The Study Of Signal Transduction Mechanism Of Estrogen Modulation In EAAC1 Protein Expression

Excitatory amino acid transporter 3 (EAAT3/EAAC1) is a primary glutamate and cystein transporter in central nervous system (CNS), whose dysfunction would cause glutathione deficiency, increased oxidative stress and multiple neurodegenerative diseases like Alzheimer disease and Parkinson disease. In addition, enhanced expression of EAAC1 which is an adaptive response to spinal cord injury and multiple sclerosis may subserve neuroprotective mechanisms occurring after these disorders. Elucidating the molecular pathway involving EAAC1 regulation, therefore, can provide powerful therapeutic targets that affect a wide spectrum of pathological conditions in CNS. Previous studies mainly focoued on signaling pathways relevent with EAAC1 function. However, the mechanisms underlying the regulation of EAAC1 expression have not been clearly characterized. In the present study, using C6, an astroglial cell line which specifically expresses EAAC1, we explored a novel signaling transduction mode for E2 modulating EAAC1 expression.It has been well-established that 17β-estradiol (E2) pocesses a neuroprotection effect against glutamate toxcity and oxidative stress. To begin with, by MTT assay, we showed that the low expression of EAAC1 was correlated with cell vulnerabilities to hydrogen peroxide, 61202)-Then we discovered E2 robustly increased EAAC1 expression, while knock down of its expression could depress protection effects of E2 against oxidative stress. At the same time, we chose Gl, one agonist of GPR30 (a membrane-associated G protein-coup led receptor), to mimic E2 effects on EAAC1 expression and on protection from H2O2 cytotoxicity, which were inhibited by applying with G15, the antagonist of GPR30. Consequently, the above results indicate that it is through GPR30 that E2 increased EAAC1 expression to protecte cells against oxidative stress.The lip id kinase sphingosine kinase-1 (Sphkl) is contributive to promote cell survival and growth. It has been reported that E2 enhanced cell viability via Sphkl in the nervous system. Hence, we hope to explore whether Sphk1 modulates EAAC expression induced by E2. We firstly tested that E2 and G1 via GPR30 activated Sphkl by triggering Sphk1 phosphorylationon Ser-225. Then, using pharmatheutical inhibitor to restrain Sphk1 activity, we detected the declined capability of E2 regulating EAAC1 expression. In addition, siRNA technique were performed to evaluate the effect of Sphkl on EAAC1, and we found Sphkl inhibition blocked E2/G1-induced EAAC1 expression. Moreover, sphingosine-1-phosphate (SIP), the catalytic product of Sphk1, exhibited a role in increasing EAAC1 protein level. Together, these findings indicate that it is dependent on Sphkl activation through GPR30 that E2 promotes EAAC1 expression.SIP is also an important second messenger which mediates the transactivations of some growth factor receptors, such as EGFR and PDGFR. Consequently, we investigated the downstream signaling events after Sphkl activation by E2. We characterized SIP, as a second messenger of GPR30, could upregulate FGF2 mRNA level. Then FGF2, E2,G1 and SIP all activated FRS2a to stimulate ERK1/2 signaling and subsequent EAAC1 expression. In addition, we discovered that FGF2 siRNA and inhibitors of tyrosine kinase or ERK1/2 repressed EAAC1 expression induced by E2, G1 or SIP. These results demonstrate FRS2α-ERK signaling pathway is involved in the increase of EAAC1 protein induced by E2, G1 or SIP. In line with these results, Sphk1 inhibition supressed E2/G1-induced both FGF transcription and FRS2α-ERK signaling transactivations. The above results indicate that FRS2α-ERK signal transduction mediated by Sphkl plays an indispensable role in regulating E2/G1-indcued EAAC1 expression.In conclusion, we prove that E2 promotes EAAC1 expression through GPR30- and Sphk1-mediated FRS2α-ERK cascade activation and the enhanced EAAC1 protein may contribute to E2’s effect against glutamate toxicity. Moreover, three independent messeage molecules, E2, SIP and FGF2 form a crosstalk to modulate EAAC1 expression. Above all, we unveiled a novel signaling pathway for E2 regulation on EAAC1 expression. This finding may cast light on understanding the action of E2 neuroprotective effect and provides new targets for controlling oxidative toxicity in various neurodegenerative diseases.