Abstract
Purpose :
Progression of age-related macular degeneration (AMD) is linked to an increase in cellular stress, especially in the endoplasmic reticulum (ER). ER proteins that maintain cellular homeostasis, such as 78 kDa glucose-regulated protein (GRP78) and protein kinase-like endoplasmic reticulum kinase (PERK), and that mediate apoptosis, such as C/EBP homologous protein (CHOP) determine the cell’s longevity through the unfolded protein response (UPR). Bax Inhibitor 1 (BI-1) is an ER-associated protein that protects against BAX-mediated cell-death. In the current study, we investigated the effect of BI-1 overexpression on ER stress proteins in RPE cells subjected to tunicamycin, an RPE cell death-inducing drug.
Methods :
Cultured human RPE cells were infected with adenovirus encoding either LacZ or BI-1 at a multiplicity of infection (MOI) of 8 for 24 hours. Media were replaced with 1% FBS-MEM, the infected cells were cultured for additional 4 days in 1% FBS-MEM, and then treated with tunicamycin at various concentrations for 24 hours. Cell morphology was assessed by light microscopy. Cell death was determined by lactose dehydrogenase (LDH) assay. GRP78, PERK, and CHOP protein levels were evaluated by Western blot.
Results :
On morphological analysis, and by LDH assay, BI-1 infected cells when compared to LacZ infected cells were more resistant to tunicamycin-induced death, regardless of concentration; LacZ infected cells released nearly twice as much LDH into media when compared to BI-1 infected cells. Tunicamycin induced increased levels of CHOP and GRP78 when compared to untreated cells, and produced higher CHOP and PERK levels in BI-1-treated cells than in LacZ-treated cells. In contrast, similar levels of GRP78 were observed in LacZ and BI-1 infected cells treated with tunicamycin.
Conclusions :
When exposed to tunicamycin, BI-1 induces increased levels of specific ER stress proteins, while at the same time protecting cells from tunicamycin induced cell death. Understanding how BI-1 relates to the adaptive unfolded protein response characteristic of ER stress proteins may guide methods to limit RPE cell death in eyes with AMD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.