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 initiated Nongenomic 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 Ca 2+, K +, cAMP, and NO levels Activation of G-protein-mediated events Stimulation 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 E 2 (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 structure The current theory is that they accomplish this by binding to estrogen receptors (ER’s).

Negative effects of xenoestrogens Declined sperm quality in fish Interference with sexual development in reptiles Disruption of pregnancies in lab animals Interference with blastocyst implantation Inappropriate induction of progesterone receptor expression Uterine weight increase Inhibit the human sperm acrosomal reaction Suspected of inducing breast cancer and proliferation of vaginal epithelium

Selected estrogens used in the study Estradiol (E 2 ) – a natural estrogen Coumestrol - a phytoestrogen P-nonylphenol and bisphenol A – detergent byproducts of plastic manufacturing Endosulfan, Dieldrin, and DDE – Organochlorine pesticides and metabolites

Purpose of the study Previous 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 pathway These studies concluded that vast amounts (1,000-10,000x) of xenoestrogens were required for transcription, as compared to E 2

Purpose of the study To 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 GH 3 /B6/F10 prolactinoma cell line.

GH 3 /B6/F10 prolactinoma cell line Pituitary tumor cells cultured from rats High expression of mER-α, a receptor with high affinity for E 2 that elicits rapid ERK responses

Methods An ELISA was performed to estimate the level of ERK phosphorylation quantitatively Cells were deprived of steroids 48 hours prior to the experiments, and plated in 96-well plates Cells were treated with each of the estrogen compounds for various time frames and in different concentrations After treatment, a primary Ab, Anti-pERK was added A secondary Ab, tagged with para-nitrophenol was added to indicate the amount of pERK present

Methods A Crystal Violet assay was then used to determine the amount of cells present in each well The 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 phosphorylation E2, the natural estrogen, produced rapid and bimodal ERK phosphorylation Xenoestrogens 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 levels Bisphenol A was not tested due to its lack of ERK activation Coumestrol and p-nonylphenol showed similar, dual-range, activation to E 2 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 action ICI, AG14, and Nystatin are inhibitors of specific membrane components B-TA, PP2, and LY are inhibitors of specific cytoplasmic components RESULTS

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

Conclusions None of the tested xenoestrogens were able to precisely repeat the activation pattern of E 2, which would explain why they cause disruption to estrogen- mediated endocrine functions All xenoestrogens tested were able to activate ERK, but via different pathway subsets The 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 pathways Only 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 action Specifically, the subsets of pathways upstream of ERK activation need to be determined for each compound

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