ABSTRACT: Estradiol (E2) is a central regulator of ovarian follicle development and granulosa cells (GCs) function, acting predominantly through estrogen receptor β (ERβ) in GCs. Human GCs co-express multiple ERβ isoforms (ERβ1, ERβ2, ERβ4 and ERβ5), yet how their relative abundance and crosstalk determine estrogen signaling remains poorly understood. Here, we investigated the contribution of nuclear and extra-nuclear ERβ signaling in human GC growth. Using the human GC line HGrC1, which lacks endogenous estrogen receptors, we generated stable cell lines expressing individual or combined ERβ isoforms, allowing dissection of isoform-specific and cooperative functions. ERβ1 and ERβ4 reduced GC proliferation, whereas ERβ2 and ERβ5 promote growth. Co-expression of all ERβ isoforms resulted in growth inhibition, highlighting that the balance between ERβ isoforms, rather than the activity or the expression level of a single isoform, determines cellular outcomes. Reporter assays showed that only ERβ1 directly activates transcription via estrogen response elements, while all ERβ isoforms inhibit E2-dependent ERα transactivation in overexpression conditions. In contrast, ERβ2, ERβ4 and ERβ5 indirectly activated transcription through activator protein-1 (AP-1) sites in a ligand-independent manner, and attenuated ERβ1-mediated repression, identifying AP-1-signaling as a central point of ERβ isoform crosstalk under the experimental conditions tested. To define ERβ1-dependent transcription in a physiological context, we performed RNA sequencing in primary human GCs (hGCs) following selective ERβ1 depletion. Knockdown abolished E2-induced gene expression, confirming ERβ1 as a central contributor to E2 responsiveness in hGCs. ERβ1 target genes were enriched in pathways regulating plasma membrane electrical potential, ion transport and cytoskeletal organization, including CACNA1A, ANO2 and GRIK4, which are critical for ion fluxes that regulate steroidogenesis, oocyte maturation and ovulation. Proteomic and biochemical analyses further revealed isoform-specific interactomes and distinct extra-nuclear signaling pathways preferences (ERK1/2, Akt or CaMKII), supporting scaffold-like signaling functions beyond transcription. Together, our study demonstrates that ERβ isoform balance orchestrates estrogen signaling in hGCs, integrating nuclear and extra-nuclear pathways to control cellular growth and function, and provide a framework for understanding ERβ isoform crosstalk in endocrine physiology. Trial registration number “2021-1”.
Guillaume CHEVREUX