Activation of ERβ increases levels of phosphorylated nNOS and NO production through a Src/PI3K/Akt-dependent pathway in hypothalamic neurons

Estrogen plays a role in restoring homeostatic balance during the stress response by altering hypothalamic function and NO production in the brain. While we know that estrogen acts on the hypothalamus to stimulate the NO system through an ERβ-dependent mechanism in neurons, the molecular mechanisms responsible for these effects are unknown. Because phosphorylation of nNOS at Ser1412 increases nNOS activity which leads to increased NO production, we investigated the effects of ERβ activation on nNOS phosphorylation at Ser1412 and NO production in primary hypothalamic neurons. Using the selective ERβ agonist, DPN (10 nM), we show that activation of ERβ rapidly increases phosphorylation levels of nNOS at Ser1412 and NO production. We also show that the PI3K pathway, but not the MAPK pathway, mediates the increases in levels of Ser1412 phosphorylation and NO production induced by ERβ activation, as the selective PI3K inhibitor, LY294002 (10 μM), blocked the effects of ERβ activation. Finally, we demonstrate that Src kinase acts upstream of the PI3K/Akt pathway based on our finding that the selective Src inhibitor, PP2 (10 μM), blocked the increases in nNOS phosphorylation levels, NO production, and PI3K/Akt activity induced by ERβ activation. Together, our results show that Src kinase mediates ERβ-induced increases in phosphorylation levels of nNOS at Ser1412 and NO production by activating the PI3K/Akt pathway. These findings provide novel insight into the signaling mechanisms through which E2 stimulates the NO system in hypothalamic neurons.