Paolo Mannella

Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion.

Background Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted. Methodology/Principal Findings In estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17β-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ERα with the G protein Gα13, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The Gα13/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane. Conclusions/Significance These results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear Gα13/RhoA/ROCK/moesin signaling cascade as a target of ERα in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

Rapid signaling of estrogen to WAVE1 and moesin controls neuronal spine formation via the actin cytoskeleton.

Estrogens are important regulators of neuronal cell morphology, and this is thought to be critical for gender-specific differences in brain function and dysfunction. Dendritic spine formation is dependent on actin remodeling by the WASP-family verprolin homologous (WAVE1) protein, which controls actin polymerization through the actin-related protein (Arp)-2/3 complex. Emerging evidence indicates that estrogens are effective regulators of the actin cytoskeleton in various cell types via rapid, extranuclear signaling mechanisms. We here show that 17beta-estradiol (E2) administration to rat cortical neurons leads to phosphorylation of WAVE1 on the serine residues 310, 397, and 441 and to WAVE1 redistribution toward the cell membrane at sites of dendritic spine formation. WAVE1 phosphorylation is found to be triggered by a Galpha(i)/Gbeta protein-dependent, rapid extranuclear signaling of estrogen receptor alpha to c-Src and to the small GTPase Rac1. Rac1 recruits the cyclin-dependent kinase (Cdk5) that directly phosphorylates WAVE1 on the three serine residues. After WAVE1 phosphorylation by E2, the Arp-2/3 complex concentrates at sites of spine formation, where it triggers the local reorganization of actin fibers. In parallel, E2 recruits a Galpha(13)-dependent pathway to RhoA and ROCK-2, leading to activation of actin remodeling via the actin-binding protein, moesin. Silencing of WAVE1 or of moesin abrogates the increase in dendritic spines induced by E2 in cortical neurons. In conclusion, our findings indicate that the control of actin polymerization and branching via moesin or WAVE1 is a key function of estrogen receptor alpha in neurons, which may be particularly relevant for the regulation of dendritic spines.

Rapid Estrogen Actions in the Cardiovascular System

Abstract:  In the last two decades, several studies have unveiled a series of original signaling mechanisms through which so‐called “nuclear” receptors can mediate rapid actions of steroid hormones. These rapid signaling actions are independent of the synthesis of mRNA or protein, and are therefore known as “nontranscriptional” or “nongenomic” as opposed to the classical genomic mechanisms. Nongenomic signaling of estrogens plays a prominent role in nonreproductive tissues, and between these is the vascular wall. At this level, estrogen triggers rapid vasodilatation, exerts anti‐inflammatory effects, stimulates endothelial growth and migration, and protects the vessels from atherosclerotic degeneration. Nongenomic signaling mechanisms have been involved in many of these actions and are increasingly considered to be of importance for vascular function in physiological and pathophysiological conditions. Rapid actions of steroid hormones have been implicated with vascular as well as with myocardial protection in animal experimental models. Moreover, the nongenomic signaling of estrogens is tightly interconnected with the nuclear pathways, and there are several indications that, through nongenomic modulation of signaling cascades, estrogens are also able to modulate the expression of several relevant genes in endothelial cells. In conclusion, while we are still in an early phase of the investigations of the nontranscriptional actions of steroid hormone receptors, it is clear that this newly recognized category of signaling mechanisms is responsible for critical steroid actions in nonreproductive tissues.

Activation of nitric oxide synthesis in human endothelial cells by red clover extracts

Objective: The unexpected findings of the Womens Health Initiative trial, where surrogate cardiovascular risk markers have failed to predict the cardiovascular performance of hormone therapy, showing no reduction of cardiovascular disease in postmenopausal women receiving hormonal preparations inducing a favorable lipid profile, raise the interest on how molecules with hormone-like activity used for the treatment of menopausal symptoms act on vascular cells. This is particularly important for estrogen-like compounds such as phytoestrogens, whose mechanisms of action may significantly differ from those of other estrogenic compounds. Design: Because endothelial-derived nitric oxide (NO) is a key regulator of vascular tone and atherogenesis as well as a well-characterized estrogen-regulated molecule, we studied the regulation of NO synthesis in cultured human endothelial cells by phytoestrogens contained in red clover extracts. Results: We show that red clover extracts activate NO synthesis in endothelial cells by recruiting transcriptional pathways but are not capable of inducing rapid NO synthesis through nongenomic mechanisms. During prolonged exposures, red clover extracts enhance the expression as well as the activity of endothelial nitric oxide synthase. These effects are mediated by a recruitment of estrogen receptor-β. Moreover, we show that red clover-derived isoflavones synergize with 17β-estradiol in increasing endothelial nitric oxide synthase activity and expression, therefore being devoid of antiestrogenic effects in human endothelial cells. Conclusions: These results help to understand the mechanisms of action of phytoestrogens on the cardiovascular system and have relevant clinical implications.

The female pelvic floor through midlife and aging

Female pelvic floor is a complex functional unit involved in multiple functions that extend beyond the sole support of pelvic organs. Pelvic floor dysfunction globally affects micturition, defecation and sexual activity. Evolutionary modifications such ad adaptation to upright standing, walking and the need to deliver fetuses with larger head diameters made the fascial and muscle support of the pelvic floor vulnerable, therefore predisposing women to pelvic organ prolapse and incontinence. Different than in males, the female pelvic floor undergoes a number of adaptive changes related to life and endocrine events. Most of the clinical manifestations of these changes become apparent after menopause and throughout aging in women. This review article summarizes the key aspects of the pathophysiology and the clinics of the modifications of the pelvic floor in women through midlife and beyond. A particular focus is given to the relationship between urinary and bowel dysfunction.

Actin Cytoskeleton Remodelling by Sex Steroids in Neurones

Cell morphology and its interaction with the extracellular environment are integrated processes involving a number of intracellular controllers orchestrating cytoskeletal proteins and their interaction with the cell membrane and anchorage proteins. Sex steroids are effective regulators of cell morphology and tissue organisation, and recent evidence indicates that this is obtained through the regulation of the actin cytoskeleton. Intriguingly, many of these regulatory actions related to cell morphology are achieved through the rapid, nonclassical signalling of sex steroid receptors to kinase cascades, independently from nuclear alteration of gene expression or protein synthesis. The identification of the mechanistic basis for these rapid actions on cell cytoskeleton has special relevance for the characterisation of the effects of sex steroids under physiological conditions, such as for the development of neurone/neurone interconnections and dendritic spine density. This is considered to be critical for gender‐specific differences in brain function and dysfunction. Recent advancements in the characterisation of the molecular basis of the extranuclear signalling of sex steroids help to clarify the role of oestrogen and progesterone in the brain, and may turn out to be of relevance for clinical purposes. This review highlights the regulatory effects of oestrogens and progesterone on actin cytoskeleton and neurone morphology, as well as recent progresses in the characterisation of these mechanisms, providing insights and working hypotheses on possible clinical applications for the modulation of these pathways in the central nervous system.

Oestrogen and progestins differently prevent glutamate toxicity in cortical neurons depending on prior hormonal exposure via the induction of neural nitric oxide synthase

Sex steroids are important for brain function and protection. However, growing evidence suggests that these actions might depend on the timing of exposure to steroids. We have studied the effects of steroid administration on the survival of neural cells and we have partially characterized the possible mechanisms. The effect of a 24h pre-treatment with 17beta-estradiol or 17beta-estradiol plus progesterone or medroxyprogesterone acetate on the toxic action of l-glutamate was used to test the experimental hypothesis. Pre-exposure to either steroid combinations turned in enhanced cell survival. Instead, addition of sex steroids together with l-glutamate, in the absence of a pre-exposure had no protective effect. Pre-treatment with the steroid combinations resulted in increased neural NOS expression and activity and blockade of NOS abolished the cytoprotective effects of steroids. These results suggest that NOS induction might be involved in sex steroid-induced neuroprotection. Furthermore, these data supports the hypothesis that prolonged and continued exposure to oestrogen and progesterone, leading to changes in gene expression, is necessary to obtain neuroprotection induced by sex steroids.

Feto-placental nitric oxide, asymmetric dimethylarginine and vascular endothelial growth factor (VEGF) levels and VEGF gene polymorphisms in severe preeclampsia

Objective: To measure plasma nitric oxide (NO), asymmetric dimethylarginine (ADMA) and vascular endothelial growth factor (VEGF) levels and VEGF gene polymorphisms in fetal circulation in severe preeclampsia. Methods: Cord vessels of singleton gestations complicated with severe preeclampsia 36 weeks or more (n = 31) and controls were sampled upon delivery for analyte measuring. Additionally, DNA was extracted from umbilical vein whole blood to determine the frequency of VEGF gene single nucleotide polymorphisms (SNPs): −2578 A/C, −1498 C/T, −1154 A/G, −634 C/G and +936 C/T. Coefficient correlations between analyte levels and placental and neonatal weight were calculated. Results: NO plasma levels in umbilical vessels (artery and vein) were significantly higher in preeclampsia cases as compared to controls (4.67 ± 3.0 vs. 0.82 ± 0.90; 4.46 ± 3.0 vs. 0.82 ± 0.99 mmol/L, respectively, p = 0.0001 both). ADMA levels displayed a similar increased trend in both fetal vessels, but this did not reach statistical significance (2.57 ± 1.03 vs. 2.34 ± 0.57; 2.74 ± 0.94 vs. 2.42 ± 0.59 mmol/L, respectively, p > 0.05). VEGF was significantly lower in artery but not in vein in preeclampsia cases (200.48 ± 225.62 vs. 338.61 ± 287.03 pg/mL, p = 0.04). A significant positive correlation was found between NO and ADMA levels (artery and vein) among preeclampsia cases. Overall, the frequency of the studied VEGF gene SNPs did not differ among pre-eclamptic cases and controls; nevertheless, a significant trend toward lower umbilical vein VEGF levels was observed in pre-eclampsia cases in the presence of -2578 CC and −1154 AG genotypes. Conclusion: Near term gestations complicated with severe preeclampsia presented higher NO levels in fetal circulation, which correlated to ADMA and lower artery VEGF values. More research is warranted to confirm that selected VEGF SNPs may be associated with lower umbilical vein VEGF.

Activation of nitric oxide synthesis in human endothelial cells using nomegestrol acetate

OBJECTIVE: Recent clinical trials indicate that synthetic progestins may be unexpectedly relevant for the development of cardiovascular disease. The aim of this study was to establish whether nomegestrol acetate induces signaling events in human endothelial cells that differ from those of other progestins, such as natural progesterone or medroxyprogesterone acetate. METHODS: We used human endothelial cells to study the action of nomegestrol acetate (either alone or in the presence of estradiol [E2]) on the synthesis of nitric oxide (NO) and on the activity or expression of endothelial nitric oxide synthase (eNOS). We compared the effects of nomegestrol acetate with those of progesterone or medroxyprogesterone acetate. In addition, we characterized the signaling events recruited by these compounds. RESULTS: Progesterone and nomegestrol acetate increase NO synthesis by transcriptional and nontranscriptional mechanisms, whereas medroxyprogesterone acetate lacks such effects. When used together with physiological E2 concentrations, progesterone and nomegestrol acetate do not interfere with (or even enhance) E2 effects, whereas medroxyprogesterone acetate impairs E2 signaling. A marked difference in the recruitment of mitogen-activated protein kinase and phosphatidylinositol-3 kinase explains the divergent effects of the three gestagens. CONCLUSION: Our findings show significant differences in the signal transduction pathways recruited by progesterone, nomegestrol acetate, and medroxyprogesterone acetate in human endothelial cells that may have relevant clinical implications.

Nitric oxide and pro-inflammatory cytokine serum levels in postmenopausal women with the metabolic syndrome

Background: The metabolic syndrome (METS) increases after the menopause which may enhance cardiovascular risk in part explained by a pro-inflammatory state. Objective: Measure nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) serum levels in postmenopausal women with and without the METS (Adult Treatment Panel III criteria). Methods: Analyte levels were compared among those with and without the syndrome and each of its diagnostic components. Rho Spearman coefficients were also calculated to determine correlations between analyte levels and various numeric variables. Results: Median age of all studied women (n = 88) was 54.4 years, 62.5% had abdominal obesity, 14.8% hyperglycemia, 59.1% high triglycerides (TG) and 44.3% hypertension. Women with the METS (n = 44) displayed higher body mass index values and higher rates of abdominal obesity, hyperglycemia, hypertriglyceridemia, hypertension and low HDL-C levels. Median NO and IL-6 levels were significantly higher in women with the METS as compared to controls (p < 0.05). Independent of presenting the METS, analytes were higher in those displaying abdominal obesity (IL-6), hypertension (IL-6 and TNF-α) and more METS diagnostic criteria and abnormal HDL-C, TG and glucose levels (NO). Both cytokines positively correlated with the number of METS criteria, age and time since menopause, IL-6 positively with waist circumference and TNF-α positively with blood pressure levels. NO levels inversely correlated with HDL-C values and positively with the number of METS criteria, glucose, and TG levels; correlation with the latter being the highest (r2 = 0.65, p = 0.0001). Conclusion: Postmenopausal women with the METS displayed higher IL-6 and NO levels, with significant correlations found between studied analytes and some of the components of the syndrome.