17Beta-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 17beta-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 Ca(2+) 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 Ca(2+) imaging, we demonstrated that E2 induced a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)) within minutes of exposure which was blocked by an L-type Ca(2+) channel antagonist. Inhibition of L-type Ca(2+) 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 Ca(2+) imaging combined with pERK immunofluorescence, demonstrated that E2 induced [Ca(2+)](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 Ca(2+) influx via the L-type Ca(2+) 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.
Wu
T-W
TW
Neuroscience Program, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA.
Wang
J M
JM
Chen
S
S
Brinton
R D
RD
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D019254:Genes, bcl-2 / Q000502:physiology*; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007150:Immunohistochemistry; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018696:Neuroprotective Agents; D011247:Pregnancy; D034741:RNA, Small Interfering; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D019061:src-Family Kinases / Q000502:physiology*
D020746:Calcium Channels, L-Type; D017362:Cyclic AMP Response Element-Binding Protein; D018696:Neuroprotective Agents; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D004958:Estradiol; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium