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Upregulation of GADD45 α in light-damaged retinal pigment epithelial cells

View Article: PubMed Central - PubMed

ABSTRACT

To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10 000 Lux at 37 °C for 9 h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45α (GADD45α) expression was markedly upregulated. Transient knockdown of GADD45α partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45α overexpression dramatically increased it. These results demonstrate the critical function of GADD45α in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45α was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45α gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45α expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45α expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment.

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AKT dephosphorylation exacerbates apoptosis and enhances GADD45α expression. (a) Western blotting demonstrated increased dephosphorylation of AKT after light exposure and Ly294002 treatment of RPE cells after 4.5 h of exposure to 10 000 Lux white light. The β-actin loading control is from the same experiment as shown in Figure 3e and is reproduced in this figure for ease of reference. (b) ARPE-19 cells treated with Ly294002 or exposed to 10 000 Lux white light for 4.5 h showed significant changes in mitochondrial membrane potential, as measured by JC-1. Bar=100 μm. (c) RT-PCR showed that GADD45α expression was significantly upregulated after 4.5 h of light exposure and/or Ly294002 treatment. Results are given as mean value±S.E.M. (n=3, ***P<0.001, *P<0.05).
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fig5: AKT dephosphorylation exacerbates apoptosis and enhances GADD45α expression. (a) Western blotting demonstrated increased dephosphorylation of AKT after light exposure and Ly294002 treatment of RPE cells after 4.5 h of exposure to 10 000 Lux white light. The β-actin loading control is from the same experiment as shown in Figure 3e and is reproduced in this figure for ease of reference. (b) ARPE-19 cells treated with Ly294002 or exposed to 10 000 Lux white light for 4.5 h showed significant changes in mitochondrial membrane potential, as measured by JC-1. Bar=100 μm. (c) RT-PCR showed that GADD45α expression was significantly upregulated after 4.5 h of light exposure and/or Ly294002 treatment. Results are given as mean value±S.E.M. (n=3, ***P<0.001, *P<0.05).

Mentions: As shown in the GO term analysis presented in Figure 3b, the PI3K-AKT network was downregulated in light-damaged ARPE-19 cells. Phosphorylated AKT (p-AKT) levels (Figure 5a and Supplementary Figures 3 and 4) and cell viability were also remarkably decreased in light-damaged ARPE-19 cells (Figure 2b). We next wanted to address whether inhibition of AKT phosphorylation aggravated RPE cellular apoptosis. To do so, we used the AKT phosphorylation inhibitor Ly294002. ARPE-19 cells pretreated with 20 μM Ly294002 followed by exposure to light displayed higher levels of AKT dephosphorylation (Figure 5a) and exacerbated apoptosis (Figure 5b). Surprisingly, higher expression of GADD45α was also observed after Ly294002 treatment, either with or without light exposure (Figure 5c). Collectively, these findings support the idea that AKT activation plays a key role in light-induced apoptosis in ARPE-19 cells. Furthermore, that the PI3K-AKT pathway might play an important role in the upregulation of GADD45α.


Upregulation of GADD45 α in light-damaged retinal pigment epithelial cells
AKT dephosphorylation exacerbates apoptosis and enhances GADD45α expression. (a) Western blotting demonstrated increased dephosphorylation of AKT after light exposure and Ly294002 treatment of RPE cells after 4.5 h of exposure to 10 000 Lux white light. The β-actin loading control is from the same experiment as shown in Figure 3e and is reproduced in this figure for ease of reference. (b) ARPE-19 cells treated with Ly294002 or exposed to 10 000 Lux white light for 4.5 h showed significant changes in mitochondrial membrane potential, as measured by JC-1. Bar=100 μm. (c) RT-PCR showed that GADD45α expression was significantly upregulated after 4.5 h of light exposure and/or Ly294002 treatment. Results are given as mean value±S.E.M. (n=3, ***P<0.001, *P<0.05).
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fig5: AKT dephosphorylation exacerbates apoptosis and enhances GADD45α expression. (a) Western blotting demonstrated increased dephosphorylation of AKT after light exposure and Ly294002 treatment of RPE cells after 4.5 h of exposure to 10 000 Lux white light. The β-actin loading control is from the same experiment as shown in Figure 3e and is reproduced in this figure for ease of reference. (b) ARPE-19 cells treated with Ly294002 or exposed to 10 000 Lux white light for 4.5 h showed significant changes in mitochondrial membrane potential, as measured by JC-1. Bar=100 μm. (c) RT-PCR showed that GADD45α expression was significantly upregulated after 4.5 h of light exposure and/or Ly294002 treatment. Results are given as mean value±S.E.M. (n=3, ***P<0.001, *P<0.05).
Mentions: As shown in the GO term analysis presented in Figure 3b, the PI3K-AKT network was downregulated in light-damaged ARPE-19 cells. Phosphorylated AKT (p-AKT) levels (Figure 5a and Supplementary Figures 3 and 4) and cell viability were also remarkably decreased in light-damaged ARPE-19 cells (Figure 2b). We next wanted to address whether inhibition of AKT phosphorylation aggravated RPE cellular apoptosis. To do so, we used the AKT phosphorylation inhibitor Ly294002. ARPE-19 cells pretreated with 20 μM Ly294002 followed by exposure to light displayed higher levels of AKT dephosphorylation (Figure 5a) and exacerbated apoptosis (Figure 5b). Surprisingly, higher expression of GADD45α was also observed after Ly294002 treatment, either with or without light exposure (Figure 5c). Collectively, these findings support the idea that AKT activation plays a key role in light-induced apoptosis in ARPE-19 cells. Furthermore, that the PI3K-AKT pathway might play an important role in the upregulation of GADD45α.

View Article: PubMed Central - PubMed

ABSTRACT

To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10&thinsp;000&thinsp;Lux at 37&thinsp;&deg;C for 9&thinsp;h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45&alpha; (GADD45&alpha;) expression was markedly upregulated. Transient knockdown of GADD45&alpha; partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45&alpha; overexpression dramatically increased it. These results demonstrate the critical function of GADD45&alpha; in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45&alpha; was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45&alpha; gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45&alpha; expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45&alpha; expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment.

No MeSH data available.


Related in: MedlinePlus