TY - JOUR
T1 - Complement factor C3a alters proteasome function in human RPE cells and in an animal model of age-related RPE degeneration
AU - Ramos de Carvalho, J. Emanuel
AU - Klaassen, Ingeborg
AU - Vogels, Ilse M. C.
AU - Schipper-Krom, Sabine
AU - van Noorden, Cornelis J. F.
AU - Reits, Eric
AU - Gorgels, Theo G. M. F.
AU - Bergen, Arthur A. B.
AU - Schlingemann, Reinier O.
PY - 2013
Y1 - 2013
N2 - 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 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. In the retinal pigment epithelial cells (RPE) of monocyte chemoattractant protein-1-deficient CCL2(-/-) 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 (β5) and immuno (β5i) subunit expression. Subsequently, proteasome overall activity was determined using the BodipyFl-Ahx3L3VS 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 α7, PA28α, β5, and β5i were also studied in RPE cells after exposure to IFN-γ, C3a, and C5a by real-time PCR and Western blotting. Retinal pigment epithelial cells of CCL2(-/-) 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. 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
AB - 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 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. In the retinal pigment epithelial cells (RPE) of monocyte chemoattractant protein-1-deficient CCL2(-/-) 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 (β5) and immuno (β5i) subunit expression. Subsequently, proteasome overall activity was determined using the BodipyFl-Ahx3L3VS 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 α7, PA28α, β5, and β5i were also studied in RPE cells after exposure to IFN-γ, C3a, and C5a by real-time PCR and Western blotting. Retinal pigment epithelial cells of CCL2(-/-) 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. 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
U2 - https://doi.org/10.1167/iovs.13-12374
DO - https://doi.org/10.1167/iovs.13-12374
M3 - Article
C2 - 23982842
SN - 0146-0404
VL - 54
SP - 6489
EP - 6501
JO - Investigative Ophthalmology & Visual Science
JF - Investigative Ophthalmology & Visual Science
IS - 10
ER -