Vasodilatory effects of the selective GPER agonist G-1 is maximal in arteries of postmenopausal women.

G-protein-coupled estrogen receptors (GPERs) have been proposed to mediate estrogen-mediated vasodilation. The presence of GPER-dependent vasodilation in human resistance-sized arteries (HRAs) or its signal transduction pathways have not been investigated. HRAs in subcutaneous fat tissues (biopsies from postmenopausal women (PMW), age-matched men (M) and pregnant women (PGW)) were mounted for in vitro isometric force recording. Vasodilation induced by G-1 (selective GPER-agonist, 3 µM) from HRAs pre-contracted with norepinephrine amounted to 40±5% in PMW, significantly larger than those obtained from M (20±5%) or PGW (20±5%). L-NAME (nitric oxide (NO) synthase inhibitor) abolished these relaxations in PGW, attenuated them in PMW and had no effect in M. Immunohistochemical analysis confirmed the presence of GPER in both smooth muscle and endothelial cells of HRA with maximum expression in PGW. In cultured human umbilical vein endothelial cells (HUVECs), G-1 increased NO-synthesis concentration-dependently through higher expressions of endothelial NO-synthase (eNOS) and through enhanced phosphorylation of eNOS on Ser(1177). In conclusion, GPER vasodilates human resistance arteries through various activating mechanisms of the eNOS-signaling pathway.

Estrogen regulates endothelial migration via plasminogen activator inhibitor (PAI-1).

Endothelial plasminogen activator inhibitor (PAI-1) controls vascular remodeling, angiogenesis and fibrinolysis. PAI-1 blood levels in women are related to estrogen. The aim of this study was to characterize the signaling pathways through which estrogen regulates PAI-1 in endothelial cells. Furthermore, we aimed to investigate whether PAI-1 is implicated in the control of endothelial migration by estrogen. Cultured human umbilical vein endothelial cells (HUVECs) and ovariectomized rats were used to test the effects of 17ß-estradiol (E(2)) on PAI-1 expression and its role on endothelial migration. At physiological concentrations, E(2) increases the expression of PAI-1 in HUVEC within 6-12 h through activation of a signaling cascade initiated by estrogen receptor α and involving G proteins, phosphatidylinositol-3-OH kinase and Rho-associated kinase II. ROCK-II activation turns into an over-expression of c-Jun and c-Fos that is required for E(2)-induced expression of PAI-1. Estrogen-induced PAI-1 expression is implicated in HUVEC horizontal migration. PAI-1 regulation is found also in vivo, in female rats, where ovariectomy is associated with reduced PAI-1 expression, while estrogen replacement counteracts this change. In conclusion, E(2) increases PAI-1 synthesis in human endothelial cells and in rodent aorta through a G protein-initiated signaling that targets early-immediate gene expression. This regulatory pathway is implicated in endothelial cell migration. These findings describe new mechanisms of action of estrogens in the vessels, which may be important for vascular remodeling and hemostasis.

Estrogen-like effects of wine extracts on nitric oxide synthesis in human endothelial cells.

Endothelial dysfunction frequently ensues during the climacteric due to hormonal and metabolic changes. Non-pharmacological interventions such as lifestyle and dietary modifications are emerging as valuable strategies to counteract the cardiovascular consequences of ageing. A number of chemical components of wine, including alcohol and some polyphenols, are known to be active on the vessels. However, the molecular mechanisms through which they modulate endothelial function are largely unclear. The aim of this study was to investigate the effects of non-alcoholic wine fractions from five different wines on the synthesis of nitric oxide (NO) via the expression and enzymatic activation of the endothelial nitric oxide synthase (eNOS) in human endothelial cells. All non-alcoholic fractions studied increased NO synthesis, although with different potencies. All wine extracts maximally enhanced NO production at doses in the range achieved with a moderate wine intake, with decreasing effects with further increases of the dose. Interestingly, a part of these actions was recruited via estrogen receptors (ERs). Within the polyphenols with known binding activity for ERs contained in the tested wines, resveratrol, epicatechin, syringic acid, apigenin, malvidin and ellagic acid were largely responsible for eNOS activation. These findings show that some of the non-alcoholic components of wine enhance the production of NO by the vessels acting on ERs, and suggest that a moderate intake of wine may benefit the cardiovascular system through estrogen-like effects.

Estrogen receptor-alpha promotes breast cancer cell motility and invasion via focal adhesion kinase and N-WASP.

The ability of cancer cells to move and invade the surrounding environment is the basis of local and distant metastasis. Cancer cell movement requires dynamic remodeling of the cytoskeleton and cell membrane and is controlled by multiprotein complexes including focal adhesion kinase (FAK) or the Neural Wiskott-Aldrich Syndrome Protein (N-WASP). We show that 17ß-estradiol induces phosphorylation of FAK and its translocation toward membrane sites where focal adhesion complexes are assembled. This process is triggered via a Gα/Gß protein-dependent, rapid extranuclear signaling of estrogen receptor α interacts in a multiprotein complex with c-Src, phosphatidylinositol 3-OH kinase, and FAK. Within this complex FAK autophosphorylation ensues, and activated FAK recruits the small GTPase cdc42, which, in turn, triggers N-WASP phosphorylation. This results in the translocation of Arp2/3 complexes at sites where membrane structures related to cell movement are formed. Recruitment of FAK and N-WASP is necessary for cell migration and invasion induced by 17ß-estradiol in breast cancer cells. Our findings identify an original mechanism through which estrogen promotes breast cancer cell motility and invasion. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

Progesterone receptor enhances breast cancer cell motility and invasion via extranuclear activation of focal adhesion kinase.

While progesterone plays multiple roles in the process of breast development and differentiation, its role in breast cancer is less understood. We have shown previously that progestins stimulate breast cancer cell migration and invasion because of the activation of rapid signaling cascades leading to modifications in the actin cytoskeleton and cell membrane that are required for cell movement. In this study, we have investigated the effects of progesterone on the formation of focal adhesion (FA) complexes, which provide anchoring sites for cell attachment to the extracellular matrix during cell movement and invasion. In T47-D breast cancer cells, progesterone rapidly enhances FA kinase (FAK) phosphorylation at Tyr(397) in a time- and concentration-dependent manner. As a result, exposure to progesterone leads to increased formation of FA complexes within specialized cell membrane protrusions. The cascade of events required for this phenomenon involves progesterone receptor interaction with the tyrosine kinase c-Src, which activates the phosphatidylinositol-3-kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase complex. In the presence of progesterone, T47-D breast cancer cells display enhanced horizontal migration and invasion of three-dimensional matrices, which is reversed by small interfering RNAs abrogating FAK. In conclusion, progesterone promotes breast cancer cell movement and invasion by facilitating the formation of FA complexes via the rapid regulation of FAK. These results provide novel mechanistic views on the effects of progesterone on breast cancer progression, and may in the future be helpful to develop new strategies for the treatment of endocrine-sensitive breast cancers.

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.

Effects of raloxifene on breast cancer cell migration and invasion through the actin cytoskeleton.

Raloxifene (RAL) is a selective oestrogen receptor modulator (SERM) approved for the prevention and treatment of osteoporosis and for the prevention of breast cancer in postmenopausal women. However, little is known on the effects of this SERM on breast cancer cell metastasis, which is the main cause of morbidity and death. Cell movement is critical for local progression and distant metastasis of cancer cells. These processes rely on the dynamic control of the actin cytoskeleton and of cell membrane morphology. The aim of the present study was to characterize the effects of RAL or of 17beta-estradiol (E2) plus RAL on oestrogen receptor (ER) positive T47-D breast cancer cell cytoskeletal remodelling, migration and invasion. Our findings show that, when given alone, RAL induces a weak actin cytoskeleton remodelling in breast cancer cells, with the formation of specialized cell membrane structures implicated in cell motility. However, in the presence of physiological amounts of estradiol, which potently drives breast cancer cell cytoskeletal remodelling and motility, RAL displays a powerful inhibitory effect on oestrogen-promoted cell migration and invasion. These actions are plaid through an interference of RAL with an extra-nuclear signalling cascade involving G proteins and the RhoA-associated kinase, ROCK-2, linked to the recruitment of the cytoskeletal controller, moesin. Hence, in the presence of estradiol, RAL acts as an ER antagonist. These results highlight a novel mechanism of action of the SERM raloxifene that might be important for the interference of breast cancer progression or metastasis induced by oestrogens in postmenopausal women.

Comparative actions of progesterone, medroxyprogesterone acetate, drospirenone and nestorone on breast cancer cell migration and invasion.

BACKGROUND: Limited information is available on the effects of progestins on breast cancer progression and metastasis. Cell migration and invasion are central for these processes, and require dynamic cytoskeletal and cell membrane rearrangements for cell motility to be enacted. METHODS: We investigated the effects of progesterone (P), medroxyprogesterone acetate (MPA), drospirenone (DRSP) and nestorone (NES) alone or with 17beta-estradiol (E2) on T47-D breast cancer cell migration and invasion and we linked some of these actions to the regulation of the actin-regulatory protein, moesin and to cytoskeletal remodeling. RESULTS: Breast cancer cell horizontal migration and invasion of three-dimensional matrices are enhanced by all the progestins, but differences are found in terms of potency, with MPA being the most effective and DRSP being the least. This is related to the differential ability of the progestins to activate the actin-binding protein moesin, leading to distinct effects on actin cytoskeleton remodeling and on the formation of cell membrane structures that mediate cell movement. E2 also induces actin remodeling through moesin activation. However, the addition of some progestins partially offsets the action of estradiol on cell migration and invasion of breast cancer cells. CONCLUSION: These results imply that P, MPA, DRSP and NES alone or in combination with E2 enhance the ability of breast cancer cells to move in the surrounding environment. However, these progestins show different potencies and to some extent use distinct intracellular intermediates to drive moesin activation and actin remodeling. These findings support the concept that each progestin acts differently on breast cancer cells, which may have relevant clinical implications.

Effects of phytoestrogens derived from red clover on atherogenic adhesion molecules in human endothelial cells.

OBJECTIVE: In the search for safer approaches to address menopausal symptoms, the administration of plant-derived estrogens has gained popularity. Recent evidence suggests that these compounds may act neutrally or even beneficially on surrogate cardiovascular risk markers in postmenopausal women. However, little is known of the effects of phytoestrogens on vascular cells. DESIGN: Endothelial expression of leukocyte adhesion molecules plays a critical role in the development of atherosclerosis and in plaque destabilization, and estrogen reduces the expression of these proatherogenic molecules. We studied the regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) and of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells by phytoestrogens contained in red clover extracts. Moreover, we characterized the mechanistic basis for these actions. RESULTS: Red clover extracts, particularly genistein and daidzein, inhibit the endothelial expression of ICAM-1 and VCAM-1 induced by bacterial lipopolysaccharide. The addition of red clover extracts to reproductive life or menopausal concentrations of 17beta-estradiol results in an additive decrease in expression of endothelial adhesion molecules. The reduction of ICAM-1 and VCAM-1 expression in the presence of red clover extracts is paralleled by a cytoplasmic stabilization of the proinflammatory transcription factor nuclear factor-kappaB. CONCLUSIONS: Red clover extracts act as anti-inflammatory and antiatherogenic agents on human endothelial cells by reducing the expression of the leukocyte adhesion molecules ICAM-1 and VCAM-1. On the basis of these results, red clover extracts may induce beneficial actions on human vessels.

Progestogens regulate endothelial actin cytoskeleton and cell movement via the actin-binding protein moesin.

The endothelial effects of progestogens are poorly investigated. Actin remodeling and cell movement are fundamental for endothelial function and are controlled by the actin-binding protein moesin. In this work, we studied the effects of progesterone and medroxyprogesterone acetate (MPA) on actin remodeling, moesin activation and cell movement in human endothelial cells. Our findings show that progesterone and MPA trigger a rapid endothelial actin rearrangement, with the formation of cortical actin complexes, pseudopodia and membrane ruffles. Both progestogens trigger a rapid progesterone receptor (PR)-dependent moesin activation via a non-genomic signaling cascade involving G proteins, the small GTPase RhoA and the Rho-associated kinase (ROCK-2). In addition, MPA signaling also requires the recruitment of phosphatidylinositol-3 kinase (PI3K). Both progestogens enhance endothelial cell migration, which is prevented by moesin silencing or by blockade of PR, G proteins, PI3K, mitogen-activated protein kinases or ROCK-2. Progesterone and MPA potentiate 17beta-estradiol (E2) induced-moesin activation. However, they partially reduce cell migration induced by E2. In conclusion, progesterone and MPA regulate endothelial cell movement by rapidly signaling to the actin-binding protein moesin and to the actin cytoskeleton. These findings provide new information on the biological actions of progestins on human endothelial cells that are relevant for vascular function.