Purpose The ubiquitin-proteasome pathway and lysosomal pathway including autophagy are the master clearance systems in cells. The p62/sequestosome 1 (p62) and LC3 have been observed to be key players linking the proteasomal and lysosomal clearance systems. In this study, crosstalk of proteasomes and autophagy was examined. Methods Effect of autophagy activator (AICAR) and inhibitor (bafilomycin) on protein aggregation and autophagy markers were studied in the ARPE-19 cells treated with proteasome inhibitor (MG-132, 5µM) with or without AICAR (2mM) or bafilomycin (50nM) for 24h hours. Autophagy gene activation was studied by cDNA PCR array. The protein levels of LC3 were evaluated by western blotting. The localization and movement of p62 and LC3 were analyzed by live confocal microscopy. Cellular organelles were visualized by transmission electron microscopy. Results MG-132 clearly increased perinuclear protein aggregation, while AICAR robustly decreased the amount of aggregates together with LC3 activation. AICAR upregulates the most important autophagy genes. We show that p62 and LC3 colocalizes with the protein aggregates that all are finally degraded in autophagy. Conclusion Autophagy is effective clearance machine in RPE cells that might be a novel therapy target to prevent cell degeneration.
Purpose Urocanic acid (UCA) is a major UV-absorbing chromophore in the epidermis and has been suggested to act as one of the initiators of UV-induced immunosuppression. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in human corneal and conjunctival epithelial cells in response to UVB-irradiation in vitro. Methods Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCEC) were exposed to 10, 100, 1000, and 5000 µg/ml concentrations of cis-UCA (BioCis Pharma, Turku, Finland) with and without UVB-radiation (4 x Philips TL 20W/12 lamps; total irradiation dose 153 mJ/cm2). Secreted interleukin-6 (IL-6) levels were analyzed with ELISA assay. Cell viability was measured by a colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results The 100 µg/ml and 1000 µg/ml concentrations of cis-UCA significantly suppressed IL-6 secretion induced by UVB-irradiation in both cell types. In addition, the same concentrations improved the viability of the UVB-irradiated cells when analyzed by MTT assay. No significant alterations in IL-6 expression levels or viability were observed in response to 10, 100, and 1000 µg/ml cis-UCA only, while 5000 µg/ml cis-UCA evoked cytotoxicity in both cell types. Conclusion Our findings suggest that cis-UCA is a promising novel drug to suppress UVB-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.
Purpose Recent findings reveal that Toll-like receptors (TLRs) and innate immunity participate in the pathology of age-related macular degeneration (AMD). Many studies indicate that estrogens and selective estrogen modulators (SERMs) modulate inflammatory responses, but their effect on the development of AMD is weakly understood. In this study, we investigated the regulatory role of various SERMs (provided by Hormos Medical Ltd.) on IL-6 expression in human retinal pigment epithelial cells (ARPE-19). Methods ARPE-19 cells were exposed to lipopolysaccharide (LPS; TLR 4 agonist) with simultaneous exposure to various SERMs and the secretion of IL-6 cytokine was analyzed by ELISA. The estrogen receptor alpha and beta were qualitatively measured by RT-PCR. To study the effect of various SERM treatments of estrogen response element (ERE) -mediated transcription, the ARPE-19 cells were transiently transfected with ERE-luciferase vector. The activity of ERE was measured by Luciferase assay. Results Simultaneous exposures to LPS and SERM-320 reduced the IL-6 expression levels in ARPE-19 cells compared to LPS exposure alone. The RT-PCR analysis showed that ARPE-19 cells expressed estrogen receptor alpha but not beta proteins. Interestingly, SERM-320 did not increase the activity of ERE in ARPE cells. This reveals that SERM-320 is implicated in regulation of IL-6 expression, but is not mediated through estrogen response element. Conclusion Our findings reveal that SERM-320 is a novel compound to suppress innate immunity response in human retinal pigment epithelial cells.
Abstract Purpose Prior to proteolysis, heat shock proteins (HSPs) attempt to refold misfolded proteins. If this process is not successful proteins are degradated in proteasomes or in lysosomes. In the present study, the roles of the HSP70 and the p62/SQSTM 1 in autophagy clearance were evaluated in ARPE‐19 cells after proteasome inhibitor treatment. Methods The HSP70, p62/SQSTM 1 and ubiquitin localization were analyzed by immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to analyze the morphological changes. HSP70 and p62/SQSTM 1 levels were modulated using RNA interference techniques. Cell viability was measured by colorimetric assay. Results The proteasome inhibitor MG‐132 evoked the accumulation of perinuclear aggregates positive for HSP70, p62/SQSTM 1 and ubiquitin‐protein conjugates. We observed that the aggregation was reversible: a cessation of proteasome inhibition led to clearance of the deposits via autophagy. Interestingly, p62/SQSTM 1 mRNA depletion delayed autophagy clearance and significantly increased cell death in conjunction with proteasome inhibition. The hsp70 RNA interference did not change autophagy clearance, but increased cell death in response to proteasome inhibition. Conclusion The HSP70 and p62/SQSTM 1 regulate differently autophagy clearance in RPE cells. Autophagy seems to be an important mechanism to clean proteasome inhibitor –induced protein aggregates.
Purpose The pathogenesis of AMD involves impaired protein degradation in RPE cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In this study, expression and trafficking of p62 was examined. Methods To study the effect of autophagy activator (AICAR) and inhibitor (bafilomycin) on p62 expression levels, the ARPE-19 cells were treated with proteasome inhibitor (MG-132, 5µM) with or without AICAR (2mM) or bafilomycin (50nM) for 24h hours. The protein levels of p62 were evaluated by western blotting. The localization and movement of p62 were analyzed by live confocal microscopy. Results MG-132 increased the p62 protein levels, while AICAR robustly decreased the p62 levels. When autophagy was inhibited with bafilomycin the p62 was highly accumulated. We show that p62 binds irreversibly to protein aggregates that are finally degraded in autophagy. Conclusion The p62/sequestosome 1 function as a linker protein between proteasomes and autophagy and can be used as a autophagy flux marker. Autophagy is effective clearance machine that may be disturbed in aged RPE cells.
