RAGE inhibition blunts insulin-induced oncogenic signals in breast cancer.

The receptor for advanced glycation end products (RAGE) is implicated in diabetes and obesity complications, as well as in breast cancer (BC). Herein, we evaluated whether RAGE contributes to the oncogenic actions of Insulin, which plays a key role in BC progression particularly in obese and diabetic patients. Analysis of the publicly available METABRIC study, which collects gene expression and clinical data from a large cohort (n = 1904) of BC patients, revealed that RAGE and the Insulin Receptor (IR) are co-expressed and associated with negative prognostic parameters. In MCF-7, ZR75 and 4T1 BC cells, as well as in patient-derived Cancer-Associated Fibroblasts, the pharmacological inhibition of RAGE as well as its genetic depletion interfered with Insulin-induced activation of the oncogenic pathway IR/IRS1/AKT/CD1. Mechanistically, IR and RAGE directly interacted upon Insulin stimulation, as shown by in situ proximity ligation assays and coimmunoprecipitation studies. Of note, RAGE inhibition halted the activation of both IR and insulin like growth factor 1 receptor (IGF-1R), as demonstrated in MCF-7 cells KO for the IR and the IGF-1R gene via CRISPR-cas9 technology. An unbiased label-free proteomic analysis uncovered proteins and predicted pathways affected by RAGE inhibition in Insulin-stimulated BC cells. Biologically, RAGE inhibition reduced cell proliferation, migration, and patient-derived mammosphere formation triggered by Insulin. In vivo, the pharmacological inhibition of RAGE halted Insulin-induced tumor growth, without affecting blood glucose homeostasis. Together, our findings suggest that targeting RAGE may represent an appealing opportunity to blunt Insulin-induced oncogenic signaling in BC.

Estrogen Receptors and Chronic Venous Disease.

OBJECTIVE/BACKGROUND: Chronic venous disease (CVD) is a common and relevant problem affecting Western people. The role of estrogens and their receptors in the venous wall seems to support the major prevalence of CVD in women. The effects of the estrogens are mediated by three estrogen receptors (ERs): ERα, ERß, and G protein-coupled ER (GPER). The expression of ERs in the vessel walls of varicose veins is evaluated. METHODS: In this prospective study, patients of both sexes, with CVD and varicose veins undergoing open venous surgery procedures, were enrolled in order to obtain vein samples. To obtain control samples of healthy veins, patients of both sexes without CVD undergoing coronary artery bypass grafting with autologous saphenous vein were recruited (control group). Samples were processed in order to evaluate gene expression. RESULTS: Forty patients with CVD (10 men [25%], 30 women [75%], mean age 54.3 years [median 52 years, range 33-74 years]) were enrolled. Five patients without CVD (three men, two women [aged 61-73 years]) were enrolled as the control group. A significant increase of tissue expression of ERα, ERß and GPER in patients with CVD was recorded (p < .01), which was also related to the severity of venous disease. CONCLUSION: ERs seem to play a role in CVD; in this study, the expression of ERs correlated with the severity of the disease, and their expression was correlated with the clinical stage.

A Bodipy as a luminescent probe for detection of the G protein estrogen receptor (GPER).

We report the rational design, based on docking simulations, and synthesis of the first fluorescent and selective probe of GPER for bioimaging purposes and functional dissecting studies. It has been conceived as a Bodipy derivative and obtained by accessible and direct synthesis. Its optical properties have been measured in different solvents, showing insensitivity to their polarity. Its binding to GPER was achieved by competition assays with [3H]E2 and [5,6-3H] nicotinic acid in ER-negative and GPER-positive SkBr3 breast cancer cells. SkBr3 cells, transfected with a GPER expression vector containing a FLAG tag, were used to confirm that the fluorophore binds to GPER in a specific manner.

Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax.

Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERß agonists, 4,4',4"-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.

Adrenal androgens stimulate the proliferation of breast cancer cells as direct activators of estrogen receptor alpha.

Estrogens stimulate the proliferation of many breast tumors and cell lines derived from them. Antiestrogens have therefore become a powerful therapeutic agent to treat breast tumors that express estrogen receptor (ER) alpha. In addition, aromatase inhibitors are now used in postmenopausal women to block the in situ conversion of adrenal androgens to estrogens. This approach can only be successful if ER-alpha in a particular tumor is not directly stimulated by adrenal androgens. We have examined this possibility using several different cell lines as model systems: (a) wild-type MCF7 cells, an ER-alpha-dependent human breast cancer cell line; (b) MCF7SH cells, an estrogen-independent MCF7 variant; (c) Ishikawa cells, an ER-alpha-containing human uterine cell line; (d) ER-negative HeLa cells; and (e) budding yeast. Transactivation assays with transfected ER-alpha reporter genes reveal a direct activation of ER-alpha by dehydroepiandrosterone (DHEA), 5alpha-androstene-3beta,17beta-diol, testosterone, and the two nonaromatizable androgens, dihydrotestosterone and 5alpha-androstane-3beta,17beta-diol. The involvement of other steroid receptors could be ruled out with specific antihormones. Moreover, the same set of ligands stimulates the proliferation of the two breast cancer cell lines. At subsaturating and physiologically relevant concentrations of DHEA, DHEA stimulates the proliferation of MCF7SH cells, which correlates with a substantial, albeit submaximal, transcriptional response. Thus, adrenal androgens must also be considered as risk factors in postmenopausal women.

Antiestrogens upregulate estrogen receptor beta expression and inhibit adrenocortical H295R cell proliferation.

The molecular mechanisms involved in adrenocortical tumorigenesis are still not completely understood. In this study, using the H295R cell line as a model system, we investigated the role of estrogens and estrogen receptor (ER) alpha and ER beta in the growth regulation of adrenocortical tumors. We demonstrated that H295R cells are able to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein and express ER beta to a greater extent than ER alpha. Moreover, physiological concentrations of 17beta-estradiol (E2) determined an increase of thymidine incorporation, suggesting the presence of an autocrine mechanism in maintaining H295R cell proliferation. Evaluating the response to ER antagonists like 4-hydroxytamoxifen (OHT) and ICI 182 780 (ICI), we observed an up-regulation of ER beta and a dose-dependent inhibition of H295R cell proliferation. Whereas ICI determined the growth arrest of H295R cells, OHT induced morphological changes that were characteristic of apoptosis. According to the above-mentioned observations, OHT but not ICI clearly induced a marked expression of FasL and the cleavage of both caspase-8 and caspase-3. Interestingly, the apoptotic effects of OHT in H295R cells may be consequent to the enhanced levels of ER beta which stimulate the expression of FasL interacting with activating protein (AP)-1 sites located within its promoter sequence. In conclusion, we have demonstrated that H295R cells are able to transform androgens to estrogens that activate an autocrine mechanism, mediated by their own receptors, and contribute to regulate the proliferation of these cells. Moreover, this study points towards a role for ER beta as an important mediator of the repressive effects exerted by antiestrogens on H295R cells; however, further studies are needed to clarify its role in the control of adrenocortical cell proliferation and on the potential benefits of antiestrogens for treatment of adrenocortical cancer.

The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

SIRT1 is involved in oncogenic signaling mediated by GPER in breast cancer.

A number of tumors exhibit an altered expression of sirtuins, including NAD+-dependent histone deacetylase silent information regulator 1 (SIRT1) that may act as a tumor suppressor or tumor promoter mainly depending on the tumor types. For instance, in breast cancer cells SIRT1 was shown to exert an essential role toward the oncogenic signaling mediated by the estrogen receptor-α (ERα). In accordance with these findings, the suppression of SIRT1 led to the inhibition of the transduction pathway triggered by ERα. As the regulation of SIRT1 has not been investigated in cancer cells lacking ER, in the present study we ascertained the expression and function of SIRT1 by estrogens in ER-negative breast cancer cells and cancer-associated fibroblasts obtained from breast cancer patients. Our results show that 17ß-estradiol (E2) and the selective ligand of GPER, namely G-1, induce the expression of SIRT1 through GPER and the subsequent activation of the EGFR/ERK/c-fos/AP-1 transduction pathway. Moreover, we demonstrate that SIRT1 is involved in the pro-survival effects elicited by E2 through GPER, like the prevention of cell cycle arrest and cell death induced by the DNA damaging agent etoposide. Interestingly, the aforementioned actions of estrogens were abolished silencing GPER or SIRT1, as well as using the SIRT1 inhibitor Sirtinol. In addition, we provide evidence regarding the involvement of SIRT1 in tumor growth stimulated by GPER ligands in breast cancer cells and xenograft models. Altogether, our data suggest that SIRT1 may be included in the transduction network activated by estrogens through GPER toward the breast cancer progression.

Dietary calcium and bone density in adolescent girls and young women in Europe.

The objective of this study was to investigate the association between dietary calcium intake and radial bone density among young women, over the whole range of intake and at different levels of calcium intake. The study design was a cross-sectional, observational multicenter investigation in six European countries. One thousand one hundred and sixteen healthy Caucasian girls aged 11-15 years and 526 women aged 20-23 years participated, after having been selected from larger population samples to represent a large range in calcium intake. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry at the ultradistal and middistal radius. Calcium intake was assessed with 3-day food records. Other potential determinants of BMD were measured by anthropometry or questionnaires. Mean calcium intake among the girls varied between 609 mg/day in Italy and 1267 mg/day in Finland; intakes for women were in a similar range. After adjustment for height, weight, and age at menarche for the women, and adjustment for age, height, weight, Tanner stage, and bone area for the girls, radial BMD at both sites did not significantly vary among quartiles of calcium intakes for both age groups. In multivariate linear regression, calcium was weakly positively associated with BMD at both sites in the girls (per 100 mg of calcium: beta = 0.57 mg/cm2, p = 0.03 for middistal BMD and beta = 0.56 mg/cm2, p = 0.01 for ultradistal BMD). For middistal BMD, the association was observed predominantly in pre-menarcheal girls. The associations were no longer statistically significant after full adjustment for all determinants of BMD, except again in pre-menarcheal girls. Radial BMD in the women was not associated with calcium intake, except after full adjustment for determinants of BMD, when ultradistal BMD became inversely associated with calcium intake (per 100 mg beta = -1.02, p = 0.03); this finding was due to results in one of the countries and not found in other countries. There was no evidence for a different relation between calcium and BMD at different levels of intake; although there was a positive association at calcium intake levels < 600 mg/day, the interaction was not significant and there was no consistent trend over intake categories. These results do not support the hypothesis that dietary calcium is a determinant of peak BMD in European women, for a wide range of intake. This study does not provide evidence that Recommended Dietary Allowances for calcium should be increased.

The in-vitro transformation of [3H]dehydroepiandrosterone into its principal metabolites in the adrenal cortex of adult castrated male rats and following steroid treatment.

The adrenal gland of castrated adult male rats metabolized [3H]dehydroepiandrosterone in vitro to delta 4-androsten-3,17-dione (4AD), testosterone, dihydrotestosterone (DHT) and 5 alpha-androstane-3,17-dione (5 alpha AD). Despite the low testosterone values, DHT and 5 alpha AD were higher 30 and especially 60 days after castration, with raised 4AD:testosterone and decreased testosterone:DHT ratios. The 5 alpha-reductase activity thus appears to increase with time after castration. Fourteen days after castration, 4AD was the only metabolite that was raised compared with intact animals, and testosterone was comparable in sham-operated and castrated rats. The administration of testosterone propionate to castrated rats restored testosterone values to those of intact rat adrenals, whereas 4AD values were greater. The administration of dihydrotestosterone propionate also yielded higher levels of 4AD, in the presence of a lower testosterone value. After administration of oestradiol benzoate, 4AD values were lower especially compared with the other hormone-treated groups, and there was an unexpectedly high testosterone value. These data indicate that the adrenal gland contributes to the production of androgens, as previously noted by Andò, Canonaco, Beraldi et al. (1988) who showed increased plasma 4AD and testosterone levels in adult male rats 30 days after castration. Furthermore, adrenal androgen production in castrated animals is differentially regulated by sex steroids.

Breast cancer: from estrogen to androgen receptor.

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

The direct proliferative stimulus of dehydroepiandrosterone on MCF7 breast cancer cells is potentiated by overexpression of aromatase.

In women after menopause aromatization of adrenal androgens represents the main source of estrogens, which may promote the development of hormone-dependent breast tumor. Several studies have attempted to determine the cell type within carcinomas that is responsible for 'in situ' estrogen biosynthesis and whether the amount produced may sustain relevant biological effects. Here we show P450arom mRNA and protein expression together with immunocytochemical localization of aromatase in the epithelial MCF7 breast cancer cell line. Moreover, we demonstrate that the enhanced aromatization of dehydroepiandrosterone in aromatase transfected MCF7 cells confers biological advantages such as proliferative stimulation similar to that induced by estradiol. Our results suggest that aromatase inhibitors may be particularly effective in the treatment of hormone-dependent breast cancer disease in postmenopausal women.

Follow-up study on the effects of thyroid hormone administration on androgen metabolism of peripubertal rat Sertoli cells.

The inhibitory effect of triiodothyronine (T3) given in early postnatal life on Sertoli cell proliferative activity, leading to their precocious terminal differentiation, has been demonstrated previously. However, data concerning the role of thyroid hormone on androgen metabolism of Sertoli cells during the same period are still lacking. In this study we performed a time-course investigation on the effects of T3 treatment on testosterone metabolism in Sertoli cells isolated from 2-, 3- and 4-weeks-old euthyroid rats. Triiodothyronine (3 micrograms/100 g body wt) was given ip., during the last week prior to sacrifice. Sertoli cells from all animal groups initially were cultured under basal conditions during the first 24 h and subsequently in the presence of testosterone (0.5 mumol/l) with or without T3 (1 nmol/l) for an additional 24 h. This treatment given to 2-week-old animals resulted in reduced testicular growth. As far as androgen metabolism is concerned, T3 in vivo and in vitro treatment in 2- and 3-week-old animals induced a lowering of dihydrotestosterone + 3 alpha-diol with an enhancement of the two other 5 alpha-reduced androgens. The effect was much less pronounced in the oldest group. In both 2- and 3-week-old treated rats a marked reduction of oestradiol was observed, which indicates an inhibition of aromatase activity, mainly in younger animals. This enzyme has been reported to be extremely active in Sertoli cells of rats (of the same strain) between the age of 5 and 20 days, but it decreases rapidly thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dietary calcium intake on bone mineral density in healthy adolescent girls and young women in southern Italy.

BACKGROUND: The association between forearm bone mineral areal density (BMD) and dietary calcium, anthropometric characteristics, puberty, and physical activity was studied for the first time in 200 girls (aged 11-15 years) and 100 women (aged 20-23 years) living in Southern Italy. METHODS: The BMD was assessed by dual energy x-ray absorptiometry at ultradistal (ud) and proximal (pr) radial sites and dietary calcium was evaluated using Food Frequency Questionnaires and detailed 3-day food records. RESULTS: For population samples grouped according to low and high calcium intake levels, forearm densities were quite similar among both girls and women. Independently of calcium intake, girls displayed strong correlations between ud/pr-BMD and age, bone age, weight, height and BMI. Furthermore, in girls of similar age and BMI, radial densities were substantially increased following menarche. Positive relationships between weight, BMI and both ud/pr-BMD were only evident in women with high calcium intake. CONCLUSIONS: This study showed that different calcium intake values do not appear to affect forearm mineral densities at the ages investigated, however puberty represents the major event in radial bone mass acquisition during adolescence.

Effect of oestradiol and insulin on the proliferative pattern and on oestrogen and progesterone receptor contents in MCF-7 cells.

In a long-term experiment MCF-7 cells showed an exponential proliferation rate in fetal calf serum while they become quiescent in growth-factor- and steroid-free serum. The mitogenic activity of this cell line was increased by oestradiol and insulin, exposure to both hormones showing a synergic effect. These mitogen-mediated effects are inhibited by genistein a phytoestrogen which, by itself at the doses used, did not affect either the basal proliferation rate or the total protein content. Immunoblotting and Scatchard analysis reveal respectively that insulin increases the oestrogen receptor (ER) content and its binding capacity. The presence of genistein decreases the ER content and completely abolishes the binding capacity of this protein. Insulin is able to increase progesterone receptor (PR) levels while, in the presence of genistein, the above-reported effect was completely abolished. On the basis of the latter data the authors suggest that insulin is able to affect the phosphorylation status of ER and PR, inducing functional changes in both proteins, although this was in the absence of their natural ligands. Indeed, the presence in the medium of a tyrosine kinase inhibitor such as genistein could substantially decrease both ER and PR levels in these cells.

Epididymal and sex accessory gland secretions in transfusion-dependent beta-thalassemic patients: evidence of an impaired prostatic function.

Neutral alpha-glucosidase levels as epididymal marker, fructose levels as vesicular marker, zinc, citric acid and prostate specific antigen levels as prostatic markers were measured in the seminal plasma of eight transfusion-dependent beta-thalassemic patients in order to study epididymal and sex accessory gland secretions (eighteen subjects served as controls). FSH and LH as well as total and free testosterone were detected displaying unaltered serum values. Ejaculate of patients showed normal sperm count and low sperm motility, in the meantime seminal plasma exhibited unaltered both neutral alpha-glucosidase and fructose values but low levels of zinc, citric acid and prostate specific antigen were noticed as well. These data suggest an impaired prostatic secretion in the thalassemic patients studied. A local iron toxicity on the prostatic tissue could be supported by the decrease of its specific markers observed only in the subgroup of patients with high ferritin serum levels.

A time course study on the "in vitro" effects of T3 and testosterone on androgen and estrogen receptors in peripuberal primary rat Sertoli cells.

The effect of Tri-iodothyronine (T3) administration leading to the precocius differentiation of Sertoli cell in prepuberal rats has been previously shown. The functional maturation of Sertoli cells is associated with changes in androgen metabolism. We have recently demonstrated that T3 influences androgen metabolism in Sertoli cells by inhibiting aromatase activity and reduces drastically the ER contents in peripubertal hypothyroid rats. To better understand the role of T3 in modulating steroid action on Sertoli cells, we performed a time course study evaluating the in vitro effects of T3 and testosterone (T) on androgen (ARs) and estrogen (ERs) receptor content in Sertoli cells isolated from two weeks old Wistar rats. ARs and ERs basal levels did not change during the time course study indicating that the exposure to culture medium per se did not affect either receptor type. After 24 hrs of incubation with either T3 or T, a decrease of ERs in both nucleus and cytosol was observed. Such a decrease was augmented by the simultaneous administration of both hormones. ARs displayed a different temporal pattern in the two cellular compartments and exhibited an earlier rise in the cytosol induced by either T3 or T. At 36 hrs, ARs were significantly enhanced in both compartments in response to either T or T3 exposure while combined hormonal treatment caused an additive increase compared with the single treatment group. As a consequence of the opposite behaviour pattern displayed by ARs and ERs, the ratio between total ARs and ERs contents was increased after 24 hrs of exposure to hormonal treatment. To evaluate if treatments performed induced a functional maturation of Sertoli cells, transferrin levels in culture medium were measured. The increase of this protein paralleled that of ARs content as well as that of ARs/ERs ratio. This study demonstrates that thyroid hormone induces a progressive increase of (AR)/(ER) ratio in the differentiating Sertoli cells bringing them to a prevalent androgen dependency along their functional maturation.

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs.

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.

Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.