Human retinal pigment epithelial cells are protected against hypoxia by BNIP3

Abstract

Background: Hypoxia has been implicated in the process of retinal pigment epithelium (RPE) dysfunction. However, recent studies suggest that hypoxia contributes to survival rather than cell death through induction of Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3)-dependent autophagy. In contrast, persistent oxidative stress was found to result in autophagy dysregulation in RPE cells. These seemingly contradictory findings led us to investigate the potential role of BNIP3, a crucial mediator of hypoxia-induced autophagy, in the context of hypoxic RPE cells.

Methods: Human RPE D407 cells were treated with low-oxygen conditions, and cell growth, apoptosis, and autophagy was assessed by Cell Counting Kit-8 assay, flow cytometry analysis and immunofluorescence staining, respectively.

Results: Hypoxic conditions simultaneously triggered a large amount of apoptosis and inhibited autophagy. Moreover, hypoxia led to severe impairments, including the stimulation of reactive oxygen species, and reduction of mitochondrial membrane potential, and adenosine triphosphate production. The stimulation of autophagy by rapamycin inhibited hypoxia-induced severe impairments to a great extent. Interestingly, similar results were observed for BNIP3 overexpression, which can be largely blocked by 3-MA, a well-defined inhibitor of autophagy. Moreover, BNIP3 knockdown further aggravated hypoxia-induced impairments in D407 cells, which can be reversed by rapamycin.

Conclusions: Collectively, these results indicated that BNIP3 can protect human retinal pigmented epithelial cells under hypoxic conditions by inducing autophagy.

Keywords: Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3); Retinal pigment epithelium (RPE); apoptosis; autophagy; hypoxia.

Figure 1

Hypoxia induced apoptosis and inhibited autophagy in D407 cells. (A) Cell growth was inhibited by hypoxia (1% O₂) over 72 h, and was partially recovered by the administration of rapamycin (100 nM). (B) Hypoxia increased apoptosis compared to normoxia, which was inhibited by the addition of 100 nM rapamycin. After having been subjected to hypoxia in the presence or absence of rapamycin for 48 h, human D407 RPE cells were collected and analyzed with fluorescence-activated cell sorting (FACS). (C) The autophagy marker LC3 was measured with fluorescence microscopy after treatment with hypoxia for 48 h. Hypoxic treatment decreased the immunofluorescence of LC3 (green), which was reversed by rapamycin (100 nM). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in blue. Scale bar: 50 μm. Asterisks indicate significant difference (***, P<0.001).

Figure 2

The expressions of apoptosis and autophagy associated proteins were investigated with western blotting. (A) The expression of Bcl2 decreased and Bax increased after hypoxic treatment for 48 h. The administration of rapamycin (100 nM) reversed these effects in D407 cells. (B) The expressions of Beclin-1, Atg5, and the ratio of LC3-II/LC3-I decreased in hypoxia-treated D407 cells. The administration of rapamycin (100 nM) reversed these effects in D407 cells. Asterisks indicate significant difference (***, P<0.001).

Figure 3

The mitochondrial function was impaired by hypoxia and partially recovered by the administration of 100 nM rapamycin. (A) Mitochondrial membrane potential decreased in hypoxia-treated D407 cells. (B) The intracellular ATP concentration decreased in hypoxia-treated D407 cells. (C) Reactive oxygen species (ROS) level increased in hypoxia-treated D407 cells. Asterisks indicate significant difference (**, P<0.01; ***, P<0.001).

Figure 4

Overexpression of BNIP3 in D407 cells alleviated the impairments of hypoxia. (A) Decreased expression of BNIP3 was investigated with quantitative RT-PCR (top) and western blotting (bottom). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Quantitative RT-PCR (top) and western blotting (bottom) results to verify the overexpression of BNIP3 in D407 cells after infection with BNIP3 or lentivirus vector. GAPDH was used as an internal control. (C) BNIP3 overexpression promoted the cell growth in hypoxic conditions, which was inhibited by the administration of 50 µM 3-MA. (D) Hypoxia induced apoptosis in D407 cells, which was inhibited by BNIP3 overexpression. (E) Overexpression of BNIP3 recovered the inhibited autophagy by hypoxia to some extent. The immunofluorescence of LC3 (green) in D407 cells with or without BNIP3 overexpression was measured with fluorescence microscopy. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in blue. Scale bar: 50 μm. (F) Overexpression of BNIP3 alleviated the impairments of hypoxia. Asterisks indicate significant difference (***, P<0.001).

Figure 5

The hypoxic impairments in mitochondrial function were recovered by BNIP3 overexpression, which can be counteracted by 3-MA (50 µM). (A) Mitochondrial membrane potential. (B) Intracellular ATP concentration. (C) Reactive oxygen species (ROS) level. D407 cells infected with BNIP3 or lentivirus vector were treated with hypoxia in the presence or absence of 50 µM 3-MA for 48 h, then mitochondrial membrane potential, intracellular ATP concentration and ROS level were examined using different assays. Asterisks indicate significant difference (**, P<0.01; ***, P<0.001).

Figure 6

The hypoxic impairments to D407 cells were further aggravated by BNIP3 knockdown, which was alleviated by the administration of 100 nM rapamycin. (A) Quantitative RT-PCR (top) and western blot analysis (bottom) of BNIP3 expressions in D407 cells after infection with BNIP3 shRNA (shBNIP3) or control shRNA lentivirus (scrambled). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Knockdown of BNIP3 further inhibited the cell growth under hypoxic conditions. (C) The number of apoptotic cells was increased by BNIP3 knockdown. (D) LC3 immunofluorescence signals (green) were diminished by BNIP3 knockdown. The nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI) in blue. Scale bar: 50 μm. Asterisks indicate significant difference (***, P<0.001).