In estrogen target cells, estrogen receptor-α(ERα) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERα is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERα stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERα protein degradation was investigated. In ER-negative HeLa cells transfected with ERα and CHIP, ERα proteasomal degradation increased, whereas ERα-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERα accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERα down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERα and CHIP associate through the CHIP TPR domain. In ERα-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERα protein and attenuation of ERα-mediated gene expression. Furthermore, the ERα-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERα degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERα dissociation from CHIP by various ERα ligands, including 17β-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERα degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERα degradation. Furthermore, based on our observations that CHIP promotes ERα degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERα stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.
ASJC Scopus subject areas
- Molecular Biology