Rapid non-classical effects of steroids on the membrane receptor dynamics and downstream signaling in neurons.

Contribution to Special Issue on Fast effects of steroids. Although rapid effects of steroid hormones on membrane receptors and intracellular signaling molecules have been extensively studied in neurons, we are only beginning to understand the molecular mechanisms behind these non-classical steroid actions. Single molecule tracking (SMT) studies on live cells demonstrated that surface trafficking of membrane receptors determines their ligand binding properties and downstream signaling events. Recent findings suggest that one of the underlying mechanisms of non-classical steroid actions is the alteration of receptor movements on the membrane surface. In order to highlight this novel aspect of steroid effects, we first address the types of receptor movements in the plasma membrane and the role of cortical actin dynamics in receptor movement. We then discuss how single molecules and the surface movements of receptors can be detected in live cells. Next, we review the fundamental processes, which determine the effect of steroids on the plasma membrane: steroid movement through the lipid bilayer and the role of steroid membrane receptors. Using glutamate and neurotrophin receptors (NTRs) as examples, we demonstrate the features of receptor dynamics in the membrane. In addition, we survey the available data of rapid steroid actions on membrane receptor trafficking: we discuss how glucocorticoids act on the surface diffusion of glutamate receptor molecules and how estradiol acts on NTRs and gamma-aminobutyric acid type A receptors (GABAARs) and their related signaling events as well as on cortical actin. Finally, we address the physiological relevance of rapid steroid action on membrane receptors dynamics.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17ß-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Phosphorylation of PTEN (phosphatase and tensin homologue deleted on chromosome ten) protein is enhanced in human fibromyomatous uteri.

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.

Effect of estrogen and inhibition of phosphatidylinositol-3 kinase on Akt and FOXO1 in rat uterus.

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.

Differential expression of Akt/protein kinase B, Bcl-2 and Bax proteins in human leiomyoma and myometrium.

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.

Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus.

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 (1-100 microg/100g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr(308) and Ser(473) regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser(473) residue in the untreated, control uteri, while phosphorylation of Thr(308) was observed only after estradiol 17beta (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, Ser(473) and Thr(308), increased, the first response was detected 2h after treatment, reaching the highest rate at 6h. The rate of phosphorylation was stronger at Ser(473) residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr(308) seems to be entirely estrogen dependent, while the phosphorylation of Ser(473) is regulated by other factors as well as estrogen.

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment.

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-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) 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 ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta 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 ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.

Akt/protein B and estradiol receptor alpha expressions in postmenopausal endometrium.

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.

Involvement of FKHR (FOXO1) transcription factor in human uterus leiomyoma growth.

OBJECTIVE: To study the expression of FOXO1 and pSer256-FOXO1 parallel to Akt and pSer473-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 pSer473-Akt, 14-3-3 gamma proteins and expression and subcellular distribution of FOXO1 and pSer256-FOXO1 during the menstrual cycle and at menopause. RESULT(S): The present study demonstrates the expression of FOXO1 and pSer256-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 pSer256-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.