| Background: Age-related macular degeneration (AMD) remains high incidence and accounts for a main cause of blindness in aging people, but its mechanism is still poorly understood. Aging and associated dysfunction of retinal pigment epithelial (RPE) cells are believed to be the pathological onset of AMD. The 20S proteasome has been tightly correlated with cell aging due to its fundamental role in maintaining cellular homeostasis, but its implication in human RPE cell aging was seldom concerned.Purpose: This study aimed to demonstrate the interconnections between proteasome and RPE aging by characterizing age-dependent changes of the 20S proteasome in primarily cultured human RPE cells, and further investigate the impact of specific proteasome inhibition on RPE aging process.Methods: (1) A replicative aging RPE cell model was established based on previous work of our lab. The primarily cultured human RPE cells were maintained and subcultured as usual. Passages 2~4, 12~14 and 22~24 were selected and defined as the young, middle and old passage group, respectively. For each passage group, RPE cells went through inverted phase contrast microscope and electro-microscope observation, XTT test for cell viability,β-galactosidase staining for senescence detection, and flow cytometry assay for cellular autofluorescence intensity. (2) The fluorogenic substrates (LLE-AMC, suc-LLVY-AMC, LSTR-AMC and FITC-casein) were used to determine the proteolytic activities of the 20S proteaosome in RPE lysates. Colorimetric carbonyl assay and western blot were employed for measurement of oxidized protein and 20S proteasome content, respectively. Immunofluorescence assay was applied for intracellular localization and quantification of both the oxidized proteins and 20S proteasome. Real-time PCR was used to detect the gene expression of the proteasomal proteolytic subunits. (3) RPE cells of early passage group were treated with MG132, a specific proteasome inhibitor, and the age-related characters were investigated through microscope observation, XTT test andβ-galactosidase staining. The proteasome function and intracellular oxidized protein content was determined with aforementioned methods.Results: (1) RPE cells in culture exhibited typical features of senescent at the late stage of cell growth, as they were enlarged, did not line up in parallel arrays, showed plasmic vaculation, had reduced cell viability, turned positive toβ-galactosidase senescence biomarker, and had elevated cellular autofluorescence. (2) Significant decline in all the three specific activities and degradation of FITC-casein of the 20S proteasome was found in aging RPE cells. The malfunctions were accompanied with remarkably increased content of oxidized proteins in the old RPE cells, and stable content of the 20S core. Immunofluorescence assay revealed that a most significant elevation of the oxidized proteins content occurred in the peri-nuclear regions and nucleus, while the 20S proteasome was concentrated in RPE nucleus regardless of passages. Proteasome-to-oxidized protein ratio indicated functional efficiency of the 20S proteasome. The highest value of this ratio was found in the nucleus of young RPE passages, which declined to less than 50% in the old passages. Real-time PCR assay revealed an up-regulated expression of the proteasomalβ5 subunit followed by a down-regulated level during RPE aging process. (3) Treatment with sub-toxic dose of MG132 elicited irreversible senescence-like features in young RPE cells, including growth arrest, typical senescence morphology, plasmic vaculation, and enhancedβ-galactosidase staining. These inducible features appeared to be dose- and time-dependent. While 5μM of MG132 inhibited a large part of PGPH activity, CT-L activity and FITC-casein degradation of the proteasome, the same dosage exhibited little effect on T-L activity and intracellular oxidized protein level. 10μM of MG132 diminished proteasomal activities and lead to significantly higher content of oxidized proteins in young RPE cells.Conclusions: An aging model of human RPE cells was validated for age- and senescence-related investigation in vitro. In such a model, we testified age-related malfunction of the 20S proteasome, concomitant with increased oxidized protein level. Partial inhibition of proteasomes in young RPE cells caused by treatment with specific inhibitors induced a senescence-like phenotype. Thus we demonstrated the fundamental importance of the proteasome in human RPE cells aging. Future researches on the mechanism of these events are still strongly recommended.To sum up, our research supported a fundmental role of the proteasome in RPE cell aging. To the best of our knowledge, similar findings have not been reported yet at home and abroad.
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