Research On The Role Of One Novel Estrogen Receptor, ER-α36, In Estrogen Neuroprotection Using Cell Models

Estrogen is one of the most important endocrine hormones for female reproduction. There are three types of estrogen:estrone (E1),17a-estradiol (E2α)、17β-estradiol (E2β) and estriol (E3). Estrogen also plays essential roles in non-reproductive systems including cardiovascular system, liver, bones and neural system. Many in vivo and in vitro studies have demonstrated the important role of estrogen in the neuronal growth and differentiation, neuroprotection, cognition, and regulation of mood. Here, we mainly focus on the neuroprotection of estrogen in the nervous system.It is well-known that estrogen works through the estrogen receptors, which are classified into ER-α and ER-β. ER-α is mainly referred to ER-α66. One novel 36KD variant of ER-a66, ER-a36, was first identified and cloned in 2005 by Dr. Wang. As a novel type of estrogen receptor, ER-a36 has been found to function in the genesis and progression of some tumors. However, there are no reports on ER-α36’s function in the nervous system till now.Although many studies have demonstrated that estrogen has neuroprotective function, but the mechanism is not fully understood, and the involved estrogen receptor is still a controversy. The goal of our present study was to determine the involved estrogen receptor, and also the mechanism in estrogen neuroprotection using the neuronal cells injured by H2O2. Our study may provide some theoretical basis and experimental evidence for estrogen’s therapeutic effect in neurodegenerative diseases.[Background]There are abundant in vitro and in vivo evidences indicating the neuroprotection of estrogen. Post-menopausal women in estrogen-deprived state are at high risk for stroke and other neurodegenerative diseases. Some researchers found that estrogen could ameliorate the progression of Parkinson’s disease (PD). Epidemiological evidence suggested that postmenopausal estrogen therapy reduce the risk, and also delay the onset of AD. The loss of estrogen from natural or surgical menopause is associated with a decline in cognitive function which could be reversed by estrogen therapy. Mortality rate after stroke is reduced in post-menopausal patients who were taking estrogen therapy during the recovery time of stroke. In addition, estrogen is reported to play important roles in recovery from brain injury.Neurodegenerative diseases are commonly accompanied by the occurrence of neuroinflammation, and estrogen has been found to have anti-neuroinflammation effects. With the decline of physiological estrogen concentration, post-menopausal women tend to suffer several neuroinflammation diseases which can be treated by appropriate estrogen therapy. Therefore, estrogen-induced neuroprotection and anti-neuroinflammation, is an important research field of modern neurobiology. It is of great significance to reveal the biological function of estrogen and develop targeted drugs for the therapy of neurodegenerative diseases.Estrogen exerts its biological effects through two signal pathways, the genomic and non-genomic pathway:1) Genomic pathway involves estrogen that binds to the steroid hormone receptors in the cytosol, transported to the nucleus, where these hormone-receptor dimmers traditionally function as transcription factors; 2) Nongenomic responses are initiated at the cell membrane, involving the rapid activation of kinases and intracellular signal pathways. Classical estrogen receptors include ERa (ER-α66 and ER-α46) and ERβ, which work through the genomic pathways. Although classic genomic mechanism are well studied, membrane-initiated non-genomic pathways, which plays an important role in estrogen’s neuroprotection, are not completely understood till now,[Objective]The goal of our study was to determine and evaluate the role of ER-α36 in E2β neuroprotection using SH-SY5Y cells and IMR-32 cells injured by H2O2. We also established neuronal cells with forced expression of recombinant ER-α36 and knocked-down levels of ER-α36 expression, respectively, to explore the role of ER-α36 in the neuroprotection and its concrete mechanisms.[Methods]SH-SY5Y and IMR-32 cells were respectively treated by 250μM of H2O, to construct the neuronal cell models of H2O2 injury, and the cell models were treated with 10nM of E2β and G1 respectively. Cell survival rate was determined by MTT assay, and cell apoptosis was quantified by flow cytometric analysis, which preliminarily identified whether ER-a36 was involved in estrogen’s neuroprotection. The phosphorylation of AKT or ERK molecule stimulated by E2β and G1 in different time points was examined by Western blotting to study the non-genomic signaling pathways in estrogen’s neuroprotection. We also established neuronal cells with knocked-down levels of ER-a36 expression and with forced expression of recombinant ER-a36, observing the changes in estrogen associated non-genomic pathway stimulated by E20 and G1 to demonstrate the involved mechanism mediated through ER-a36 in estrogen neuroprotection.[Results](1) MTT results demonstrated that E2β and G1 could markedly attenuate cell loss induced by H2O2 in the cultured neuronal cells, demonstrating the neuroprotective effect. However, this effect was markedly inhibited in the neuronal cells with knocked-down levels of ER-a36 expression.In cultured neuronal cells with knocked-down levels of ER-α66 expression, this protective effect was not affected.(2) Flow cytometric analysis demonstrated that E2β and G1 could markedly reduce the number of apoptotic and necrotic cells induced by H2O2 in cultured neuronal cells. The effect was markedly inhabited in the neuronal cells with knocked-down levels of ER-a36 expression. In cultured neuronal cells with knocked-down levels of ER-a66 expression, the protective effect was not affected.(3) Western blotting results indicated that E2β and G1 could activate the PI3K/AKT and MAPK/ERK pathways in five minutes, which was markedly inhibited in the neuronal cells with knocked-down levels of ER-a36 expression. However, this activation effects were not affected in cultured neuronal cells with knocked-down levels of ER-a66 expression. The above results indicated that ER-a36 was involved in estrogen associated non-genomic signaling pathways.(4)We established cells with knocked-down level of GPR30 using GPR30-specific small interfering RNA (siRNA) in SH-SY5Y cells with constant forced expression of recombinant ER-α36. Western blotting assays indicated that E2β and G1 could also activate PI3K/AKT and MAPK/ERK pathways in these cells, which indicated GPR30 is not involved in ER-α36 meidated non-genomic signaling pathways.[Conclusion]Our results demonstrated that ER-α36 is involved in estrogen’s neuroprotection and also the estrogen associated non-genomic signaling pathways such as PI3K/AKT and MAPK/ERK signals. GPR30 is not involved in ER-α36 meidated non-genomic signaling pathways.