Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a...

Using hippocampal primary cell cultures at 14 days in vitro (div), we have investigated actions of 17-beta estradiol (E; 10 nM) on the phosphorylation of CREB and on signaling pathways that regulate CREB phosphorylation. After demonstrating that 14 div is optimal for these studies, we examined the time course of E induction of CREB phosphorylation (pCREB) at serine residue 133. The induction of pCREB occurs as early as 1 h following E treatment, presumably via a mechanism involving an E-stimulated signal transduction system, which is sustained for at least 24 h but inhibited by 48 h. The early activity may represent an initial signal required for events leading to phosphorylation of CREB while the sustained signal may lead to CREB-mediated gene expression for cell survival and synapse formation. Furthermore, we examined the pathways for E action preceding pCREB induction by blocking three major kinases (protein kinase; mitogen activated protein kinase, MAPK; and calcium-calmodulin kinase II, CaMKII) upstream of pCREB. We found that E stimulates each pathway at 24 h and that phosphorylation of CREB is dependent on both MAPK and CaMK activities, but less dependent on the Akt pathway. Because CREB has been linked to E induction of excitatory spine synapses, we used a spine marker, spinophilin, to establish E effects on spine formation. Spinophilin expression was up-regulated in response to E and this effect was blocked by an inhibitor of (CaMKII). These studies demonstrate the central role played by CaMKII pathway in the actions of E on both transcriptional regulation and structural reorganization in neurons.