Estrogen induces phosphorylation of cyclic AMP response element binding (pCREB) in primary hippocampal cells in a time-dependent manner.
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.
Lee
S J
SJ
Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. leesus@rockefeller.edu
Campomanes
C R
CR
Sikat
P T
PT
Greenfield
A T
AT
Allen
P B
PB
McEwen
B S
BS
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects / Q000378:metabolism*; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004967:Estrogens / Q000378:metabolism* / Q000494:pharmacology; D005333:Fetus; D005809:Genes, Regulator / Q000187:drug effects / Q000235:genetics; D006624:Hippocampus / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D000099273:Neurabins
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D004967:Estrogens; D008840:Microfilament Proteins; D019950:Mitogen-Activated Protein Kinase 1; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000099273:Neurabins; C494920:Akt1 protein, rat