Role of the proteasome in the regulation of estrogen receptor α turnover and function in MCF-7 breast carcinoma cells

Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of S-35-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E-2) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [H-3]E-2, even in the presence of MG-132, indicating that the regulation of ER level and E-2 binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E-2 to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments. (c) 2005 Elsevier Ltd. All rights reserved.