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Endocrinology, doi:10.1210/en.2007-0659
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Endocrinology Vol. 148, No. 10 5020-5029
Copyright © 2007 by The Endocrine Society

Role and Regulation of the Serum- and Glucocorticoid-Regulated Kinase 1 in Fertile and Infertile Human Endometrium

Fakhera Feroze-Zaidi, Luca Fusi, Masashi Takano, Jenny Higham, Madhuri S. Salker, Tomoko Goto, Seby Edassery, Karin Klingel, Krishna Murthy Boini, Monica Palmada, Rick Kamps, Patrick G. Groothuis, Eric W.-F. Lam, Stephen K. Smith, Florian Lang, Andrew M. Sharkey and Jan J. Brosens

Institute of Reproductive and Developmental Biology (F.F.-Z., L.F., M.T., J.H., M.S.S., T.G., E.W.-F.L., S.K.S., J.J.B.), Imperial College London, Hammersmith Hospital, London W12 ONN, United Kingdom; Proteomics Core Laboratory (S.E.), Rush University Medical Centre, Chicago, Illinois 60612; Department of Obstetrics and Gynaecology (R.K., P.G.G.), University Hospital Maastricht, University of Maastricht, 6202 AZ Maastricht, The Netherlands; Institute of Physiology (K.M.B, M.P., F.L.) and Molecular Pathology (K.K.) University of Tübingen, D-76072 Tübingen, Germany; and the Department of Pathology (A.M.S), Cambridge CB2 1QP, United Kingdom

Address all correspondence and requests for reprints to: Jan Brosens, M.D., Ph.D., Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, United Kingdom. E-mail: j.brosens{at}imperial.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
COMPARED WITH OTHER mammals, the early human conceptus is deeply embedded in the uterine mucosa with placental trophoblast invading as far as the inner third of the uterine muscle wall (1). To accommodate implantation and deep invasion by the conceptus, human endometrium undergoes extensive remodeling in response to the postovulatory rise in circulating progesterone (P4) levels. This is a sequential process first characterized by secretory transformation of the glandular compartment, followed by influx of specialized uterine natural killer cells in response to locally produced chemokines and subsequently decidualization of the stromal compartment and its spiral arteries (2, 3).

Implantation requires synchronized endometrial and embryo development. The first step is thought to involve apposition and attachment of an implantation-competent blastocyst to a receptive endometrial luminal epithelium (4, 5, 6, 7). The period of endometrial receptivity is restricted, probably confined in humans to d 20–24 of a regular 28-d cycle (8). In recent years, microarray analyses have been extensively used to identify gene networks that underpin the receptive and unreceptive states of the endometrium (7, 9, 10, 11, 12, 13). Although each study has generated numerous candidate genes, the number of common endometrial receptivity genes identified in these studies is relatively small (for review see Ref. 6). This largely reflects differences in experimental approach, timing of endometrial sampling, and array platforms. Once attached to the luminal epithelium, invasion of the blastocyst and subsequent placental development is thought to be critically dependent upon decidualization of the endometrial stromal compartment. This differentiation process transforms stromal cells into secretory, epithelioid-like cells, capable of regulating trophoblast invasion and attenuating the maternal immune response to the semiallogeneic conceptus (2, 14). Decidualization of human endometrium is not dependent upon the presence of an implanting embryo, in contrast to many other mammalian species, and the appearance of decidual stromal cells surrounding the terminal spiral arteries around d 24 of the menstrual cycle heralds the end of the period of endometrial receptivity.

Failure of the endometrium to establish a receptive phenotype is thought to be a major cause of infertility, a prevalent condition that affects approximately 9% of couples in both developed and less developed countries (15). Conversely, delayed implantation, presumably reflecting an inordinately long implantation window and impaired decidual process, is associated with an exponential increase in miscarriage rate (16). Despite the considerable progress in defining the factors that confer endometrial receptivity, a molecular definition of the endometrial defects responsible for infertility or early pregnancy loss is still lacking. Although targeted gene ablation studies in mice have identified several endometrial factors indispensable for implantation (17, 18, 19), it has proven difficult to implicate these factors in clinical reproductive failure.

In this study, we used cDNA microarrays to screen for differences in gene expression in midsecretory endometrial samples from fertile women and infertility patients without overt uterine, tubal, or pelvic pathology or male-factor infertility. We identified SGK1 as a gene aberrantly expressed specifically in luminal epithelial cells during the midsecretory receptive phase of the cycle in women with unexplained infertility or recurrent implantation failure after in vitro fertilization treatment. This gene encodes for serum- and glucocorticoid-regulated kinase 1 (SGK1), an intermediate in the phosphoinositide 3-kinase (PI3K) signaling pathway closely related to the Akt (PKB) family of kinases (20, 21). SGK1 is probably best characterized for its ability to regulate the expression and activity of epithelial Na+ channels (ENaCs), raising the possibility that impaired uterine fluid homeostasis contributes to implantation failure in these patients. We further show that SGK1 is also induced and activated in differentiating human endometrial stromal cells (HESCs) where it controls the expression of prolactin (PRL), a major decidual marker gene (2, 14).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Patient selection and sample collection
The Local Research and Ethics Committees of Hammersmith and Addenbrooke’s Hospitals NHS Trusts, UK, approved the study. Written informed consent was obtained from all patients before endometrial sampling. For microarray analysis, endometrial biopsies were obtained from women with proven fertility (control group 1; n = 8) and patients with unexplained infertility (case group 1; n = 6). Unexplained infertility was defined as conception delay of 24 months or more after excluding anovulation, tubal blockage, pelvic adhesions and endometriosis, or impaired semen quality. The demographic details of the patients are summarized in Table 1Go. For validation studies, samples were obtained from women with proven fertility (control group 2; n = 20), and patients with either unexplained infertility or recurrent implantation failure, defined as at least three failed in vitro fertilization treatment cycles with transfer of good quality embryos (case group 2; n = 15) (Table 2Go). All patients had regular cycles and monitored daily urinary LH levels using an ovulation prediction kit (Assure Ovulation Predictor, San Diego, CA). Endometrial biopsies, timed between 5 and 10 d after the preovulatory LH surge (LH+5 to LH+10), were taken using a pipelle catheter. Each biopsy was divided and one portion snap-frozen in liquid nitrogen for RNA analysis. The other portion was fixed in formalin for histological dating using standard criteria. A venipuncture was also performed and P4 levels measured to ensure that ovulation had occurred.


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  		TABLE 1. Demographic details of control and case groups used for microarray analysis

 

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  		TABLE 2. Demographic details of control and case groups used for RTQ-PCR validation

 
RNA extraction
Total RNA extracted from frozen tissue using Trizol reagent (Invitrogen, Paisley, UK) was treated with DNase I (Ambion, Inc., Austin, TX), according to the manufacturer’s instructions. RNA quality was assessed by loading 200 ng total RNA onto an RNA Labchip (Agilent Technologies, Waldbronn, Germany) and analyzed on an A2100 bioanalyzer (Agilent).

Microarray analysis
Microarray analysis was performed using a custom-made array representing 16,000 different human cDNA clones, spotted in duplicate. The microarrays were produced in the microarray core facility, Department of Pathology, University of Cambridge, UK. A full list of the cDNAs is available online (http://www.path.cam.ac.uk/resources/microarray/microarrays/).

One microgram of total RNA was used to synthesize double-stranded cDNA (ds-cDNA) with a SMART PCR cDNA synthesis kit (Clontech, Oxford, UK) according to the manufacturer’s instructions. The ds-cDNA from each sample in the case and control groups was labeled with Cy3-deoxyuridine triphosphate and Cy5-deoxyuridine triphosphate using the Bioprime DNA labeling kit (Invitrogen) with random hexamers (Amersham, Little Chalfont, UK). A common reference cDNA was made from pooled endometrial total RNA samples from the control individuals and was similarly labeled with Cy5-deoxyuridine triphosphate. Each Cy3-labeled case or control sample was combined with an equal amount of the pooled Cy5-labeled common reference. These samples were purified using Autoseq G50 columns (Amersham), pooled with 5 µg human Cot-1 DNA (Invitrogen) and 1 µg poly dA (Amersham) and hybridized to the cDNA microarray at 50 C for 16 h in a final volume of 60 µl. The arrays were washed twice in 2x standard saline citrate (SSC), 0.5% SDS; twice in 0.1x SSC, 0.1% SDS for 5 min; and twice with 0.1x SSC at room temperature. The fluorescence signals were acquired with a Genepix 4100 microarray scanner (Axon Instruments, Foster City, CA) allowing the Cy3 hybridization signal for each spot to be compared with the Cy5 signal from the common reference hybridized to the same slide. The images were processed using GenePix Pro 3.0 software (Axon Instruments).

Array data analysis
Microarray analysis was performed using the BRB Array Tools package developed by the U.S. National Cancer Institute. The raw data were normalized per spot and per chip with intensity-dependent (Lowess) normalization (percentage of the data used for smoothing 10%). Low hybridization signals were removed to yield an average of 8000 different genes expressed above background. Genes differentially expressed between the control and case groups were initially identified using the class comparison tool in BRB, which combines a univariate F test and permutation test (n = 2000) with a randomized variance model. A significance level of 0.05 was chosen to limit the number of false-negative results.

Real-time quantitative (RTQ)-PCR
Total RNA extracted from biopsy samples or primary cultures was reversed transcribed and the resulting cDNA amplified using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) and the following gene-specific primer pairs: L19 sense (5'-GCG GAA GGG TAC AGC AAT-3') and L19 antisense (5'-GCA GCC GGC GCA AA-3'), SGK1 sense (5'-TTC CTA TCG CAG TGT TTC AGT TCT T-3') and SGK1 antisense (5'-CAC ACT CAC ACG ACG GTT CAC-3'), LRRC32 sense (5'-CTG GTT TGG TGC GGT GAG T-3') and LRRC32 antisense (5'-CAA ACA GTC TGC TTG GAA ATG TCT-3'), PTPRR sense (5'-GCA AGT TCA CAT TTA CTC CAA AGT G-3') and PTPRR antisense (5'-CAT GAC GCG GAA TAT CAA TTT C-3'), PKIA sense (5'-GGG AGA AGC AGC AAA ATC TGA-3') and PKIA antisense (5'-ATA CTC CTG GAG ATT TGA GAC ATT C-3'), G2 sense (5'-TTG GAC GTG CTC ACA ATC TGA-3') and G2 antisense (5'-TGA GCC AAG CGG ACA ACA TAC-3'), PRL sense (5'-AAG CTG TAG AGA TTG AGG AGC AAA C-3') and PRL antisense (5'-TCA GGA TGA ACC TGG CTG ACT A-3'). 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 and the data analyzed using Student’s t test with P < 0.05 considered significant.

Laser microdissection (LCM)
Endometrial biopsies were embedded in Tissue-Tek and frozen in liquid nitrogen. Cryosections (6 µm) were fixed in 70% ethanol and counterstained with Mayer’s hematoxylin, and distinct cell populations (luminal epithelial, stromal, and glandular cells) were isolated using P.A.L.M. Robot-Microbeam version 4.0 (P.A.L.M. Microlaser Technologies AG, Bernried, Germany). LCM was performed with a 15- to 30-µm laser beam with laser power of 50 mV and a laser power duration of 4–6 msec. For each cell population, an average of 150 laser shots were transferred onto a 0.5-ml tube cap and stored at –70 C until used for total RNA extraction.

In situ hybridization
Tissue specimens of human endometrium were fixed in 4% paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.2) for 4 h and embedded in paraffin. Tissue sections (4 µm) were dewaxed and hybridized with mixture containing either the 35S-labeled RNA antisense or sense control hSGK1 probe (22) (500 ng/ml) in 10 mM Tris-HCl (pH 7.4), 50% (vol/vol) deionized formamide, 600 mM NaCl, 1 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.05% BSA, 10% dextran sulfate, 10 mM dithiothreitol, denatured sonicated salmon sperm DNA at 200 µg/ml rabbit liver tRNA at 100 µg/ml. Hybridization with RNA probes proceeded at 42 C for 18 h. Slides were then washed followed by 1 h incubation at 55 C in 2x SSC. Nonhybridized single-stranded RNA probes were digested by RNase A (20 µg/ml) in 10 mM Tris-HCl (pH 8.0), 0.5 M NaCl for 30 min at 37 C. Tissue slide preparations were autoradiographed (23) and stained with hematoxylin and eosin.

Immunohistochemistry
Formalin-fixed, paraffin-embedded samples were stained for SGK1 expression. Briefly, after mounting, the samples were deparaffinized and rehydrated in graded concentrations of ethanol, and endogenous peroxidase activity was blocked by immersion of the slides for 30 min in a freshly prepared solution of 2 ml 30% hydrogen peroxide diluted in 200 ml methanol. The slides were then washed in PBS, preincubated in 1.5% nonimmune rabbit serum in PBS for 30 min at room temperature, and incubated overnight at 4 C with a primary rabbit polyclonal antibody specific to SGK1 (1:50 dilution; Upstate Biotechnology, Dundee, UK), and staining was visualized using the Universal LSAB Plus Kits (Dako North America, Inc, Carpinteria, CA). Renal tissue sections were included as positive control, and the primary antibody was omitted as a negative control. Immunostaining was assessed, blinded to the clinical history, in each cell compartment using a semiquantitative histological scoring system (HSCORE) as described previously (24). Briefly, HSCORE was calculated using the following equation: HSCORE = {Sigma}Pi(I + 1), where I represents staining intensity (1 = weak, 2 = moderate, and 3 = strong) and Pi is the percentage of stained cells for each intensity. HSCOREs were analyzed by ANOVA for multiple comparisons with P < 0.05 considered significant.

Primary HESC cultures and endometrial explants
HESCs were isolated from normal proliferative endometrial tissues obtained from cycling women as previously described (25, 26, 27, 28). Samples were collected in DMEM/F-12 containing 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma Chemical Co., St. Louis, MO). After enzymatic digestion, the stromal cells were separated from epithelial cells and passed into culture. Proliferating HESCs were cultured in maintenance medium of DMEM/F-12 containing 10% dextran-coated charcoal-treated FBS and 1% antibiotic-antimycotic solution. Confluent monolayers were treated in DMEM/F-12 containing 2% dextran-coated charcoal-treated FBS with 0.5 mM 8-bromo-cAMP (8-br-cAMP) (Sigma) with or without 10–6 M medroxyprogesterone acetate (MPA) (Sigma) to induce a differentiated phenotype. All experiments were carried out before the third cell passage. Explants cultures were also prepared from proliferative endometrium as recently described in detail (29). Briefly, 24 endometrial fragments, measuring approximately 2–3 mm2 each, were placed in Millicell-CM culture inserts in six-well plates containing DMEM/F-12 medium supplemented with L-glutamine (1%) and penicillin and streptomycin (1%). The explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), 17ß-estradiol (E2; 10 nM), P4 (10 nM), or a combination.

Western blot analysis
Whole-cell extracts or nuclear and cytoplasmic protein fractions were immunoblotted as described (30). Antibodies to FOXO1, Akt, and phospho-Akt (Ser473) were purchased from Cell Signaling Technology (Beverly, MA), phospho-FOXO1 (Ser319) from Abcam (Cambridge, UK), and SGK1 and phospho-SGK1 (Ser255/Thr256) from Upstate Biotechnology. Primary antibodies were used at 1:1000 dilution except for antibody to ß-actin (Abcam), which was diluted 1:100,000.

Transient transfection and siRNA
HESCs cultured in 24-well plates were transiently transfected, using calcium phosphate precipitation, with 400 ng/well promoter-reporter constructs and 100 ng/well expression vector. A ß-galactosidase control expression vector (50 ng/well) was cotransfected to control for transfection efficiency. The promoter-reporter constructs (dPRL-3000/luc3 and 6xDBS/luc3) and expression vectors [pcDNA3.1/FOXO1, pcDNA3.1/FOXO1(A3), and pIRES2EGFP/hSGK1] have been previously described (26, 31). Transfections were performed in triplicate and repeated at least three times. For gene silencing, undifferentiated or decidualized HESCs were transiently transfected with 50 nM SGK1 siGENOME SMARTpool or siCONTROL nontargeting siRNA pool (Dharmacon, Lafayette, CO).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Microarray analysis and validation
To identify factors that may impair endometrial receptivity, cDNA microarray analysis was performed on timed endometrial samples (LH+6 to +10) from women with unexplained infertility (n = 6) and fertile controls (n = 8). The case and control groups were matched for age, cycle length, P4 levels, and time of biopsy in the cycle (P > 0.05; Table 1Go). Using Class Prediction Tool, 51 genes were identified that differentiated between fertile and infertile women (data not shown). When these genes were used for cluster analysis and cross-validation, 12 of the 14 samples could be assigned to the correct clinical group. However, examination of the hybridization data revealed low signal intensities for many of these genes, suggesting that the corresponding transcripts are expressed at low abundance. Moreover, only 12 genes were either up- or down-regulated more than 1.5-fold in the infertile group when compared with controls. Based on this fold change and a significant signal intensity, the expression of only four up-regulated (SGK1, LRRC32, PTPRR, and C11orf41) and two down-regulated (PKIA and TYRP1) genes was confirmed to be significantly different between the groups using a two-tailed unpaired t test (P < 0.05). To verify these findings, we examined the expression of these putatively discriminatory genes in a larger, independent sample set, consisting of timed endometrial biopsy samples from 20 infertile women and 15 fertile controls, matched for age, cycle length, circulating P4 levels, and time of biopsy in the cycle (P > 0.05; Table 2Go). RTQ-PCR confirmed up-regulation of SGK1, LRRC32, and PTPRR and down-regulation of PKIA transcripts in infertile patients compared with fertile controls (Fig. 1Go, A–C). The abundance of C11orf41 (also termed G2) and TYRP1 mRNA was too low for meaningful analysis (data not shown). Moreover, only the transcript levels of SGK1 [median 5.73 arbitrary units (au), SD 3.8, vs. 9.1 au, SD 3.7) and LRRC32 (median 9.21 au, SD 11.3, vs. 18.78 au, SD 25.3) were statistically significantly different between both groups (P < 0.05).


Figure 1
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  		FIG. 1. Microarray validation. RTQ-PCR analysis of SGK1, LRRC32, PTPRR, and PKIA mRNAs in timed endometrial biopsies (d 19–24) from fertile (n = 15) and infertile (n = 20) patients. The abundance of each transcript was normalized to that of L19 and expressed in au. The horizontal bar indicates the median expression in each patient group. *, P < 0.05.

 
Thus, validation of the array data yielded only two transcripts that were differentially expressed in midsecretory endometrium of a second cohort of infertile patients and fertile controls. This is not entirely surprising because accurate timing of endometrial samples is fraught with difficulties and endometrial gene expression is known to change rapidly during the midsecretory phase of the cycle (32, 33, 34). Moreover, unexplained infertility is by definition a heterogeneous, multifactorial disorder. One of the identified genes, LRRC32, also termed GARP or GARPIN, encodes for a 72-kDA transmembrane protein consisting of 21 extracellular leucine-rich repeats (LRR) and a short 13-amino-acid cytoplasmic tail (35, 36). No function has as yet been ascribed to LRRC32, and the lack of commercially available antibodies precluded further analysis of its expression in human endometrium.

Regulation of SGK1 expression in cycling human endometrium
We thus focused on the expression and regulation of SGK1 in human endometrium. First, we used RTQ-PCR to determine SGK1 transcript levels throughout the cycle. As shown in Fig. 2AGo, the abundance of SGK1 transcripts in the early secretory endometrium was approximately 3-fold higher than levels in proliferative samples, although this did not reach statistical significance (P > 0.05). Another 3-fold increase in SGK1 mRNA expression was observed during the midsecretory phase, and the levels remained elevated throughout the late secretory phase (Fig. 2AGo). Together these observations indicate that endometrial SGK1 gene expression is highly regulated upon the postovulatory rise in circulating P4 levels. To confirm that SGK1 expression is under sex hormone control, we cultured endometrial explants, established from proliferative endometrium, in the presence or absence of E2 (10 nM), P4 (10 nM), or a combination for 24 h. RTQ-PCR demonstrated that treatment with P4 yielded a 2-fold induction in SGK1 mRNA (Fig. 2BGo). Notably, treatment of explants with E2 alone had little or no effect on SGK1 transcript levels (Fig. 2BGo).


Figure 2
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  		FIG. 2. Cycle-dependent expression and hormonal regulation of SGK1 mRNA in human endometrium. A, Expression of SGK1 mRNA was examined in endometrial biopsies obtained during different phases of the cycle: proliferative (P; n = 12), early-secretory (ES; n = 7), midsecretory (MS; n = 8), and late-secretory (LS; n = 14) using RTQ-PCR. The abundance of SGK1 transcripts was normalized to L19 and expressed in au. Data show the mean ± SEM. *, P < 0.05. B, Endometrial explants were cultured in the presence or absence of E2 (E; 10 nM), P4 (P; 10 nM), or a combination (E+P) for 24 h. Subsequently, the explants were harvested and SGK1 mRNA levels determined using RTQ-PCR. The abundance of SGK1 transcripts was normalized to that of L19 and expressed in au. The data show the mean ± SD from one representative experiment.

 
Increased endometrial SGK1 expression in infertile patients
The differential expression of SGK1 mRNA in fertile and infertile women suggested a possible role for this kinase in coordinating the interaction between the blastocyst and endometrial surface epithelium. This prompted us to examine the spatial expression of SGK1. In situ hybridization with a 35S-labeled hSGK1-specific antisense RNA probe demonstrated abundant expression of SGK1 transcripts in secretory endometrium with especially strong expression in the glandular and luminal epithelial compartments (Fig. 3AGo). Next, we used LCM to isolate enriched surface epithelial cells, underlying stromal cells, and glandular epithelium. Defined cell populations were captured from 14 midsecretory endometrium samples (d 19–24) and SGK1 mRNA levels determined by RTQ-PCR. Variation in SGK1 transcript levels was considerably less in enriched cell populations when compared with whole biopsy samples (Fig. 3BGo). SGK1 transcripts were detected in both stromal and epithelial compartments. However, the abundance of SGK1 mRNA in stromal cells was approximately 6-fold lower when compared with surface epithelium (mean 0.12 au, SEM 0.02 and mean 0.69 au, SEM 0.08, respectively; P = 0.0001). Glandular epithelial cells also expressed SGK1 transcripts albeit at lower levels than luminal epithelial cells (mean 0.42 au, SEM 0.05; P = 0.003).


Figure 3
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  		FIG. 3. Tissue distribution of SGK1 mRNA and protein in human endometrium. A, In situ hybridization demonstrates abundance of SGK1 transcripts in secretory endometrium, especially in the epithelial glandular and luminal compartments. B, Enriched surface epithelial cells (SE), glandular cells (GLD), and stromal cells (STR) were isolated from midsecretory phase endometrial biopsies (d 19–24; n = 14) using LCM and expression of SGK1 mRNA determined by RTQ-PCR. The abundance of SGK1 transcripts was normalized to L19 and expressed in au. Data show mean ± SEM; *, P < 0.05. C, SGK1 immunoreactivity in midsecretory endometrium (d 19–24) from fertile (top) and infertile (bottom) patients. Staining was found to be predominately cytoplasmic and most intense at the apical border of epithelial cells. D, Relative SGK1 staining was quantified in different cellular compartments in midsecretory endometrial samples from fertile (n = 9) and infertile (n = 9) patients using HSCORE. Data show mean ± SEM; *, P < 0.05.

 
The cellular distribution of SGK1protein was determined by immunohistochemistry. SGK1 immunoreactivity was prominent in luminal epithelial cells, and staining intensity decreased noticeably upon transition of surface to glandular epithelium (Fig. 3CGo). There was also a clear gradient in SGK1 staining within endometrial glands, which decreased toward the basal layer (data not shown). SGK1 localized predominantly to the cytoplasm of epithelial cells, but there was also staining of the apical membrane, which was much more pronounced when compared with the basolateral borders (Fig. 3CGo). To quantify our results, we used a semiquantitative histological scoring system (HSCORE). Immunostaining was assessed, blinded to clinical history, in 18 midsecretory biopsy samples from fertile (n = 9) and infertile patients (n = 9). As shown in Fig. 3DGo, SGK1 immunoreactivity in different cellular compartments correlated well with transcript levels, characterized by significantly higher expression in surface epithelium when compared with either glandular or stromal compartments. Furthermore, the luminal HSCORE, but not that of glands or stroma, was significantly higher in infertile samples compared with fertile controls (P = 0.008).

The mechanism whereby increased SGK1 expression would lead to implantation failure is as yet unclear. However, cyclic regulation of the fluid environment of the uterus is essential for key reproductive events, including sperm transport, embryo development and transport, and implantation (37). A predominance of estrogens causes fluid secretion, whereas P4 promotes fluid absorption, mediated by amiloride-sensitive ENaC expressed on the apical surface of luminal and glandular epithelial cells. Fluid absorption results in uterine closure, characterized by complete apposition of the epithelium of the opposing uterine walls (37, 38, 39, 40). This closure response is thought to be essential for implantation by allowing embryos to establish and maintain contact with the luminal epithelium. In the kidney, SGK1 participates in the regulation of ENaC-mediated Na+ transport by aldosterone and presumably fully accounts for the stimulating effect of insulin on Na+ transport (20, 21, 41, 42). The underlying mechanism by which SGK1 increases ENaC-mediated Na+ transport is complex and involves both inhibition of Nedd4-2-mediated ubiquitination, internalization, and degradation of channels as well as enhanced transcription of the {alpha}-subunit of ENaC (43, 44, 45). Together, the data suggest that aberrant endometrial SGK1 expression, and presumably activity, in infertile patients may disturb uterine fluid handling, resulting in premature or perturbed uterine closure and implantation failure. Furthermore, SGK1 has been shown to promote cell survival in many different cell types (46, 47, 48, 49). It is well documented, at least in rodents, that the uterine epithelium undergoes apoptosis in response to signals derived from an implanting blastocyst (50, 51). Moreover, a single intraluminal injection of a caspase-3 inhibitor in the uterine horn of mice or hamsters has been shown to virtually abolish implantation without conferring embryo toxicity (51). However, it is not known whether this type implantation, referred to as displacement penetration, is relevant to humans where the invasive potential of the conceptus is much higher than in rodents.

SGK1 expression in decidualizing human endometrial stromal cells
We noted that SGK1 is also expressed in decidualizing HESCs underlying the luminal epithelium (Fig. 3CGo). In fact, SGK1 immunoreactivity in the stroma appeared also more pronounced in infertile women compared with fertile controls, although the difference in HSCOREs was not statistically significant (Fig. 3Go, C and D). The molecular changes associated with the decidual process in vivo are closely recapitulated in primary HESC cultures made to differentiate in response to activation of the cAMP pathway and P4 signaling (2, 14). To investigate whether SGK1 plays a role in the decidual process, primary HESC cultures were treated with the progestin MPA, 8-br-cAMP, or a combination for 3 d, and whole-cell lysates were analyzed by Western blotting. MPA alone induced a modest up-regulation of total SGK1 protein, which was enhanced upon cotreatment with 8-br-cAMP (Fig. 4AGo). Notably, treatment of HESCs with 8-br-cAMP had little or no effect on SGK1 expression levels. SGK1 is a serine-threonine kinase that functions as a downstream effector in the PI3K pathway (20, 52, 53). Using a phosphospecific antibody (Ser225/Thr256), we found that SGK1 is not only expressed but also activated in differentiated HESCs in response to MPA and 8-br-cAMP treatment (Fig. 4AGo). These observations are consistent with findings in other cell systems, demonstrating that P4 up-regulates SGK1 expression (54) and that this kinase can be activated through the PI3K as well as the cAMP pathway (20, 55).


Figure 4
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  		FIG. 4. SGK1 inhibits the expression of decidual PRL in differentiating HESCs. A, Primary HESCs were cultured in the presence or absence of 8-br-cAMP (0.5 mM), MPA (10–6 M), or a combination for 72 h. Whole-cell lysates were used to examine expression of total and phospho-SGK by Western blot analysis. B, Primary HESCs were first mock transfected or transfected with SGK1 siRNA and then treated with 8-br-cAMP (0.5 mM) plus MPA (10–6 M) for 72 h. Parallel cultures were harvested for RTQ-PCR analysis and Western blotting, as indicated. C, HESCs were transfected with an empty expression vector or a plasmid encoding for wild-type SGK1 and treated with 8-br-cAMP (0.5 mM) for 48 h. PRL transcripts were measured by RTQ-PCR and normalized to the level of L19 mRNA expression. The data, expressed in au, represent mean ± SD of triplicate measurements. D, HESCs were transfected with nontargeting (NT) siRNA or SGK1 siRNA and treated with 8-br-cAMP (0.5 mM) and MPA (10–6 M) for 96 h. PRL transcripts were measured by RTQ-PCR and normalized to the level of L19 mRNA expression. The data, expressed in au, represent mean ± SD of triplicate measurements. E and F, primary HESC cultures were transfected with the decidual PRL promoter coupled to luciferase (dPRL3000-Luc) and an empty expression vector, wild-type SGK1, nontargeting (NT) siRNA, or SGK1 siRNA and treated as indicated with 8-br-cAMP or 8-br-cAMP plus MPA for 24 h. Luciferase activity was normalized to ß-galactosidase activity. The data show the mean fold change (±SD) in normalized luciferase activity of triplicate measurements relative to untreated cells. *, P < 0.05.

 
The induction of SGK1 protein in response to 8-br-cAMP plus MPA was reflected at the RNA level (Fig. 4BGo). Moreover, transfection of HESCs with small interfering RNA (siRNA) targeting SGK1 attenuated the induction of this kinase upon decidualization. Interestingly, we consistently found that siRNA was more efficient in lowering the abundance of SGK1 transcripts when compared with protein (Fig. 4BGo), suggesting that SGK1 expression may in part be regulated posttranslationally in decidual cells.

SGK1 attenuates decidual prolactin (PRL) expression
PRL, a well-established decidual marker gene, is induced in HESCs in response to 8-br-cAMP treatment and its expression further enhanced upon cotreatment with MPA (25, 31). As shown in Fig. 4AGo, SGK1 is not induced in HESCs upon treatment with 8-br-cAMP alone. Thus, to examine the role SGK1 in HESC differentiation, primary cultures were first transfected with an empty expression vector or a plasmid encoding for wild-type SGK1 and then treated with or without 8-br-cAMP for 72 h. RTQ-PCR was used to measure PRL mRNA levels. Overexpression of SGK1 nearly abolished the induction of PRL in response to 8-br-cAMP treatment (Fig. 4CGo). Conversely, we transfected primary HESCs with either nontargeting siRNA or siRNA targeting SGK1 and decidualized the cultures with 8-br-cAMP plus MPA for 72 h. RTQ-PCR analysis demonstrated that SGK1 knockdown enhances PRL mRNA expression by approximately 50% (Fig. 4DGo). To validate these findings, we transiently transfected HESCs with dPRL3000/Luc, a luciferase reporter construct coupled to 3 kb of the decidua-specific PRL promoter region, and examined the effect of SGK1 overexpression or knockdown on promoter activity in differentiating HESCs. In agreement with the RTQ-PCR results, cAMP-induced dPRL3000/Luc activity was abolished upon cotransfection of SGK1 (Fig. 4EGo). In the reverse experiment, inhibition of endogenous SGK1 expression by siRNA led to a greater than 2-fold increase in decidual PRL promoter activity in cells treated with 8-br-cAMP and MPA (Fig. 4FGo).

SGK1 phosphorylates FOXO1 in decidualizing HESCs
The FOXO family (FOXO1, FOXO3a, and FOXO4) is a downstream target of the PI3K pathway, and targeted phosphorylation of these transcription factors by Akt or other kinases, including SGK1, results in their nuclear exclusion and loss of activity (56, 57, 58). FOXO proteins have emerged as important mediators of cell fate decision because of their ability to regulate either proapoptotic genes (59) or genes involved in differentiation, cell cycle arrest (60), oxidative defenses, and DNA repair (61, 62). In fact, the survival function of SGK1 in many cell types exposed to environmental stress is mediated by phosphorylation and inactivation of FOXO3a (46, 47, 48, 56), thereby disabling the activation of the apoptotic machinery. However, FOXO3a expression is repressed in differentiating endometrium, but FOXO1 is induced and accumulates in the nuclei of HESCs in response to cAMP signaling (26, 27, 28). Cotreatment with MPA induces phosphorylation and partial cytoplasmic translocation of FOXO1, which can be blocked by the PI3K inhibitor LY294002 (27). Yet, the residual nuclear FOXO1 in 8-br-cAMP plus MPA-treated cells has been shown to be essential for the expression of many decidua-specific genes, including PRL. We speculated that SGK1 may play a role in regulating FOXO1 phosphorylation and nucleo-cytoplasmic shuttling in differentiating HESCs. To test this hypothesis, primary cultures were first treated with 8-br-cAMP and MPA for 2, 4, or 8 d, and total and phosphorylated Akt, SGK1, and FOXO1 levels were examined by Western blot analysis using pan- and phosphospecific antibodies, respectively (Fig. 5Go). As expected, decidualization was associated with a sustained increase in total SGK1 and FOXO1 levels, whereas total Akt levels remained unchanged. Phospho-SGK1 and -FOXO1 levels also increased upon treatment of cells with 8-br-cAMP plus MPA. In contrast, there was a striking reduction in phospho-Akt levels in undifferentiated HESCs maintained in prolonged cultures, which was much more pronounced in cells treated with 8-br-cAMP plus MPA. Thus, the phosphorylation status of FOXO1 correlated with SGK1 but not Akt activation in decidualizing HESCs. The mechanism of Akt inactivation in differentiating HESCs is as yet unclear. One possibility is that decidualization is associated with the induction of a specific inhibitor of Akt phosphorylation such as TRB3, a pseudokinase capable of binding and inactivating the kinase domain of Akt (63).


Figure 5
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  		FIG. 5. SGK1 but not Akt activation correlates with FOXO1 phosphorylation in decidualizing HESCs. Primary cultures were decidualized with 8-br-cAMP (0.5 mM) and MPA (10–6 M) and harvested at the indicated times. Whole-cell lysates were subjected to Western blot analysis and the expression levels of total and phosphorylated (p-) SGK1, Akt, and FOXO1 examined using pan- and phosphospecific antibodies, respectively. ß-Actin served as a loading control.

 
We next transfected SGK1 in primary cultures and monitored the induction of total and phospho-FOXO1 levels in response to 8-br-cAMP treatment. As shown in Fig. 6AGo, exogenous SGK1 expression in 8-br-cAMP-treated cells had no effect on total FOXO1 levels but triggered phosphorylation and a relative decrease in nuclear FOXO1 levels. Conversely, SGK1 knockdown in HESCs treated with 8-br-cAMP and MPA for 72 h decreased phosphorylation of FOXO1 and increased the relative nuclear levels of this transcription factor (Fig. 6BGo). Moreover, coexpression of SGK1 inhibited the ability of wild-type FOXO1 to trans-activate a reporter construct containing six FOXO-response elements coupled to luciferase (6xDBS/Luc). In contrast, SGK1 failed to attenuate the transcriptional activity of FOXO1(A3), a constitutively active FOXO1 mutant in which the three conserved PI3K phosphorylation sites are changed to alanine (Fig. 6CGo). Combined, the data demonstrate that SGK1 is major regulator of FOXO1 activity in differentiating HESCs.


Figure 6
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  		FIG. 6. Phosphorylation and transcriptional inactivation of FOXO1 by SGK1 in HESCs. A, Mock-transfected HESCs or cells transfected with an expression vector encoding for SGK1 were treated with or without 8-br-cAMP (0.5 mM) for 48 h. Whole-cell, cytosolic, and nuclear protein fractions were examined for expression of total and phosphorylated (p-) FOXO1 by Western blot analysis. ß-Actin served as a loading control. B, HESCs were transfected with SGK1 siRNA or a nontargeting siRNA (NT siRNA) and treated with 8-br-cAMP (0.5 mM) and MPA (10–6 M) for 48 h. Whole-cell, cytosolic, and nuclear protein fractions were examined for expression of total FOXO1 or phospho-FOXO1 by Western blot analysis. C, HESCs were transfected with a luciferase reporter construct coupled to six FOXO response elements (6xDBS/luc), a constitutively active ß-galactosidase expression vector, and expression vectors encoding for wild-type FOXO1 (FOXO1 WT), a constitutively active FOXO1 mutant (FOXO1 A3) in which the three Akt phosphorylation sites (Thr-24, Ser-256, and Ser-319) are mutated to alanines, or wild-type SGK1 (SGK1 WT). Luciferase activity was measured 24 h later and normalized to ß-galactosidase activity. Data show the mean ± SD of triplicate measurements. *, P < 0.05.

 
In summary, the distinct temporal-spatial expression of SGK1 in human endometrium during the secretory phase of the cycle, characterized first by expression in luminal epithelial cells followed by induction in decidualizing stroma, suggests a role for this kinase in mediating early implantation events. We demonstrated that SGK1 is a P4-dependent gene in endometrial explant cultures, although expression and activation in primary HESCs requires both cAMP and P4 signaling. In women with unexplained infertility, the increased SGK1 expression in luminal epithelial cells during the midsecretory phase may block implantation either by dysregulating ENaC-mediated Na+ and water transport or by preventing focal apoptosis. These proposed mechanisms require further investigation, and we are currently establishing a mouse model in which sgk1 can be conditionally overexpressed to examine the consequences on female fertility. Finally, we showed that SGK1 fine-tunes the nucleocytoplasmic ratio of FOXO1 in differentiating HESCs, thereby controlling the expression of decidual marker genes.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance provided on this project by Dr. Julia Francis. We are also grateful to Dr. Birgit Gellersen (Endokrinologikum Hamburg, Germany) for providing the decidual PRL promoter-reporter construct.


   Footnotes
 
J.J.B. was supported by grants from the Great Britain Sasakawa Foundation and the IOG Trust. A.S. was supported by the Wellcome Trust Grant GR076856.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 19, 2007

Abbreviations: ar, Arbitrary units; 8-br-cAMP, 8-bromo-cAMP; ds-cDNA, double-stranded cDNA; E2, 17ß-estradiol; ENaC, epithelial Na+ channel; HESC, human endometrial stromal cell; LCM, laser microdissection; MPA, medroxyprogesterone acetate; P4, progesterone; PI3K, phosphoinositide 3-kinase; PRL, prolactin; RTQ, real-time quantitative; SGK1, serum- and glucocorticoid-regulated kinase 1; siRNA, small interfering RNA; SSC, standard saline citrate.

Received May 17, 2007.

Accepted for publication July 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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