Complement Factor C3a Alters Proteasome Function in Human RPE Cells and in an Animal Model of Age-Related RPE Degeneration

J. Emanuel Ramos de Carvalho, Ingeborg Klaassen, Ilse M. C. Vogels, Sabine Schipper-Krom, Cornelis J. F. van Noorden, Eric Reits, Theo G. M. F. Gorgels, Arthur A. B. Bergen, Reinier O. Schlingemann
2013 Investigative Ophthalmology and Visual Science  
Citation: Ramos de Carvalho JE, Klaassen I, Vogels IMC, et al. Complement factor C3a alters proteasome function in human RPE cells and in an animal model of age-related RPE degeneration. Invest Ophthalmol Vis Sci. PURPOSE. Complement activation plays an unequivocal role in the pathogenesis of age-related macular degeneration (AMD). More recent evidence suggests an additional role in AMD for the ubiquitin proteasome pathway (UPP), a protein-degradation nanomachinery present in all types of
more » ... all types of eukaryotic cells. The purpose of this study was to elaborate on these findings and investigate whether the complement system directly contributes to derangements in the UPP through the activated complement components C3a and C5a. METHODS. In the retinal pigment epithelial cells (RPE) of monocyte chemoattractant protein-1deficient CCL2 À/À mice, a mouse model that may serve as a model for age-related atrophic degeneration of the RPE, proteasome function was investigated by immunohistochemistry of household (b5) and immuno (b5i) subunit expression. Subsequently, proteasome overall activity was determined using the BodipyFl-Ahx 3 L 3 VS probe in primary-cultured human retinal pigment epithelial cells (HRPE) cells that were exposed to different stimuli including C3a and C5a, using confocal laser scanning microscopy and flow cytometry. Gene expression and protein levels of proteasome subunits a7, PA28a, b5, and b5i were also studied in RPE cells after exposure to IFN-c, C3a, and C5a by real-time PCR and Western blotting. RESULTS. Retinal pigment epithelial cells of CCL2 À/À mice showed immunoproteasome upregulation. C3a, but not C5a supplementation, induced a decreased proteasome overall activity in HRPE cells, whereas mRNA and protein levels of household proteasome and immunoproteasome subunits were unaffected. CONCLUSIONS. In HRPE cells, C3a induces decreased proteasome-mediated proteolytic activity, whereas in a mouse model of age-related RPE atrophy, the immunoproteasome was upregulated, indicating a possible role for complement-driven posttranslational alterations in proteasome activity in the cascade of pathologic events that result in AMD.
doi:10.1167/iovs.13-12374 pmid:23982842 fatcat:spmvrwoy7jdf7ljwgicy5nlfae