-terminal kinases (JNK) stress signalling pathway and promote nuclear import and activation of FoxO4 [17e19] resulting in enhancement of MMP-9 gene transcription and enzymatic activity [18]. In podocytes, exposure to advanced glycation endproducts (AGEs) is associated with FoxO4 transcriptional activation, leading to increased expression of effector proteins of apoptosis [20]. Inflammatory cytokines and the transcriptional activity of nuclear factor kappa B (NF-kB) are up-regulated in FoxO4 knockout mice [21]. We have previously profiled the expression of FoxO1 proteins in human gestational tissues [22], and shown increased expression of acetylated (ac)-FoxO1 in fetal membranes overlying the cervix [23],

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which may be involved in the increased apoptosis and extracellular matrix (ECM) degradation associated with this site [11,24e28]. There is however, no data available on the expression of FoxO3 and FoxO4 in human placenta and fetal membranes. Thus, the aims of this study are to (i) characterise the localisation, gene and protein expression of FoxO3 and FoxO4 in human placenta, amnion and choriodecidua from normal term pregnancies; and (ii) elucidate the effect of supracervical (SC) apposition on FoxO3 and FoxO4 expression in human fetal membranes in the absence of labour. 2. Materials and methods 2.1. Sample collection The Research Ethics Committee of Mercy Health and Aged Care approved this study. Written, informed consent was obtained from participating women. Human placenta and attached fetal membranes were obtained (with Institutional Research and Ethics Committee approval) from women who delivered healthy, singleton infants at term (37 weeks gestation) before labour undergoing elective Caesarean section (indications for Caesarean section were breech presentation and/or previous Caesarean section). A placental lobule (cotyledon) was removed from the central region of the placenta. The basal plate and chorionic surface were removed from the cotyledon, and villous tissue was obtained from the middle cross-section. Placental tissue was blunt dissected to remove visible connective tissue and calcium deposits. In samples from non-labouring women, amnion and choriodecidua were obtained from the SC site (SCS) and a distal site (DS). Identification of the SCS was performed as we have previously detailed [23,27,29]. Briefly, prior to commencement of the Caesarean section, a swab soaked in Bonney’s Blue dye (1:1 mixture of brilliant green (0.5%) and crystal violet (0.5%) dissolved in 90% ethanol) is introduced through the cervix onto the chorion lying above the internal os of the cervix. Upon delivery of the placenta, a blue mark is obvious on the membrane where the dye had been applied. The fetal membranes were washed briefly in PBS and three membrane samples were taken from the identified SCS (Bonney’s Blue staining) and three membrane samples from a distal site (DS, no Bonney’s Blue staining). The placenta and fetal membranes were collected within 30 min of application of Bonney’s Blue and transferred within 10 min of delivery to the laboratory within the hospital. Those patients with a low lying placenta were excluded from the study. Tissue samples were fixed and paraffin embedded for immunohistochemical analysis, or snap frozen in liquid nitrogen and immediately stored at 80  C for RNA and protein analysis. 2.2. RNA extraction and quantitative RT-PCR (qRT-PCR) Total RNA was extracted from approximately 100 mg of tissue using Tri Reagent according to manufacturer’s instructions (SigmaeAldrich, Saint LouisMissouri). RNA concentrations were quantified using a spectrophotometer (Smart Spec, Bio-Rad). RNA quality and integrity was determined via the A260/A280 ratio and agarose gels electrophoresis. One mg of RNA was converted to cDNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. The cDNA was diluted ten-fold, and 2 ml of cDNA was used to perform RT-PCR using Sensimix Plus SYBR green (Quantace, Alexandria, NSW, Australia) and 100 nM of QuantiTect Primer Assays (Qiagen, Germantown, Maryland, USA). The FOXO3 (catalogue number QT00031941) gene ID is 2309 and FOXO4 (catalogue number QT00029141) gene ID is 4303. Average gene CT values were normalised to the average actin CT values of the same cDNA sample. There was equal efficiency of PCR amplification of target (FoxO3 and FoxO4) and reference (actin) mRNA (average between 98 and 103%). There was no significant difference in the mRNA expression of actin between any of the sample groups. The specificity of the product was assessed from melting curve analysis. RNA without reverse transcriptase during cDNA synthesis as well as PCR reactions using water instead of template showed no amplification. A positive control sample was also used in each run on each plate. Fold differences were determined using the 2DDCt method [30]. 2.3. Immunohistochemistry Tissues were placed in embedding cassettes (Techno-Plas, SA, Australia) fixed in buffered formaldehyde solution (4%) for 48 h and embedded in paraffin. Serial sections (4 mm thick), were cut and mounted on sections onto superfrost plus slides. Four slides were prepared consecutively for each sample. Each site was immunolabelled with each of the antibodies and 2 were used as a negative control slide. Sections were deparaffinised followed by an antigen retrieval step (boiled in 10 mM Tris and 1 mM EDTA, pH 9.0 for 10 min followed by 20 min incubation). Endogenous peroxidase activity was removed using 3% H2O2 in methanol for 10 min. Sections were transferred to TBS, and incubated in a humidity chamber for 1 h in antibody diluted in 1% BSA in TBS. Rabbit polyclonal anti-FoxO3 (H-144): sc-11351 and goat polyclonal anti-FoxO4 (N-19): sc-5221, purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used at 2 mg/ml FoxO3 recognises the product of the

human FOXO3 (FKHRL1) gene (ID: 2309, Chromosome 6q21), whereas FoxO4 recognises the product of the human FOXO4 gene (ID: 4303, Chromosome Xq13.1). After incubation the binding sites were labelled with Dako Envision þ polymer linked secondary reagent and visualized using Dako DABþ (Dakocytomation). Nuclei were counterstained with Mayer’s haematoxylin and the sections were dehydrated and cover slipped using a resinous mounting agent. Positive controls, which were composite slides with tonsil, breast tumour and ovarian tumour, were included in each run. Negative control slides, where primary antibody was replaced with normal rabbit or goat IgG serum were also included. Sections were assessed microscopically for both intensity and extent of staining. The evaluation of all immunohistochemical staining was done as a blind assessment and independently scored by an experienced pathologist (CR). The entire tissue section was scored and the extent of staining was determined on a scale of 0e5 according to the estimated percentage of cells stained: 0 < 10%; 1 ¼ 11e25%; 2 ¼ 26e50%; 3 ¼ 51e75%; 4 ¼ 76e90%; 5 > 90%. Staining intensity was assessed on a scale of 0e3: 0 ¼ no staining, negative; 1 ¼ pale brown, weak; 2 ¼ brown, moderate; 3 ¼ dark brown, strong [31]. The average of the extent and intensity score was taken for each case and subsequently used in the final analysis. 2.4. Western blotting Cytosplamic and nuclear protein extracts were preformed as we have previously described [32]. Assessment of FoxO3 and FoxO4 cytoplasmic and nuclear protein expression was analysed by Western blotting. Rabbit polyclonal anti-FoxO3 and goat polyclonal anti-FoxO4 (N-19) (as detailed above) were used at 1 mg/ml for 24 h. Forty micrograms of protein was separated on polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PVDF. Protein expression was identified by comparison with the mobility of protein standard. Membranes were viewed and analysed using the Chemi-Doc system (Bio-Rad). Quantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad). Data were corrected for background, and expressed as optical density (OD/mm2). 2.5. Statistical analysis Statistical analyses were performed using a commercially available statistical software package (Statgraphics Plus version 3.1, Statistical Graphics Corp., Rockville, Maryland, USA). Two sample comparisons were analysed by paired sample comparison, Student’s t-test or ManneWhitney (Wilcoxon) test. For all other comparisons, analysis was performed using a one-way ANOVA (using Tukey HSD correction to discriminate among the means); homogeneity of data was assessed by Bartlett’s test, and when significant, data were logarithmically transformed before further analysis. Statistical significance was ascribed to P value <0.05. Data was expressed as mean  standard error of the mean (SEM).

3. Results 3.1. Expression and localisation of FoxO3 in human gestational tissues 3.1.1. FoxO3 mRNA expression in term placenta and fetal membranes FoxO3 mRNA expression in human gestational tissues at term was analysed using qRT-PCR. Actin mRNA was used for normalisation of the data. FoxO3 mRNA was detected in all placenta, amnion and choriodecidua samples, with the data presented in Fig. 1A. FoxO3 mRNA expression was significantly higher in placenta and amnion compared to choriodecidua (p ¼ 0.004). The gene expression of FoxO3 was similar between placenta and amnion. 3.1.2. Immunohistochemical localisation of FoxO3 expression in term placenta and fetal membranes Placenta exhibited FoxO3 staining only in the syncytiotrophoblast layer, that was both cytoplasmic and nuclear (Fig. 2A). FoxO3 was not present within the cytotrophoblasts, villous structure or endothelial cells. Amnion and chorion exhibited, both cytoplasmic and nuclear FoxO3 (Fig. 2B). FoxO3 was present in the amnion epithelium, chorion trophoblast and fibroblast cells of the spongy layer. Decidua, also exhibited cytoplasmic FoxO3. No staining was present in the negative controls for FoxO3 in placenta (Fig. 2C) and fetal membranes (Fig. 2D).

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Fig. 1. (A) FoxO3 and (B) FoxO4 mRNA expression in term human placenta, amnion and choriodecidua. qRT-PCR was performed on mRNA extracted from term placenta, amnion and choriodecidua from women not-in-labour (n ¼ 6). Actin mRNA expression was used for the normalisation of the data. FoxO mRNA expression is displayed as mean fold change ratio (relative to placenta) as calculated by the 2DDCT method. Data is displayed as the mean  SEM. *P < 0.05 vs. placenta; xP < 0.05 vs. choriodecidua.

3.1.3. FoxO3 protein expression in term placenta and fetal membranes To further evaluate the expression of FoxO3 in human gestational tissues, placenta, and fetal membrane cytoplasmic and nuclear extracts were prepared and Western blot analysis performed. FoxO3 protein expression was detected in both cytoplasmic and nuclear preparations of all tissue extracts with a band detected at approximately 97 kD (Fig. 3). Cytoplasmic FoxO3 protein expression was highest in placenta, followed by choriodecidua and then amnion (p ¼ 0.03 for amnion vs. placenta, and p ¼ 0.02 for placenta vs. choriodecidua). On the other hand, nuclear FoxO3 protein expression was not statistically different between placenta, amnion and choriodecidua.

3.1.4. Effect of supracervical apposition on FoxO3 expression in fetal membranes in the absence of labour Comparisons between the SCS and a DS of fetal membranes were performed in order to better understand the mechanisms that may be involved in rupture of fetal membranes. We utilised the technique we have established for the identification of SCS in fetal membranes obtained from non-labouring women [23,27,29]. Immunohistochemistry was used to examine the intensity and extent of FoxO3 protein expression fetal membranes from the SCS and a DS. A summary of the intensity and extent of cytoplasmic FoxO3 staining is presented in Table 1. When compared to the DS, the extent of cytoplasmic FoxO3 staining was significantly higher in amnion and chorion obtained from the SCS (Fig. 4).

Fig. 2. Immunohistochemical localisation of FoxO3 in human placenta and fetal membranes. (A) Cytoplasmic and nuclear FoxO3 staining was contained only in the syncytiotrophoblast layer. No staining was detected in the cytotrophoblasts or endothelial cells. Original magnification 250, magnification at insert 100. (B) Cytoplasmic and nuclear FoxO3 is localised primarily to the trophoblasts of the chorion layer, amnion epithelium and decidua. Original magnification 250, magnification at insert 100. (C) No specific staining for FoxO3 is seen in the negative control for placenta. Magnification 100. (D) No specific staining for FoxO3 is seen in the negative control for fetal membranes. Magnification 100. syncytiotrophoblast cells (sy), chorion trophoblast cells (ct), amnion epithelial cells (ae), connective tissue layer (cl), deciduas (dec).

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Fig. 3. Western blot of cytoplasmic and nuclear protein expression of FoxO3 in term human placenta (P), amnion (A) and choriodecidua (C) collected from non-obese women at term Caesarean section (n ¼ 5 samples per tissue). Each bar represents the peak optical density of FoxO3 relative to b-actin expression. *P < 0.05 vs. cytoplasmic placenta FoxO3 expression; xP < 0.05 vs. cytoplasmic amnion FoxO3 expression.

3.2. Expression and localisation of FoxO4 in human gestational tissues 3.2.1. FoxO4 mRNA expression in term placenta and fetal membranes FoxO4 mRNA was detected in all samples of placenta and choriodecidua. On the other hand, there was minimal FoxO4 mRNA in amnion, with some samples having no detectable FoxO4 (Fig. 1B). FoxO4 mRNA expression was significantly higher in placenta compared to amnion and choriodecidua (p ¼ 0.001 and p ¼ 0.002, respectively). Additionally, FoxO4 gene expression was significantly higher in choriodecidua compared to amnion (p < 0.001). 3.2.2. Immunohistochemical localisation of FoxO4 expression in term placenta and fetal membranes The cellular localisation of FoxO4 protein in human placenta and fetal membranes (combined amnion and choriodecidua) was performed by immunohistochemistry. FoxO4 staining was manly Table 1 Extent and intensity of staining of FoxO3 and FoxO4 in SCS and DS fetal membranes obtained from non-labouring women. Antigen

FoxO3

FoxO4

Tissue

Amnion Chorion Decidua Amnion Basal Chorion Superficial Chorion Decidua

Intensity of staining

Extent of staining

SCS

SCS

1.0 1.8 e 0.0 2.3 1.2 e

DS  0.0  0.2  0.0  0.2  0.2

1.0 1.3 1.2 0.0 2.0 1.2 0.8

      

0.0 0.2 0.2 0.0 0.0 0.2 0.5

4.0 4.3 e 0.0 4.5 1.8 e

DS  0.4  0.3  0.0  0.2  0.4

2.8 3.5 2.8 0.0 3.3 0.7 0.5

      

0.3* 0.4* 0.2 0.0 0.2* 0.4* 0.2

Extent and intensity of staining was scored as described in Materials and methods. All data are expressed mean  SEM (n ¼ 6 per group). *P < 0.05 vs. SCS (paired sample comparison).

cytoplasmic, with very little or no nuclear FoxO4 staining. Cytoplasmic FoxO4 in the placenta was moderate and was confined to the syncytiotrophoblast layer (Fig. 5A), with no FoxO4 within the villous structure and endothelium. There was no FoxO4 in the amnion epithelium and in the cells within the spongy layer (Fig. 5B). On the other hand, cytoplasmic FoxO4 was detected in the trophoblast layer of the chorion; more so in the basal layer than the superficial layer (Fig. 5B). Decidua, where present, exhibited fairly weak FoxO4 that was mainly cytoplasmic (Fig. 5B). As expected, the negative controls for placenta (Fig. 5C) and fetal membranes (Fig. 5D) exhibited no staining for FoxO4. 3.2.3. FoxO4 protein expression in term placenta and fetal membranes The cytoplasmic expression of FoxO4 for placenta, amnion and choriodecidua is shown in Fig. 6, where a band of approximately 60 kD was detected. There was no detectable band for FoxO4 in the cytoplasmic fractions of amnion. There was no difference in the expression of FoxO4 in cytoplasmic protein fractions of placenta and choriodecidua. In all three tissues, there was no detectable FoxO4 in the nucleus (data not shown). 3.2.4. Effect of supracervical apposition on FoxO4 expression in fetal membranes in the absence of labour To determine the effect of SC apposition on the intensity and extent of FoxO4 protein expression in human fetal membranes from non-labouring women, immunohistochemistry was used. A summary of the intensity and extent of staining is presented in Table 1. There was no FoxO4 detected in the amnion epithelium layer. There was no difference in the intensity of FoxO4 staining in SC and distal chorion. However, FoxO4 staining was significantly higher in both basal (p < 0.001) and superficial (p ¼ 0.02) chorion obtained from SCS compared to the DS (Fig. 7).

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Fig. 4. Immunohistochemical expression of FoxO3 in term SCS and DS fetal membranes obtained from non-labouring women (magnification 250). These sections are representative of 1 patient sample. ae, amnion epithelial layer; cl, connective tissue layer; ct, chorionic trophoblast layer; dec, decidua.

4. Discussion This is the first study to report on the expression and localisation of FoxO3 and FoxO4 in human placenta and fetal membranes. FoxO3 mRNA and protein were detected in placenta, amnion and choriodecidua. Immunohistochemistry and Western blotting localised FoxO3 protein in both nuclear and cytoplasmic fractions. On the other hand, FoxO4 protein was limited to cytoplasm in placenta and choriodecidua. FoxO3 and FoxO4 protein expression was higher in fetal membranes obtained in the absence of labour from the SCS compared to those obtained from a DS. Of note, in choriodecidua, FoxO3 protein in nuclear fractions had higher mobility than amnion and placenta, and multiple bands were detected in the cytoplasmic extracts. It is possible these represent multiple modified forms of FoxO3. Indeed, several important covalent

post-translational modifications have been observed for FoxO3, including phosphorylation, ubiquitination, methylation, and acetylation. Additionally, different splice variants may be being detected. It has been reported that there up to at least six suggested splice variants for FoxO3, although the two documented variants encode the same protein [33]. The regulation of FoxO proteins is complex. Post-translational modifications such as phosphorylation, acetylation/deacetylation and ubiquitination regulate the subcellular localisation and transcriptional activity of FoxO proteins [34]. The localisation of FoxO3 and FoxO4 in cytoplasmic and nuclear fractions of placenta, amnion and choriodecidua suggests several potential functions. Nuclear localisation of FoxO proteins suspends cell cycle progression [6], promotes apoptosis [7], and negatively regulate angiogenesis [8]. Upstream kinases phosphorylate FoxO3 and FoxO4 proteins

Fig. 5. Immunohistochemical localisation of FoxO4 in human placenta and fetal membranes. (A) Cytoplasmic FoxO4 staining was contained only in the syncytiotrophoblast layer. No staining was detected in the cytotrophoblasts or endothelial cells. Original magnification 250, magnification at insert 100. (B) FoxO4 is localised primarily to the trophoblasts of the chorion layer, decidual cells. There was no FoxO4 staining in the amnion epithelium. Original magnification 250, magnification at insert 100. (C) No specific staining for FoxO4 is seen in the negative control for placenta. Magnification 100. (D) No specific staining for FoxO4 is seen in the negative control for fetal membranes. Magnification 100. Syncytiotrophoblast cells (sy), cytotrophoblast cells (cy).

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Fig. 6. Western blot of cytoplasmic protein expression of FoxO4 in term human placenta (P), amnion (A) and choriodecidua (C) collected from non-obese women at term Caesarean section (n ¼ 5 samples per tissue). Each bar represents the peak optical density of FoxO4 relative to b-actin expression. *P < 0.05 vs. cytoplasmic amnion FoxO4 expression.

resulting in nuclear export and cytoplasmic sequestration effectively inhibiting its transcriptional function [33]. On the other hand, TNFa has been shown to activate the JNK stress signalling pathway and promote nuclear import and thus activation of FoxO4 [17e19]. Current functional studies are now underway in our laboratory to determine the regulation and role of FoxO proteins in human gestational tissues. We document increased expression of FoxO3 and FoxO4 in SC fetal membranes obtained from non-labouring women. This is in keeping with our previous data demonstrating increased ac-FoxO1 in fetal membranes overlying the cervix [29]. In fetal membranes obtained from non-labouring women, the SCS is a region that undergoes significant remodelling and apoptosis [11]. Previous studies have shown that FoxO proteins regulate both ECM remodelling enzymes and apoptosis. FoxO3 is thought to function as a trigger for apoptosis through upregulation of genes necessary for cell death [3,35e38], and by regulating cell cycle arrest [39e41].

Additionally, FoxO3 downregulates anti-apoptotic proteins [38]. FoxO3 induces MMP-2 and MMP-9 enzymatic activities [13]. Likewise, increased expression of FoxO4 is associated with activation of MMP-9 gene transcription [18]. Conversely, inhibition of FoxO4 expression by siRNA or gene knockout reduces MMP-9 gene expression and enzymatic activity [18]. However, the promoter sequences of MMP-2 or MMP-9 do not contain the consensus binding site for the forkhead transcription factors. Therefore, increased enzymatic activity of MMP-2 or MMP-9 observed after FoxO3 activation may involve indirect regulation through changes in the expression of other transcription factors such as NF-kB, or recruitment and interaction with either co-factor or co-repressor proteins [42e45]. For example, FoxO proteins interact with cofactors proteins, including CBP/p300, to promote histone acetylation [46]. This is in keeping with our previous studies demonstrating increased expression of NF-kB and CBP/p300 at SC fetal membranes [29].

Fig. 7. Immunohistochemical expression of FoxO4 in term SCS and DS fetal membranes obatied from non-labouring women (magnification 250). These sections are representative of 1 patient sample. ae, amnion epithelial layer; cl, connective tissue layer; ct, chorionic trophoblast layer; dec, decidua.

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The expression of FoxO3 and FoxO4 in the placental syncytiotrophoblast layer suggests important and specialised roles. The syncytiotrophoblast layer consists of terminally differentiated and fused cytotrophoblasts. It surrounds the chorionic villi and is in permanent contact with maternal blood and external agents. The syncytiotrophoblast layer is the major site for many placental functions required for maintenance of pregnancy and for fetal growth and development. Both preeclampsia and IUGR are associated with increased apoptosis in the syncytiotrophoblast [47], and as mentioned above FoxO proteins regulate apoptosis. Further, IUGR is characterised by reduce the flow of nutrients from mother to fetus [48,49], and activation of FoxO impairs glucose metabolism [50]. As such, it could be suggested that FoxO3 and FoxO4 may play a role in these pregnancy complications. However, in preliminary studies, when compared to gestationally-age mateched preterm controls, there was no effect of preeclampsia or IUGR on FoxO3 and FoxO4 mRNA expression in placenta (Lappas, personal communications). This is in keeping with our previous data demonstrating no difference in FoxO1 gene and protein expression in placenta from term preeclamptic pregnancies [22]. Further studies, are however, required to fully elucidate the role of FoxO proteins in pregnancy complications. Unlike homozygous FoxO1/ mice, both FoxO3 and FoxO4 null mice are viable, and indistinguishable from their wild-type littermates; although FoxO3 appears to have a role in ovarian follicular physiology [51]. However, the identification of the FoxO3 and FoxO4 in human gestational tissues suggests that they may play functional roles in these tissues. Further studies of the role and regulation of FoxO3 and FoxO4 in human gestational tissues, and investigation of their expression in additional pathological conditions will be important to delineate the exact role of these transcription factors in human pregnancy. Certainly, various inflammatory disease states are associated with altered FoxO expression. FoxO3 mRNA and protein expression levels are increased in polymorphonuclear cells from rheumatoid arthritis patients compared to controls [52] and increased expression of FoxO3 in the brain of patients with Parkinson’s disease [53]. Conflict of interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Funding Dr. Martha Lappas is in recipient of a National Health and Medical Research Council (NHMRC) RD Wright Fellowship (grant no. 454777). The work described in this manuscript was funded by the Melbourne Research Grant Scheme and ANZ Charitable Trust (Medical Research and Technology Grant). Funding for the Leica Qwin Image Analysis System was provided by theMedical Research Foundation for Women and Babies. Acknowledgements The authors gratefully acknowledge the assistance of the Clinical Research Midwives Gabrielle Fleming, Astrid Tiefholz and Anne Beeston; and the Obstetrics and Midwifery staff of the Mercy Hospital for Women for their co-operation. References [1] Anderson MJ, Viars CS, Czekay S, Cavenee WK, Arden KC. Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 1998;47(2):187e99.

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