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p62/sequestosome 1 as a regulator of proteasome inhibitor‐induced autophagy in human retinal pigment epithelial cells
Author(s) -
KAARNIRANTA K
Publication year - 2010
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2010.2133.x
Subject(s) - autophagy , proteasome , microbiology and biotechnology , sequestosome 1 , viability assay , proteasome inhibitor , hsp70 , aggresome , gene silencing , biology , ubiquitin , lysosome , retinal pigment epithelium , protein aggregation , retinal , chemistry , cell , heat shock protein , biochemistry , apoptosis , gene , enzyme
Purpose The pathogenesis of age‐related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress –induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates.The functional roles of p62 and HSP70 were evaluated in conjunction with protesome inhibitor ‐induced autophagy in human RPE cells (ARPE‐19). Methods The p62, 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. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results Proteasome inhibition evoked the accumulation of p62 and HSP70 that strongly co‐localized with each other in perinuclear aggregates. The p62 accumulation was time and concentration dependent after MG‐132 proteasome inhibitor loading. Interestingly, autophagy induction was p62 and Hsp70 independent. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently we showed that hsp70 mRNA depletion increased cell death in ARPE‐19 cells. Here we now demonstrate that p62 mRNA silencing has similar effects on cellular viability. Conclusion The p62 and HSP70 are central molecules in the regulation of protein turnover in human retinal pigment epithelial cells in proteasome inhibitor‐ induced autophagy.