Molecular Human Reproduction, Vol. 9, No. 5, 265-269,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Akt as a possible intracellular mediator for decidualization in human endometrial stromal cells
Submitted on September 11, 2002; accepted on January 27, 2003
1 Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655 and 2 CREST Japan Science and Technology, Kawaguchi 332-0012, Japan
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. e-mail: yutakaos-tky{at}umin.ac.jp
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ABSTRACT |
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To gain an insight into intracellular mechanisms involved in differentiation of human endometrial cells into decidual cells, we examined the presence of Akt, an emerging intracellular mediator in human endometrial stromal cells (ESC). We explored the mechanisms regulating Akt phosphorylation during the process of progesterone-induced decidualization using a primary cell culture system of ESC. Both Akt and phosphorylated Akt (phospho-Akt) were present in ESC. The total Akt level in ESC cultured for 12 days in the absence of ovarian hormones was similar to ESC treated with estradiol (E2) at 10 ng/ml, progesterone at 100 ng/ml or E2 plus progesterone (E2 progesterone), whereas the levels of phospho-Akt were markedly decreased with progesterone or E2 progesterone, compared to control cells. Notably, phospho-Akt levels increased during 12 days of culture in parallel with an increase in total Akt in untreated cells. An increase of phospho-Akt in the E2 progesterone-treated cells was marginal. The level of phospho-Akt in E2 progesterone-treated cells was markedly reduced compared to control cells at all time points examined. Treatment of the cells with 8-bromo-cAMP decreased the amount of phospho-Akt in ESC in as short a period as 15 min, while no discernible change was observed in the untreated cells. Conversely, H89, an inhibitor of protein kinase A (PKA), significantly increased the amount of phospho-Akt. The addition of H-89 reversed the decrease in the level of phospho-Akt seen in the cells treated with E2 progesterone. Thus, we demonstrated the presence of Akt and its phosphorylated form in human ESC. We further suggest that Akt phosphorylation through the cAMP/PKA signal transduction pathway may regulate cellular functions coupled with decidualization.
Key words: Akt/cAMP/decidualization/endometrium/protein kinase A/B
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Introduction |
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Decidualization, a process of endometrial differentiation, is essential for embryo implantation and maintenance of pregnancy. During the process of decidualization, characteristics of endometrial cells change profoundly. This was demonstrated by marked variation in the expression of numerous genes in decidualized human endometrial stromal cells (ESC) in vitro (Popovici et al., 2000). It is well known that progesterone induces decidualization of ESC, though the effect is relatively weak. Ligands that activate protein kinase A (PKA) pathways, such as prostaglandin E2 (PGE2) (Frank et al., 1994) and relaxin (Tseng et al., 1992), are also known to induce decidualization in the absence of progesterone. However, our knowledge regarding the global picture of intracellular signalling pathways involved in decidualization is preliminary at best.
In recent years, several lines of evidence have been accumulated to demonstrate the importance of Akt, which is also known as protein kinase B (PKB), as an intracellular mediator (Staal et al., 1977; Bellacosa et al., 1991; Kandel and Hay, 1999; Brazil and Hemmings, 2001). Growth factors such as insulin-like growth factor-I, insulin and platelet-derived growth factor phosphorylate Akt in a phosphatidylinositol 3-kinase (PI3K)-dependent way. The activated Akt, in turn, phophorylates its target proteins (Blume-Jensen and Hunter, 2001). These substrate proteins are shown to regulate various cellular processes, including cell growth, apoptosis, glycogen synthesis, protein synthesis, transcription, etc.
Despite a large body of data suggesting Akt is a pivotal player in a variety of physiological and pathophysiological processes, it is unknown whether Akt has a role in the endometrium. As a first step toward addressing this, we studied the expression of Akt in ESC using an in-vitro decidualization model. We further explored intracellular mechanisms involved in Akt phosphorylation in decidualization.
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Materials and methods |
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Patients and samples
Endometrial tissue was collected from 20 patients undergoing hysterectomy for benign gynaecological conditions. All patients had regular menstrual cycles, and none of the patients had received hormonal treatment before surgery. All endometrial samples were collected during the mid to late proliferative phase of the cycle. The study was approved by our Institutional Review Board, and written informed consent was obtained from each patient.
Isolation and culture of human endometrial stromal cells
Endometrial biopsies were collected into sterile medium and rinsed to remove blood cells. The tissue was minced into small pieces and digested by incubation in DMEM/Ham’s F-12 culture medium containing type I collagenase (0.25%; Sigma, St Louis, MO, USA), DNase I (15 IU/ml; Takara Shuzo, Tokyo, Japan) for 60 min at 37°C with agitation. The dispersed endometrial cells were separated by filtration through a 40 µm Nylon Cell Strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA). The dispersed ESC passed through the strainer into the filtrate, whereas the epithelial glands, which remained intact, were retained by the strainer. Stromal cells in the filtrate were collected by centrifugation and resuspended in phenol-red free DMEM/Ham’s F-12 containing 10% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were plated in a 100 mm culture plate and kept at 37°C in a humidified 5% CO2/95% air environment. At the first passage, ESC were plated at a density of 2x105 cells/ml in 60 mm culture plates. The purity of the stromal preparations was confirmed by positive cellular staining for vimentin.
In-vitro decidualization was performed as described previously (Koga et al., 2001). Briefly, ESC grown to subconfluence in 60 mm plates were rinsed and incubated in the medium with 2.5% charcoal-stripped FBS with either estradiol (E2, 10 ng/ml), progesterone (progesterone, 100 ng/ml) or E2 plus progesterone (E2 progesterone), or vehicle (0.1% ethanol) for up to 12 days. During the time-course experiments, the cells were treated with E2 progesterone, which appeared to be a more physiological and potent stimulus for decidualization than progesterone (Irwin et al., 1989; Koga et al., 2001). Culture medium was collected and replaced every 3 days. Decidualization was assessed by measurement of prolactin (PRL) in conditioned media using an SR1 analyser (Stat Profile Ultra; Nova Biomedical Boston, MA, USA). The sensitivity limit of this assay was 500 pg/ml and the intra-assay and inter-assay coefficients of variance were <10%. The concentration of cAMP in the conditioned media was measured using a cyclic AMP immunoassay kit (R&D, Minneapolis, MN). The sensitivity of the assay was 0.3 pmol/ml. The intra-assay and inter-assay coefficients of variance were <5%.
In some experiments, the cells were treated with 8-bromo-cAMP (1 mmol/l; Sigma) for up to 60 h or protein kinase A (PKA) inhibitor, H-89 (Calbiochem, San Diego, CA, USA) (5 µmol/l) for up to 6 h at 37°C.
Western blot analysis
Cultured cells were homogenized in lysis buffer [50 mmol/l Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/l dithiothreitol]. Homogenized cells were further diluted to give a final concentration of 1 mg total protein/ml with lysis buffer containing 0.1% bromophenol blue. Samples were subjected to Western blot analysis. Samples of 20 µg protein per lane were resolved by 10% SDS–PAGE in parallel lanes. Proteins were transferred onto a nitrocellulose membrane and incubated with anti-rabbit Akt antibody (recognizing total human Akt) or anti-rabbit phospho-specific (Ser473) Akt antibody (1:1000; New England BioLabs, Beverly, MA, USA) as primary antibody, and anti-rabbit horseradish peroxidase antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as secondary antibody. Phosphorylation of Ser473 in Akt is known to correlate with the activation of the molecule (Alessi et al., 1996). Immune complexes were visualized by the ECL Western blotting system (Amersham, Buckinghamshire, UK).
Statistical analysis
Student’s t-test was used for the statistical analysis of concentrations of PRL and cAMP in culture media. P < 0.05 was considered to be statistically significant.
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Results |
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Akt immunoreactivity was demonstrated in ESC. Immunoreactivity of phosphorylated Akt (phospho-Akt) was also detected in ESC. Figure 1 illustrates the presence of both total and phospho-Akt in ESC cultured for 12 days in the absence or presence of either E2, progesterone or E2 progesterone. Total Akt protein levels were the same in ESC cultured in the absence or presence of ovarian hormones. Interestingly, the amounts of phospho-Akt in ESC treated with progesterone or E2 progesterone were markedly decreased compared with ESC cultured with E2 alone or in the absence of hormones.
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The cells treated with E2 progesterone for 12 days underwent decidualization as judged by their ability to secrete PRL. As shown in Figure 2A, PRL production by the cells treated with E2 progesterone increased significantly compared with control cells from day 6 to day 12 of the culture period. Figure 2B shows the changes in total Akt and phospho-Akt in the cells during the culture period. The amount of total Akt was virtually the same in control cells and E2 progesterone-treated cells at any time point examined. However, the amount of total Akt in the ESC increased with time. Likewise, the amount of phospho-Akt increased throughout the culture period in the untreated cells. The increase in the treated cells was marginal. Again, the level of phospho-Akt was reduced in the E2 progesterone-treated cells compared with the relevant control. The cAMP concentration in culture media from E2 progesterone-treated cells was increased significantly (P < 0.05) as compared to control cells (Figure 2C).
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We then examined the effect of 8-bromo-cAMP in this system. As shown in Figure 3A, significantly increased concentrations of PRL were observed in steroid-treated cells compared with control cells after 24h of culture. The amount of total Akt was not afffected by treatment with 8-bromo-cAMP throughout a culture period of 6 h. On the other hand, the addition of 8-bromo-cAMP resulted in a decrease in the amount of phospho-Akt as early as 15 min. The decrease was sustained up to 6 h, whereas the amount of phospho-Akt in the untreated cells was constant during the culture period (Figure 3B). To see whether PKA is involved in the regulation of Akt phosphorylation, we tested the effect of H89, a PKA inhibitor (Figure 4). ESC treated with H89 showed an increase in the amounts of phospho-Akt, which was observed at 15 min. The increase was sustained up to 6 h, whereas no changes were observed without H89.
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We wanted to test whether PKA had a role in the E2 progesterone-induced reduction in phospho-Akt in decidualized ESC. The addition of H89 completely prevented the E2 progesterone-induced decrease in phospho-Akt (Figure 5).
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Discussion |
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Decidualization is a process during which endometrial cells undergo drastic change in their biochemical properties as well as morphological appearance. For instance, increased expression of PRL and insulin-like growth factor-binding protein 1 takes place in parallel with decidualization (Giudice et al., 1992; Telgmann and Gellersen, 1998). In addition, we have shown that angiogenic molecules such as platelet-derived endothelial cell growth factor and angiogenin are upregulated in the process of decidualization (Osuga et al., 1995; Koga et al., 2001). However, intracellular signalling pathways leading to decidualization are still far from clear.
In the present study, we demonstrated the presence of Akt in human endometrial cells. The level of phospho-Akt was found to decrease in cells undergoing in-vitro decidualization although the level of total Akt was the same as that in non-decidualized endometrial cells. Given the current fashionable concept of Akt as an intracellular regulator of a variety of cellular functions, modulation of Akt phosphorylation may therefore also play a role in decidualization.
Phosphorylation of Akt is known to be induced by activated PI3K through the conversion of PI(4,5)P2 to PI(3,4,5)P3. PTEN (gene, phosphatase and tensin homologue deleted on chromosome ten) can counteract the action of PI3K by its phosphatase activity to convert PI(3,4,5)P3 to PI(4,5)P2 (Cantley and Neel, 1999). Interestingly, PTEN has been shown to be highly expressed in decidualized stromal cells (Mutter et al., 2000). Therefore, the increased PTEN during decidualization could diminish the effect of PI3K, which may subsequently reduce Akt phosphorylation in ESC.
Multiple lines of evidence have suggested that the cAMP/PKA signal transduction pathway mediates the action of progesterone to induce decidualization (Tabanelli et al., 1992; Tang and Gurpide, 1993; Tang et al., 1993; Brar et al., 1997; Telgmann et al., 1997). Indeed, increased production of cAMP was observed in progesterone-induced decidualized cells in the present study. In addition, cAMP stimulated PRL production by ESC faster than progesterone. In the present study, both progesterone and 8-bromo-cAMP suppressed Akt phosphorylation whereas an inhibitor of PKA induced phospho-Akt. These results are compatible with the view that the level of phosphorylation of Akt, i.e. whether it is phosphorylated or not, might be intimately coupled with decidualization.
Cyclic AMP has been shown to exert different effects on Akt activation in a cell-type dependent manner, i.e. stimulatory effects in thyroid cells (Cass et al., 1999; Tsygankova et al., 2001) and ovarian granulosa cells (Gonzalez-Robayna et al., 2000), or inhibitory effects in Swiss 3T3, HEK293, COS and Rat2 cells (Kim et al., 2001). A recent study has shown that opposing effects on Akt activation are mediated by Epac and PKA, both of which are stimulated by cAMP (Mei et al., 2002). Activation of Epac leads to a PI3K-dependent Akt activation, while stimulation of PKA inhibited Akt activity. Another study reported that cAMP inhibits Akt phosphorylation by lowering Rap1 activity in a PKA-independent manner in rat C6 glioma cells (Wang et al., 2001), suggesting that cAMP-induced Akt activation might be quite complex. Our study indicated that cAMP induced a PKA-dependent inhibitory effect on Akt phosphorylation in ESC.
Interestingly, a recent study showed that phosphorylation of Akt stimulated PRL promoter activity via a CREB-dependent mechanism in GH3 rat pituitary tumour cells (Hayakawa et al., 2002). On the other hand, Akt phosphorylation was decreased in ESC undergoing decidualization despite increased PRL production by these cells. In addition, it has recently been reported that FKHR (FOXO1), a possible target protein of Akt, is involved in cAMP-induced decidual PRL gene expression while the mechanism is suggested to be Akt-independent (Christian et al., 2002). The disparities might be due to the difference in PRL promoters between pituitary and decidual PRL (DiMattia et al., 1990; Berwaer et al., 1994; Gellersen et al., 1994) and/or cell type-specific downstream effectors of Akt.
Akt phosphorylation is a stimulator of differentiation in muscle cells and adipocytes (Magun et al., 1996; Jiang et al., 1999). Conversely, Akt phosphorylation may play a role in inhibiting neuronal differentiation by preventing the growth-arresting effect of nerve growth factor (NGF) (Bang et al., 2001).
In the present study, reduced phosphorylation of Akt was associated with decidualization, i.e. differentiation of ESC. Therefore, it is reasonable to speculate that alterations in the status of Akt phosphorylation serve to regulate cellular functions in a range of tissues.
Recently, Zhang and colleagues reported the presence of Akt in ESC (Zhang et al., 2002). In their study, however, only non-phosphorylated Akt (not phospho-Akt) was detected in non-decidualized ESC. This finding is different from our study in which phospho-Akt was detected in non-decidualized ESC. Although the reason for the inconsistency is unknown at the present time, it might be due to different experimental conditions, e.g. differences in the antibodies utilized or exposure times in Western blot analysis.
The present study may have experimental limitations. A time-dependent increase in total Akt observed in this study might be influenced by culture conditions. Nevertheless, phospho-Akt was clearly reduced in decidualized ESC compared with control ESC. We therefore believe that phosphorylation of Akt is reduced in the process of decidualization.
In summary, we have shown the presence of Akt in ESC. We have further demonstrated that diminution of Akt phosphorylation was coupled with decidualization through the cAMP/PKA signalling pathway.
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Acknowledgements |
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We thank Emi Nose for technical assistance.
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