p62/sequestosome 1 as a regulator of proteasome inhibitor-induced autophagy in human retinal pigment epithelial cells.
OBJECTIVE
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. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome 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 perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time- and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. 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 demonstrate that p62 mRNA silencing has similar effects on cellular viability.


CONCLUSIONS
Our findings open new avenues for understanding the mechanisms of proteolytic processes in retinal cells, and could be useful in the development of novel therapies targeting p62 and HSP70.
Viiri
Johanna
J
Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland.
Hyttinen
Juha M T
JM
Ryhänen
Tuomas
T
Rilla
Kirsi
K
Paimela
Tuomas
T
Kuusisto
Erkki
E
Siitonen
Ari
A
Urtti
Arto
A
Salminen
Antero
A
Kaarniranta
Kai
K
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D000071456:Sequestosome-1 Protein
D048868:Adaptor Proteins, Signal Transducing; D018840:HSP70 Heat-Shock Proteins; D007976:Leupeptins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D061988:Proteasome Inhibitors; D011993:Recombinant Fusion Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D049452:Green Fluorescent Proteins; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde