The involvement of ATF4 and S-opsin in retinal photoreceptor cell damage induced by blue LED light

Abstract

Purpose: Blue light is a high-energy emitting light with a short wavelength in the visible light spectrum. Blue light induces photoreceptor apoptosis and causes age-related macular degeneration or retinitis pigmentosa. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress induced by blue light-emitting diode (LED) light exposure in murine photoreceptor cells.

Methods: The murine photoreceptor cell line was incubated and exposed to blue LED light (464 nm blue LED light, 450 lx, 3 to 24 h). The expression of the factors involved in the unfolded protein response pathway was examined using quantitative real-time reverse transcription (RT)-PCR and immunoblot analysis. The aggregation of short-wavelength opsin (S-opsin) in the murine photoreceptor cells was observed with immunostaining. The effect of S-opsin knockdown on ATF4 expression in the murine photoreceptor cell line was also investigated.

Results: Exposure to blue LED light increased the bip, atf4, and grp94 mRNA levels, induced the expression of ATF4 protein, and increased the levels of ubiquitinated proteins. Exposure to blue LED light in combination with ER stress inducers (tunicamycin and dithiothreitol) induced the aggregation of S-opsin. S-opsin mRNA knockdown prevented the induction of ATF4 expression in response to exposure to blue LED light.

Conclusions: These findings indicate that the aggregation of S-opsin induced by exposure to blue LED light causes ER stress, and ATF4 activation in particular.

Figure 1

Unfolded protein response (UPR)-related mRNAs induced in response to exposure to blue LED light in murine photoreceptor cells. Induction of bip (A), grp94 (B), or atf4 (C) mRNA after blue light-emitting diode (LED) light exposure time with real-time reverse transcription (RT)–PCR. Gapdh mRNA was used as the control. Data are represented as the means ± standard error of the mean (SEM; n=4). **p<0.01, *p<0.05 versus control (Student t test).

Figure 2

Change in protein levels induced in response to exposure to blue LED light in murine photoreceptor cells. Immunoblotting shows the protein levels of the unfolded protein response (UPR) factors. The bands indicate protein expression levels at 3, 6, 9, 12, and 24 h for the control and light-exposed samples. Representative image (upper) and quantitative data (lower) of the BiP (A), GRP94 (B), and ATF4 (C) immunoblots. Data are presented as the mean ± standard error of the mean (SEM; n=6 to 14). *p<0.05, **p<0.01 versus control (Student t test).

Figure 3

Changes in ubiquitinated protein level induced by exposure to blue LED light. Immunoblotting shows the ubiquitinated protein levels. A: Representative image. B: Quantitative data of the immunoblot. Data are presented as the mean ± standard error of the mean (SEM; n=6). *p<0.05 versus control (Student t test).

Figure 4

The aggregation of S-opsin induced by exposure to blue LED light or ER stress inducers. A: Representative immunostaining images of short-wavelength opsin (S-opsin) after exposure to blue light-emitting diode (LED) light for 6 h. B: 2 μg/ml tunicamycin treated for 12 h. C: 0.5 mM dithiothreitol (DTT) treated for 6 h. D: Quantitative analysis of the immunostaining images. The ratio of aggregated S-opsin cells. Data are presented as the mean ± standard error of the mean (SEM; n=3 or 4). *p<0.05, * * p<0.01 versus control (one-way ANOVA followed by Dunnett’s test). Scale bar=50 μm. Arrowheads indicate the aggregation of S-opsin.

Figure 5

Effects of S-opsin siRNA on exposure to blue LED light in murine photoreceptor cells. A: Representative immunoblotting images of short-wavelength opsin (S-opsin), ATF4, and β-actin. B: Expression level of S-opsin. C: Expression level of ATF4. Data are presented as the mean ± standard error of the mean (SEM; n=7 to 9). **p<0.01, #p<0.05 versus normal (control); †p<0.05, † † p<0.01 versus normal (blue light-emitting diode [LED]); (Student t test). N, normal; NC, negative control siRNA.