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

ABSTRACT: Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ER? appears to be the critical isoform. The role of ER? in reproduction has been found to involve a nuclear ER? that induces a genomic mechanism of action. More recently, a plasma membrane ER? 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 ER? involves receptor trafficking. The present study examined the insertion of ER? 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 ER? and a 52kDa ER? spliced variant missing the fourth exon - ER??4. Using surface biotinylation, we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ER? protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ER? 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 (PKC?) and the release of intracellular calcium. These results indicate that membrane ER? levels in N-38 neurons are dynamically autoregulated by estradiol.

SUBMITTER: Dominguez R

PROVIDER: S-EPMC3636190 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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

Dominguez Reymundo R Dewing Phoebe P Kuo John J Micevych Paul P

Steroids 20130105 6

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 ...[more]

PMID: 23296142

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Project description:BackgroundOvernutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood.MethodsHere we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis.ResultsImportantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor-β-activated kinase 1 binding protein (TAB1) interaction as identified in the periphery.ConclusionsTaken together, GPR120 is functionally active in the hypothalamic neuronal line, rHypoE-7, wherein it mediates the anti-inflammatory actions of DHA to reduce the inflammatory response to TNFα.

Dataset Information

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

Publications

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

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