Distinct regulation of insulin receptor substrate-1 and -2 by 90-kDa heat-shock protein in adrenal chromaffin cells

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 μM 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-3β, and Raf-1 levels, without changing extracellular signal-regulated kinase and its upstream kinase levels. Chronic (≥12 h) treatment with 0.1–10 μM Hsp90 inhibitor (geldanamycin, 17-allylamino-17-demethoxy-geldanamycin, herbimycin A, and radicicol) decreased insulin receptor substrate-1 level by ∼66%, while increasing insulin receptor substrate-2 level by ∼160%. These effects of geldanamycin (IC50 155 nM, EC50 177 nM) and 17-allylamino-17-demethoxy-geldanamycin (IC50 310 nM, EC50 260 nM) were time- and concentration-dependent. Geldanamycin-induced decrease of insulin receptor substrate-1 was attenuated by lactacystin, β-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 ∼39%, while raising insulin receptor substrate-2 mRNA level by ∼109% between 3 and 24 h, 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.