Identification of ER-α/ER-β Mediated Phosphorylation Activation State of eNOS in Ovine Uterine Artery Endothelial Cells from Pregnant Sheep
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July 28 2011
Mentor: Ronald Magness, Ph.D.
Background: Estrogen concentrations increase dramatically during pregnancy. The rises of estrogen help maintain the required uterine blood flow delivery of nutrients and oxygen to the growing fetus. Estrogen significantly increases the expression and activity of endothelial nitric oxide synthase (eNOS) thereby increasing the production of Nitric Oxide (NO), a potent uterine artery vasodilator. The enzyme eNOS is under complex post-translational modification that modulates its activity. Phosphorylation at Ser 1177 and Ser 635 have been shown to increase activity, while phosphorylation at Thr 495 decreases it. Plasma membrane estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β) bind to estrogen in order to initiate rapid nongenomic estrogenic actions. These ER isoforms may initiate phosphorylation of eNOS; which leads to the enzyme activation. However it is unknown in uterine artery endothelial cells from pregnant sheep (P-UAECs) which ER subtypes ER-α and/or ER-β are specifically responsible for the estrogen-mediated activation of eNOS. This can be tested using specific pharmacologic antagonist. We, therefore, tested the hypothesis that estrogen-mediated eNOS activation will be abrogated by either ER-α and/or ER-β specific antagonist.
Methods: P-UAECs (n=3) were plated in T-75 flasks to grow to ~80-90% confluence in growth media. P-UAECs were then re-plated into 6 well plates and pretreated with antagonist (or vehicle) for 60 min and the effects of 10nM E2β (or vehicle) were tested for 10 min with or without pretreatment with either ER-α (MPP; 1uM) and/or ER-β (PHTPP; 1uM) specific antagonist. The nonspecific ER-α+ER-β antagonist ICI 182780 (1uM) was used as a positive control. We utilized Western Analysis to assess eNOS “activation state” by identifying changes in phosphorylation at Ser 1177, Ser 635, and Thr 495 relative to total eNOS and β-actin.
Results: Compared to controls, Estrogen increased phosphorylation sites on Ser 1177 and Ser 635 and decreased phosphorylation site Thr 495, suggesting that there is an increase in eNOS activation in addition to lowering eNOS auto-inhibition. This was ER mediated as ICI 182780 abrogated these estrogen-mediated responses. Using the ER subtype specific antagonists, it appeared that on Ser 1177 and Thr 495 MPP and PHTPP also blocked the estrogen-induced changes in phosphorylation, suggesting that both ER-α and ER-β are involved in estrogen-induced eNOS activation. In contrast, data for Ser 635 indicated that only the ER-α antagonist MPP abrogated the estrogen-induced increase in this phosphorylation site.
Conclusion: Although our overall hypothesis was supported these data are only preliminary meaning further studies with more cell lines should be done to further investigate if ER-α and/or ER-β are responsible for eNOS activation. Support NIH HL49210, HD38843, HL87144.
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