Molecular mechanisms of alternative estrogen receptor signaling

Abstract

Estrogen is a key regulator of growth, differentiation and function in a broad range of target tissues, including the male and female reproductive tracts, mammary gland, bone, brain and the cardiovascular system. The biological effects of estrogen are mediated through estrogen receptor a (ERalpha) and estrogen receptor beta (ERbeta), which belong to a large superfamily of nuclear receptors that act as ligand-activated transcription factors. The classical mechanism of ER action depends on hormone binding to the receptors, after which the receptors dimerize and bind to estrogen response elements (EREs) located in the promoters of estrogen-responsive genes. However, evidence for signaling pathways that deviate from this classical model has emerged, and it is now accepted that ERalpha and ERbeta may regulate gene expression by a number of alternative mechanisms. The work presented in this thesis provides further insight into the alternative mechanisms of ER signaling.

We show that ERalpha and ERbeta efficiently potentiate the transcriptional activity of Stat5 when Stat5 is bound to the beta-casein promoter upon prolactin stimulation. The potentiation of Stat5 activity did not require the presence of the AF-1 and AF-2 domains of the receptor, and was observed not only in the presence of estrogen, but also in the presence of estrogen antagonists such as tamoxifen and ICI 182,287, suggesting that the interaction of ER with classical co-activators is not required. ERalpha and ERbeta were able to interact with Stat5 in vitro through a defined region of the receptor comprising the DBD and the hinge domain. Furthermore, we found that specific residues within the second helical structure of the ER DBD were essential for ER-mediated potentiation of Stat5 activity. Whereas disruption of the second zinc finger abrogated the potentiation of Stat5 activity, the structure of the first zinc finger was not important. In contrast to the situation concerning cross-talk with Stat5, the integrities of both zinc finger structures were essential for ER-mediated modulation of AP-1 dependent transcription. Thus, we propose that distinct parts of the ER DBD distinguish between the classical mechanism of action and cross-talk with Stat5 and AP-1.

We also show that ERalpha and ERbeta mediate activation of Stat proteins via non-genomic actions. In endothelial cells, estrogen rapidly induced phopsphorylation, nuclear translocation and DNA-binding activity of Stat3 and Stat5. Furthermore, agonist-bound ERs activated transcription from Stat3- and Stat5-regulated promoters in a manner that correlated with cytoplasmic localization of the receptors and required intact MAPkinase, P13- kinase and Src-kinase signaling pathways. The ER-mediated activity neither required the DBD nor the AF-2 functional domain of the receptor, hence did not involve the classical mechanism of ER action.

ERalpha and ERbeta modulate AP-1-dependent transcription via protein-protein interactions on DNA. We provide evidence for an additional mechanism by which ERs modulate the transcriptional activity of AP-1. ERalpha and ERbeta located in the cytoplasm activated transcription from AP-1 sites in response to estrogen, whereas receptors located in the nucleus repressed transcription under the same conditions. We propose that the reversed response to estrogen was due to the ability of ERs to induce AP-1 activity via non-genomic actions when present in the cytoplasm.

These results highlight alternative signaling pathways by which estrogen may regulate the expression of genes in which no consensus ERE has been found, and they contribute to understanding the molecular mechanisms by which ERs act in cross-talk with non-related transcription factors.