Peptide inhibitors disrupt the serotonin 5-HT2C receptor interaction with phosphatase and tensin homolog to allosterically modulate cellular signaling and behavior.

Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R. A peptide derived from the third intracellular loop of the human 5-HT(2C)R [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT(2C)R-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT(2C)R signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT(2C)R allostery and therapeutics for 5-HT(2C)R-mediated disorders.

Direct estradiol and diethylstilbestrol actions on early- versus late-stage prostate cancer cells.

BACKGROUND: Diethylstilbestrol (DES) and other pharmaceutical estrogens have been used at ≥ µM concentrations to treat advanced prostate tumors, with successes primarily attributed to indirect hypothalamic-pituitary-testicular axis control mechanisms. However, estrogens also directly affect tumor cells, though the mechanisms involved are not well understood. METHODS: LAPC-4 (androgen-dependent) and PC-3 (androgen-independent) cell viability was measured after estradiol (E2) or DES treatment across wide concentration ranges. We then examined multiple rapid signaling mechanisms at 0.1 nM E2 and 1 µM DES optima including levels of: activation (phosphorylation) for mitogen-activated protein kinases, cell-cycle proteins, and caspase 3, necroptosis, and reactive oxygen species (ROS). RESULTS: LAPC-4 cells were more responsive than PC-3 cells. Robust and sustained extracellular-regulated kinase activation with E2 , but not DES, correlated with ROS generation and cell death. c-Jun N-terminal kinase was only activated in E2-treated PC-3 cells and was not correlated with caspase 3-mediated apoptosis; necroptosis was not involved. The cell-cycle inhibitor protein p16(INK4A) was phosphorylated in both cell lines by both E2 and DES, but to differing extents. In both cell types, both estrogens activated p38 kinase, which subsequently phosphorylated cyclin D1, tagging it for degradation, except in DES-treated PC-3 cells. CONCLUSIONS: Cyclin D1 status correlated most closely with disrupted cell cycling as a cause of reduced cell numbers, though other mechanisms also contributed. As low as 0.1 nM E2 effectively elicited these mechanisms, and its use could dramatically improve outcomes for both early- and late-stage prostate cancer patients, while avoiding the side effects of high-dose DES treatment.

Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.

BACKGROUND: Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line. METHODS: For each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens. RESULTS: Individual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen's response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the vehicle control, suggesting a possible apoptotic response. Extrinsic caspase 8 activity was suppressed by estradiol, elevated by bisphenol S, and unaffected by mixtures. Intrinsic caspase 9 activity was inhibited by estradiol, and by xenoestrogen combinations (at 10-14 and 10-8 M). Mixtures of xenoestrogens impeded the estradiol-induced release of prolactin. CONCLUSIONS: In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

Environmental estrogen exposures alter molecular signaling in immune cells that promote the development of childhood asthma.

Environmental estrogens (EEs) are associated with an increased prevalence of asthma. These epigenetic alterations of the immune cells may explain the multigenerational effects on asthma development. We hypothesized that exposure to immune cells enhances allergic sensitization by initiating signaling in these cells. Human T cell lines (TIB-152, CCL-119) were exposed to varying concentrations of estradiol, bisphenol A, bisphenol S, or bisphenol A + estradiol. H3K27me3, phosphorylations of EZH2 (pEZH2), AKT (pAKT), and phosphatidylinositide 3-kinase (pPI3K) were assessed. pAKT and pPI3K were decreased in response to some of the concentrations of these exposures in both cell lines. It is likely that EEs exposure to immune cells is one of the factors in the increase in the prevalence of asthma.

Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2C receptors.

BACKGROUND: The serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands. RESULTS: We optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines. CONCLUSIONS: These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.

Nongenomic effects of estradiol vs. the birth control estrogen ethinyl estradiol on signaling and cell proliferation in pituitary tumor cells, and differences in the ability of R-equol to neutralize or enhance these effects.

Ethinyl estradiol (EE2, the active component of many birth control formulations) persists in treated waste waters and it has become a concerning endocrine-disrupting contaminant throughout the world. Previous studies have not examined the behavior of EE2 in nongenomic signaling pathways and the subsequent functional responses (either alone or in mixtures) or conducted comparisons with the physiological estrogen estradiol (E2). In this study, mitogen-activated protein kinases (MAPKs), ERK, and JNK were activated in pituitary tumor cells by fM EE2, but p38 activation was insensitive to

Serotonin 5-HT2C receptor protein expression is enriched in synaptosomal and post-synaptic compartments of rat cortex.

The action of serotonin (5-HT) at the 5-HT(2C) receptor (5-HT(2C)R) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5-HT(2C)R protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5-HT(2C)R in synaptosomes and the post-synaptic density, an electron-dense thickening specialized for post-synaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, post-synaptic density), and controls (peptide neutralization, 5-HT(2C)R stably-expressing cells), we established the selectivity of two commercially available 5-HT(2C)R antibodies and employed the antibodies in western blot and immunoprecipitation studies of prefrontal cortex (PFC) and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5-HT(2C)R in post-synaptic density-enriched fractions from both PFC and motor cortex. Co-immunoprecipitation studies revealed the presence of post-synaptic density-95 within the 5-HT(2C)R protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5-HT(2C)R is localized within the post-synaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.

Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells.

BACKGROUND: Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens. METHODS: We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERß, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively). RESULTS: All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses. CONCLUSIONS: Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.

Nongenomic mechanisms of physiological estrogen-mediated dopamine efflux.

BACKGROUND: Neurological diseases and neuropsychiatric disorders that vary depending on female life stages suggest that sex hormones may influence the function of neurotransmitter regulatory machinery such as the dopamine transporter (DAT). RESULTS: In this study we tested the rapid nongenomic effects of several physiological estrogens [estradiol (E2), estrone (E1), and estriol (E3)] on dopamine efflux via the DAT in a non-transfected, NGF-differentiated, rat pheochromocytoma (PC12) cell model that expresses membrane estrogen receptors (ERs) alpha, beta, and GPR30. We examined kinase, ionic, and physical interaction mechanisms involved in estrogenic regulation of the DAT function. E2-mediated dopamine efflux is DAT-specific and not dependent on extracellular Ca2+-mediated exocytotic release from vesicular monoamine transporter vesicles (VMATs). Using kinase inhibitors we also showed that E2-mediated dopamine efflux is dependent on protein kinase C and MEK activation, but not on PI3K or protein kinase A. In plasma membrane there are ligand-independent associations of ERalpha and ERbeta (but not GPR30) with DAT. Conditions which cause efflux (a 9 min 10(-9) M E2 treatment) cause trafficking of ERalpha (stimulatory) to the plasma membrane and trafficking of ERbeta (inhibitory) away from the plasma membrane. In contrast, E1 and E3 can inhibit efflux with a nonmonotonic dose pattern, and cause DAT to leave the plasma membrane. CONCLUSION: Such mechanisms explain how gender biases in some DAT-dependent diseases can occur.

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated kinases and caspases.

BACKGROUND: Phytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high levels of the membrane estrogen receptor-alpha. METHODS: We measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities. RESULTS: Four phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH3/B6/F10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens. CONCLUSION: Phytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary.

Nongenomic actions of estradiol compared with estrone and estriol in pituitary tumor cell signaling and proliferation.

Physiological estrogens, including estrone (E(1)), estradiol (E(2)), and estriol (E(3)), fluctuate with life stage, suggesting specific roles for them in biological and disease processes. We compared their nongenomic signaling and functional actions in GH3/B6/F10 rat pituitary tumor cells. All hormones caused prolactin release at 1 min; the lowest effective concentrations were 10(-11) M E(2), 10(-10) M E(1), and 10(-7) M E(3). All estrogens increased the oscillation frequency of calcium (Ca) spikes, with the same time delay (approximately 200 s) at all levels (10(-15) to 10(-9) M). At some concentrations, E(1) and E(3) provoked more Ca-responding cells than E(2). The amplitude and volume of Ca peaks were elevated by all hormones at > or = 10(-15) M. All hormones caused cell proliferation, with the lowest effective concentrations of E(2) (10(-15) M) > E(1) (10(-12) M) > E(3) (10(-10) M); E(2) caused higher maximal cell numbers at most concentrations. All estrogens caused oscillating extracellular-regulated kinase (ERK) activations, with relative potencies of E(1) and E(2) > E(3). All estrogens were ineffective in activation of ERKs or causing proliferation in a subline expressing low levels of membrane estrogen receptor-alpha. Dose-response patterns were frequently nonmonotonic. Therefore, the hormones E(1) and E(3), which have been designated "weak" estrogens in genomic actions, are strong estrogens in the nongenomic signaling pathways and functional responses in the pituitary.

Xenoestrogen interference with nongenomic signaling actions of physiological estrogens in endocrine cancer cells.

Rapid nongenomic signaling by estrogens (Es), initiated near the cell membrane, provides new explanations for the potent actions of environmental chemicals that imperfectly mimic physiological Es. These pathways can affect tumor growth, stabilization, or shrinkage via a number of signaling streams such as activation/inactivation of mitogen-activated protein kinases and caspases, generation of second messengers, and phospho-triggering of cyclin instability. Though prostate cancers are better known for their responsiveness to androgen deprivation, ∼17% of late stage tumors regress in response to high dose natural or pharmaceutical Es; however, the mechanisms at the cellular level are not understood. More accurate recent measurements show that estradiol (E2) levels decline in aging men, leading to the hypothesis that maintaining young male levels of E2 may prevent the growth of prostate cancers. Major contributions to reducing prostate cancer cell numbers included low E2 concentrations producing sustained ERK phospho-activation correlated with generation of reactive oxygen species causing cancer cell death, and phospho-activation of cyclin D1 triggering its rapid degradation by interrupting cell cycle progression. These therapeutic actions were stronger in early stage tumor cells (with higher membrane estrogen receptor levels), and E2 was far more effective compared to diethylstilbestrol (the most frequently prescribed E treatment). Xenoestrogens (XEs) exacerbated the growth of prostate cancer cells, and as we know from previous studies in pituitary cancer cells, can interfere with the nongenomic signaling actions of endogenous Es. Therefore, nongenomic actions of physiological levels of E2 may be important deterrents to the growth of prostate cancers, which could be undermined by the actions of XEs.

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells.

The effects of 17beta-estradiol (E(2)) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E(2) at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) alpha, ERbeta, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of (3)H-DA from pre-loaded cells; a 9-15 min 10(-9 )M E(2) treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E(2)-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose-responses for E(2) were non-monotonic, also characteristic of non-genomic estrogenic actions. ERalpha siRNA knockdown abolished E(2)-mediated DA efflux, while ERbeta knockdown did not, and GPR30 knockdown increased E(2)-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERalpha is the predominant mediator of E(2)-mediated DA efflux, with inhibitory contributions from GPR30 and ERbeta. E(2) also caused trafficking of ERalpha to the plasma membrane, trafficking of ERbeta away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERalpha is largely responsible for non-genomic estrogenic effects on DAT activity.

Estradiol activates mast cells via a non-genomic estrogen receptor-alpha and calcium influx.

BACKGROUND: Allergic airway diseases are more common in females than in males during early adulthood. A relationship between female hormones and asthma prevalence and severity has been suggested, but the cellular and molecular mechanisms are not understood. OBJECTIVE: To elucidate the mechanism(s) by which estrogens enhance the synthesis and release of mediators of acute hypersensitivity. METHODS: Two mast cell/basophil cell lines (RBL-2H3 and HMC-1) and primary cultures of bone marrow derived mast cells, all of which naturally express estrogen receptor-alpha, were examined. Cells were incubated with physiological concentrations of 17-beta-estradiol with and without IgE and allergens. Intracellular Ca(2+) concentrations and the release of beta-hexosaminidase and leukotriene C(4) were quantified. RESULTS: Estradiol alone induced partial release of the preformed, granular protein beta-hexosaminidase from RBL-2H3, BMMC and HMC-1, but not from BMMC derived from estrogen receptor-alpha knock-out mice. The newly synthesized LTC(4) was also released from RBL-2H3. Estradiol also enhanced IgE-induced degranulation and potentiated LTC(4) production. Intracellular Ca(2+) concentration increased prior to and in parallel with mediator release. Estrogen receptor antagonists or Ca(2+) chelation inhibited these estrogenic effects. CONCLUSION: Binding of physiological concentrations of estradiol to a membrane estrogen receptor-alpha initiates a rapid onset and progressive influx of extracellular Ca(2+), which supports the synthesis and release of allergic mediators. Estradiol also enhances IgE-dependent mast cell activation, resulting in a shift of the allergen dose response.

Novel Bivalent 5-HT2A Receptor Antagonists Exhibit High Affinity and Potency in Vitro and Efficacy in Vivo.

The 5-HT2A receptor (5-HT2AR) plays an important role in various neuropsychiatric disorders, including substance use disorder and schizophrenia. Homodimerization of this receptor has been suggested, but tools that allow direct assessment of the relevance of the 5-HT2AR:5-HT2AR homodimer in these disorders are necessary. We chemically modified the selective 5-HT2AR antagonist M100907 to synthesize a series of homobivalent ligands connected by ethylene glycol linkers of varying lengths that may be useful tools for probing 5-HT2AR:5-HT2AR homodimer function. We tested these molecules for 5-HT2AR antagonist activity in a cell line stably expressing the functional 5-HT2AR and quantified a downstream signaling target, activation (phosphorylation) of extracellular regulated kinases 1/2 (ERK1/2), in comparison to in vivo efficacy of altering spontaneous or cocaine-evoked locomotor activity in rats. All of the synthetic compounds inhibited 5-HT-mediated phosphorylation of ERK1/2 in the cellular signaling assay; the potency of the bivalent ligands varied as a function of linker length, with the intermediate linker lengths being the most potent. The Ki values for the binding of bivalent ligands to 5-HT2AR were only slightly lower than the values for the parent (+)-M100907 compound, but significant selectivity for 5-HT2AR over 5-HT2BR or 5-HT2CR binding was retained. In addition, the 11-atom-linked bivalent 5-HT2AR antagonist (2 mg/kg, intraperitoneally) demonstrated efficacy on par with that of (+)-M100907 in inhibiting cocaine-evoked hyperactivity. As we develop further strategies for ligand-evoked receptor assembly and analyses of diverse signaling and functional roles, these novel homobivalent 5-HT2AR antagonist ligands will serve as useful in vitro and in vivo probes of 5-HT2AR structure and function.

Environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators.

BACKGROUND: Prevalence and morbidity of allergic diseases have increased over the last decades. Based on the recently recognized differences in asthma prevalence between the sexes, we have examined the effect of endogenous estrogens on a key element of the allergic response. Some lipophilic pollutants have estrogen-like activities and are termed environmental estrogens. These pollutants tend to degrade slowly in the environment and to bioaccumulate and bioconcentrate in the food chain; they also have long biological half-lives. OBJECTIVES: Our goal in this study was to identify possible pathogenic roles for environmental estrogens in the development of allergic diseases. METHODS: We screened a number of environmental estrogens for their ability to modulate the release of allergic mediators from mast cells. We incubated a human mast cell line and primary mast cell cultures derived from bone marrow of wild type and estrogen receptor alpha (ER-alpha)-deficient mice with environmental estrogens with and without estradiol or IgE and allergens. We assessed degranulation of mast cells by quantifying the release of beta-hexosaminidase. RESULTS: All of the environmental estrogens tested caused rapid, dose-related release of beta-hexosaminidase from mast cells and enhanced IgE-mediated release. The combination of physiologic concentrations of 17beta-estradiol and several concentrations of environmental estrogens had additive effects on mast cell degranulation. Comparison of bone marrow mast cells from ER-alpha-sufficient and ER-alpha-deficient mice indicated that much of the effect of environmental estrogens was mediated by ER-alpha. CONCLUSIONS: Our findings suggest that estrogenic environmental pollutants might promote allergic diseases by inducing and enhancing mast cell degranulation by physiologic estrogens and exposure to allergens.

Xenoestrogens are potent activators of nongenomic estrogenic responses.

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E(2)) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-alpha on pituitary cells, and can provoke Ca(2+) influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E(2). It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.

In vitro molecular mechanisms of bisphenol A action.

Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS# 80-05-7) is a chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and as a non-polymer additive to other plastics. Recent evidence has demonstrated that human and wildlife populations are exposed to levels of BPA which cause adverse reproductive and developmental effects in a number of different wildlife species and laboratory animal models. However, there are major uncertainties surrounding the spectrum of BPA's mechanisms of action, the tissue-specific impacts of exposures, and the critical windows of susceptibility during which target tissues are sensitive to BPA exposures. As a foundation to address some of those uncertainties, this review was prepared by the "In vitro" expert sub-panel assembled during the "Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health" workshop held in Chapel Hill, NC, Nov 28-29, 2006. The specific charge of this expert panel was to review and assess the strength of the published literature pertaining to the mechanisms of BPA action. The resulting document is a detailed review of published studies that have focused on the mechanistic basis of BPA action in diverse experimental models and an assessment of the strength of the evidence regarding the published BPA research.

Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

INTRODUCTION: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells. METHODS: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory. RESULTS: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A. CONCLUSION: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.