Estrogen Induces Estrogen Receptor α-Dependent cAMP Response Element-Binding Protein Phosphorylation via Mitogen Activated Protein Kinase Pathway in Basal Forebrain Cholinergic NeuronsIn Vivo

Abstract

In addition to classical genomic mechanisms, estrogen also exerts nonclassical effects via a signal transduction system on neurons. To study whether estrogen has a nonclassical effect on basal forebrain cholinergic system, we measured the intensity of cAMP response element-binding protein (CREB) phosphorylation (pCREB) in cholinergic neurons after administration of 17␤-estradiol to ovariectomized (OVX) mice. A significant time-dependent increase in the number of pCREB-positive cholinergic cells was detected after estrogen administration in the medial septum-diagonal band (MS-DB) and the substantia innominata (SI). The increase was first observed 15 min after estrogen administration. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The estrogeninduced CREB phosphorylation in cholinergic neurons was present in ER␤ knock-out mice but completely absent in ER␣ knock-out mice in MS-DB and SI. A series of in vitro studies demonstrated that estrogen acted directly on cholinergic neurons. Selective blockade of the mitogen activated protein kinase (MAPK) pathway in vivo completely prevented estrogen-induced CREB phosphorylation in cholinergic neurons in MS-DB and SI. In contrast, blockade of protein kinase A (PKA) was effective only in SI. Finally, studies in intact female mice revealed levels of CREB phosphorylation within cholinergic neurons that were similar to those of estrogen-treated OVX mice. These observations demonstrate an ER␣-mediated nonclassical effect of estrogen on the cholinergic neurons and that these actions are present under physiological conditions. They also reveal the role of MAPK and PKA-MAPK pathway activation in nonclassical estrogen signaling in the basal forebrain cholinergic neurons in vivo.

Membrane-initiated estrogen signaling in hypothalamic neurons

“…However, it is now widely accepted that some of the actions of estrogen are quite rapid and cannot be attributed to the classical nuclear-initiated steroid signaling of ERα or ERβ. One view is that both nuclear and plasma membrane-associated ERs might be products of the same genes (Razandi et al, 1999, Boulware et al, 2005, Pedram et al, 2006, Szegõ et al, 2006, Dewing et al, 2007. This belief stems primarily from the fact that many of the rapid effects of E2 can be induced by selective ERα or ERβ ligands, antagonized by the ER antagonist, ICI 182,780, or are lost in animals bearing mutations in ERα and/or ERβ genes (Couse and Korach, 1999, Singer et al, 1999, Dubal et al, 2001, Wade et al, 2001, Abraham et al, 2003, Boulware et al, 2005.…”

Modulation of αCaMKII signaling by rapid ERα action

“…Like αCaMKII, all three proteins are involved in neuronal differentiation, and ERK1/2 and CREB have also been shown to play a role in LTP and memory processes (Trifilieff, 2006). E2 action has previously been linked to ERK1/2 and CREB phosphorylation in various populations of neurons and in vivo (Lee, 2004;Bryant DN, 2005;Szego, 2006), and we have provided evidence that αCaMKII action mediates their phosphorylation by E2 in NLT and primary hippocampal neurons. CREB can be directly phosphorylated by αCaMKII (Wu, 2001), although in our studies it appears that CREB is indirectly affected by αCaMKII-mediated ERK1/2 activity since its phosphorylation by E2 was blocked by U0126 pre-treatment.…”

“…Furthermore, E2 has been reported to stimulate their activation in various populations of neurons (Lee, 2004;Szego, 2006), although the role of αCaMKII in this activation has not been thoroughly investigated. To examine the effect of E2-induced αCaMKII activity on ERK1/2 and CREB phosphorylation, NLT cells were either unstimulated or treated for 10 min with vehicle, E2, or E2 plus 30 min pretreatment with KN-62, ICI, Nifedipine, or U0126, a commonly used inhibitor of ERK1/2 activity.…”

“…E2 also rapidly induces c-Src activation in explanted mouse cortical neurons to influence neuronal differentiation (Nethrapalli, 2001). CREB phosphorylation is stimulated by E2 through the activation of ERK1/2 in forebrain cholinergic neurons, which requires ERα but not ERβ (Szego, 2006). Additionally, Sawai (2002) reported that E2 induces αCaMKII activity in hippocampal neurons in an ICI-sensitive manner, implicating the involvement of ER; however, the mechanism was not addressed.…”

Modulation of hypothalamic neuronal activity through a novel G-protein-coupled estrogen membrane receptor

“…The membrane-associated ERs might be derived from the same genes as ERα and ERβ [45][46][47][48]. This belief stems primarily from the fact that many of the rapid effects of E 2 can be induced by selective ERα or ERβ ligands, antagonized by the ER antagonist, ICI 182,780 or are lost in animals bearing mutations in ERα and/or ERβ genes [15,46,[49][50][51][52].…”