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Ellis R Levin

Publications and source records attributed to Ellis R Levin.

At least 19 recordsLinked to original sources
PubMed2006

Estrogen signaling in the cardiovascular system.

Estrogen exerts complex biological effects through the two isoforms of estrogen receptors (ERs): ERalpha and ERbeta. Whether through alteration of gene expression or rapid, plasma membrane-localized signaling to non-transcriptional actions, estrogen-activated ERs have significant implications in cardiovascular physiology. 17-beta-estradiol (E2) generally has a protective property on the vasculature. Estrogen treatment is anti-atherogenic, protecting injured endothelial surfaces and lowering LDL oxidation in animal models. Increased NO production stimulated by E2 results in vasodilation of the coronary vascular bed, and involves rapid activation of phosphotidylinositol-3 kinase (PI3K)/Akt signaling to eNOS in carotid and femoral arteries. Both isoforms of ERs impact various vascular functions, modulating ion channel integrity, mitigating the response to arterial injury, inducing vasodilation, and preventing development of hypertension in animal models. In addition to reducing afterload by vasodilation, ERs have a direct antihypertrophic effect on the myocardium. E2-activated ERs (E2/ER) antagonize the hypertrophic pathway induced by vasoactive peptides such as angiotensin II by activating PI3K, subsequent MICIP gene expression, leading to the inhibition of calcineurin activity and the induction of hypertrophic genes. In models of ischemia-reperfusion, E2/ER is antiapoptotic for cardiomyocytes, exerting the protective actions via PI3K and p38 MAP kinases and suppressing the generation of reactive oxygen species. In sum, E2-activated ERs consistently and positively modulate multiple aspects of the cardiovascular system.

Journal Article↗
PubMed2006

Nature of functional estrogen receptors at the plasma membrane.

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.

Amino Acid Sequence↗
PubMed2006

Functional estrogen receptors in the mitochondria of breast cancer cells.

Steroid hormones have been reported to indirectly impact mitochondrial functions, attributed to nuclear receptor-induced production of proteins that localize in this cytoplasmic organelle. Here we show high-affinity estrogen receptors in the mitochondria of MCF-7 breast cancer cells and endothelial cells, compatible with classical estrogen receptors ERalpha and ERbeta. We report that in MCF-7, estrogen inhibits UV radiation-induced cytochrome C release, the decrease of the mitochondrial membrane potential, and apoptotic cell death. UV stimulated the formation of mitochondrial reactive oxygen species (mROS), and mROS were essential to inducing mitochondrial events of cell death. mROS mediated the UV activation of c-jun N-terminal kinase (JNK), and protein kinase C (PKC) delta, underlying the subsequent translocation of Bax to the mitochondria where oligomerization was promoted. E2 (estradiol) inhibited all these events, directly acting in mitochondria to inhibit mROS by rapidly up-regulating manganese superoxide dismutase activity. We implicate novel functions of ER in the mitochondria of breast cancer that lead to the survival of the tumor cells.

Breast Neoplasms↗
PubMed2005

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms.

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.

Animals↗
PubMed2005

Estrogen modulates the mechanical homeostasis of mouse arterial vessels through nitric oxide.

We have recently shown that estrogen causes vessel dilation through receptor-mediated stimulation of nitric oxide (NO) production. Here, we hypothesize that estrogen modulates the mechanical homeostasis in the blood vessel wall through NO production. The mechanical properties of female ovariectomized (ovx) mice, female mice lacking the gene for endothelial NO synthase (eNOS(-/-)), and control female and male mice were studied to test the hypothesis. The femoral and carotid arteries and aorta were cannulated in situ and mechanically distended. The stress, strain, elastic modulus, and wall thickness of vessels in ovx and eNOS(-/-) mice, as well as intact female and male mice, were determined. Western blot and immunohistochemistry were used to assess eNOS protein expression in the aorta. Moreover, NO by-products of the femoral and carotid artery were determined by measuring the levels of nitrite and nitrate. Our results show that ovariectomy and eNOS(-/-) significantly decrease the strain in all arteries. Furthermore, the eNOS protein was significantly reduced in ovx mice. Finally, the NO metabolites were significantly decreased both in ovx and eNOS(-/-) mice. We found statistically significant correlations between the structural (wall thickness), mechanical (stress, strain, and elastic modulus), and biochemical parameters (NO by-products). These novel results connect NO to the structural and mechanical properties of the vessel wall. Hence, the effect of endogenous estrogen on the arterial mechanical properties is mediated by the regulation of NO derived from eNOS.

Animals↗
PubMed2005

Estrogen inhibits cardiomyocyte hypertrophy in vitro. Antagonism of calcineurin-related hypertrophy through induction of MCIP1.

Evidence from in vivo studies suggests that some inputs to cardiac hypertrophy are opposed by the actions of estrogen. However, the mechanisms of E2 action in this respect are mainly unknown. An important pathway that is utilized by multiple hypertrophic stimuli involves the activation of the tyrosine phosphatase, calcineurin (PP2B). Here we show that 17beta-estradiol (E2) significantly prevents angiotensin II (AngII)- or endothelin-1 (ET-1)-induced new protein synthesis, skeletal muscle actin expression, and increased surface area in cultured rat cardiomyocytes. ET-1 stimulated calcineurin phosphatase activity, resulting in new protein synthesis, and both were prevented by E2. E2 induced the MCIP1 gene, an inhibitor of calcineurin activity, via phosphatidylinositol 3-kinase, transcriptional, and mRNA stability mechanisms. Small interfering RNA for MCIP1 significantly reversed both the E2 restraint of protein synthesis and the inhibition of AngII-induced calcineurin activity. AngII-induced the translocation of the hypertrophic transcription factor, NF-AT, to the nucleus of the cardiomyocyte and stimulated NF-AT transcriptional activity. Both were prevented by E2. AngII also stimulated the activation of ERK and protein kinase C, contributing to cardiac hypertrophy. E2 inhibited these pathways, related to the stimulation of atrial natriuretic peptide production and secretion. Thus, restraint of calcineurin and kinase signaling to the hypertrophic program underlie these important effects of E2.

Angiotensin II↗
PubMed2005

Requirements for estrogen receptor alpha membrane localization and function.

The estrogen receptor alpha (ERalpha) exists as a functional receptor at the plasma membrane. The structural requirements for localization and function are not well understood. Several laboratories have recently elucidated certain requirements. We recently found the translocation of ERalpha to the membrane in the absence of estrogen is dependent on caveolin-1 and serine 522 of the ERalpha protein. Mutation of serine 522 to alanine results in a 62% decrease in membrane localization and association with caveolin-1. Similarly, deletion of the caveolin-1 scaffolding domain (amino acids 60-100) largely prevents the localization of ERalpha at the plasma membrane. In the presence of estradiol (E2), ERalpha, Src-homology and collagen homology (Shc), and insulin-like growth factor receptor-1 proteins associate with and increase the localization of ERalpha at the membrane. Membrane-localized ERalpha functions as an atypical G-protein coupled receptor. There is no good evidence that ERalpha spans the membrane or contains an extracellular domain. E2/ERalpha activates different G-proteins in cell context-related fashion. These G-proteins lead to the activation of Src through PLC, PKC, IP3 and calcium influx. In breast cancer, Src activates matrix metalloproteinase-2 and -9, which cleaves heparin binding epidermal growth factor, and thus activates EGFR. This leads to downstream signaling through ERK and PI3 kinase, imparting cell growth and survival.

Alanine↗
PubMed2005

Estrogen induces vascular wall dilation: mediation through kinase signaling to nitric oxide and estrogen receptors alpha and beta.

Estrogen has been shown to affect vascular cell and arterial function in vitro and in vivo. Here we examined the ability of estradiol (E(2)) to cause rapid arterial dilation of elastic and muscular arteries in vivo and the mechanisms involved. E(2) administration caused a rapid increase in the outer wall diameter of both types of arteries in ovariectomized female mice. This resulted from estrogen receptor (ER)-mediated stimulation of nitric oxide production, demonstrated by preinjecting the mice arteries with a soluble inhibitor of nitric oxide (monomethyl l-arginine) and by showing the absence of E(2) action in eNOS-/- mice. Rapid activation of both ERK/MAP kinase and phosphatidylinositol 3-kinase activity was found in the E(2)-exposed arteries, and inhibiting either kinase prevented the vasodilatory action of E(2). Kinase activation and vasodilator responses to E(2) were absent in either ERalpha or ERbeta knock-out mice, implicating both receptor subtypes as mediating this E(2) action. These results indicate that E(2) modulation of arterial tonus through plasma membrane ER and rapid signaling could underlie many previously observed actions of estrogen reported to occur in women.

Animals↗
PubMed2005

Integration of the extranuclear and nuclear actions of estrogen.

Estrogen receptors (ERs) are localized to many sites within the cell, potentially contributing to overall estrogen action. In the nucleus, estrogen mainly modulates gene transcription, and the resulting protein products determine the cell biological actions of the sex steroid. In addition, a small pool of ERs localize to the plasma membrane and signal mainly though coupling, directly or indirectly, to G proteins. In response to steroid, signal transduction modulates both nontranscriptional and transcriptional events and impacts both the rapid and more prolonged actions of estrogen. Cross-talk from membrane-localized ERs to nuclear ERs can be mediated through growth factor receptor tyrosine kinases, such as epidermal growth factor receptor and IGF-I receptor. Growth factor receptors enact signal transduction to kinases such as ERK and phosphatidylinositol 3-kinase that phosphorylate and activate nuclear ERs, and this can also occur in the absence of sex steroid. A complex relationship between the membrane and nuclear effects of estrogen also involves membrane-initiated phosphorylation of coactivators, recruiting these proteins to the nuclear transcriptosome. Finally, large pools of cytoplasmic ERs exist, and some are localized to mitochondria. The integration of sex steroid effects at distinct cellular locations of its receptor leads to important cellular physiological outcomes and are manifest in both reproductive and nonreproductive organs.

Animals↗
PubMed2004

Plasma membrane estrogen receptors exist and functions as dimers.

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.

Animals↗
PubMed2004

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer.

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.

BRCA1 Protein↗
PubMed2003

Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane.

Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ERalpha protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ERalpha. Upon transfection in CHO cells, the S522A mutant ERalpha resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ERalpha. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ERalpha S522A than from those of CHO cells expressing wt ERalpha. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ERalpha in binding caveolin 1, which facilitates ER transport to the PM. All functions of ERalpha mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ERalpha S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G1/S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ERalpha at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER.

Animals↗
PubMed2002

Bidirectional signaling between the estrogen receptor and the epidermal growth factor receptor.

Interactions between the estrogen receptor (ER) and the epidermal growth factor receptor (EGFR) contribute to the biological effects of these binding protein families. EGFR stimulates DNA synthesis and gene transcription in the uterus, related in part to estrogen-independent activation of the nuclear ER. This results from signal transduction enacted by the plasma membrane tyrosine kinase growth factor receptor, leading to 1) phosphorylation and activation of the nuclear ER, and 2) phosphorylation of coregulator proteins. More recently, it has been shown that a pool of ERalpha resides in or associates with the plasma membrane as a cytoplasmic protein. These ERs utilize the membrane EGFR to rapidly signal through various kinase cascades that influence both transcriptional and nontranscriptional actions of estrogen in breast cancer cells. This is congruent with a general theme of receptor signaling, where membrane G protein-coupled receptors activate tyrosine kinase growth factor receptors (EGFR, IGF-I receptor) that subsequently signal to MAPKs and other pathways. Overall, the bidirectional cross-talk between EGFR and cellular pools of ER contributes to reproductive organ physiology and pathophysiology.

Animals↗
PubMed2002

Proximal events in signaling by plasma membrane estrogen receptors.

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.

Animals↗
PubMed2002

Integration of the non-genomic and genomic actions of estrogen. Membrane-initiated signaling by steroid to transcription and cell biology.

Estrogen binds to receptors that translocate to the plasma membrane and to the nucleus. The rapid, non-genomic actions of this sex steroid are attributed to membrane action, while gene transcription occurs through nuclear receptor function. However, gene transcription can also result from estrogen signaling initiated at the membrane, but the relative importance of this mechanism is not known. In vascular endothelial cells (EC), estradiol (E(2)) activates several kinase cascades, including phosphatidylinositol 3-phosphate (PI3K)/Akt, a signaling pathway that impacts EC biology. We determined here by DNA microarray that 40-min exposure to E(2) significantly increased 250 genes in EC, up-regulation that was substantially prevented by the PI3K inhibitor, LY294002. This coincided with maximum E(2)-induced PI3K activity at 15-30 min. An important vascular gene strongly up-regulated by E(2) in our array produces cyclooxygenase-2 (Cox-2). In cultured EC, E(2) induced both Cox-2 gene expression and new Cox-2 protein synthesis by 40 and 60 min, respectively, and rapidly stimulated the secretion of prostaglandins PGI(2) and PGE(2). The up-regulation of gene expression reflected transcriptional transactivation, shown using Cox-2 promoter/luciferase reporters in the EC. Soluble inhibitors or dominant negative constructs for PI3K and Akt prevented all these actions of E(2). Functionally, EC migration was induced by the sex steroid, and this was significantly reversed by NS-398, a Cox-2 inhibitor. Gene transcription and cell biological effects of estrogen emanate from rapid and specific signaling, integrating cell surface and nuclear actions of this steroid.

Base Sequence↗
PubMed2002

Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide.

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology.

Actins↗
PubMed2002

Cellular functions of plasma membrane estrogen receptors.

Strong evidence now exists for the presence and importance of plasma membrane estrogen receptors (ER) in a variety of cells that are targets for steroid action. When estradiol (E2) binds cell surface proteins, the initiation of signal transduction triggers downstream signaling cascades that contribute to important functions. These functions include cell growth and survival, migration, and new blood vessel formation. In some instances these effects result from the initiation of gene transcription, upregulated through signaling from the membrane. The membrane ER probably originates from the same gene and transcript that produces the nuclear receptor. In the membrane, ER appear to localize mainly to discrete domains of the plasma membrane, known as caveolae, but the mechanisms by which this small pool of ER translocates to this site are currently unknown. At the caveolae, a cross talk with signaling molecules facilitates E2/ER cell biologic actions. This both includes direct stimulation of signaling via G protein activation, and a cross-activation of the epidermal growth factor receptor (EGFR). This review article highlights some of the important advances in understanding the cell biology of estrogen action that emanates from the membrane.

Animals↗