G Protein-Coupled Estrogen Receptor Regulates Mesenchymal Stem Cell Mechanotransduction and Differentiation

G protein-coupled estrogen receptor (GPER) is a heptahelix estrogen-binding G protein-coupled receptor, and a potential therapeutic target for estrogen-related cancers and diseases. Recently, GPER has been recognized as a key mechano-regulator, but the effects on cell adhesion, spreading, morphology, migration, and differentiation are inconsistent or even contradicting in literature, due to the variations across cell lines and complex crosstalks with ER, and non-genomic actions of other hormones. Here, we focus on investigating the GPER effect on mesenchymal stem cell (MSC) mechanotransduction and differentiation.

MSCs treated by synthetic agonist G1 and untreated cells were cultured on fibronectin-coated surfaces. Cell migration was studied by both chemokinesis and chemotaxis experiments. After two-week differentiation, MSC adipogenesis and osteogenesis were evaluated by staining lipid droplets in adipocytes with Oil-Red O and alkaline phosphatase in osteocytes with NBT/BCIP, respectively. In particular, since micropatterns have been widely used to mimic extracellular matrix (ECM) cues, modulate MSC mechanotransduction and differentiation, we investigate the GPER effect on both single-cell and sub-cellular fibronectin microline patterns prepared by microcontact printing.

GPER activation regulates cytoskeleton organization, with reduced cell polarization, thinner ventral stress fibers, and reduced RhoA signaling; reduces MSC migration speed; significantly promotes osteogenesis and inhibits adipogenesis. Cell elongation by micropatterns and the reduction of cell polarization by GPER coexist in a sophisticated interplay.

GPER directly mediates MSC mechanotransduction by RhoA inactivation, while its sustained effect on MSC differentiation promotes osteogenesis and inhibits adipogenesis despite reduced cell polarization and tension, suggesting potential mechanisms other than RhoA signaling. Our findings pave the way towards a deep understanding of GPER's role and its interplay with ECM cues in mechanotransduction and differentiation, which will be important for developing GPER as a new therapeutic target, as well as considering its important effects in stem cell therapies and hormonal therapies.