Phosphorylation Alters Oestrogen Receptor ß-Mediated Transcription in Neurones.

Nuclear steroid hormone receptors are ubiquitously expressed transcription factors whose activity can be altered by post-translational modifications, such as phosphorylation. The consequences of post-translational modifications have been described for several members of the nuclear steroid hormone receptor superfamily; however, little is known about the effects of oestrogen receptor (ER)ß phosphorylation in the brain. Moreover, to our knowledge, the presence of phosphorylated ERß has not been detected in the brain of any species to date. Oestrogen receptor ß is highly expressed in several regions of the brain and in vitro studies have demonstrated that it can be phosphorylated at two serine residues (S87 and S105) in the N-terminal AF-1 region. The present study aimed to determine whether phosphorylated ERß is detectable in the hippocampus of aged female rats, as well as the functional consequences of ERß S87 and S105 phosphorylation on transcriptional activity in neuronal cells. First, we used a novel PhosTag(™) approach to detect phosphorylated forms of ERß in the dorsal hippocampus of aged female rats. The data obtained demonstrated abundant forms of phosphorylated ERß in the dorsal hippocampus, suggesting that this post-translational modification might be an important regulator of ERß function. To assess the functional consequences of ERß phosphorylation in neuronal cells, we created phospho-mimetic (S87E, S105E) and phospho-null (S87A, S105A) ERß receptors that were transiently transfected in a hippocampal-derived cell line. Collectively, our results showed that phosphorylation of S87 and S105 altered both ligand-independent and ligand-dependent ERß transcriptional regulation. Overall, these data demonstrate that phosphorylated forms of ERß are present in the brain of aged female rats and that phosphorylation of ERß could differentially alter ERß-mediated gene expression.

Characterisation of human oestrogen receptor beta (ERß) splice variants in neuronal cells.

Oestrogen receptor (ER)α and ERß are members of the ligand-activated superfamily of nuclear receptors and mediate most facets of oestrogen signalling. Several naturally occurring splice variants of each ER have been identified in the human brain, yet the biological significance of these splice variants in the brain remains unknown. In the present study, we exploit the unique structural differences of the human ERß splice variants to determine the functional significance of individual ER domains in the brain. We previously established that full-length rodent ERß (i.e. rERß1) has constitutive transcriptional activity in neuronal cells in the absence of ligand. By contrast to the rodent splice variants, the human ERß splice variants used in the present study contain varying length truncations of exon 8, which encodes for the E/F domains. Our results reveal that, in neuronal cells, each human-specific ERß splice variant constitutively activated promoters mediated by a canonical oestrogen response element and repressed promoters mediated by activator protein-1 sites via p38 activity. From these data, we conclude that the C-terminus, encoding the AF-2 region and F domain, is not essential for the constitutive properties of human ERß. Taken together, these studies show that human-specific ERß variants are constitutively active and also provide novel insight into the contributions of the functional domains of ERß towards mediating constitutive transcription at various promoters in neuronal cells.