Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan.
Neurochem Int. 2010 Jan;56(1):42-50. doi: 10.1016/j.neuint.2009.08.013. Epub 2009 Sep 6.
Multiple signaling pathways via insulin receptor substrate-1 and -2 play crucial roles in health, diseases, and therapeutics (i.e., longevity, tumorigenesis, and neuroprotection). The 90-kDa heat-shock protein (Hsp90) is an emerging target molecule of therapeutics, Hsp90 inhibitors being promising against various diseases (e.g., cancer, brain and cardiac ischemia, and neurodegenerative diseases). Much remains, however, unknown whether Hsp90 could regulate insulin receptor substrate-1 and -2 signaling pathways. In cultured bovine adrenal chromaffin cells, we observed that 24-h treatment with 1 microM geldanamycin (an inhibitor of Hsp90) decreased insulin receptor substrate-1 level, while increasing insulin receptor substrate-2 level; besides, geldanamycin lowered phosphoinositide 3-kinase, phosphoinositide-dependent kinase-1, Akt, glycogen synthase kinase-3beta, and Raf-1 levels, without changing extracellular signal-regulated kinase and its upstream kinase levels. Chronic (>or=12h) treatment with 0.1-10 microM Hsp90 inhibitor (geldanamycin, 17-allylamino-17-demethoxy-geldanamycin, herbimycin A, and radicicol) decreased insulin receptor substrate-1 level by approximately 66%, while increasing insulin receptor substrate-2 level by approximately 160%. These effects of geldanamycin (IC(50) 155 nM, EC(50) 177 nM) and 17-allylamino-17-demethoxy-geldanamycin (IC(50) 310 nM, EC(50) 260 nM) were time- and concentration-dependent. Geldanamycin-induced decrease of insulin receptor substrate-1 was attenuated by lactacystin, beta-lactone or MG132 (proteasome inhibitor), but not by calpastatin (calpain inhibitor) or leupeptin (lysosome inhibitor); geldanamycin did not affect heteroprotein complex formation between insulin receptor substrate-1 or -2 and Hsp90. Geldanamycin-induced increase of insulin receptor substrate-2 was prevented by cycloheximide or actinomycin D. Geldanamycin lowered insulin receptor substrate-1 mRNA level by approximately 39%, while raising insulin receptor substrate-2 mRNA level by approximately 109% between 3 and 24h, without changing the stability of insulin receptor substrate-1 and -2 mRNAs. Nuclear run-on assay revealed that geldanamycin retarded insulin receptor substrate-1 gene transcription by 42%, while accelerating insulin receptor substrate-2 gene transcription by 41%. Hsp90 inhibitors oppositely altered insulin receptor substrate-1 and -2 levels via proteasomal degradation and gene transcription.
多种信号通路通过胰岛素受体底物-1 和 -2 在健康、疾病和治疗（即长寿、肿瘤发生和神经保护）中发挥关键作用。90kDa 热休克蛋白 (Hsp90) 是治疗的新兴靶分子，Hsp90 抑制剂对各种疾病（如癌症、脑和心脏缺血以及神经退行性疾病）具有很大的治疗作用。然而，Hsp90 是否可以调节胰岛素受体底物-1 和 -2 信号通路仍有许多未知之处。在培养的牛肾上腺嗜铬细胞中，我们观察到 24 小时用 1 microM 格尔德霉素（Hsp90 的抑制剂）处理会降低胰岛素受体底物-1 水平，同时增加胰岛素受体底物-2 水平；此外，格尔德霉素降低了磷酸肌醇 3-激酶、磷酸肌醇依赖性激酶-1、Akt、糖原合酶激酶-3β和 Raf-1 水平，而不改变细胞外信号调节激酶及其上游激酶水平。用 0.1-10 microM Hsp90 抑制剂（格尔德霉素、17-烯丙基-17-脱甲氧基格尔德霉素、海兔霉素 A 和雷帕霉素）慢性（>或=12 小时）处理会使胰岛素受体底物-1 水平降低约 66%，同时使胰岛素受体底物-2 水平增加约 160%。格尔德霉素（IC50 为 155 nM，EC50 为 177 nM）和 17-烯丙基-17-脱甲氧基格尔德霉素（IC50 为 310 nM，EC50 为 260 nM）的这些作用是时间和浓度依赖性的。格尔德霉素诱导的胰岛素受体底物-1 减少被乳胞素、β-内酰胺或 MG132（蛋白酶体抑制剂）减弱，但不被钙蛋白酶抑制剂 calpastatin 或溶酶体抑制剂亮肽素减弱；格尔德霉素不影响胰岛素受体底物-1 或 -2 与 Hsp90 之间的异源蛋白复合物形成。格尔德霉素诱导的胰岛素受体底物-2 增加被环己酰亚胺或放线菌素 D 阻止。格尔德霉素在 3 至 24 小时内使胰岛素受体底物-1 mRNA 水平降低约 39%，使胰岛素受体底物-2 mRNA 水平升高约 109%，而不改变胰岛素受体底物-1 和 -2 mRNA 的稳定性。核转录物检测显示，格尔德霉素使胰岛素受体底物-1 基因转录延迟 42%，同时使胰岛素受体底物-2 基因转录加速 41%。Hsp90 抑制剂通过蛋白酶体降解和基因转录相反地改变胰岛素受体底物-1 和 -2 水平。
