Abstract
Purpose: :
Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly. Cigarette smoking is the strongest epidemiologic risk factor associated with AMD, yet we do not understand how smoking affects the RPE. The purpose herein was to determine the response by RPE cells to cigarette smoke (CS).
Methods: :
Confluent ARPE-19 cells were treated with 100-500 ug/ml CS extract for 24h. Total RNA was extracted and processed on the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. RNA was also collected for RT-qPCR confirmation and protein was extracted for Western Blot analysis.
Results: :
ARPE-19 cells exposed to 100-500 ug/ml of CS for 24h remained viable. Microarray analysis of cells exposed to 100 and 250 ug/ml CS for 24h showed an increase in the antioxidant response and ER stress. The expression of antioxidant genes (i.e.NQO1, HO1 and GCLM) increased after CS (p<0.002). The number of antioxidant genes with augmented expression from CS increased from 23 at 100 ug/ml to 36 at 250ug/ml. The number of ER stress genes with augmented expression also increased with CS dose. For example, lipid biosynthesis genes with increased expression increased from 10 to 21 genes, and protein processing genes increased from 35 to 65 when CS increased from 100-250 ug/ml. At 250 ug/ml CS, there was increased expression of genes related to autophagy and apoptosis, not seen at lower concentrations. We also observed a decrease in the number of DNA repair and cell cycling genes with decreased expression from 23 to 13 genes, as the dose of CS increased. CS produced a dose dependent increase in protein carbonylation, a marker of oxidative protein injury. Western blot of IRE1-α, was increased in cells treated with all 3 CS doses (p<0.05). CHOP, was increased only at 500 ug/ml CS (p= 0.05). PSMB6, a proteosomal sub-unit, was increased at 100-250 ug/ml of CS (P= 0.05), but decreased at 500ug/ml CS, consistent with progression of ER stress.
Conclusions: :
CS generates a protective transcriptional antioxidant and a prominent ER stress defense response in ARPE-19 cells. This expression profile was associated with changes in the proteosomal protein PSMB6, IRE1- α, and CHOP. This acute response could be an important protective response by RPE cells to CS exposure. Future studies will determine the impact of ER stress from prolonged CS exposure.
Keywords: retinal pigment epithelium • antioxidants • gene microarray