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

1. 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

2. 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.

3. 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

4. Nongenomic effects of steroids • Changes in Ca2+, 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 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

5. 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).

6. 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

7. Selected estrogens used in the study • Estradiol (E2) – a natural estrogen • Coumestrol - a phytoestrogen • P-nonylphenol and bisphenol A – detergent byproducts of plastic manufacturing • Endosulfan, Dieldrin, and DDE – Organochlorine pesticides and metabolites

8. 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 E2

9. 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 GH3/B6/F10 prolactinoma cell line.

10. GH3/B6/F10 prolactinoma cell line • Pituitary tumor cells cultured from rats • High expression of mER-α, a receptor with high affinity for E2 that elicits rapid ERK responses

11. 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

12. 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

13. RESULTS 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

15. 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 E2

17. 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

20. 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

21. Conclusions • None 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 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

22. 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

23. 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

24. QUESTIONS/COMMENTS?