Abstract
Purpose:
We have previously shown that oxidative stress and alternative pathway (AP) complement attack, both of which have been implicated in age-related macular degeneration (AMD), synergistically damage RPE cells. While oxidative stress has been shown to induce ER stress in RPE cells, the role of complement activation in ER stress has not yet been explored. This study was undertaken to clarify the role of AP complement activation on ER stress in RPE cells.
Methods:
Cultured RPE cells were incubated with serum-free MEM or 125 uM hydroquinone (HQ) for 1.5 hours, primed with a sheep anti-ARPE-19 antibody (Allergan, Inc.) for 30 minutes, and then incubated in either 6% C1q-depleted human serum or 6% heat inactivated normal human serum for 2, 3, 4.5, and 6 hours. Quantitative RT-PCR was performed to test the ER stress markers CHOP, PERK, and GRP78 gene expression levels. Western blot was performed to compare the protein expression of CHOP in cells primed with the anti-ARPE-19 antibody to that of untreated age-matched cells at 2, 3 and 6 hours.
Results:
Both CHOP and PERK gene expression at 2 and 3 hours was up-regulated over 2-fold and over 4-fold after treatment with AP complement attack alone and in combination with HQ 125 uM, respectively. By 4.5 hours, the up-regulation of these genes had decreased. GRP78 gene expression increased at 3 hours, and its up-regulation persisted at 4.5 hours. CHOP protein levels were increased over untreated controls by AP complement attack alone and in combination with HQ 125 uM. These increased protein levels were diminished by 6 hours.
Conclusions:
AP complement attack on human RPE cells, either alone or in combination with hydroquinone, increases ER stress marker gene expression and protein levels. The effect of AP complement activation and HQ on ER stress markers was additive. These results suggest that oxidant-enhanced AP complement-mediated ER stress marker activation may induce RPE cell death and contribute to AMD.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
533 gene/expression