KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation

Abstract

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-β2-induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-β2-induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.

Keywords: autophagy; epithelial-mesenchymal transition; keratin 8; proliferative vitreoretinopathy; retinal pigment epithelial cells.

Figure 1

Expression of KRT8 and its phosphorylated form, and autophagy marker in human PVR membranes. A, Representative fluorescence microscopy images show the distributions of immunoreactive KRT8 (green fluorescence) and LC3B (red fluorescence) within the subretinal and epiretinal membranes from three independent PVR patients. Yellow or orange fluorescence resulted from the overlay of green and red fluorescence, which indicates the co‐localization of KRT8 with LC3B. Nuclei were stained with DAPI and are represented with blue fluorescence. The upper panel shows the representative immunofluorescence staining of negative control using mouse and rabbit control IgG. Scale bar = 10 µm. B, Western blot analysis of p‐KRT8 in the retina from normal donor eye and subretinal and epiretinal membranes from two independent PVR patients. GAPDH levels were used as loading control

Figure 2

KRT8 phosphorylation and autophagic flux are increased during TGF‐β2–induced EMT in RPE cells. A, Western blot analysis of p‐KRT8, KRT8, autophagy hallmark proteins (LC3‐II, p62, Atg5‐12 and Beclin 1) and EMT markers (α‐SMA, fibronectin and collagen IV) in ARPE‐19 cells treated with TGF‐β2 (10 ng/mL) for the indicated hours. GAPDH levels were used as loading control. B, Western blot analysis of LC3‐II in ARPE‐19 cells treated with TGF‐β2 (10 ng/mL, 24 h) either in the presence or absence of the autophagy inhibitor Baf‐A1 (10 nmol/L). GAPDH was used as loading control. C, Bar graph shows the relative expression level of LC3‐II (normalized to GAPDH) in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. *P < .05 and ****P < .0001. D, The localization of GFP‐LC3B puncta in ARPE‐19 and human primary RPE cells treated with TGF‐β2 (10 ng/mL) for 24 h. Nuclei were stained with Hoechst33258 and are represented with blue fluorescence. Scale bar = 5 µm. E, Bar graph indicates the average number of LC3B puncta per cell obtained in fluorescence analysis. The data are presented as mean ± SEM, n = 20. ****P < .0001

Figure 3

Autophagy inhibitors attenuate TGF‐β2–induced cell migration and EMT markers synthesis. A, Wound healing assays of TGF‐β2 (10 ng/mL) stimulated ARPE‐19 cells co‐treatment with or without 3‐MA (10 mmol/L). Phase‐contrast microphotographs (4 × objective) were acquired at 0, 24 and 48 h after scratching. B, Graph shows the percentage of wound healing area relative to 0 h. The data are presented as mean ± SEM, n = three independent experiments. ****P < .0001. C, D, Western blot analysis of α‐SMA, fibronectin and collagen IV in ARPE‐19 cells treated with TGF‐β2 (10 ng/mL) in the absence or presence of either 3‐MA (10 mmol/L) or Baf‐A1 (10 nmol/L) for 24 h. GAPDH was used as loading control. E, F, Bar graphs indicate the relative expression levels of α‐SMA, fibronectin and collagen IV (normalized to GAPDH) in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. *P < .05, **P < .01 and ****P < .0001

Figure 4

Knockdown of ATG5 or Beclin 1 attenuates enhanced cell migration and EMT markers synthesis induced by TGF‐β2. A, B, Wound healing assays were performed on ATG5‐ and Beclin 1‐depleted ARPE‐19 cells (si‐ATG5 and si‐BECN1, respectively) and on control cells (NC siRNA) with or without TGF‐β2 (10 ng/mL) treatment. Phase‐contrast microphotographs (4 × objective) were obtained at 0, 24 and 48 h after scratching. C, D, Graph shows the relative wound healing area normalized to 0 h. The data are presented as mean ± SEM, n = three independent experiments. ****P < .0001. E, F, Western blot analysis of α‐SMA, fibronectin, collagen IV, Atg5‐12 and Beclin 1 in NC siRNA, si‐ATG5 or si‐BECN1 transfected ARPE‐19 cells with or without TGF‐β2 (10 ng/mL) treatment for 24 h. GAPDH was used as loading control. G, H, Bar graphs indicate the relative expression level of each protein (normalized to GAPDH) in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. *P < .05, **P < .01 and ***P < .001

Figure 5

KRT8 phosphorylation is correlated with autophagy during TGF‐β2 mediated EMT in RPE cells. A, Western blot analysis of p‐KRT8, KRT8, LC3‐II and p62 present in ARPE‐19 cells exposed to TGF‐β2 (10 ng/mL) in the absence or presence of either 3‐MA (10 mmol/L) or Baf‐A1 (10 nmol/L) for 24 h. GAPDH was used as loading control. B, Bar graph shows the p‐KRT8:KRT8 ratio in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. **P < .01 and ****P < .0001. C, Representative fluorescence microscopy images show the expression of p‐KRT8 (red fluorescence) in ARPE‐19 and human primary RPE cells. Cells were exposed to TGF‐β2 (10 ng/mL) in the absence or presence of either 3‐MA (10 mmol/L) or Baf‐A1 (10 nmol/L) for 24 h. Nuclei were stained with DAPI and are represented with blue fluorescence. Scale bar = 10 µm. D, Western blot analysis of p‐KRT8, KRT8 and Atg5‐12 in NC siRNA or si‐ATG5 transfected ARPE‐19 cells treated with or without TGF‐β2 (10 ng/mL) for 24 h. GAPDH was used as loading control. E, Bar graph shows the p‐KRT8:KRT8 ratio in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. *P < .05 and **P < .01

Figure 6

Phosphorylated KRT8 is critical to autophagosome‐lysosome fusion in RPE cells. A, Western blot analysis of p‐KRT8, KRT8, LC3‐II, p62 and EMT markers (α‐SMA, fibronectin, collagen IV) in NC siRNA or si‐KRT8 transfected ARPE‐19 cells treated with or without TGF‐β2 (10 ng/mL) for 24 h. GAPDH was used as loading control. B, Bar graphs indicate the relative expression of each protein (normalized to GAPDH) in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. NS represents no significance, *P < .05, **P < .01 and ***P < .001. C, Western blot analysis of p‐KRT8, KRT8, LC3‐II and p62 in ARPE‐19 cells transfected with empty vector, wild‐type KRT8 or KRT8‐S74A mutant with or without TGF‐β2 (10 ng/mL, 24 h) treatment. GAPDH was used as loading control. D, Bar graphs show the relative expression of each protein (normalized to GAPDH) in Western blot analysis. The data are presented as mean ± SEM, n = three independent experiments. NS represents no significance, *P < .05, **P < .01, ***P < .001 and ****P < .0001. E, Representative fluorescence microscopy images of autophagosomes (yellow puncta) and autolysosomes (red puncta) in the Ad‐mRFP‐GFP‐LC3 infected ARPE‐19 cells transfected with empty vector, wild‐type KRT8 or KRT8‐S74A mutant with or without TGF‐β2 (10 ng/mL, 24 h) stimulation. Scale bar = 10 µm. F, Bar graph indicates the average number of autophagosomes and autolysosomes in each cell obtained in fluorescence analysis. The data are presented as mean ± SEM, n = three independent experiments. **P < .01. G, Representative fluorescence microscopy images of LC3B‐labelled autophagosomes (red fluorescence) and LAMP2‐labelled lysosomes (green fluorescence) in ARPE‐19 cells transfected with empty vector, wild‐type KRT8 or KRT8‐S74A mutant with or without TGF‐β2 (10 ng/mL, 24 h) treatment. Scale bar = 10 µm. H, Bar graph indicates the average number of autophagosomes and autolysosomes in each cell obtained in fluorescence analysis. The data are presented as mean ± SEM, n = three independent experiments. ***P < .001