GPER-independent effects of estrogen in rat aortic vascular endothelial cells

• The effects of estrogen reflect a balance between those actions mediated by the “classical” estrogen receptors (ER) and GPER.
• The nature of that balance in endothelial cells with endogenous expression of both ERα and GPER was unclear.
• In rat aortic endothelial cells estradiol inhibits ERK phosphorylation and apoptosis via an ERα-dependent pathway.
• With ERα downregulation, estradiol stimulates ERK phosphorylation and apoptosis via a GPER-dependent mechanism.
• In summary, in rat aortic endothelial cells estradiol mediates its effects via an ER-dependent pathway, with GPER-dependent effects only detectable in the setting of ER inhibition.

GPER (aka GPR30) has been identified as an important mechanism by which estrogen mediates its effects. Previous studies from our laboratories and those of others have demonstrated that GPER activation mediates a range of vascular contractile and growth regulatory responses. However, the importance of GPER in mediating the actions of estradiol (E2) in rat aortic endothelial cells is unclear. Therefore, we sought to determine the importance of GPER vs. the “classical” estrogen receptor (ER) in mediating the endothelial growth regulatory effects of E2. To do this we assessed the effect of E2 in regulating phosphoERK content and apoptotic rates in rat aortic endothelial cells and the role of GPER in mediating these effects.E2 mediated a concentration-dependent inhibition of both ERK phosphorylation and serum deprivation-induced apoptosis with a maximal effect at a concentration of 10 nM. Pretreatment with the ER antagonist ICI 182780 abolished E2-mediated inhibition of both ERK phosphorylation and apoptosis. In contrast, pretreatment with GPER antagonist G15 had no significant effect on E2-mediated inhibition of ERK phosphorylation or on apoptosis. Further, downregulation of GPER expression with a GPER shRNA adenovirus did not block E2-mediated inhibitory effects on ERK phosphorylation and apoptosis. In fact, these inhibitory effects of E2 were further enhanced by GPER downregulation.Downregulation of ERα expression reversed the E2-mediated inhibitory effects to stimulatory effects. E2's phosphoERK and apoptosis stimulatory effects seen with ERα downregulation are attenuated by pretreatment with G15.In conclusion, in rat aortic endothelial cells, E2-mediated endothelial effects are predominantly driven by ER and not by GPER.