Kálmán A. Kovács

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.

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.

Ghrelin and acyl ghrelin in preterm infants and maternal blood: relationship with endocrine and anthropometric measures

OBJECTIVE The objective of the present study was to examine the association of acylated and total ghrelin levels at birth in preterm infants with anthropometric features and with related hormones in infants and their mothers. DESIGN Prospective, descriptive study. METHODS In total 23 pregnant women and their 26 preterm infants were involved in the study (3 twin pregnancies; gestational age, 25-35 weeks). Maternal and umbilical vein blood samples were taken after the delivery. Serum acylated and total ghrelin, leptin, cortisol, insulin, GH, and glucose were determined. RESULTS The mean level of acylated ghrelin concentration was higher in the maternal than in the cord blood (P<0.01) and there was a significant correlation between the fetal and maternal acylated ghrelin levels (P<0.01). The total ghrelin concentration was higher in neonates than in mothers (P<0.01), but there was no correlation between them. The multivariate regression analysis for fetal acylated and maternal total ghrelin as dependent variables shows that the fetal acylated ghrelin has two independent predictors, the maternal acylated ghrelin (P<0.01) and the fetal cortisol (P<0.05), whereas the maternal total ghrelin has only one independent predictor, the maternal glucose (P<0.05). CONCLUSIONS These data provide the first evidence that umbilical cord acylated ghrelin concentrations are lower than in maternal blood and support the hypothesis that the acylation process in the fetus is partly affected by cortisol and the placenta may play a role in this process.

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.

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.

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.

Assessment of cells in the ascitic fluid of women with ovarian hyperstimulation syndrome: The clinical implications for subsequent ovarian malignancy

BackgroundAlthough some studies have reported a potential connection between ovulation induction therapy (OIT) and malignant ovarian diseases, the results have been inconclusive. In the present study, we sought to determine whether women undergoing OIT at our in vitro fertilization (IVF) clinic, especially those with severe ovarian hyperstimulation syndrome (OHSS) and suspicious cytologic findings, were at risk for developing malignant ovarian tumours after treatment.MethodsPatients who underwent OIT at our IVF clinic were enrolled in this study and assessed for any evidence of malignant ovarian tumours. Patients who developed severe OHSS as a result of OIT were treated with a culdocentesis. Cells from the ascitic fluid were cytologically scored for abnormality and malignancy. Peripheral blood samples were obtained from patients with severe OHSS to determine serum levels of the tumour markers (CA-125 and HE4) that were used to calculate the Risk for Ovarian Malignancy Algorithm (ROMA) index.ResultsFollow-up data were available for 1,353 of the 1,587 patients (85%) who underwent OIT at our IVF clinic between January 2006 and December 2012. Twenty-three patients (1.4%) were hospitalized with OHSS. Culdocentesis was performed 16 times in nine patients with severe OHSS (age range, 23–34 years; mean, 27.1 years). Although cytological examination of the ascitic cells of these patients suggested malignant ovarian neoplasia, over the course of the observation period, the ovarian volume gradually decreased and became normal. Subsequent cytological and histological examinations failed to find evidence of any malignant tumours in these nine patients. None of the 1,353 participants who underwent OIT developed any malignant ovarian tumours during the study period. Moreover, none of the 462 patients who were in our ovarian tumour registry were also participants in the IVF program.ConclusionsThe presence of atypical cells in the ascitic fluid of women with severe OHSS does not likely indicate malignancy; therefore, radical surgical intervention is not justified. The risk of malignancy is minimal shortly after OIT. At our centre, OIT has not been associated with any cases of ovarian tumour.

Regulation of activator protein-1-DNA binding activity by opioid peptides in estrogen-sensitive cells of rat hypothalamus and 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.

Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides.

Abstract.  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 Dulbeccos modified Eagles 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.