Expression of Endoplasmic Reticulum Stress Markers GRP78 and CHOP Induced by Oxidative Stress in Blue Light-Mediated Damage of A2E-Containing Retinal Pigment Epithelium Cells

Abstract

Aims: Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. Methods: ARPE-19 cells were incubated with A2E (10, 25, 50 μ<smlcap>M</smlcap>) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. Results: After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. Conclusion: RPE cells exhibited ROS accumulation and subsequent elevation of GRP78 and CHOP expression after A2E and blue light-induced damage. The ROS scavenger NAC diminished ER stress protein expression, suggesting a connection between ER and oxidative stress in blue light-mediated damage of A2E-containing RPE cells. Besides, GRP78 may play a protective role in it.

Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp

“…Phosphorylated PERK helps to block protein synthesis and to upregulate the expression of Bcl-2, leading to the survival of cells (34)(35)(36). In contrast, CHOP/GADD153 could be upregulated to start the ER apoptosis pathway when the accumulated misfolded protein from long-term ER stress exceeds the capacity of the unfolded protein response (37). However, our data showed that no significant change in CHOP expression was observed between HepG2/IR and the parental HepG2 cells, suggesting that the development of IR only promoted the protective ER stress response and the activation of the PERK pathway was one of the mechanisms leading to the tolerance to chemotherapeutic drugs of the IR liver tumor cells.…”

A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2

“…Previous research also indicated that A2E and its precursor alltrans retinal could generate ROS inside the RPE cells. [45][46][47] The possible mechanisms were related to the C/EBP homologous protein (CHOP) and GRP18 pathways 32 and cell damage. 48 Moreover, oxidative stress and inflammation are interrelated.…”

“…32,33 Isolated mouse primary RPE cells in passage 2 were collected with or without the stimulation of A2E at a concentration of 10 lM for 6 hours. The RPE cells or the A2E-laden RPE (A2E-RPE) cells were collected and washed three times with PBS, followed by co-culture with naïve T cells under different polarization conditions as indicated.…”

“…Human ARPE19 cells from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured and treated with A2E as previously described. 32 Isolation and Culture of T Cells…”

“…32 Briefly, total protein was collected from cultured RPE cells as indicated. Proteins were electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) and immunoblotted for COX-2 (Abcom, Cambridge, MA, USA).…”