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The Journal of Neuroscience, June 18, 2008, 28(25):6473-6482; doi:10.1523/JNEUROSCI.0514-08.2008

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Cellular/Molecular
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

Danny Titolo,¹ Christopher M. Mayer,¹ Sandeep S. Dhillon,¹ Fang Cai,¹ and Denise D. Belsham^1,2,3,4

Departments of ¹Physiology, ²Obstetrics and Gynecology, and ³Medicine, University of Toronto, and ⁴Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8

Correspondence should be addressed to Dr. Denise D. Belsham, Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Email: d.belsham{at}utoronto.ca

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 17β-estradiol (E₂) 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 E₂ 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 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ERβ 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 –97 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 estrogen-mediated 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.

Key words: neuropeptide Y; membrane signaling; neurons; hypothalamic cell lines; estrogen receptor; transcription

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Received Feb. 4, 2008; revised April 2, 2008; accepted May 1, 2008.

Correspondence should be addressed to Dr. Denise D. Belsham, Department of Physiology, University of Toronto, Medical Sciences Building 3247A, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8. Email: d.belsham{at}utoronto.ca

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