17 beta-Estradiol induced Ca2+ influx via L-type calcium channels activates the Src/ERK/cyclic-amp response element binding protein signal pathway and Bcl-2 expression in rat hippocampal neurons: A potential initiation mechanism for estrogen-induced neuroprotection

Our group and others have demonstrated that 17(beta-estradiol (E2) induces neurotrophic and neuroprotective responses in hippocampal and cortical neurons which are dependent upon the Src/extracellular signal-regulated kinase (ERK) signaling pathways. The purpose of this study was to determine the upstream mechanism(s) that initiates the signaling cascade leading to E2-inducible neuroprotection. We tested the hypothesis that E2 activates rapid Ca2+ influx in hippocampal neurons, which would lead to activation of the Src/ERK signaling cascade and up-regulation of Bcl-2 protein expression. Using fura-2 ratiometric Ca2+ imaging, we demonstrated that E2 induced a rapid rise of intracellular Ca2+ concentration ([Ca2+](i)) within minutes of exposure which was blocked by an L-type Ca2+ channel antagonist. Inhibition of L-type Ca2+ channels resulted in a loss of E2 activation of the Src/ERK cascade, activation of cyclic-AMP response element binding protein (CREB) and subsequent increase in Bcl-2. Real-time intracellular Ca2+ imaging combined with pERK immunofluorescence, demonstrated that E2 induced [Ca2+](i) was coincident with ERK activation in the same neuron. Small interfering RNA knockdown of CREB resulted in a loss of E2 activation of CREB and subsequent E2-induced increase of Bcl-2 expression. We further demonstrated the presence of specific membrane E2 binding sites in hippocampal neurons. Together, these data indicate that E2-induced Ca2+ influx via the L-type Ca2+ channel is required for E2 activation of the SrC/ERK/CREB/Bcl-2 signaling. Implications of these data for understanding estrogen action in brain and use of estrogen therapy for prevention of neurodegenerative disease are discussed. (c) 2005 IBRO. Published by Elsevier Ltd. All rights reserved.