Estrogen Facilitates both Phosphatidylinositol 3-Kinase/Akt and ERK1/2 Mitogen-Activated Protein Kinase Membrane Signaling Required for Long-Term Neuropeptide Y Transcriptional Regulation in Clonal, Immortalized Neurons

Abstract

It is established that increases in neuropeptide Y (NPY) expression are associated with hyperphagia and obesity. These effects can be reversed by estrogen, a recognized anorexigen. We found that 17␤-estradiol (E 2 ) regulates biphasic NPY gene expression in a clonal, immortalized hypothalamic cell line, N-38, through estrogen receptor (ER) action at the level of the NPY promoter. However, rapid, nongenomic actions of estrogen, linked to the phosphatidylinositol 3-kinase (PI3-K)/Akt and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, may also play a role. We therefore examined the changes in the phosphorylation status of Akt, ERK1/2, and cAMP response element-binding protein (CREB) after treatment with 10 nM E 2 in the N-38 neurons and found activation of these signaling proteins within 5-30 min. We also demonstrated possible cross talk between the estrogen-activated PI3-K/Akt and MAPK/extracellular signal-regulated kinase pathways using pharmacological inhibitors. We find that only ER␣ is involved in the early signaling events using the ER␣ agonist 4,4Ј,4Љ-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ER␤ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. Furthermore, we can detect colocalization of ER␣ and caveolin-1, a membrane-associated signaling protein. Remarkably, we find that the membrane-mediated events are critical for the long-term estrogen-mediated repression of NPY gene expression that can be mapped to within Ϫ97 bp of the NPY promoter. To link the early signaling events to downstream effectors, we detected induction of c-fos and inactivation of MSK-1 by estrogen and binding of CREB to this minimal promoter region. These observations suggest that rapid estrogenmediated signaling is mediated by ER␣, and the signal transduction events potentiate the genomic actions of estrogen on NPY gene expression in the N-38 NPY neurons.

Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons

“…The experiments completed in this study were inspired by earlier work from our lab that showed that E 2 decreased NPY mRNA expression in the mHypoE-38 and mHypoE-42 neurons based through PI3K-and MAPK-dependent mechanisms. 19,23 Our findings extend these studies by providing the first evidence that acute E 2 exposure can directly regulate NPY secretion. Here, using clonal, immortalized, hypothalamic, NPY-expressing cell lines we investigated the direct regulation of the orexigenic neuropeptide NPY as a central target for E 2 .…”

Estrogen augments the T cell-dependent but not the T-independent immune response

“…Intracellular regulatory cascades such as extracellular signal-regulated kinase/ mitogen-activated protein kinases (ERK/MAPK) and PI3-K have been shown to be stimulated by estrogen in diverse cell types, such as tumor cells and neurons [30,39,40,43]. A non-transcriptional mechanism for ER signaling in human as well as in animal endothelial cells was also described, showing that ER can physically and functionally couple to PI3K.…”

Genetic rescue of nonclassical ERα signaling normalizes energy balance in obese Erα-null mutant mice

“…Additionally, E 2 can activate PI3K through protein-protein interactions between ERα and p85 (a regulatory subunit of PI3K), which in turn activate Akt in a variety of cell types, including hypothalamic neurons, a process that is completely inhibited by the PI3K inhibitors wortmannin and LY294004 (43,44). We therefore tested whether E 2 activates Akt via ERE-independent ERα signaling in Erα +/+ , Erα -/-, and Erα -/AA mice by immunocytochemistry for pAkt in the ARC and vlVMN of the hypothalamus.…”