Regulation of the membrane estrogen receptor-α: Role of cell density, serum, cell passage number, and estradiol

We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor-α (mERα) in a rat pituitary tumor cell line, GH₃/B6/F10. We studied the regulation of mERα vs. levels of intracellular ERα (iERα) using our 96-well plate immunoassay. The anti-ERα antibody C542 was used to label the ERα (via conjugated alkaline phosphatase) in fixed permeabilized (for iERα) vs. nonpermeabilized cells (for mERα). Expression of mERα was highest at low cell densities (<1000 cells/well) and decreased significantly at densities where cellular processes touched, whereas the more abundant iERα increased with increasing cell density over the same range. Serum starvation for 48 h caused increases in mERα, whereas iERα levels showed no significant changes. A large decline in mERα and iERα levels with cell passage number was observed. Minutes after nM 17β-estradiol (E₂) treatment, a portion of the cells rounded up and detached from the culture plate, whereas nM cholesterol had no such effect. Although E₂ treatment did not change mERα levels, the antigen was reorganized from a fine particulate to aggregation into asymmetric large granules of staining. That common culturing conditions favor down-regulation of mERα may explain the relatively few reports of this protein in other experimental systems.