Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Molecular and intra-cellular mechanisms involved in the regulation of apoptosis processes in endometrial cells are poorly understood and documented. We have investigated the possibility that Akt survival pathway might be involved in the regulation of apoptosis in the uterus during the estrous cycle. Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) or endometrial protein extracts collected for Western analysis. TUNEL analysis revealed that apoptosis was mainly found at estrus compared to other day of estrous cycle. TUNEL positive cells were apparent in luminal epithelial cells only. No apoptotic cells were observed at proestrus. In contrast, proliferation was maximal at proestrus as confirmed with the expression of CDC47/MCM7 (a cell proliferation marker). Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus. Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells. PTEN protein, a phosphatase involved in the regulation of Akt phosphorylation, was present at all days of estrous cycle and showed no significant regulation in relation to cycle. Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus. Akt protein expression was maximal at estrus. IHC revealed that Akt expression was high in both stromal and epithelial cells at estrus. Further studies using ovariectomized rats demonstrated that 17β-estradiol increased endometrial cell proliferation which was accompanied by an increase of both Akt expression and phosphorylation. These results suggest that increased Akt expression and activity in response to estradiol may be an important mechanism to protect endometrial cells from apoptotic triggering and to induce endometrial cell proliferation, whereas inhibition of Akt activity leads to caspase-3 activation and apoptosis in endometrial cells

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus

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

<p>Abstract</p> <p>Background</p> <p>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-β 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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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