CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-α
CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-α
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DOI：
10.1210/me.2005-0111
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发表时间：
2005-12-01
影响因子：
--
通讯作者：
Nephew, KP
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文献类型：
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作者：
Fan, MY;Park, A;Nephew, KP
在雌激素靶细胞中，雌激素受体-α(ER-α)蛋白水平受到严格调控。尽管受体的更新是一个连续的过程，但受体水平的动态波动主要由泛素-蛋白酶体途径调节，随着细胞条件的变化而发生。在缺乏配体的情况下，ERα被隔离在由热休克蛋白90(Hsp90)和辅伴侣组成的稳定的伴侣蛋白复合体中。然而，调控ERα稳定性和周转的分子机制(S)仍不清楚。一种可能的机制涉及CHIP，Hsc70相互作用蛋白的羧基末端，以前被证明针对泛素化和蛋白酶体降解的Hsp90相互作用蛋白。本研究探讨了CHIP在ERα蛋白降解中的作用。在转ERα和CHIP的ER阴性HeLa细胞中，ERα蛋白酶体降解增加，而ERα介导的基因转录减少。相反，小干扰RNA耗尽芯片导致ERα积聚和报告基因反式激活增加。突变的CHIP构建体的转染表明，CHIP内的U-box(包含泛素连接酶活性)和四肽重复(TPR，伴侣结合所必需的)结构域都是CHIP介导的ERα下调所必需的。此外，免疫共沉淀实验证明ERα和CHIP通过CHIP TPR结构域结合在一起。在ERα阳性的乳腺癌MCF7细胞中，CHIP过表达导致内源性ERα蛋白水平降低，ERα介导的基因表达减弱。此外，Hsp90抑制剂格尔达那霉素(GA)刺激ERα与芯片的相互作用，导致ERα降解增强；这种GA作用因CHIP的过度表达而进一步增强，但被CHIP耗尽所消除。最后，ERα被17β-雌二醇、4-羟基三苯氧胺和ICI 182,780等多种ERα配体从CHIP解离，阻断了CHIP介导的ERα降解。这些结果表明，CHIP在基础和GA诱导的ERα降解中都发挥了作用。此外，根据我们的观察，CHIP促进ERα的降解并减弱受体介导的基因转录，我们认为CHIP通过调节ERα的稳定性，有助于调节雌激素靶细胞的功能性受体水平，从而调节激素的反应性。
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.