title: Geldanamycin activates Hsp70 response and attenuates okadaic acid-induced cytotoxicity in human retinal pigment epithelial cells.
abstract: Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein-protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition.
authors.affiliation: Department of Ophthalmology, University of Kuopio, P.O. BOX 1627, 70211 Kuopio, Finland. kaarnira@messi.uku.fi
authors.forename: Kai
authors.initials: K
authors.lastname: Kaarniranta
authors.forename: Tuomas
authors.initials: T
authors.lastname: Ryhänen
authors.forename: Reijo K
authors.initials: RK
authors.lastname: Sironen
authors.forename: Tiina
authors.initials: T
authors.lastname: Suuronen
authors.forename: Mika A
authors.initials: MA
authors.lastname: Elo
authors.forename: Hannu M
authors.initials: HM
authors.lastname: Karjalainen
authors.forename: Mikko J
authors.initials: MJ
authors.lastname: Lammi
authors.forename: Markku
authors.initials: M
authors.lastname: Teräsvirta
authors.forename: Hannu
authors.initials: H
authors.lastname: Uusitalo
authors.forename: Antero
authors.initials: A
authors.lastname: Salminen
mesh_terms: D016227:Benzoquinones; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000187:drug effects* / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D019319:Okadaic Acid / Q000037:antagonists & inhibitors* / Q000633:toxicity; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology*; D012162:Retinal Degeneration / Q000201:enzymology / Q000503:physiopathology; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D016227:Benzoquinones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C001277:geldanamycin