OBJECTIVE:Estrogen receptor interacts in several types of cells with phosphatidyl-inositol 3-kinase/Akt pathway regulating cell survival and apoptosis. No data are available of the Akt/PKB signaling and its role in the endometrial homeostasis of the postmenopausal uterus. The aim of the present investigation was to study the Akt/protein kinase B signaling in tissue samples retrieved from postmenopausal endometrium of the human uterus with parallel observation of the changes of the expression and phosphorylation of ERalpha. STUDY DESIGN:Sixty disease-free postmenopausal women were enrolled in the study. Endometrial tissue samples were obtained from diagnostic curettage or direct from the uterus after hysterectomy done for benign uterine lesions other than endometrial disease. For comparison, the studied parameters were also analyzed in endometrial samples of women with regular menstrual cycles (n=16). In each individual tissue sample the expression and phosphorylation of ERalpha, Akt, and cyclin D1 was analyzed by Western blotting. RESULTS:The level of Akt protein did not show significant change, however, the activation of Akt proteins and the expression of ERalpha increased parallel with serum estrogen (E2) levels, suggesting the role of E2 in Akt activation and ERalpha expression. The level of pERalpha(Ser167) changed parallel with pAkt(Ser473) levels. Significant correlation was found between the changes of pERalpha and ERalpha (r=0.650399, p<0.005), and in that of pERalpha and pAkt (r=0.639643, p<0.007), respectively. The expression of cyclin D1 was increased in samples with elevated pAkt levels. CONCLUSION:The results are indicating that the postmenopausal endometrium responds to E2 by both genomic and nongenomic mechanism. The interaction between ERalpha and Akt plays crucial role in the regulation of proliferative activity in postmenopausal endometrium.

Objective: To study the expression of FOXO1 and pSer(256)-FOXO1 parallel to Akt and pSer(473)-Akt in leiomyoma compared with adjacent myometrium from human uteri.Design: Prospective study.Setting: University departments.Patient(s): Thirty-eight cyclic and 20 menopausal women who underwent hysterectomy for benign indications.Intervention(s): None.Main Outcome Measure(s): Western blot analyses were used for evaluation in leiomyoma and adjacent myometrium of Akt and pSer(473)-Akt, 14-3-3 gamma proteins and expression and subcellular distribution of FOXO1 and pSer(256)-FOXO1 during the menstrual cycle and at menopause.Result(s): The present study demonstrates the expression of FOXO1 and pSer(256)-FOXO1 at the tissue level in the human uterus leiomyoma and adjacent myometrium. The level of phosphorylated FOXO1 in leiomyoma was higher than in matched myometrium. The pSer(256)-FOXO1 in leiomyoma during menstrual phases was located mostly in the nuclear fraction comparison to that of the myometrium. The reason for this difference is presumably the simultaneously detected lower level of 14-3-3 protein.Conclusion(s): Abundant level of the phosphorylated FOXO1, its impaired nucleocytoplasmic shuttling, and the lowered expression of 14-3-3 protein in leiomyoma induces a shift in the cellular machinery toward a prosurvival execution program and thus presents a potential therapeutic target for treatment of leiomyoma. (Fertil Steril (R) 2010;94:1491-5. (C) 2010 by American Society for Reproductive Medicine.)

Patkany uterusban E2 kezeles hatasara az Akt foszforilacioja a Ser473 es a FOXO1 Ser 256 csoportokon fokozodik, amit ICI 182, 780 es Wortmannin gatol. Az Akt expresszio nem valtozott, a pAkt myomaban jelentősen fokozodott. A pAkt a proliferacios fazisban volt a legmagasabb. Myomakban a ciklus alatti Akt foszforilaciő fokozodasaval parhuzamosan az ER foszforilacioja, a cyclin D1 es Bcl-2 proteinek expresszioja is fokozodott. Myomaban az inaktiv pPTEN-Ser380 fokozodott a pAkt valtozasokkal megegyező modon. A myometriumokban pPTEN szintekben ciklus fazisoktol fuggő es pAkt-tol fuggetlen valtozasokat talaltunk. A PTEN inaktivacioja es Akt aktivacioja myomaban a menstruacios ciklus alatt parhuzamosan valtozott. A pFOXO1 (Ser256) protein szintje magasabb volt myomaban es valtozott a menstruacios ciklus alatt ill. menopauzaban. Myomaban a pFOXO-Ser256 elsősorban a magban volt kimutathato, feltehetően a nucleocytoplasmatikus transport zavara miatt, aminek az oka valoszinűleg a parhuzamosan meghatarozott 14-3-3 protein expresszio csokkenese.Myomaban a megvaltozott PTEN/ER/PI3K/Akt/FOXO1 jelatvitel a sejtekben sejttulelest biztosito (prosurvival) programok előterbe keruleset serkenti. Az ERalpha expresszio postmenopauzas endometriumban irregularisan valtozott. Az ER(Ser167) es Akt foszforilacioja magasabb ver E2 szintnel (>50pmol/L) fokozodott. A pAkt pozitivan korrelalt se E2 koncentracioval (r: 0.811, p 50pmol/L). The pAkt (Ser473) was abundant in endometrium of women with higher (>50 pmol/L) E2 concentration. The pAkt significantly correlated with Se E2 (r: 0.811, p<0.001) and pERalpha (r: 0.879, p< 0.001).

Az opioid peptidek sejtproliferaciot gatlo hatasat vizsgaltuk tenyesztett patkany es human uterus sejtekben. Mereseinkhez sejtszamlalast, DNS meghatarozast, MTT sejtproliferacios assay-t es immunoblot modszereket alkalmaztunk. Adataink alapjan az opioid peptidek patkany uterus sejtek osztodasat gatlo hatasaban ket elterő fazis kulonithető el az egyedfejlődes alatt, melyeket egy erzeketlen periodus valaszt el egymastol. Az opioid peptid es progeszteron receptor rendszerek kozott ketiranyu kapcsolat műkodik felnőtt patkany uterus sejtek, valamint human myometrium es endometrium sejtek szaporodasanak gatlo jellegű szabalyozasaban. Fiatal, eretlen patkanyok uterus sejtjeiben e molekularis kapcsolat meg nem mutathato ki. A naloxon- es az RU486-hatas időfuggesenek elteresei alapjan e ketiranyu kapcsolat elterő tamadaspontokon keresztul valosulhat meg. Az opioid peptidek sejtosztodast gatlo mechanizmusa es az opiat-progeszteron kolcsonhatas műkodőkepes human leiomyoma sejtekben is. Azonban [D-Met2-Pro5]-enkefalinamid (ENK) a leiomyoma sejtek nem-stimulalt szaporodasat is gatolta, es az ENK es DAMGO mellett itt [Met5]-enkefalin is hatasosnak bizonyult a 7 napos patkany uterusban leirtakhoz hasonloan. A myometrium sejtekben talaltakkal ellentetben, RU486 onmagaban nem serkentette a leiomyoma sejtek a szaporodasat, azonban az ENK gatlo hatasat kivedte. Az elteresek a sejtosztodas szabalyozasanak reszleges dedifferentaciojat mutathatjak, ami a pathomechanizmusban is szerepet jatszhat. | The inhibitory action of opioid peptides on the proliferation of cultured rat and human uterine cells was investigated. Cell counting, DNA determination, MTT cell proliferation assay and immunoblot technique were used to obtain data. Our data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between. A bidirectional interaction exists between the opioid peptide and progesterone signaling systems in the inhibitory regulation of cell proliferation in adult rat uterus and in human myometrium and endometrium. Uteri of immature rats lack this molecular connection. The difference between the time dependence of naloxon and RU486 actions suggests that, this bidirectional action is realized by targeting different elements in the mechanism of action. The opioid peptides' growth inhibitory system and the opiate-progesterone interaction are functional in human uterine leiomyoma cells. However, [D-Met2-Pro5]-enkephalinamide (ENK) inhibited the non-stimulated proliferation of leiomyoma cells and, besides ENK and DAMGO, [Met5]-enkephalin was effective as well, similar to that of found in 7-day-old rats. In contrast to myometrial cells, RU486 alone did not stimulate the proliferation of leiomyoma cells however; it antagonized the inhibitory effect of ENK. These alterations suggest a partial dedifferentiation of cell-division regulation and, might play a role in the pathomechanism.

The importance of FOXO transcription factors in regulating different aspects of cellular homeostasis and apoptosis has become apparent. Akt/protein kinase B has been shown to phosphorylate and inactivate members of FOXO family of transcription factors. Akt and its upstream regulator, phosphatidylinositol-3 kinase (PI3K) are involved in rapid action of estrogen (E2) in different cells and tissues. The aim of the present study was to analyze the E2/PI3K/Akt/FOXO pathway in rat uterus. In response to E2, phosphorylation of Akt/PKB on Ser473 and FOXO1 on Ser256 and Thr24 residues increased but with distinct kinetics, regulating the activation and inactivation of Akt and FOXO1 proteins, respectively. The antiestrogen ICI 182,780 prevented E2 induced Akt activation suggesting that estrogen receptors mediate this effect of E2. Intrauterine injection of Wortmannin caused a decrease in the phosphorylation of Ser473 of Akt, and attenuated phosphorylation of its downstream target FOXO1 at Ser256 and at Thr24. However, the effect of E2 on phosphorylation of Thr24 showed a kinetic pattern distinct from that of Ser256. Our results suggest that the E2/PI3K/Akt/FOXO1 pathway in rat uterus is functioning even at the lack of ovarian hormones and responses to E2 treatment. Estradiol increases Akt phosphorylation through a Wortmannin sensitive way, presumably involving PI3K. The present work shows that PI3K plays a crucial role in the phosphorylation and inactivation of FOXO1 in vivo, indicating that the regulation of this transcription factor is a more complex event in uterine cells requiring further investigations.

PTEN phosphatase, a product of PTEN tumor suppressor gene, exists in cells in phosphorylated and unphosphorylated form and has a central role in regulation of PI3K/Akt signalling which is involved in non-genomic action of estradiol. The purpose of this study was to analyze the level of total PTEN and phosphoPTEN parallel to phosphoAkt in leiomyoma and adjacent myometrium during menstrual cycle and at menopause. The expression of total PTEN in leiomyoma and myometrium did not change throughout the experiments. However, the level of phosphoPTEN was increased in leiomyoma during menstrual cycle. The phosphorylation of PTEN in myometrium was lower during secretory phase than that of proliferative phase. The phosphoAkt was abundant in leiomyoma, and its expression was higher during menstrual cycle than in myometrium. The phosphorylation of PTEN was directly related to phosphoAkt, suggesting a direct link between the inactivation of PTEN and activation of Akt. At the decline of sexual steroids, at menopause, no differences were observed in the expression of studied proteins between the two types of tissues. Our results suggest that the altered phosphorylation of PTEN protein and the consequent activation of survival signals may contribute to the pathomechanism of leiomyoma.

This study investigated the expression and activation of Akt/PKB in developing and adult rat uterus. Expression of Akt was observed in uteri from adult ovariectomized and 7–35-day-old rats and no changes were observed in response to in vivo estradiol treatment (1–100μg/100g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr308 and Ser473 regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser473 residue in the untreated, control uteri, while phosphorylation of Thr308 was observed only after estradiol 17β (E2) treatment. The effects of E2 treatment were age dependent, no response was induced in 11-day-old uteri, while in 28 days and older rats the activation of Akt at both regulatory sites, Ser473 and Thr308, increased, the first response was detected 2h after treatment, reaching the highest rate at 6h. The rate of phosphorylation was stronger at Ser473 residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr308 seems to be entirely estrogen dependent, while the phosphorylation of Ser473 is regulated by other factors as well as estrogen.

Adult ovariectomized rats were implanted with [d-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17β (E2) at a dose 10 μg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERα) and beta (ERβ) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52±11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24–48 h following ENK implantation, expression of ERα and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERβ level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERα and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.

Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.

The expression and activation of serine/threonine protein kinase, Akt, in leiomyoma and in adjacent myometrium of human uteri was studied parallel with the changes of Bcl-2, Bax proteins, estrogen and progesterone receptors during menstrual cycle and early stage of the menopause. Abundant expression of Akt protein was detected in the studied tissues during menstrual cycle, the rate of increase was higher in leiomyoma than in corresponding myometrium. The expression of estrogen receptor alpha, progesterone receptor and of Bcl-2 protein changed parallel with that of Akt protein. The level of phosphorylated Akt (pAkt(473)) was seen only in leiomyoma samples from the growing period of tumors. At early stage of menopause levels of all studied proteins were lower than that in the menstrual cycle with the exception of Bax protein expression, which was high in leiomyoma. Our data suggest the involvement of phosphatidylinositol 3-kinase/Akt signaling in the pathomechanism of leiomyoma.

Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.

The aim of the present experiment was to investigate the effect of [D-Met2,Pro5] enkephalinamide (ENK) implantation on the development of the uterus during 8–33 days of age and the involvement of epidermal growth factor (EGF) in the effect. Administration of ENK was attained by osmotic minipumps (5 μg/h) implanted intraperitoneally. ENK resulted in a decrease in the EGF content of the uterus, which was already significant after 48 h of the implantation. The DNA content 24 and 48 h after the treatment decreased, no change at 72 h was found, however the protein/DNA ratio on the effect of ENK treatment was significantly decreased at this time in all examined age groups. High affinity and lower capacity competitive naloxone binding sites were demonstrated in the membrane fraction of the uteri. Seventy-two h after ENK treatment the binding capacity of these sites significantly dropped. The present results suggest a novel multiple interaction between estrogen and two probably paracrine hormones, EGF and opioid peptide, in the regulation of growth and development of the uterus.

The present studies demonstrate, for the first time, that the binding of activator protein-1 (AP-1)-DNA in rat uterus and the estrogen-sensitive areas of the hypothalamus, as measured by electrophoretic mobility shift assay, is increased 2 h after intraperitoneal injection of [D-Met(2),Pro(5)]enkephalinamide. The effect was prevented by the opiate antagonist naltrexone given 30 min before the administration of [D-Met(2),Pro(5)]enkephalinamide, suggesting the involvement of opioid peptide receptors in the observed effects. The present findings support the role of opioid peptides in the regulation of transcription in estrogen-sensitive cells.

The effects of a single injection or continuous infusion of opioid peptide, [d-Met2,pro5]enkephalinamide (ENK) on the hormone binding and transcriptional properties of estrogen receptors were investigated in estradiol (E2) treated rat uterus. The level of estrogen- (ER) and progesterone receptor (PR) proteins, the hormone binding of E2 receptors and the effects of single injection of ENK with or without naltrexone (NAL) on the E2-induced changes in the level of Fos and Jun proteins and the binding of AP-1 proteins to DNA were studied. The receptor proteins levels were determined by Western blots and the binding of AP-1 to DNA by electrophoretic mobility shift assay. Both the ER and PR protein concentrations and the [3H]Estradiol binding to the high affinity nuclear receptors decreased after ENK treatment during the first two days. At 72 h the PR concentration decreased further, while no significant changes were found in the level of ER, however, at this time the former competitive E2 binding turned into positive cooperativity. The E2-induced increase in the level of Fos proteins and the binding of AP-1 proteins to DNA was inhibited by a single injection of ENK. We conclude that the endogenous opioid peptides may interact with E2 in the gene regulation of rat uterus.