Inhibiting proteasomal proteolysis sustains estrogen receptor-α activation

Estrogen receptor-alpha (ERalpha) is a ligand-dependent transcription factor that mediates physiological responses to 17beta-estradiol (E2). Ligand binding rapidly down-regulates ERalpha levels through proteasomal proteolysis, but the functional impact of receptor degradation on cellular responses to E2 has not been fully established. In this study, we investigated the effect of blocking the ubiquitin-proteasome pathway on ERalpha-mediated transcriptional responses. In HeLa cells transfected with ERalpha, blocking either ubiquitination or proteasomal degradation markedly increased E2-induced expression of an ER-responsive reporter. Time course studies further demonstrated that blocking ligand-induced degradation of ERalpha resulted in prolonged stimulation of ER-responsive gene transcription. In breast cancer MCF7 cells containing endogenous ERalpha, proteasome inhibition enhanced E2-induced expression of endogenous pS2 and cathepsin D. However, inhibiting the proteasome decreased expression of progesterone receptor (PR), presumably due to the heterogeneity of the PR promoter, which contains multiple regulatory elements. In addition, in endometrial cancer Ishikawa cells overexpressing steroid receptor coactivator 1, 4-hydroxytamoxifen displayed full agonist activity and stimulated ERalpha-mediated transcription without inducing receptor degradation. Collectively, these results demonstrate that proteasomal degradation is not essential for ERalpha transcriptional activity and functions to limit E2-induced transcriptional output. The results further indicate that promoter context must be considered when evaluating the relationship between ERalpha transcription and proteasome inhibition. We suggest that the transcription of a gene driven predominantly by an estrogen-responsive element, such as pS2, is a more reliable indicator of ERalpha transcription activity than a gene like PR, which contains a complex promoter requiring cooperation between ERalpha and other transcription factors.