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Female Reproductive Tract |
Division of Reproductive Endocrinology,2 Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520-8063
Departments of Medical Biology and Genetics3
Histology and Embryology,4 School of Medicine, Akdeniz University, Antalya 07070, Turkey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß classically activate gene expression after binding to the estrogen response element in the promoter regions of target genes. Estrogen also has rapid, nongenomic effects. It activates several membranous or cytoplasmic kinase cascades, including the phosphatidylinositol 3-phosphate (PI3K/Akt) cascade, a signaling pathway that plays a key role in cell survival and apoptosis. Normal human endometrium is exposed to variable levels of steroid hormones throughout the menstrual cycle. We hypothesized that Akt phosphorylation in human endometrium may vary with the menstrual cycle and in early pregnancy and that fluctuations in estrogen level may play a role in Akt activation in endometrial cells. We analyzed Akt phosphorylation using in vivo and in vitro techniques, including Western blot, immunohistochemistry, and immunocytochemistry. Estradiol significantly increased Akt phosphorylation in endometrial cells. Rapid stimulation of Akt activation in cultured stromal cells was observed. Akt phosphorylation by estradiol was inhibited by the PI3K inhibitor, wortmannin, but not by the ER antagonist, ICI 182 780. The maximal effect on Akt activity was observed following 5–15 min of estradiol treatment. Our results suggest that estradiol may directly affect PI3K-related signaling pathway by increasing the phosphorylation of Akt in endometrial cells. Thus, estradiol may exert part of its proliferative and antiapoptotic effects by a nongenomic manner through the Akt signaling pathway.
early development, estradiol, female reproductive tract, menstrual cycle, phosphatases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß, which belong to the steroid/thyroid hormone superfamily of transcription factors [2]. Estradiol-bound ER homodimerizes or heterodimerizes, translocates to the cell nucleus, and binds the estrogen response element (ERE) in the promoter region of target genes. This is the classical, or genomic, pathway of the estrogen effect [3, 4]. In contrast with this genomic effect, a nongenomic mechanism of estrogen action causes a rapid regulation of intracellular signaling pathways through protein-protein interactions that do not appear to require de novo gene transcription [5]. Nongenomic estrogenic effects have been described in several mammalian tissues and cell lines. In such cells, estrogen has been shown to activate distinct intracellular signaling pathways such as protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3-kinase (PI3K), the last of which activates the Akt signaling pathway [6, 7]. Recent studies have demonstrated that there are cell membrane-localized ER, which may be responsible for the observed activation of MAPK and PI3K signaling [8, 9].
The serine/threonine kinase (Akt), also known as protein kinase B (PKB), is the cellular homologue of the viral oncogene, v-Akt. It is activated by multiple growth factors and functions as a downstream regulator of PI3K signaling. Akt is phosphorylated on serine 473 and/or threonine 308 residues [10]. Phosphorylated Akt is an important regulator of multiple biological processes, including the regulation of apoptosis, cell survival, the cell cycle, and glucose uptake [11, 12].
Human endometrium is capable of many physiological processes, such as blastocyst attachment and implantation, regulation of trophoblast invasion, immune surveillance, and efficient disposal of blood and desquamated cellular debris with menstruation. All of these events require some measure of regulated cellular proliferation, differentiation, and apoptosis. Akt signaling pathways likely play an important role in regulating these processes.
In the present study, we hypothesized that Akt phosphorylation in human endometrium is variable throughout the menstrual cycle and in early pregnancy and that estrogen may play a role in Akt activation through nongenomic estrogen signaling. We analyzed Akt phosphorylation using in vivo and in vitro techniques, including Western blots, immunohistochemistry, and immunocytochemistry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ishikawa (a well-differentiated endometrial adenocarcinoma cell line) and MCF-7 (breast cancer cell line) cells were provided by Dr. R. Hochberg (Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT) as a frozen stock. RL-95 cells were purchased from American Type Culture Collection (Manassas, VA). All cells were grown in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12; Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Rockville, MD). At 80% confluence, the medium was replaced with serum-free and phenol red-free DMEM to remove endogenous steroids.
Tissue Collection
Endometrial tissue was obtained from human uteri after hysterectomy or from endometrial biopsies, conducted for benign diseases excluding endometrial disease (n = 18). Decidual tissues (n = 5) were collected from clinically normal pregnancies, which were voluntarily terminated by dilation and curettage during the first trimester. Informed consent, in writing, was obtained from each patient before surgery; consent forms and protocols were approved by the Human Investigation Committee of Yale University. The mean age of patients was 35 (range 27–39) years. All patients had regular menstrual cycles and none of the patients had received hormonal treatment immediately before surgery. The day of the menstrual cycle was established from the patient's menstrual history and was verified by histological examination of the endometrium. Samples were grouped according to menstrual cycle phases: early proliferative (days 1–6), late proliferative (days 7–14), early secretory (days 15–20), and late secretory (days 21–28). Tissues were embedded in paraffin for immunohistochemistry. Some samples were placed in Hanks balanced salt solution (HBSS; Sigma) and transported to the laboratory for isolation and culture of endometrial stromal cells.
Isolation and Culture of Human Endometrial Stromal Cells
Endometrial stromal and glandular cells isolated from mid to late proliferative samples of endometrium were separated and maintained in monolayer culture, as previously described [13]. Endometrial tissue was rinsed in HBSS to remove blood and debris. Endometrial tissue was digested by incubation of tissue minces in HBSS that contained HEPES (25 mmol; Sigma), penicillin (200 U/ml; Sigma), streptomycin (200 mg/ml; Sigma), collagenase (1 mg/ml, 15 U/mg; Sigma), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg; Sigma) for 30 min at 37°C with agitation. The dispersed endometrial cells were separated by filtration through a wire sieve (73-µm-diameter pore; Sigma). The endometrial glands (largely undispersed pieces) were retained by the sieve, whereas the dispersed stromal cells passed through the sieve into the filtrate.
Stromal cells were plated in DMEM/F12 containing 10% FBS and antibiotics-antimycotics (1% v/v; Gibco BRL) in T-75 plastic flasks (Falcon, Franklin Lakes, NJ). Cells were plated in plastic flasks (75 cm2, Falcon), maintained at 37°C in a humidified atmosphere (5% CO2 in air), and allowed to replicate to confluence. Thereafter, the stromal cells were passed by standard methods of trypsinization and plated in culture dishes (100-mm diameter) and were allowed to replicate to confluence, which takes approximately 7–10 days. Endometrial stromal cells after first passage were assayed immunocytochemically using cell-surface-specific markers and were previously found to contain 0–7% epithelial cells, no detectable endothelial cells, and 0.2% macrophages [13, 14]. Experiments were commenced 1–3 days after confluence was attained. The confluent cells were treated with serum-free, phenol red-free media (Sigma) for 24 h before treatment with steroids. Stromal cells were treated with 17ß-estradiol in ethanol for various time points and harvested for Western blot analysis and immunocytochemistry.
Western Blot Analysis
Both Akt and phospho-Akt antibodies were purchased from Cell Signaling (Beverly, MA). Anti-Akt antibody recognizes phosphorylated and unphosphorylated forms of Akt; however, anti-phospho-Akt binds to phospho-Akt only when phosphorylated at serine 473 residue. Total protein from the cells was extracted using T-PER tissue protein extraction reagent (Pierce, Rockford, IL), supplemented with protease inhibitor cocktail (1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF; Calbiochem, San Diego, CA). The protein concentration was determined by a detergent-compatible protein assay (Pierce). Twenty micrograms protein was loaded into each lane, separated electrophoretically by SDS-PAGE using 10% Tris-HCl Ready Gels (Bio-Rad Laboratories, Hercules, CA), and electroblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween-20 (TBS-T) for 1 h to reduce the nonspecific binding. Then the membrane was incubated overnight with rabbit polyclonal anti-human phospho-Akt (Ser 473) antibody (Cell Signaling) at 4°C. The membrane was then washed three times with TBS-T for 20 min and then incubated for 1 h with peroxidase-labeled anti-rabbit IgG (Vector Laboratories, Burlingame, CA), subsequently washed with TBS-T three times for 20 min. Immunodetection was performed with chemiluminescent detecting reagents (NEN Life Science, Boston, MA), and BioMax film (Kodak, Rochester, NY).
Nitrocellulose membranes were stripped with stripping solution (Pierce) and reprobed with polyclonal rabbit anti-human Akt antibody (Cell Signaling). Thereafter, the membranes were exposed to the same steps as described above. Equal loading of protein in each lane was confirmed by probing the membranes with mouse anti-human glyceraldehyde phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoblot bands were quantified using a laser densitometer. Akt and phospho-Akt band intensities were normalized to those of GAPDH.
Immunohistochemistry and Immunocytochemistry
Immunocytochemistry refers to the immunostaining of cultured cells plated on chamber slides, while immunohistochemistry refers to the immunostaining of tissue sections.
Paraffin-embedded tissue samples were cut and mounted on SuperFrost Plus slides (Erie Scientific Company, Portsmouth, NH). Following deparaffinization, slides were rinsed with PBS for 10 min. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 for 20 min and followed by a rinse with PBS. For antigen retrieval, slides were placed in 10 mM of citrate buffer and were microwaved twice for 5 min. Sections were incubated with 5% normal goat serum for 30 min at room temperature to block nonspecific staining. Thereafter, sections were incubated overnight at 4°C with polyclonal rabbit anti-human phospho-Akt antibody (1:100; Cell Signaling). For negative control slides, normal rabbit IgG (2.8 µg/ml; Vector) and blocking peptide preabsorbed phospho-Akt (10-fold higher concentration) was used instead of primary antibody for phospho-Akt. The sections were washed in PBS, incubated with biotinylated goat anti-rabbit IgG (Vector), and then incubated with streptavidin-peroxidase complex using the Vectastain ABC Elite kit (Vector). The chromogenic reaction was carried out with 3-amino 9-ethyl carbazole (Vector) and the reaction was terminated with tap water. Slides were counterstained with hematoxylin before permanent mounting and then evaluated under a light microscope.
For immunocytochemistry staining, endometrial stromal cells were plated on four-chamber slides (Falcon). Cells were treated with estradiol (10–8 M) for 5–15 min and slides were fixed in 4% paraformaldehyde at 4°C for 20 min and washed three times with TBS for 5 min at room temperature. Cells were permeabilized with 0.2%Triton X-100 in PBS for 5 min at room temperature and rinsed in TBS. Endogenous peroxidase activity was quenched by incubation in 3% H2O2 for 20 min and followed by a rinse in TBS. Chamber slides were incubated with 5% normal goat serum for 30 min at room temperature to reduce nonspecific binding. Thereafter, slides were incubated overnight at 4°C with rabbit polyclonal anti-human phospho-Akt (1:100) and rabbit polyclonal anti-human Akt (1: 100) antibodies (Cell Signaling). For negative control slides, normal rabbit IgG (Vector) was used instead of primary antibodies. Slides were washed in TBS and incubated with biotinylated goat anti-rabbit IgG (Vector), followed by incubation with streptavidin-peroxidase complex using the Vectastain ABC Elite kit (Vector). Subsequently, the chromogenic reaction was carried out with 3-amino 9-ethyl carbazole (Vector) and the reaction was terminated with tap water. Slides were counterstained with hematoxylin before permanent mounting and evaluated under a light microscope.
HSCORE evaluation was used as described previously [15, 16]. The intensity for phospho-Akt immunoreactivity in endometrial tissues was semiquantitatively evaluated as positively stained cells according to nuclear and cytoplasmic immunoreactivity using the following intensity categories: – (no staining), 1+ (weak but detectable staining), 2+ (moderate or distinct staining), 3+ (intense staining). For each tissue, a HSCORE value was derived by summing the percentages of cells that stained at each intensity category and multiplying that value by the weighted intensity of the staining, using the formula HSCORE = Pi(i + l), where i represents the intensity scores and Pi is the corresponding percentage of the cells. In each slide, five different areas and 100 cells in each area were evaluated under a microscope with 40x objective magnification, the percentage of cells for each intensity within these areas were determined by two investigators blinded to the treatments and menstrual cycle phases of the samples, and the average score was then used.
Preparation of Nuclear Extracts
Nuclear extracts from endometrial stromal cells grown to confluence in 60-mm plates were obtained using a nuclear extraction kit (Aktiv Motif, Carlsbad, CA). Briefly, cells were washed with ice-cold PBS and phosphatase inhibitors, removed from the dish by scraping with a cell lifter, and transferred to prechilled tubes. Cell suspensions were centrifuged at 4°C for 5 min at 500 rpm. Pellets were resuspended in the hypotonic buffer, incubated for 15 min on ice, and, after adding a detergent, centrifuged again at 4°C for 30 sec at 14 000 x g. The pellet was resuspended in a lysis buffer and incubated for 30 min on ice on a rocking platform. The suspension was centrifuged at 4°C for 10 min at 14 000 x g and the supernatant (nuclear fraction) was aliquoted and frozen at –80°C. An aliquot of each sample was used to quantify the nuclear protein amount via Coomassie protein assay (Pierce, Rockford, IL). Five micrograms of nuclear extract sample was loaded into each well and assayed according to the manufacturer's directions using a microplate reader. Equal loading was also confirmed by performing Ponceau S staining of the nitrocellulose membranes. Ponceau S is a product designed for rapid (5 min) staining of protein bands on nitrocellulose or polyvinylidene fluoride membranes and confirms equal protein transfer to the membrane [17].
Statistical Analysis
Ratios of phospho-Akt, Akt, and GAPDH band levels and HSCORE ratios of immunohistochemistry and immunocytochemistry were normally distributed as tested by Kolmogorov-Smirnov test, and thus were analyzed by ANOVA and post hoc Tukey test for pairwise comparisons. P < 0.05 was considered to be statistically significant. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA). Each experiment was repeated three times using cells prepared from three independent endometrial tissue specimens.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endometrial tissues (early proliferative, n = 4; late proliferative, n = 5; early secretory, n = 4; late secretory, n = 5) and decidual tissues (n = 5) were evaluated by immunohistochemistry for phospho-Akt staining.
Immunohistochemical results revealed that stromal and glandular cells expressed both nuclear and cytoplasmic staining for phospho-Akt throughout the menstrual cycle and during early pregnancy (Fig. 1). The immunoreactivity in endometrial cells was mostly nuclear and reached the highest level during late proliferative phase (Figs. 1b and 2). On the other hand, in the early secretory phase, the phospho-Akt immunoreactivity was weaker and mostly cytoplasmic, similar to that observed in the early proliferative phase (Figs. 1, a and c, and 2). Moreover, during the late secretory phase and early pregnancy, glandular cells showed stronger but heterogeneous immunoreactivity for phospho-Akt compared with early secretory phase samples (Fig. 1, c–e). Specifically, some glandular cells showed nuclear immunoreactivity while others revealed weak cytosolic immunoreactivity, even from within the same gland segment. Moreover, decidual cells expressed weak and mostly cytoplasmic immunoreactivity for phospho-Akt (Figs. 1e and 2). When the nuclear:cytoplasmic (N:C) ratio of phospho-Akt for glandular and stromal cells was evaluated, the highest ratio was found in late proliferative phase compared with all other cycle phases and to early pregnancy (Fig. 2).
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Estradiol Stimulates Akt Phosphorylation in Human Endometrial Stromal Cells
To investigate the effect of estradiol on Akt activation, cultured endometrial stromal cells were treated with estradiol (10–8 M) for short (5–90 min) (Fig. 3A) and long (3– 24 h) exposures (Fig. 3B). Endometrial stromal cells in culture expressed Akt and phospho-Akt. Total Akt levels in both estradiol-treated and control cells were similar for both short- and long-term treatments. On the other hand, estradiol-treatment induced a significant increase in the phosphorylation of Akt, as detected by phospho-serine 473 Akt antibody (Fig. 3A). The increase in the phosphorylation of Akt was present at 15 min in estradiol-treated cells when compared with control cells (Fig. 3A). Phospho-Akt level in estradiol-treated cells returned to baseline levels observed in control cells at 90 min of treatment (Fig. 3A). In cells treated with estradiol, the phospho-Akt level was 46% and 53% higher at 5 and 15 min, respectively, compared with control cells (P < 0.05) (Fig. 3A). Moreover, in cells treated with estradiol for 3–24 h, both total Akt and phospho-Akt levels were no different than observed for untreated cells (Fig. 3B). Thus, long-term estrogen treatment of endometrial stromal cells did not affect phospho-Akt and Akt levels. GAPDH immunoblot analysis was performed for normalization of these results (Fig. 3, A and B).
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Estradiol Activation of Akt Phosphorylation Is Unaffected by an Estrogen Antagonist
To investigate the estradiol-mediated Akt phosphorylation, stromal cells were treated for 15 min with estradiol alone or estradiol in addition to wortmannin (10 nM), a PI3K inhibitor, and ICI 182 780, a pure estrogen receptor antagonist. While Akt phosphorylation by estradiol was blocked by wortmannin, this phosphorylation was not inhibited by ICI 182 780 (Fig. 4). When used alone, ICI 182 780 did not induce a different response in Akt phosphorylation as compared with untreated control cells (Fig. 4). This result suggests that estradiol regulates Akt phosphorylation through the PI3K signaling pathway but that this activation is ligand specific.
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Phospho-Akt Level Increases in the Nucleus Following Estradiol Stimulation
To assess the intracellular localization of phospho-Akt after estradiol treatment, we carried out immunocytochemistry of endometrial stromal cells cultured in chamber slides. Immunocytochemistry results revealed that estradiol-treated cells exhibited weak cytoplasmic but strong and mostly nuclear immunoreactivity for phospho-Akt, whereas control cells revealed weaker immunoreactivity with less nuclear localization (Fig. 5, A and B). Moreover, no difference in total Akt immunoreactivity was detected in cells treated with estradiol when compared with control cells (data not shown).
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To confirm our immunocytochemistry results, we carried out Western blot analysis of nuclear extracts obtained from estradiol- and vehicle-treated (control) stromal cells for 5 and 15 min to test the nuclear localization of phospho-Akt. Western blot results showed that estradiol treatment increased phospho-Akt levels in the nucleus compared with control (P < 0.05; Fig. 5C), similar to those observed using immunocytochemistry.
Effects of Selective Estrogen Receptor Modulators on the Phosphorylation of Akt
To determine if Akt activation is also regulated by selective estrogen receptor modulators, endometrial stromal cells were treated with estradiol (10–8 M), genistein (10–6 M), tamoxifen (10–7 M), and raloxifene (10–6 M) for 15 min. Genistein- and raloxifene-treated groups showed similar levels of Akt-phosphorylation compared with control cells. On the other hand, tamoxifen treatment increased the phospho-Akt levels when compared with control cells (P < 0.05; Fig. 6).
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The Effect of Estradiol on Akt Phosphorylation in PTEN-Mutant and Nonmutant Cells
To test whether the effect of estradiol on Akt phosphorylation is related to PTEN (phosphatase and tensin homolog deleted on chromosome 10), an upstream inhibitory protein of Akt phosphorylation, we used MCF-7 cells that have endogenous wild-type PTEN, and Ishikawa and RL-95 cells that are PTEN-mutant. Cells were treated with estradiol (10–8 M) and vehicle (control) for 5 and 15 min. Ishikawa and RL-95 cells showed extremely high levels of phospho-Akt in control cells, and estradiol treatment did not increase phospho-Akt levels any further (Fig. 7). However, when compared with control cells, estradiol treatment induced a significantly higher level of phospho-Akt in MCF-7 cells (P < 0.05; Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estradiol is thought to exert its biological effects by at least two mechanisms: one termed genomic and the other nongenomic [21]. Our in vitro findings regarding the rapid phosphorylation of Akt after exposure to estradiol and tamoxifen seem to be achieved via a nongenomic signaling pathway. This may produce activation of estrogen-mediated Akt-dependent genes, a subject of active pursuit in our laboratory. ERs also mediate many biological effects of estrogen by binding to ERE in the promoter regions of target genes or through protein-protein interactions on chromatin (termed tethering) [2]. It has been previously shown that growth factors such as epidermal growth factor and insulin-like growth factor increase the activity of protein kinases that phosphorylate different sites on ER, leading to ER-dependent transcription of target genes even in the absence of estradiol [22]. Both estrogen-dependent and estrogen-independent activation of ER are involved in the classic genomic pathway and take hours to produce maximal response through de novo protein synthesis [23].
In contrast with such genomic effects, the nongenomic effects of estrogen occur in minutes after estrogen exposure. Several signaling pathways are rapidly stimulated by estrogen in target cells expressing ER and/or ERß. However, these signaling cascades were also shown to be activated in cells without endogenous ER. Recently, Migliaccio et al. [24] showed that estrogen is able to induce a rapid activation of a signaling pathway involving Src-related tyrosine kinases and MAP kinases. Although it has been shown that estrogen stimulates the activity of Akt in ER-positive and -negative cancer cells [7, 25], this is the first report showing activation of Akt by estrogen in human endometrial stromal cells in vivo and in vitro. Two recently published studies describe Akt activation in rat endometrial and human decidual cells, supporting our in vivo and in vitro findings [26, 27]. Dery et al. [26] have shown that, in rat endometrium, phospho-Akt level decreased significantly during estrus and that estrogen exposure increases levels of Akt and phospho-Akt. Additionally, Yoshino et al. [27] recently reported that the total Akt level did not change in endometrial cells either by estrogen, progesterone, or both, even after 12 days of treatment, while the phosphorylation level significantly increased. Consistent with a previous report by Tsai et al. [25], we have observed that 5–15 min treatments of endometrial stromal cells with estrogen rapidly induce phosphorylation of Akt. We propose that estrogen-mediated Akt activation in endometrial stromal cells is a nongenomic effect because estrogen-mediated phosphorylation of Akt was maximal in only 5 min. We did not observe changes in Akt phosphorylation when compared with control cells with longer estrogen treatments. ICI 182 780 did not block estrogen-mediated phosphorylation of Akt, suggesting that ER binding to chromatin is not required for this effect. One of the ways that ICI 182 780 works as an estrogen antagonist is by producing conformational changes in the ER that preclude binding to an ERE [28]. In a recent study, Klotz et al. [29] showed that ICI 182 780 does not suppress estrogen-activated Akt in mouse endometrium, a finding in accordance with our results in human endometrial stromal cells. On the other hand, the same authors found that estrogen does not produce phosphorylation of Akt in endometrial cells of ER-knockout mice, suggesting that this activation is ER dependent. When taken with findings of Klotz et al. [29], our results suggest that ICI 182 780 may not antagonize some of the nongenomic actions of estrogen, including the effect on ER-mediated phosphorylation of Akt.
Recently, rapid effects of estrogen have been described in neurons and endothelial cells. It is suggested that estrogen may protect neurons against apoptosis and may modulate endothelial nitric oxide synthase expression and activity in vascular endothelium. These rapid effects of estrogen are proposed to be important for neurodegenerative and cardiovascular diseases [7].
Recent studies have also shown that Akt may change the estrogenic response of cells by phosphorylating the ER [30]. We speculate that Akt activation by estradiol may cause phosphorylation of ERs in vivo and in vitro. This, in turn, may represent reciprocal activation of these signaling pathways (i.e., positive feedback), suggesting a role for in vivo signaling in endometrium. Our in vivo results, showing increased nuclear localization and highest immunoreactivity of phospho-Akt in the proliferative phase, suggest that Akt phosphorylation is likely to be related to estrogen levels, which peak at this phase in the menstrual cycle. Likewise, immunocytochemistry results, showing stronger and predominantly nuclear immunoreactivity for the phosphorylated form of Akt in endometrial stromal cells, further supports this conclusion. However, another possibility is that there may be an increase in the nuclear Akt phosphorylation rather than a cytoplasmic to nuclear translocation. Nuclear translocation of activated Akt after its phosphorylation has been reported in other recent studies [30–33]. Activated Akt promotes cell survival by phosphorylating and modulating the activity of various nuclear transcription factors such as the forkhead transcription factor family, CREB, and nuclear factor-kappa B [34]. On the other hand, during the late secretory phase and in early pregnancy, increased Akt activation may depend on increased cytokine levels, such as tumor necrosis factor .
The increased Akt phosphorylation in response to estrogen in MCF-7 cells but not in PTEN-mutant cells suggests that this estrogenic effect is closely related to PTEN. Estrogen may activate Akt by inhibiting PTEN in endometrium because we have recently demonstrated that estrogen increases PTEN phosphorylation, which may inactivate the protein [16], which suggests a circumstantial link between estrogen, PTEN, and Akt phosphorylation. The inactivation of PTEN may produce increased activation of Akt. Alternatively, the balance of ER isotypes may contribute to estrogen-mediated effects on Akt signaling.
In conclusion, we have shown that estradiol stimulates Akt phosphorylation and it seems that this activation is too rapid to involve direct activation of EREs. Moreover, estrogen directly affects the Akt signaling pathway by regulating PI3K activity in endometrial cells because wortmannin eliminates estrogen-induced Akt phosphorylation. Thus, we speculate that estrogen may exert part of its proliferative and antiapoptotic effect in endometrial cells by a nongenomic mechanism that utilizes the Akt signaling pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 4 January 2004.
First decision: 14 January 2004.
Accepted: 9 April 2004.
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REFERENCES |
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