Membrane-initiated estradiol signaling in immortalized hypothalamic N-38 neurons

• Published in: Steroids Vol. 78; no. 6; pp. 607 - 613
• Main Authors: Dominguez, Reymundo, Dewing, Phoebe, Kuo, John, Micevych, Paul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2013
• Subjects: Animals; Cell Membrane - metabolism; ERα slice variant; Estradiol - metabolism; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - metabolism; Hypothalamus - cytology; Membrane-initiated estradiol signaling; Mice; N-38 neurons; Neurons - cytology; Neurons - metabolism; Receptor trafficking; RNA, Messenger - genetics; RNA, Messenger - metabolism; Signal Transduction; Surface biotinylation; ERα slice variant; N-38 neurons; Receptor trafficking; Membrane-initiated estradiol signaling; Surface biotinylation
• ISSN: 0039-128X; 1878-5867
• DOI: 10.1016/j.steroids.2012.12.008

► Different ERα splice variants are found on the cell surface of N-38 neurons. ► Estradiol increases membrane ERα levels by activation of PKC in N-38 neurons. ► Estradiol rapidly increases PKCθ phosphorylation and intracellular calcium.
Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ERα appears to be the critical isoform. The role of ERα in reproduction has been found to involve a nuclear ERα that induces a genomic mechanism of action. More recently, a plasma membrane ERα has been shown to trigger signaling pathways involved in reproduction. Mechanisms underlying membrane-initiated estradiol signaling are emerging, including evidence that activation of plasma membrane ERα involves receptor trafficking. The present study examined the insertion of ERα into the plasma membrane of N-38 neurons, an immortalized murine hypothalamic cell line. We identified, using western blotting and PCR that N-38 neurons express full-length 66kDa ERα and a 52kDa ERα spliced variant missing the fourth exon – ERαΔ4. Using surface biotinylation, we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ERα protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ERα was blocked by the ER antagonist ICI 182,780 or with the protein kinase C (PKC) pathway inhibitor bisindolylmaleimide (BIS). Downstream membrane-initiated signaling was confirmed by estradiol activation of PKC-theta (PKCθ) and the release of intracellular calcium. These results indicate that membrane ERα levels in N-38 neurons are dynamically autoregulated by estradiol.