title: Distinct regulation of insulin receptor substrate-1 and -2 by 90-kDa heat-shock protein in adrenal chromaffin cells.
abstract: 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.
authors.affiliation: Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan.
authors.forename: Norie
authors.initials: N
authors.lastname: Yoshikawa
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nemoto
authors.forename: Shinya
authors.initials: S
authors.lastname: Satoh
authors.forename: Toyoaki
authors.initials: T
authors.lastname: Maruta
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Yanagita
authors.forename: Etsuo
authors.initials: E
authors.lastname: Chosa
authors.forename: Akihiko
authors.initials: A
authors.lastname: Wada
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D010770:Phosphotransferases / Q000187:drug effects / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D055504:Insulin Receptor Substrate Proteins; D047029:Lactams, Macrocyclic; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D010770:Phosphotransferases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin