Membrane ERα-dependent activation of PKCα in endometrial cancer cells by estradiol

The purpose of this study was to investigate the role of the oestrogen receptor subtypes ERα and ERβ in mediating the non-genomic effects of 17-β-estradiol (E₂) in two human endometrial cancer cell lines (RL95-2 and HEC-1A) expressing different levels of these receptor subtypes. Western blotting analysis using phosphorylation site-specific antibodies showed that physiological concentrations of E₂ rapidly (<20 min) activated PKCα, but not PKCδ in the RL95-2 cell line. E₂ had no effect on PKCα or PKCδ activity in the HEC-1A cell line and suppressed basal levels of PKA activity in both cell lines. PKCα activation coincided with its membrane translocation. ERα was detected in the RL95-2 cell line by Western blotting and RT-PCR but not in the HEC-1A cells, which did express ERβ. A selective ERα agonist PPT had the same effect as E₂ on PKCα activation in the RL95-2 cells, but the selective ERβ agonist DPN had no such effect. A 46 kDa variant of ERα increased in abundance in the cell membrane within 20 min of E₂ treatment suggesting that ERα mediated the E₂ non-genomic effects on PKCα through the formation of a membrane associated signalling complex.