Abstract
Purpose:
Oxidative stress and complement activation are implicated in age-related macular degeneration (AMD). HO-1 is a stress-inducible protein with antioxidant and potential anti-inflammatory effects. We previously reported that complement activation through the AP attenuates HQ-mediated HO-1 mRNA induction. Multiple signaling pathways, including Nrf-2, NF-κB, AP-1, and MAPK, modulate HO-1 expression in non-ocular cells, and Nrf-2 is thought to play an important role. To better understand complement-mediated attenuation of HO-1, we investigated the role of Nrf-2 signaling on HO-1 expression in response to HQ alone, or in combination with AP complement.
Methods:
Cultured hRPE were treated with 125 uM HQ for 1.5 hours, primed with sheep anti-ARPE-19 antibody for 30 minutes and incubated in C1q-depleted human serum (C1q-Dep) for 3 hours. To study the role of Nrf-2 in HQ-mediated HO-1 induction, 80% confluent hRPE were transfected with 30 nM of small interfering RNA (siRNA) specific for Nrf-2. Once confluent, cells were exposed to 125 uM HQ for 1.5 hours and incubated in serum-free media for 3.5 hours. Cytoplasmic and nuclear protein was harvested, and protein levels were determined by Western blot. Confluent hRPE were treated with 40 ng/ml TNF-α, various doses of HQ, or AP complement at multiple time points. NF-κB translocation was examined with immunofluorescent staining and western blots of cytoplasmic and nuclear extracts.
Results:
AP complement activation attenuated HQ-induced HO-1 protein; the magnitude of this effect varied among experiments. Increased nuclear Nrf-2 accompanied HQ-mediated HO-1 induction, and AP complement attack had minimal effect on HQ-induced Nrf-2 protein. HQ-induced Nrf-2 protein was knocked down by Nrf-2 siRNA, but Nrf-2 inhibition had minimal effect on HQ-induced HO-1. TNF-α caused NF-κB nuclear translocation at 40 and 90 minutes, but HQ and AP complement alone did not cause NF-κB nuclear translocation at any dose or time point.
Conclusions:
AP complement activation attenuated HQ-mediated up-regulation of HO-1 protein expression. These phenomena appeared largely Nrf-2 and NF-κB independent. These data suggest that other transcription factors may play more significant HO-1 regulatory roles and represent further targets of study to understand the synergistic effects of oxidative stress and AP activation on RPE cell function.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
634 oxidation/oxidative or free radical damage