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. 2011;6(10):e26102.
doi: 10.1371/journal.pone.0026102. Epub 2011 Oct 17.

Up-regulation of NDRG2 in Senescent Lens Epithelial Cells Contributes to Age-Related Cataract in Human

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Free PMC article

Up-regulation of NDRG2 in Senescent Lens Epithelial Cells Contributes to Age-Related Cataract in Human

Zi-Feng Zhang et al. PLoS One. .
Free PMC article

Abstract

Background: Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer's disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract.

Methodology/principal findings: Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H(2)O(2) to simulate senescence. After being exposed to 50 µM H(2)O(2) for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50-200 µM H(2)O(2) for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses.

Conclusions/significance: NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characteristics induced by 2 weeks exposure of SRA01/04 cells to low-doses of H2O2.
A. Shows that cell viability (Left) of SRA01/04 cells decreases with increasing concentrations of H2O2 as measured by the MTT assay (Mean± SD; n = 5.). Morphologic changes (Right) of SRA01/04 cells after 2 weeks exposure to 50 µM H2O2 demonstrate gross enlargement, flattening, and the accumulation of granular cytoplasmic inclusions (Arrows indicate the nuclei of typical senescent cells, 100×). B. Shows staining for SA-β-gal activity increased in SRA01/04 cells exposed to 50 µM H2O2 for 2 weeks expressed as either SA-β-gal-positive cell index (Mean± SD; n = 3. * P<0.05. Left) or actual cellular staining (Right). C. Shows that DNA synthesis decreased in SRA01/04 cells exposed to 50 µM H2O2 for 2 weeks. BrdU labeling in SRA01/04 cells expressed either by BrdU incorporation and DAPI cell viability labeling (Mean± SD; n = 3. * P<0.05. Left) or expression of BrdU labeling of cells (Right). D. Shows that Ndrg2 protein is up-regulated in SRA01/04 cells exposed to 0–50 µM H2O2 for 2 weeks (Mean± SD; n = 3. * P<0.05 compare with normal SRA01/04 cells).
Figure 2
Figure 2. Functional changes of SRA01/04 cells infected with Ad-NDRG2.
A. Shows by Western blot that protein expression of Ndrg2 in SRA01/04 cells is induced when cells are infected with Ad-NDRG2 but not Ad-LacZ (Mean± SD; n = 3. * P<0.05 compare with normal SRA01/04 cells). B. Shows that SRA01/04 cells infected with Ad-NDRG2 after 48 h show morphological changes that include fibroblast-like appearances (100×). C. Shows that cell viability in SRA01/04 cells decreased when infected for 48 h with Ad-NDRG2, but not Ad-LacZ. Determined by the MTT assay (Mean± SD; n = 5. * P<0.05). D. Shows cell viability (normalized to control cells) of uninfected and 48-hour infected SRA01/04 cells as determined by the MTT assay. These results suggest that SRA01/04 cells were more susceptible to oxidative stress after Ad-NDRG2 infection (Mean± SD; n = 5. * P<0.05 compare with uninfected and Ad-LacZ infected SRA01/04 cells).
Figure 3
Figure 3. Differential expression of NDRG2 between HLECs from age-related cortical cataracts and clear lenses.
A. Shows that NDRG2 by RT-PCR analysis is higher in HLECs from age-related cortical cataracts (65.3±8.7 years) versus clear lenses (46.2±7.3 years). (Mean± SD; n = 3. * P<0.05). B. Differential expression of Ndrg2 protein in HLECs from age-related cortical cataracts (66.5±8.2 years) and clear lenses (47.8±7.7 years). Measured by Western blots (Mean± SD; n = 3. * P<0.05). C. Whole-mount immunofluorescence staining of Ndrg2 (green) in anterior capsules from age-related cortical cataracts (a, b, c) and clear lenses (d, e, f). HLECs were visualized by DAPI staining (blue). Images from both groups were captured by confocal microscopy under the same exposure condition. The fluorescence of Ndrg2 protein, which is localized in the cytoplasm, was higher in the cataractous compared with clear lenses (400×).

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