Modulation of αCaMKII signaling by rapid ERα action

Abstract

The estrogen receptor (ER) subtypes, ERα and ERβ, modulate numerous signaling cascades in the brain to result in a variety of cell fates including neuronal differentiation. We report here that 17β-estradiol (E2) rapidly stimulates the autophosphorylation of α-Ca²⁺/calmodulin-dependent kinase II (αCaMKII) in immortalized NLT GnRH neurons, primary hippocampal neurons, and Cos7 cells co-transfected with ERα and αCaMKII. The E2-induced αCaMKII autophosphorylation is ERα- and Ca²⁺/calmodulin (CaM)-dependent. Interestingly, the hormone-dependent association of ERα with αCaMKII attenuates the positive effect of E2 on αCaMKII autophosphorylation, suggesting that ERα plays a complex role in modulating αCaMKII activity and may function to fine-tune αCaMKII-triggered signaling events. However, it appears as though the activating signal of E2 dominates the negative effect of ER since there is a clear, positive downstream response to E2-activated αCaMKII; pharmacological inhibitors and RNAi technology show that targets of ERα-mediated αCaMKII signaling include extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding protein (CREB), and microtubule associated protein 2 (MAP2). These findings suggest a novel model for the modulation of αCaMKII signaling by ERα, which provides a molecular link as to how E2 might influence brain function.

Introduction

Estrogen is intimately involved in the development and regulation of a variety of tissues, including the brain where it functions as a potent neuroprotective agent (Tang, 1996, Slooter, 1999) and a positive influencer of mood and cognition in both humans and rodents (Duff, 2000, Ahokas et al., 2001, Estrada-Camarena et al., 2003, Sandstrom and Williams, 2004, Almeida et al., 2005). Specifically, estrogen replacement in ovariectomized female rats significantly improves spatial memory retention as tested by a delayed matching-to-place water maze task (Sandstrom and Williams, 2004), and examination of the ERα knock-out mouse model (ERαKO) demonstrated that ERα is required for learning hippocampal-dependent inhibitory avoidance tasks (Fugger et al., 2000). Clearly, a connection between estrogen and brain function exists, however, the underlying mechanisms are still an area of fierce investigation.

Estrogen influences cognition primarily by its rapid modulatory effects on multiple signaling pathways. The rapid effects of estrogen refer to those events that occur seconds to minutes after estrogen administration and are not sensitive to inhibitors of transcription or translation (Heldring et al., 2007). Wu et al. (2005) as well as Zhao et al. (2005) have shown that E2 induces Ca²⁺ influx in hippocampal neurons within seconds, initiating signaling cascades that result in varied neurotrophic responses including neurite outgrowth and neuroprotection. E2 also rapidly induces c-Src activation in explanted mouse cortical neurons to influence neuronal differentiation (Nethrapalli et al., 2001). CREB phosphorylation is stimulated by E2 through the activation of ERK1/2 in forebrain cholinergic neurons, which requires ERα but not ERβ (Szego et al., 2006). Additionally, Sawai et al. (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.

αCaMKII plays an essential role in neuronal differentiation and cognitive processes (Silva et al., 1992, Blanquet, 1999, Gaudilliere et al., 2004), thus defining the relationship between E2 action and αCaMKII signaling is imperative for a better understanding of the mechanism by which E2 exerts its effects on the brain. αCaMKII is a multi-subunit serine/threonine kinase that is exquisitely responsive to Ca²⁺ levels (Hanson et al., 1992). Under basal conditions, αCaMKII is kept inactive by its autoinhibitory domain and only the binding of Ca²⁺/CaM to this domain disrupts autoinhibition, allowing for kinase activity. Autophosphorylation of αCaMKII at residue Thr286 occurs when the duration or magnitude of the Ca²⁺ signal increases and two neighboring kinase subunits bind Ca²⁺/CaM. This permits the activation of one subunit while the second subunit undergoes a conformational change, revealing Thr286 to the active subunit for phosphorylation. CaMKII remains active until it is dephosphorylated, even when Ca²⁺ levels return to normal (Hanson and Schulman, 1992, Hudmon and Schulman, 2002).

In the present study, the hypothesis that CaMKII signaling is modulated by ER action is investigated. We show that E2 rapidly induces the autophosphorylation of αCaMKII in an ERα- and Ca²⁺ influx-dependent manner in transfected Cos7 cells, immortalized GnRH neurons, and primary hippocampal neurons. Interestingly, the physical interaction of ERα with αCaMKII counteracts the positive effect of E2 on αCaMKII activity suggesting a dual role for ERα in modulating αCaMKII function. Ultimately, the E2-evoked αCaMKII activity results in ERK1/2, CREB, and MAP2 phosphorylation in primary hippocampal neurons.