Several reports have shown the ability of ERs to interact with cytoplasmic kinases via crosstalk between cytoplasmic kinase pathways and genomic ER action. Various signalling pathways are activated upon E2 binding to ERs. These rapid events may be classified into four main signalling cascades: (i) phospholipase C (PLC)/protein kinase C (PKCs) (Ferret, et al., 2001, Incerpi, et al., 2003, Marino, et al., 2001, Marino, et al., 1998, Morley, et al., 1992, Picotto, et al., 1999), (ii) Ras/Raf/mitogen- activated protein kinase (MAPK) (Dos Santos, et al., 2002, Klinge, et al., 2005, Marino, et al., 2002, Migliaccio, et al., 2002, Russell, et al., 2000, Tanaka, et al., 2003, Watters, et al., 1997, Woo, et al., 2005), (iii) phosphatidyl inositol 3 kinase (PI3K)/AKT (Acconcia, et al., 2005, Alexaki, et al., 2006, Bjornstrom, et al., 2005, Castoria, et al., 1999, Castoria, et al., 2001, Chambliss, et al., 2005, Levin, 2005, Marino, et al., 2005, Marino, et al., 2003, Simoncini, et al., 2000) and (iv)
cAMP/protein kinase A (PKA) (Chen, et al., 1998, Farhat, et al., 1996, Gu, et al., 1996, Malyala, et al., 2005, Picotto, et al., 1996, Picotto, et al., 1999). These pathways present numerous interactions with several other pathways. For example, the ERa-E2 complex interactions with the IGF-1 receptor (IGF-1R), leading to IGF- 1R activation and hence to MAPK signalling pathway activation (Kahlert, et al., 2000). In addition, the ERa-E2 complex activates the epidermal growth factor receptor (EGFR) leading to an increase in the extracellular regulated kinases (ERK) and PI3K/AKT activities (Dos Santos, et al., 2002, Driggers, et al., 2002, Improta- Brears, et al., 1999, Kupzig, et al., 2005, Razandi, et al., 2003, Zhang, et al., 2004). These pathways also have a crucial role in the E2 action as a survival agent. They enhance the expression of bcl-2, an anti-apoptotic agent, block the activation of p38/MAPK, reducing pro-apoptotic caspase-3 activation and promote G1 to S phase transition via the enhancement of cyclin D1 expression (Fig. 1.17) (Acconcia, et al., 2005, Marino, et al., 2002, Marino, et al., 2003).
ERb has the opposite effect on these signalling pathways. It regulates E2 signalling when co-expressed with ERa, causing a concentration dependent reduction in ERa mediated transcription (Matthews, et al., 2003). It also represses cyclin D1 expression and block ERa mediated induction when both ERs isoforms are present (Kilker, et al., 2004, Liu, et al., 2002). Recently ERb has also been reported to induce persistent membrane-initiated activation of p38/MAPK without any interference on survival
proliferative pathways, thus restoring the balance with apoptosis (Acconcia, et al., 2005). Human epidermal growth factor receptor 2 (HER2) can also be down regulated by ERb and PTEN is upregulated, both of which lead to the down regulation of the PI3K/AKT pathway (Fig. 1.17) (Lindberg, et al., 2011). ERb has been considerably understudied in ER cell signalling and information is scarce. However, the
significance of ERa signalling regulation is likely to have a major impact on these pathways.
Figure 1.17: Illustration of the non-genomic signalling pathway. The ER monomer is activated upon E2 ( ) binding, facilitating dissociation from the heat shock protein (HSP) and dimerisation. The ER interacts with cell signalling pathways up/down- regulating processes such as apoptosis and cell proliferation (from Spencer, 2016 and Zhou et al., 2014 with permission).
Clearly, ER actions are complex and importantly allow for tight regulation of estrogens in the body. However, the genomic and non-genomic pathways, although often studied independently, appear to be inherently linked. Therefore, when considering the actions of ERs in the non-genomic pathway (e.g. cell signalling pathways), not only does the cell signalling pathway become activated but in turn the ER is post-translationally modified (e.g. phosphorylated) which promotes the
genomic pathway. For example, ligand bound ERa binds to SRC and Pi3K
complexes leading to AKT and MAPK activation. In turn, ERa is post translationally modified at sites such as Try537 which allows recruitment of the E3 ligase, E6 associated protein (E6AP), and initiates ER proteolysis and transcriptional activation (Fig.1.18).
Figure 1.18: Illustration of the interplay between genomic and non-genomic signalling pathways. The ER dimer is phosphorylated by kinase proteins, which is required for genomic signalling, whilst activating non-genomic signalling pathways.
E2 = , CoA= coactivator complex, Prot= proteasome, P=phosphate and
Ub=ubiquitin. (Elements of this diagram were derived from Zhou et al., 2014. The concept of the entire diagram is the author’s).
This is just one example of the many post translational modifications of the ER which influence the stability, subcellular localisation, transcriptional activity and hormone sensitivity of the ligand activated ER-transcriptional apparatus. There are
approximately 29 sites on the ER that undergo either phosphorylation, methylation, acetylation, sumoylation, palmitoylation or ubiquitylation (Le Romancer, et al., 2011). Activation of these sites drives rapid signalling kinase cascades which lead to non-genomic mitogenic effects. These post translational events modulate ER function by altering its binding to ligands, to target gene promoters or to ER coactivators (Campbell, et al., 2001, Legoff, et al., 1994, Likhite, et al., 2006, Rayala, et al., 2006).
Altering these key ER functions can influence gene expression and the genomic pathway. There are many other interactions between hormonal and growth factor signalling pathways. These multiple signalling pathways downstream of receptor tyrosine kinases (e.g. EGRF) and insulin-like growth factor 1 receptor (IGF1R)) co- ordinately regulate the dynamics of the ER mediated transcriptional regulation. MAPK mediates ER phosphorylation at S294, and cyclin E-cyclin dependent kinase 2 (CDK2) phosphorylates ER at S342 to prime the ER-S-phase kinase associated protein 2 (SKP2) interaction.
One of the key roles for post translational modifications of the ER is aiding in the formation of the coactivator complex and recruitment of proteins to the complex. There is a series of post translational modifications that lead to the recruitment of the transcriptional machinery and the degradation of the ER to the proteasome. The coactivators often have enzymatic activities which enable them to acetylate, methylate or demethylate (Lonard, et al., 2007), helping overcome the physical constraints of the highly coordinated chromatin encased template (Zhou, et al., 2014). Coactivation allows recruitment of the transcriptional machinery and thus gene transcription. The coactivators then recruit the appropriate ubiquitination proteins (E1, E2 and E3
ligases) which makes a polyubiquitin chain and thus signals to the proteasome that the ER is ready for degradation.
These actions are not isolated to endogenous estrogens and antagonists. They can be induced by compounds that mimic E2, known as xenoestrogens.