p62/sequestosome 1 as a regulator of proteasome inhibitor‐induced autophagy in human retinal pigment epithelial cells

Abstract

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. 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 . 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.Age-related macular degeneration (AMD) is the leading cause of blindness of elderly people in the developed countries. The disease affects the macula, which is located in the central area of the retina. Primarily, AMD is characterized by degeneration of the macular retinal pigment epithelial (RPE) cells [1]. The RPE cells take care of the health of rods and cones. Therefore, the degeneration and cell death of RPE cells cause secondary adverse effects on the neural cells, ultimately leading to visual loss. Chronic oxidative stress and inflammation are key factors evoking RPE degeneration and promoting the AMD process [2,3]. One hallmark of AMD is the accumulation of lysosomal lipofuscins, and extracellular drusens between RPE cells and Bruch's membrane. This cargo is a clear evidence of a disturbance in the cellular protein Correspondence to: Kai Kaarniranta, Department of Ophthalmology, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland; Phone.: +358-17-162015; FAX: +358-17-162048; email: Kai.Kaarniranta@uef.fi clearance system in aged RPE cells [1]. Recently,...

Autophagy Involvement in the Postnatal Development of the Rat Retina

“…At P14 and P18, with the presence of a fully vascularized retina, a decrease in p-ULK1 and LC3 II protein levels is determined concomitantly with an increase of p62. The observed accumulation of p62 with a decrease of LC3 II protein levels at P14 could be associated with a transition from autophagy-dependent to -independent mechanisms of retinal homeostasis, as previously suggested in retinal pigment epithelium cells [51,52].…”

Differential Expression of Inflammasome-Related Genes in Induced Pluripotent Stem-Cell-Derived Retinal Pigment Epithelial Cells with or without History of Age-Related Macular Degeneration

“…The characterization confirmed that both control-RPE cells and AMD-RPE cells showed characteristics of mature RPE cells. prevent protein clearance and induce inflammasomes in ARPE-19 cells and in embryonal stem-cell-derived RPE cells [22,33,34]. Activation of inflammasomes in iPSC-RPE cells significantly reduced cellular viability, as observed by an elevated release of lactate dehydrogenase (LDH).…”

“…To stimulate inflammasome activation, iPSC-RPE cells were first primed with IL-1α and inflammasomes were then activated by inhibiting cellular clearance mechanisms using MG-132 (inhibition of the proteasome) and bafilomycin A1 (inhibition of autophagy). The conditions and concentrations had been previously tested and found to prevent protein clearance and induce inflammasomes in ARPE-19 cells and in embryonal stem-cell-derived RPE cells [22,33,34]. Activation of inflammasomes in iPSC-RPE cells significantly reduced cellular viability, as observed by an elevated release of lactate dehydrogenase (LDH).…”