CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-α

In estrogen target cells, estrogen receptor-alpha(ER-alpha) 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, ER alpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ER alpha 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 ER alpha protein degradation was investigated. In ER-negative HeLa cells transfected with ER alpha and CHIP, ER alpha proteasomal degradation increased, whereas ER alpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ER alpha 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 ER alpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ER alpha and CHIP associate through the CHIP TPR domain. In ER alpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ER alpha protein and attenuation of ER alpha-mediated gene expression. Furthermore, the ER alpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ER alpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ER alpha dissociation from CHIP by various ER alpha ligands, including 17 beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ER alpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ER alpha degradation. Furthermore, based on our observations that CHIP promotes ER alpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ER alpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.