Estradiol inhibits GSK3 and regulates interaction of estrogen receptors, GSK3, and beta-catenin in the hippocampus P. Cardona-Gomez, a,b M. Perez, c J. Avila, c L.M. Garcia-Segura, a and F. Wandosell c, * a Laboratory of Cellular and Molecular Neuroendocrinology, Instituto Cajal, CSIC, Madrid 28002, Spain b Neuroscience Group, School of Medicine University of Antioquia, PO Box 1226 Medellin, Colombia c Centro de Biologı ´a Molecular "Severo Ochoa" CSIC-Universidad Auto ´noma de Madrid, Madrid 28049, Spain Received 30 January 2003; revised 24 September 2003; accepted 7 October 2003 Estrogens regulate a wide set of neuronal functions such as gene expression, survival and differentiation in a manner not very different from that exerted by neurotrophins or by growth factors. The best- studied hormonal action is the transcriptional activation mediated by estrogen receptors. However, the direct effects of estrogen on growth factor signaling have not been well clarified. The present data show that estradiol, in vivo, induces a transient activation of GSK3 in the adult female rat hippocampus, followed by a more sustained inhibition, as inferred from phosphorylation levels of Tau. Similar data was obtained from cultured hippocampal neurons when treated with the hormone. The transient activation was confirmed by direct measure of GSK3 kinase activity. In addition, our results show a novel complex of estrogen receptor A, GSK3, and B-catenin. The presence of the hormone removes B- catenin from this complex. There is a second complex, also affected by estradiol, in which Tau is associated with GSK3, B-catenin, and elements of the PI3 kinase complex. Considering the role of GSK3 in neurodegeneration, our data suggest that part of the neuroprotective effects of estrogen may be due to the control of GSK3. D 2004 Elsevier Inc. All rights reserved. Introduction Considerable evidence from animal studies indicates that estra- diol is neuroprotective (Garcia-Segura et al., 2001; Green and Simpkins, 2000; Lee and McEwen, 2001; Wise et al., 2001). At least part of the neuroprotective actions of estradiol are mediated by estrogen receptors, which are expressed in different brain areas, primarily, but not exclusively, in neurons (Shughrue et al., 1997; Simerly, 1993). In addition, estradiol has rapid effects in neurons that may be associated with new membrane forms of estrogen receptors or with transient association of classical estrogen recep- tors with specific membrane compartments (Toran-Allerand et al., 1999, 2002). Rapid effects of estradiol in the brain include the activation of signaling pathways coupled to tyrosine kinase receptors (Bi et al., 2000; Cardona-Gomez et al., 2002; Honda et al., 2000; Ivanova et al., 2002a; Kuroki et al., 2001; Singh, 2001; Singh et al., 1999; Zhang et al., 2001). Furthermore, one of such receptors, the insulin- like growth factor-I (IGF-I) receptor, is involved in many different actions of estradiol in the brain, including the regulation of neuronal differentiation (Duenas et al., 1996; Ma et al., 1994; Toran-Allerand et al., 1988), synaptic plasticity (Cardona-Gomez et al., 2000; Fer- nandez-Galaz et al., 1999), neuronal survival after injury (Azcoitia et al., 1999; Cardona-Gomez et al., 2001), gonadotrophin secretion, and reproductive behavior (Quesada and Etgen, 2001, 2002). The mechanisms involved in the interaction of estradiol and IGF-I receptor are not well understood. Recent studies have shown that estrogen receptor a, but not h, interacts with IGF-I receptor and with the p85 subunit of the PI3K in the brain (Mendez et al., in press). In addition, systemic administration of estradiol to adult ovariectomized female rats results in a transient increase in tyrosine phosphorylation of the IGF-I receptor, in a transient interaction of the IGF-I receptor with the estrogen receptor a and in an enhanced interaction of estrogen receptor a with p85 (Mendez et al., in press). This suggests that estrogen receptor a may affect IGF-I actions in the brain by a direct interaction with some of the components of IGF-I receptor signaling. Such interactions may explain why IGF-I and estradiol synergistically activate in the brain the kinase Akt (Cardona-Gomez et al., 2002), a component of the IGF-I/insulin signaling pathway (Ivanova et al., 2002b; Singh, 2001; Zhang et al., 2001). Downstream of Akt, there is another protein that may have a key role in IGF-I/insulin signaling: glycogen synthase kinase 3 (GSK3) (Cross et al., 1995; Pap and Cooper, 1998). GSK3 has two highly conserved isoforms, a and h; for review, see Frame and Cohen (2001) and Grimes and Jope (2001). GSK3 is involved not only in the signaling pathways of insulin/IGF-I, but also in the pathways of Wnt/Wingless (Cook et al., 1996; Welsh and Proud, 1993), FGF (Hashimoto et al., 2002), or LPA (Sayas et al., 1999). GSK3 is highly expressed in the central nervous system (Takahashi et al., 1994) where it may participate in a broad range of biological processes. In particular, its inhibition is associated with the activation of survival pathways in neurons (Cross et al., 1995). One of the substrates of GSK3 is h-catenin, a protein that is both a cell adhesion molecule as well as a transcription factor (Cadigan and Nusse, 1997; Miller and Moon, 1996). Other sub- 1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2003.10.008 * Corresponding author. Fax: +34-91-3974799. E-mail address: FWANDOSELL@cbm.uam.es (F. Wandosell). Available online on ScienceDirect (www.sciencedirect.com.) www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 25 (2004) 363 – 373