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Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells.

Ryhänen T, Hyttinen JM, Kopitz J, Rilla K, Kuusisto E, Mannermaa E, Viiri J, Holmberg CI, Immonen I, Meri S, Parkkinen J, Eskelinen EL, Uusitalo H, Salminen A, Kaarniranta K

Department of Ophthalmology, University of Kuopio, Kuopio, Finland.

ABSTRACT Purpose. The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). Methods. The Hsp70 and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. Conclusions. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human retinal pigment epithelial cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Published 19 November 2008 in J Cell Mol Med.
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