title: CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha.
abstract: In estrogen target cells, estrogen receptor-alpha (ERalpha) 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, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha 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 ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha 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 ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.
authors.affiliation: Medical Sciences, Indiana University School of Medicine, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401, USA.
authors.forename: Meiyun
authors.initials: M
authors.lastname: Fan
authors.forename: Annie
authors.initials: A
authors.lastname: Park
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Nephew
mesh_terms: D016227:Benzoquinones; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D015536:Down-Regulation; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D050883:HSC70 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D047029:Lactams, Macrocyclic; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D047628:Estrogen Receptor alpha; D004967:Estrogens; D050883:HSC70 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D034741:RNA, Small Interfering; D025801:Ubiquitin; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin