Geldanamycin activates Hsp70 response and attenuates okadaic acid-induced cytotoxicity in human retinal pigment epithelial cells.
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.
Kaarniranta
Kai
K
Department of Ophthalmology, University of Kuopio, P.O. BOX 1627, 70211 Kuopio, Finland. kaarnira@messi.uku.fi
Ryhänen
Tuomas
T
Sironen
Reijo K
RK
Suuronen
Tiina
T
Elo
Mika A
MA
Karjalainen
Hannu M
HM
Lammi
Mikko J
MJ
Teräsvirta
Markku
M
Uusitalo
Hannu
H
Salminen
Antero
A
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
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