Endocrine Abstracts

Estradiol (E2), produced by granulosa cells (GCs) of ovarian follicles, plays an essential role in folliculogenesis by controlling follicular development and selection. E2 actions are principally mediated by the two nuclear receptors, ERα and ERβ. These two receptors are differently expressed in GCs, with ERβ being predominant. After ligand binding and receptor dimerization, ERα and ERβ mainly regulate gene transcription through direct interaction with specific DNA motifs (estrogen-response elements, ERE), or by tethering onto other transcription factors such as c-Fos/c-Jun (activator protein-1, AP-1). In Human, four alternative splicing isoforms of ERβ (ERβ1, ERβ2, ERβ4 and ERβ5) are described and highly expressed in GCs. Little is known about their biological activities, except that only the longest form ERβ1 can bind E2. Therefore, the aim of our study was to identify the role of each ERβ isoform on GCs growth, as well as deciphering the nature of their respective transcriptional activities. For this purpose, we overexpressed each ERβ isoform in a human immortalized GC line (HGrC1) to assess their respective role on cell proliferation and apoptosis. By scoring BrdU incorporation and measuring Annexin-V binding to membrane apoptotic proteins, we showed that ERβ2 and ERβ5 induced cell proliferation whereas ERβ1 and ERβ4 increased GCs apoptosis. Interestingly, transfection of ERβ isoforms in HGrC1 cells exhibited various effects on GCs fate, whether they were expressed alone or together. For example, pro-apoptotic activities of ERβ1 and ERβ4 were lost when both isoforms were co-expressed. To understand the underlying mechanisms of these effects, we examined ERβ transcriptional activities, with or without ligand, on luciferase reporter constructs driven by ERE or AP-1 dependent promoters. We found that only ERβ1 elicited ligand-dependent and ligand-independent transactivation at ERE sites. ERβ2, ERβ4 and ERβ5 (alone or together) did not influence ERβ1 transactivity. In addition, ERβ2, ERβ4 or ERβ5 showed a ligand-independent transactivation at AP-1 sites that impaired ERβ1 transactivity but led to gene transcription activation when all isoforms were co-transfected. In conclusion, our study demonstrates an important role of each ERβ isoform on GCs proliferation and apoptosis. Importantly, it reveals that although being E2-insensitive and unable to transactivate gene expression through ERE sites, ERβ2, ERβ4 and ERβ5 display biological effects. Their direct activities on AP1 sites as well as their potential non-genomic signaling are under current investigation. Hence, changes in the complex crosstalk between ERβ isoform signaling could contribute to uncontrolled cell growth such as that encountered in tumor cells.