Nucleoredoxin Plays a Key Role in the Maintenance of Retinal Pigmented Epithelium Differentiation

Abstract

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/β-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release.

Keywords: RPE; differentiation; glia; hypoxia-ischemia; morphology; neurons; nucleoredoxin; redox regulation; reoxygenation; retina.

Figure 1

Changes in the retina due to H-I. (A). Immunofluorescence staining for Nrx (green) and Dapi counterstaining (blue) in SHAM and CARO retinas. White arrows show increased immunolabeling (B). Immunofluorescence staining for differentiation markers: SOX2 (green), Nestin (red), NF(s) (green) and Dapi counterstaining (blue) in SHAM and CARO retinas. White arrow shows increased immunolabeling (C). Nrx levels in retinas exposed to H-I (CARO group) and control conditions (SHAM) analyzed using immunofluorescence (Nrx in green) and Western blot (p = 0.0286; n = 4). Bars and error bars represent the mean + standard deviation (SD) and the asterisk represents a significative difference.

Figure 2

Nrx changes in ARPE-19 cells. (A). Nrx protein (p = 0.0067, 0.0015; n = 3) and mRNA levels in ARPE-19 cells exposed to hypoxia and reoxygenation (p = 0.0053, 0.0068; n = 6). (B). qRT-PCR analysis using differentiation markers (SOX2, Nestin and nNOS) in ARPE-19 cells exposed to hypoxia and reoxygenation (p = ns; n = 5). (C). MTT assay displaying cell proliferation/viability in control ARPE-19 cells (siScr) or cells with transient Nrx knock-down (siNrx) (p = 0.0002; n = 8). Bars and error bars represent the mean + standard deviation (SD). **, *** Asterisks represent different levels of significance.

Figure 3

Protein levels in ARPE-19 cells. (A). Western blot analysis of differentiation markers: GFAP (p = 0.0239; n = 3), MCT4 (p = 0.0027; n = 3), NeuroD1 (p = 0.0281; n = 3) and NF(s) in siScr and (B). siNrx ARPE-19 cells. C. Nrx protein levels analyzed using Western blot (p = 0.0361; n = 3). (C). Western blot analysis of activated β-catenin over total β-catenin levels in siScr and siNrx ARPE-19 cells exposed to H-I and reoxygenation (siScr: p = 0.0272; n = 3, siNrx: p = 0.0133; n = 3). (D). ELISA assay for VEGF levels in siScr and siNrx ARPE-19 cells (p = 0.0063; n = 6). *, ** Asterisks represent different levels of significance.

Figure 4

Nrx knock-down affects ARPE19 morphology and number of protrusions. (A). Immunofluorescence staining of the cytoskeleton: Actin (phalloidin, red), Tubulin (green) and nuclei counterstaining (Dapi, blue) in siScr and siNrx ARPE-19 cells. (B). Light microscopy area analysis of siScr and siNrx ARPE-19 (p = < 0.0001; n = 40–50). (C,D). Light microscopy photographs of 3D morphology analysis at different time points of siScr and siNrx ARPE-19 cells. (E). Sholl analysis of siScr and siNrx ARPE-19 cells at 0 h and 12 h after plating (p = 0.000128, 0.000098, 0.019188; n = 20). Bars and error bars represent the mean + standard deviation (SD). *, **** Asterisks indicate different levels of significance.