Regulation of the PI3-K/Akt survival pathway in the rat endometrium.

The occurrence of apoptosis and cell survival in the receptive uterus is intimately involved in the embryo implantation process in order to facilitate embryo attachment to the maternal endometrium. The initial stimulus leading to successful implantation might be triggered by the conceptus itself. By the end of rat embryo implantation, decidualization begins, followed by the regression of the decidua basalis on Day 14. The phosphatidylinositol 3-kinase (PI3-K) survival pathway and TGF-beta have been thought to play a role in this process. The objective of the present study was to investigate the regulation of the PI3-K/PTEN/Akt pathway in rat endometrium during pregnancy. Rats were killed on different days of pregnancy (Day 1-22 and postpartum) or pseudopregnancy (Day 1-9), and uteri were removed to collect endometrial tissues. The active form of Akt (pAkt) was increased at Day 5 of pregnancy and at Day 3 of pseudopregnancy as well as at Day 12 of pregnancy and at Day 1 postpartum. Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt3 was the only isoform phosphorylated at Day 5 during the implantation process and at postpartum as demonstrated by immunoprecipitation studies. PI3-K inhibition in vivo blocked Akt phosphorylation, reduced Smad2 phosphorylation, and reduced both TGF-beta2 and XIAP expression. PI3-K inhibition in cultured decidual cells led to inhibition of pAkt and decrease XIAP expression. These results suggest that Akt and XIAP may be important surviving signaling molecules by which apoptosis is regulated in the rat endometrium during pregnancy and that TGF-beta could be linked to this process.

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells.

BACKGROUND: During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-beta isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo. METHODS: Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1-9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms. RESULTS: Our results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner. CONCLUSION: Those results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

TGF-beta expression during rat pregnancy and activity on decidual cell survival.

BACKGROUND: During early rat pregnancy, trophoblast of the tiny embryo joins with the endometrium and epithelial cells undergo apoptosis. Near the end of pregnancy, regression of the decidua basalis (DB) is also observed (from day 14 to 20). However, little is known about the intra-cellular and molecular mechanisms involved in apoptosis regulation in the uterus during pregnancy. The objective of the present study was to investigate the presence and the developmental expression of transforming growth factor-beta isoforms (TGF-beta well known differentiation factor) in the rat endometrium throughout pregnancy and its action in vitro using cultured endometrial stromal cells. METHODS: In vivo: Rats were killed at different days of pregnancy (days 2-20) and uteri removed to collect endometrial protein extracts or the uteri were fixed, embedded and sectioned for immunohistochemistry (IHC) and in situ cell death analyses using TdT-mediated dUTP nick end labeling (TUNEL). In vitro: Rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were then collected and cultured. RESULTS: An increase of apoptosis in the DB on days 14, 16 and 18 was observed. Cleaved caspase-3 was clearly detected during regression of the DB by Western analysis and immunofluorescence. Western analyses using endometrial protein extracts demonstrated that TGF-beta1, TGF-beta2 and TGF-beta3 were highly expressed at the time of DB regression (day 14). During early pregnancy, TGF-beta1 and -beta2 expressions raised at days 5.5 to 6.5. TGF-beta3 protein was not detected during early pregnancy. IHC analyses revealed that TGF-beta1 and -2 were found surrounding both epithelium (luminal and glandular) in the stroma compartment at the implantation site, and TGF-beta3 was mainly located surrounding endometrial epithelium in the stroma compartment. Smad2 phosphorylation was increased at the time of DB regression. In vitro studies using decidual endometrial stromal cells revealed that TGF-beta1 induced apoptosis and Smad2 phosphorylation. Moreover, TGF-beta1 reduced both Akt (a well known survival factor) phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression in decidual endometrial stromal cells in vitro. CONCLUSION: Taken together, these results suggest that TGF-beta isoforms are regulated differently during pregnancy and may have an important role in the control of apoptosis and cell survival at specific stages during pregnancy.