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. 2015 Jul;56(8):4198-210.
doi: 10.1167/iovs.15-16557.

Aldose Reductase Mediates Transforming Growth Factor β2 (TGF-β2)-Induced Migration and Epithelial-To-Mesenchymal Transition of Lens-Derived Epithelial Cells

Kun-Che Chang  1 , J Mark Petrash  1
Affiliations

Aldose Reductase Mediates Transforming Growth Factor β2 (TGF-β2)-Induced Migration and Epithelial-To-Mesenchymal Transition of Lens-Derived Epithelial Cells

Kun-Che Chang et al. Invest Ophthalmol Vis Sci. 2015 Jul.

Abstract

Purpose: Cataract surgery involves removal of lens tissue, but is associated with a high complication rate due to regrowth of residual lens epithelial cells to produce posterior capsule opacification (PCO) and diminished visual acuity. As inhibitors of aldose reductase (AR) have been shown to suppress markers of PCO, our studies were designed to identify a role for AR in the pathogenesis of PCO.

Methods: Sorbinil-mediated AR inhibition was determined by measuring sorbitol accumulation. Cell migration was measured using both transwell and scratch assays. Proteins in the SMAD signaling pathway were measured by Western blotting. The interactions of AR and SMADs were demonstrated by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA). Epithelial-to-mesenchymal transition (EMT) expression was measured by Western blot and quantitative PCR (q-PCR). Matrix metalloproteinase (MMP)-2 and MMP-9 activities were measured in conditioned medium by zymography.

Results: We observed that either Sorbinil-mediated AR inhibition or siRNA-mediated AR gene knockdown prevented migration of lens epithelial cells following exposure to TGF-β2. AR inhibition or AR knockdown reduced SMAD and MMP activation triggered by TGF-β2. In addition, we demonstrated AR inhibition or AR knockdown decreased TGF-β2-induced expression of EMT markers. Co-IP studies and PLA were used to demonstrate that AR and SMAD2 interact either directly or in close concert with additional factor(s) in a nonenzymatic manner.

Conclusions: This study demonstrates that AR participates in the response of lens epithelial cells to TGF-β2. Our studies raise the possibility that AR inhibition may be effective in preventing development of PCO by disrupting the TGF-β2/SMAD pathway.

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Figures

Figure 1
Figure 1
Aldose reductase inhibitors or AR knockdown decrease TGF-β2–induced migration. Human lens epithelial B3 cells were treated with dimethyl sulfoxide (DMSO) or Sorbinil for 30 minutes followed by treatment with TGF-β2 (5 ng/mL) for 10 hours for scratch migration assay (A). Staining with 4′,6-diamidino-2-phenylindole was used to visualize cell nuclei. Number of migrated cells in central zone was performed in statistic bar chart (B). Human lens epithelial B3 cells were further treated with vehicle (DMSO) or Sorbinil (10 μM) in the absence or presence of TGF-β2 (5 ng/mL) for 24 hours for transwell assay. Cells were further transfected with control or AR siRNA for 72 hours followed by treatment with TGF-β2. The cell migration ability (C, D) with ARI treatment or AR knockdown was determined after TGF-β2 exposures using transwell assay. The width of the cell-free gap is 500 ± 50 μm. Data shown are means ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2
Figure 2
Aldose reductase inhibitors or AR knockdown decreases TGF-β2–dependent SMADs phosphorylation. Human lens epithelial B3 cells were incubated with TGF-β2 (5 ng/mL) for indicated times (A). Human lens epithelial B3 cells were pretreated with vehicle (V) or Sorbinil (S, 10 μM) for 30 minutes followed by TGF-β2 (5 ng/mL) for 30 minutes (B). Cells were also transfected with control or AR siRNA for 72 hours followed by treatment with TGF-β2 for 30 minutes (C). In all cases, the fold-activation of p-SMAD2 and p-SMAD3 were normalized to total SMAD2 and SMAD3, respectively. Fold change is compared with vehicle in the TGF-β2 group. The efficiency of AR knockdown was examined by probing with AR antibody. Changes in the level of proteins of interest were analyzed for statistical significance and are displayed in graphical form as mean values ± SEM (n = 3). *P < 0.05.
Figure 3
Figure 3
Aldose reductase inhibitors or AR knockdown limit MMP-2 and MMP-9 activation by TGF-β2. Human lens epithelial B3 cells were pretreated with vehicle (V) or Sorbinil (S, 10 μM) for 30 minutes followed by TGF-β2 (5 ng/mL) exposure for 24 hours (A). Cells were further transfected with control (siCtrl) or AR siRNA (siAR) for 72 hours and followed by TGF-β2 exposure (B). Pro-MMP-2, -9 and active MMP-2, -9 secreted in medium were detected by gelatin zymography (A, B). Fold change of pro-MMPs is compared with vehicle or siCtrl in no TGF-β group. Changes in the level of proteins of interest were analyzed for statistical significance and are displayed in graphical form as mean values ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 4
Figure 4
Aldose reductase inhibition or AR knockdown reduce TGF-β2–induced expression of EMT markers. Human lens epithelial B3 cells (A) or primary murine lens capsular epithelial cells (C) were pretreated with vehicle (V) or Sorbinil (S, 10 μM) for 30 minutes followed by TGF-β2 (5 ng/mL) for 48 hours. Cells were also transfected with control or AR siRNA for 72 hours followed by treatment with TGF-β2 (B). Values for relative band intensity are given below the relevant sets of blots. The fold-activation of α-SMA and vimentin were normalized to actin. Fold change is compared with vehicle or siCtrl in no TGF-β2 group. The efficiency of AR knockdown was examined by probing with AR antibody. Changes in the level of proteins of interest were analyzed for statistical significance and are displayed in graphical form as mean values ± SEM (n = 3). *P < 0.05; ***P < 0.001.
Figure 5
Figure 5
Aldose reductase inhibition or ablation reduces levels of gene transcripts corresponding to TGF-β2–induced EMT markers. Messenger RNA was collected from either HLE B3 cells (A–D) or murine lens culture (E, F). Human lens epithelial B3 cells were treated with the vehicle or Sorbinil in the absence or presence of TGF-β2 (5ng/mL) for 24 hours. Lenses from wild-type or AR-null mice were treated with or without TGF-β2 (5 ng/mL) for 24 hours. Real-time analysis of mRNA levels of α-SMA (A, E), fibronectin (B, F), Snail (C), and Slug (D) was performed in HLE B3 cells or murine lens culture. Data shown are means ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 6
Figure 6
Aldose reductase inhibitors inhibit AR-SMAD2 and SMAD2-SMAD4 interactions. The HLE B3 cells were treated with or without TGF-β2 (5 ng/mL) in the presence or absence of NADPH during co-immunoprecipitations (A).The HLE B3 cells were pretreated with vehicle (V) or Sorbinil (S, 10 μM) for 1 hour followed by TGF-β2 (5 ng/mL) for 1 hour (B–E). Co-immunoprecipitation was performed by using AR or SMAD2 antibody for pull down and SMAD2 (B) or SMAD4 (C) was detected by Western blot after pull-down assay. Immunoglobulin G was used for negative control. The SMAD2 and AR in whole-cell lysate (WCL) were used for loading control. Aldose reductase catalysis is irrelevant for SMAD2 interactions (D, E). Human lens epithelial B3 cells were co-transfected with either AR-V5 (D) or ARY48F-V5 (E) and Flag-SMAD2 plasmids followed by co-immunoprecipitation using Flag antibody for pull down. Western blot detected V5 after pull-down assay. Immunoglobulin G was used for negative control. Flag and V5 in WCL was used for loading control. Values for relative band intensity are given below the relevant sets of blots. Overexpression of AR or ARY48F (AR*) enhances TGF-β2–induced SMAD2-SMAD4 interactions (F). Changes in the level of proteins of interest were analyzed for statistical significance and are displayed in graphical form as mean values ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 7
Figure 7
Proximity ligation assay of AR-SMAD2 interaction. The HLE B3 cells were pretreated with DMSO or Sorbinil for 1 hour in the presence or absence of TGF-β2 (5 ng/mL) for another 1 hour. Proximity ligation assay was done by using AR and SMAD2 primary antibodies and oligonucleotide-linked PLA secondary antibodies. A spot is formed when AR and SMAD2 are separated by no more than an approximately 40-nm distance. Data shown are number of PLA spots per cell. Data shown are means ± SEM (n = 3). *P < 0.05; **P < 0.01.
Figure 8
Figure 8
Model of AR-SMAD interaction. Aldose reductase binding to NADPH induces a conformational change that exposes a SMAD binding domain. Under TGF-β2 treatment, AR-NADPH binary complex binds to SMADs (A). However, Sorbinil binding to AR blocks the SMADs binding domain of AR, preventing their interaction at the TGF-β2 receptor (B).
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