Xenoestrogen-Induced ERK-1 and ERK-2 Activation via Multiple Membrane-Initiated Signaling Pathways

Nataliya N. Bulayeva and Cheryl S. Watson, Department of Human Biological Chemistry

and Genetics, University of Texas

What are estrogens?Steroid hormones responsible for the programming of the female body for reproduction, especially the breasts, uterus, and brain.Other desirable effects include helping to maintain stable body temperature, regulating cholesterol levels, and maintaining bone density .Also has negative effects that occur with aging, including promoting breast and uterine cancer.

Mechanisms of steroid action

Genomic pathway involves direct transport of steroid hormones into the cell and straight to the nucleus, where transcription is directly initiatedNongenomic pathway involves the binding of steroid hormones to a cell membrane receptor that initiates an intracellular pathway to transcription via secondary messengers

Nongenomic effects of steroids

Changes in Ca2+, K+, cAMP, and NO levelsActivation of G-protein-mediated eventsStimulation of kinases, such as extracellular-regulated kinases (ERKs), phosphoinositide-3 kinase (PI3K), p38, and Jun kinase (Junk)While the precise mechanism of nongenomic actions are not fully understood, it is known that some rapid E2 (estradiol) effects can be initiated by binding to membrane-associated receptors (mERs), producing the same proteins via second messengers as their nuclear-receptor binding counterparts

What are xenoestrogens?Literally, “foreign estrogens”Molecular compounds found in the environment that share specific properties with biological estrogens that allow them to mimic their effects, even though they can vary greatly in structureThe current theory is that they accomplish this by binding to estrogen receptors (ER’s).

Negative effects of xenoestrogens

Declined sperm quality in fishInterference with sexual development in reptilesDisruption of pregnancies in lab animalsInterference with blastocyst implantationInappropriate induction of progesterone receptor expressionUterine weight increaseInhibit the human sperm acrosomal reactionSuspected of inducing breast cancer and proliferation of vaginal epithelium

Selected estrogens used in the study

Estradiol (E2) – a natural estrogenCoumestrol - a phytoestrogenP-nonylphenol and bisphenol A – detergent byproducts of plastic manufacturingEndosulfan, Dieldrin, and DDE – Organochlorine pesticides and metabolites

Purpose of the studyPrevious experiments involving xenoestrogen activity have focused on genomic activity (i.e. gene transcription)These studies have failed to provide a link between estradiol and xenoestrogens, with respect to their ability to cause reproductive abnormalities via the steroid pathwayThese studies concluded that vast amounts (1,000-10,000x) of xenoestrogens were required for transcription, as compared to E2

Purpose of the studyTo determine the ability of common estrogen mimetics to produce rapid activation of ERKs, which is upstream of transcriptional activity.To determine the signaling pathway(s) involved in ERK activation via these compounds, including intermediate cellular proteins, using the GH3/B6/F10 prolactinoma cell line.

GH3/B6/F10 prolactinoma cell line

Pituitary tumor cells cultured from ratsHigh expression of mER-α, a receptor with high affinity for E2 that elicits rapid ERK responses

MethodsAn ELISA was performed to estimate the level of ERK phosphorylation quantitativelyCells were deprived of steroids 48 hours prior to the experiments, and plated in 96-well platesCells were treated with each of the estrogen compounds for various time frames and in different concentrationsAfter treatment, a primary Ab, Anti-pERK was addedA secondary Ab, tagged with para-nitrophenol was added to indicate the amount of pERK present

MethodsA Crystal Violet assay was then used to determine the amount of cells present in each wellThe amount of pERK was normalized to the amount of cells present in each well by using a ratio of pNp/CV

Xenoestrogens can cause unique time-dependent patterns of ERK

phosphorylationE2, the natural estrogen, produced rapid and bimodal ERK phosphorylationXenoestrogens also caused ERK activation, but with distinct patterns, which all differed from the pattern of E2

RESULTS

Xenoestrogens exhibit unique concentration-dependent patterns of ERK

phosphorylation

All compounds tested were active at physiological levelsBisphenol A was not tested due to its lack of ERK activation Coumestrol and p-nonylphenol showed similar, dual-range, activation to E2

RESULTS

Determining possible pathways of ERK activation by selected xenoestrogens

Inhibitors of various ERK pathway intermediates were used in order to determine possible pathways of xenoestrogen actionICI, AG14, and Nystatin are inhibitors of specific membrane componentsB-TA, PP2, and LY are inhibitors of specific cytoplasmic components

RESULTS

ConclusionsAll tested xenoestrogens, except bisphenol A, elicited rapid membrane-initiated actions at very low concentrations compared to their reported potencies in genomic pathwaysDiffering chemical structure among the xenoestrogens did not seem to affect their ability to activate ERKs

ConclusionsNone of the tested xenoestrogens were able to precisely repeat the activation pattern of E2, which would explain why they cause disruption to estrogen-mediated endocrine functionsAll xenoestrogens tested were able to activate ERK, but via different pathway subsetsThe complexity of multiple signaling pathways triggered simultaneously is probably related to the organization of ERs within membrane substructures

Possible problems The authors assume that different timings of events correlate with different signaling pathwaysOnly estradiol was used for comparison, when other estrogens also exist (i.e. estrone)Compounds used to inhibit ERK phosphorylation were active before some xenoestrogen activity

What is the next step to determine these pathways?

Each xenoestrogen needs to be tested for an array of possible mechanistic routes of actionSpecifically, the subsets of pathways upstream of ERK activation need to be determined for each compound

QUESTIONS/COMMENTS?