Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 versus several XEs representing organochlorine pesticides (dieldrin, endosulfan, o′p′-dichlorodiphenylethylene), plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-α. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca2+ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca2+ elevation. Removing Ca2+ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca2+ changes, as did 10 μM nifedipine. This suggests that XEs mediate their actions via voltage-dependent L-type Ca2+ channels in the plasma membrane. None of the Ca2+ fluxes came from intracellular Ca2+ stores. E2 and each XE also caused unique time- and concentration-dependent patterns of prolactin (PRL) secretion that were largely complete within 3 min of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca2+ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular Ca2+ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies that could explain their abilities to disrupt endocrine functions.
Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane. In this study, we demonstrate that several xenoestrogens can rapidly activate extracellular-regulated kinases (ERKs) in the pituitary tumor cell line GH3/B6/F10, which expresses high levels of the membrane receptor for ER-α(mER). We tested a phytoestrogen (coumestrol), organochlorine pesticides or their metabolites (endosulfan, dieldrin, and DDE), and detergent by-products of plastics manufacturing (p-nonylphenol and bisphenol A). These xenoestrogens (except bisphenol A) produced rapid (3–30 min after application), concentration (10−14–10−8 M)-dependent ERK-1/2 phosphorylation but with distinctly different activation patterns. To identify signaling pathways involved in ERK activation, we used specific inhibitors of ERs, epidermal growth factor receptors, Ca2+ signaling, Src and phosphoinositide-3 kinases, and a membrane structure disruption agent. Multiple inhibitors blocked ERK activation, suggesting simultaneous use of multiple pathways and complex signaling web interactions. However, inhibitors differentially affected each xenoestrogen response examined. These actions may help to explain the distinct abilities of xenoestrogens to disrupt reproductive functions at low concentrations.
Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-alpha (mERalpha). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2-P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10(-14) and 10(-8)M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3-6 min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.
The role of membrane estrogen receptor-α (mERα) in rapid nongenomic responses to 17β-estradiol (E2) was tested in sublines of GH3/B6 rat prolactinoma cells selected for high (GH3/B6/F10) and low (GH3/B6/D9) mERα expression. E2 elicited rapid, concentration-dependent intracellular Ca2+ concentration ([Ca2+]i) increases in the F10 subline. Lack of inhibition by thapsigargin depletion of intracellular Ca2+ pools, together with abrogation of the response in Ca2+-free medium, suggested an extracellular source of Ca2+ for this response. The participation of voltage-dependant channels in the E2-induced [Ca2+]i increase was confirmed by the specific L-type Ca2+ channel inhibitor nifedipine. For comparison, the D9 mERα-depleted subline was insensitive to steroid action via this signaling mechanism. [Ca2+]i elevation was correlated with prolactin (PRL) release in the F10 cell line in as little as 3 min. E2 caused a much higher PRL release than KCl treatment (which caused maximal Ca2+ elevation), suggesting that secretion was also controlled by additional mechanisms. Participation of mERα in these effects was confirmed by the ability of E2-peroxidase (a cell-impermeable analog of E2) to cause these responses, blockage of the responses with the ER antagonist ICI 182 780, and the inability of the E2 stereoisomer 17α-E2 to elicit a response. Thus rapid exocytosis of PRL is regulated in these cells by mERα signaling to specific Ca2+ channels utilizing extracellular Ca2+ sources and additional signaling mechanisms.
Studies of the nuclear transcriptional regulatory activities of nonphysiological 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-α on pituitary cells, and can provoke Ca ++ 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.
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