József L. Környei

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.

Opioids regulate cell proliferation in the developing rat uterus : Effects during the period of sexual maturation

The present studies demonstrate, for the first time, that the rate of DNA synthesis in rat uterus of 21-32 days of age is inhibited by opioid peptides [D-Met2, Pro5]enkephalinamide. At around the time of vaginal opening (approximately 33 days) the opioids failed to act. High-affinity nuclear [3H]naloxone binding sites with linear Scatchard plots were detected in the uteri during the opioid-sensitive periods of DNA synthesis. Characteristics of these binding sites and the opioid sensitivity of uterine DNA synthesis are dependent on the age of the animals, the level of circulatory oestradiol and/or the maturity of the nuclear oestrogen receptor system.

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.

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.

Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17β-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation in the rat endometrium in correlation with the MIF.

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.

Opioid peptides inhibit the estradiol-induced proliferation of cultured rat uterine cells

The effect of opioid peptides on estradiol-induced cell proliferation in adult rat uterine primary cell cultures was studied. Estradiol increased cell density by 40%. This estradiol-induced stimulation of cell proliferation was decreased to control values by [D-Met2,Pro5]enkephalinamide. The opioid-induced inhibition of uterine cell proliferation was blocked completely by the specific opiate antagonist naloxone, while naloxone did not have any effect on its own. The inhibition of cell proliferation by enkephalinamide was apparent at each stimulatory estradiol concentration examined. This opioid effect was mediated mainly by the mu opiate receptor. The observed effects occurred within the physiological nanomolar concentration range. Enkephalinamide did not have any effect on the basal proliferation rate of adult rat uterine cells. However, enkephalinamide inhibited the basal rate of cell proliferation in cell cultures prepared from 7-day-old immature rats. In summary, here we present evidence of novel physiological direct cross-talk between the opioid and estrogenic signaling systems in the regulation of normal uterine growth.

Stimulation by oestradiol of soluble-protein synthesis in rat hypothalamus

The effect of oestradiol treatment on protein synthesis in the cytosol fraction (10(5) g supernatant) from ovariectomized mature and developing female rat hypothalami were studied. Results obtained on adults by double-label technique and SDS polyacrylamide gel or cellogel electrophoresis show that, as in the uterus, oestradiol-induced protein synthesis (IP) occurred in the cytosol fraction of the hypothalamus. The molecular weight of IP was about 40 000 dalton. During postnatal development, IP was also observed in cytosol fractions from the hypothalami of female rats 14, 21 and 28 days old. No clear-cut effect of oestradiol was found in the 7-day-old animals.

Antiestrogenic effect of opioid peptides in rat uterus.

The effects of a single injection or continuous infusion of opioid peptide, [D-Met(2),pro(5)]enkephalinamide (ENK) on the hormone binding and transcriptional properties of estrogen receptors were investigated in estradiol (E(2)) treated rat uterus. The level of estrogen- (ER) and progesterone receptor (PR) proteins, the hormone binding of E(2) receptors and the effects of single injection of ENK with or without naltrexone (NAL) on the E(2)-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 E(2) binding turned into positive cooperativity. The E(2)-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 E(2) in the gene regulation of rat uterus.

Nuclear [3h]naloxone binding in rat hypothalamus

High affinity (Kd approximately 1 nM) and low capacity [3H]naloxone binding sites were detected in the nuclear fraction of rat hypothalamus. The profile of this binding changed with age. In immature rats from 11 days of age until vaginal opening (approximately 33 day) the Scatchard plots of saturation data were linear and the Hill coefficient was 1, while just after vaginal opening (< 6 h) Scatchard analysis of [3H]naloxone binding gave a curvilinear component, with the Hill coefficient approximately 2, followed by an increase in binding sites and a decrease in Kd. In adults, after ovariectomy, the pattern of [3H]naloxone binding was similar to that observed in immature rats before vaginal opening. Following oestradiol treatment, binding sites with linear Scatchard plots disappeared and returned at least 24 h after hormone administration.