Lee S J, Campomanes C R, Sikat P T, Greenfield A T, Allen P B, McEwen B S
Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
Neuroscience. 2004;124(3):549-60. doi: 10.1016/j.neuroscience.2003.11.035.
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.
利用体外培养14天的海马原代细胞，我们研究了17-β雌二醇（E；10 nM）对CREB磷酸化以及调节CREB磷酸化的信号通路的作用。在证明14天体外培养对这些研究最为适宜后，我们检测了E诱导丝氨酸残基133处CREB磷酸化（pCREB）的时间进程。pCREB的诱导在E处理后1小时就开始出现，推测是通过一种涉及E刺激信号转导系统的机制，这种诱导至少持续24小时，但在48小时时受到抑制。早期活性可能代表导致CREB磷酸化的事件所需的初始信号，而持续信号可能导致CREB介导的促进细胞存活和突触形成的基因表达。此外，我们通过阻断pCREB上游的三种主要激酶（蛋白激酶；丝裂原活化蛋白激酶，MAPK；以及钙调蛋白激酶II，CaMKII）来检测pCREB诱导之前E的作用途径。我们发现E在24小时时刺激每条途径，并且CREB的磷酸化依赖于MAPK和CaMK的活性，但对Akt途径的依赖性较小。由于CREB与E诱导兴奋性棘突触有关，我们使用棘标记物亲棘蛋白来确定E对棘形成的影响。亲棘蛋白的表达响应E而上调，并且这种作用被CaMKII的抑制剂阻断。这些研究证明了CaMKII途径在E对神经元转录调控和结构重组作用中的核心作用。
