Geldanamycin Increases 4–Hydroxynonenal (HNE) –Induced Cytotoxic Effects in Human Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: Development of age–related macular degeneration is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defense systems, e.g. via increased expression of heat shock protein and activation of stress sensitive AP–1 and NF–kappaB transcription factors. We examined the accumulation of Hsp70 protein as well as activation of AP–1 and NF–kappaB transcription factors in human ARPE–19 cells subjected to a 4–hydroxynonenal (HNE) –induced oxidative stress. In addition, the influence of a specific Hsp90 inhibitor geldanamycin (GA) was studied in HNE–treated cells. Mitochondrial metabolic activity and caspase–3 activity were determined to evaluate cell death in the ARPE–19 cells. Methods: Hsp70 protein accumulation was analyzed by Western blotting and the DNA–binding activities of AP–1 and NF–kappaB were examined by gel mobility shift assay. MTT assay was used to determine cell viability by measuring their ability to metabolize 3–(4,5–dimethyldiazol–2–yl)–2,5–diphenyltetrazolium bromide. Caspase–3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate. Results: The ARPE–19 cells showed increased accumulation of Hsp70 protein while DNA–binding activities of AP–1 or NF–kappaB transcription factors were decreased in response to HNE. Interestingly, GA significantly increased cytotoxicity in the HNE–treated cells. No alterations in caspase–3 activity were observed. Conclusions: This study reveals that Hsp70 is highly expressed in response to HNE–induced oxidative stress. Furthermore, Hsp90 inhibition with GA leads to an increased cellular damage in the RPE cells. Thus, the heat shock proteins may play an important role in the cytoprotection of RPE cells subjected to oxidative stress.

Keywords: stress response • oxidation/oxidative or free radical damage • retinal pigment epithelium