**1.3 Mechanism of estrogen action**

Estrogens act on target tissues by binding to ERs. These proteins function as transcription factors when they are activated by a ligand. Biological action of ERs involves complex and broad mechanisms. For the ERs two main mechanisms of action have been described, including a genomic and a non-genomic pathway (Figure 2).

The *genomic action* of ERs occurs in the nucleus of the cell, when the receptor binds specific DNA sequences directly ("direct activation" or classical pathway) or indirectly ("indirect activation" or non-classical pathway). In the absence of ligand, ERs are associated with heatshock proteins. The Hsp90 and Hsp70 associated chaperone machinery stabilizes the ligand binding domain (LBD) and makes it accessible to the ligand. Liganded ER dissociates from the heat-shock proteins, changes its conformation, dimerizes, and binds to specific DNA sequences called estrogen responsive elements (EREs) in order to regulate transcription (Nilsson *et al.*, 2001). In the presence of the natural ligand E2, ER induces chromatin remodelling and increases transcription of estrogen regulated genes (Berno *et al.*, 2008).

Fig. 2. Mechanisms of estrogen receptor (ER) action. In the *direct activation*, ERs dimerize after ligand binding and attach to the ERE in the promoter of target genes. In the *indirect activation* manner, ligand-bound ER dimers might activate transcription of non-ERE containing genes, by binding to other transcription factors (e.g. AP1 or SP1). In the *nongenomic pathway*, ligand-bound ERs interact directly with and change the function of proteins some of which function as 'second messengers' (SM). ERs can also be activated by phosphorylation in the absence of ER ligands (*ligand-independent activation*). Based on Morani *et al*. 2008 (Morani *et al.*, 2008).

In the non-classical pathway, AP-1 (DeNardo *et al.*, 2005) and SP-1 (Kim *et al.*, 2003) are alternative regulatory DNA sequences used by both isoforms of the receptor, ERα and ERβ, to modulate gene expression. In this case, ER does not interact directly with DNA but interacts with other DNA-bound transcription factors such as c-Jun or c-Fos, or with other proteins (Kushner *et al.*, 2003). Both AF-1 and AF-2 domains of ER are required for the interaction with Fos/Jun complex and both receptors differentially affect AP-1 dependent genes. In the presence of ERα, E2 works as AP-1 agonist by enhancing activity of the proteins at AP-1 sites (Brzozowski *et al.*, 1997), while in the presence of ERβ it antagonizes

Fig. 2. Mechanisms of estrogen receptor (ER) action. In the *direct activation*, ERs dimerize after ligand binding and attach to the ERE in the promoter of target genes. In the *indirect activation* manner, ligand-bound ER dimers might activate transcription of non-ERE containing genes, by binding to other transcription factors (e.g. AP1 or SP1). In the *nongenomic pathway*, ligand-bound ERs interact directly with and change the function of proteins some of which function as 'second messengers' (SM). ERs can also be activated by phosphorylation in the absence of ER ligands (*ligand-independent activation*). Based on

In the non-classical pathway, AP-1 (DeNardo *et al.*, 2005) and SP-1 (Kim *et al.*, 2003) are alternative regulatory DNA sequences used by both isoforms of the receptor, ERα and ERβ, to modulate gene expression. In this case, ER does not interact directly with DNA but interacts with other DNA-bound transcription factors such as c-Jun or c-Fos, or with other proteins (Kushner *et al.*, 2003). Both AF-1 and AF-2 domains of ER are required for the interaction with Fos/Jun complex and both receptors differentially affect AP-1 dependent genes. In the presence of ERα, E2 works as AP-1 agonist by enhancing activity of the proteins at AP-1 sites (Brzozowski *et al.*, 1997), while in the presence of ERβ it antagonizes

Morani *et al*. 2008 (Morani *et al.*, 2008).

AP-1 activity (Nilsson *et al.*, 2001). When both receptors are present, ERβ inhibits the action of ERα on AP-1 promoters (Matthews *et al.*, 2006). Interactions of ERs with other transcription factors might be also selectively modulated by different ligands, such as genistein and quercetin, which are not able to stimulate AP-1 dependent transcription (Figtree *et al.*, 2003; Schreihofer, 2005).

Even though ERs are considered transcription factors they can act through *non-genomic* mechanisms. Rapid ER effects were first observed in 1960s when administration of a physiological dose of E2 was reported to increase uterine cAMP levels in ovariectomized rats within 15 seconds (Szego and Davis, 1967), a time scale that is considered too fast for a genomic action**.** There is still no agreement if receptors responsible for rapid actions of estrogens are the same proteins as nuclear ERs or distinct G-protein coupled steroid receptors (Funakoshi *et al.*, 2006; Maggiolini *et al.*, 2004; Pedram & Levin, 2006; Warner & Gustafsson, 2006). However, a broad range of other rapid pathways induced by E2 has been identified so far. Some of these pathways include MAPK/ERK pathway, activation of endothelial nitric oxide synthase (eNOS), PLC stimulated IP3 production, calcium influx and PI3K/Akt pathway activation (Stirone *et al.*, 2005; Virgili F, 2004; Ascenzi *et al.*, 2006). Similarly to non-classic mechanisms of activation, phytoestrogens might affect rapid pathways in a different way than E2. Quercetin for example has been shown to fail to phosphorylate ERK-2 kinase (opposite to E2) nor did it stimulate transcription of Cyclin D1, the transcription of which sometimes depends on rapid ER pathways (Virgili F, 2004). The stimulation of eNOS, which plays a role in cardiovascular health effects induced by E2 also seems to be regulated differently by phytoestrogens. Rapid activation of eNOS in the presence of E2 is dependent on ERα (Simoncini *et al.,* 2005), while both receptors are required for prolonged effects. However phytoestrogens do not activate eNOS in a rapid manner but seem to activate it through a prolonged, ERβ dependent transcriptional mechanism (Simoncini *et al.,* 2005).

In addition to ligand dependent mechanisms, ERα has ligand independent activity mediated through AF-1, which has been shown to be associated with stimulation of MAPK through growth factors such as Insulin like Growth Factor – 1 (IGF-1) and Epidermal Growth Factor (EGF). Activity of AF-1 is dependent on phosphorylation of Ser 118. A good example of the cross-talk between ER and growth factor signalling is phosphorylation of Ser 118 by MAPK in response to growth factors, such as IGF-1 and EGF (Kato *et al.*, 1995). The importance of growth factors in ER signalling is well illustrated by the fact that EGF can mimic effects of E2 in the mouse reproductive tract (Nilsson *et al.*, 2001).
