Promotive effect of the TGF-β/WNT5a/JNK signaling pathway on the epithelial-mesenchymal transition in human lens epithelial cells

Objective  To investigate the effects of the transforming growth factor-β/Wingless 5a/c-Jun N-terminal kinase (TGF-β/WNT5a/JNK) signaling pathway on fibrosis of lens epithelial cells (LECs).

Methods  The human LECs line SRA01/04 was divided into three groups, control group cultured with conventional medium, TGF-β group treated with TGF-β1 for 24 hours and WNT5a group treated with WNT5a for 24 hours.Western blot was performed to detect the relative protein expression levels of WNT5a, JNK, and phosphorylated JNK (p-JNK) in cells of the three groups.The SRA01/04 cell line was further divided into four groups, control group cultured with conventional medium, TGF-β group treated with TGF-β ₁ for 24 hours, TGF-β+ SP600125 group treated with TGF-β1 for 24 hours+ JNK inhibitor SP600125 for 2 hours, and WNT5a+ SP600125 group treated with WNT5a for 24 hours+ SP600125 for 2 hours.The relative expression of WNT5a, JNK, p-JNK, type Ⅰ collagen (Col-Ⅰ), fibronectin (FN), and α-smooth muscle actin (α-SMA) in cells of the four groups was detected by Western blot.The distribution of α-SMA in cells was determined by immunofluorescence staining.Cell migration was evaluated via Transwell assay, and Col-Ⅰ gel area ratio was measured at 8, 16, 24, and 48 hours of culture by gel contraction experiment.

Results  Western blot revealed that the relative protein expression levels of WNT5a, JNK, and p-JNK were significantly higher in the TGF-β and WNT5a groups than in the control group (all P<0.05).The expression levels of Col-Ⅰ, FN, and α-SMA were significantly higher in the TGF-β, TGF-β+ SP600125, and WNT5a+ SP600125 groups than in the control group and significantly lower in the TGF-β+ SP600125 and WNT5a+ SP600125 groups than in the TGF-β group (all P<0.05).Immunofluorescence staining showed that TGF-β-treated SRA01/04 cells transformed from columnar epithelial cells to spindle-shaped myofibroblasts in TGF-β group, whereas most cells in the TGF-β+ SP600125 and WNT5a+ SP600125 groups were still columnar epithelial cells.The relative fluorescence intensity of α-SMA was significantly higher in the TGF-β, TGF-β+ SP600125 and WNT5a+ SP600125 groups than in the control group, and the relative fluorescence intensity of α-SMA was significantly lower in the TGF-β+ SP600125 and WNT5a+ SP600125 groups than in the TGF-β group (all P<0.05).Transwell assay showed that there were more migrating cells in TGF-β group than in the control group, and the migrating cell count was lower in TGF-β+ SP600125 group and WNT5a+ SP600125 group than in the control group, with statistically significant differences (all P<0.05).Col-Ⅰ gel contraction experiment results showed that with the extension of culture time, the Col-Ⅰ gel area in each group decreased significantly, with significant overall comparison differences in the Col-Ⅰ gel area shrinkage ratios in each group at different time points ( F _time=71.599, P<0.001) and among different groups ( F _group=71.604, P<0.001).After 48 hours of culture, the Col-Ⅰ gel shrinkage ratios in TGF-β group, TGF-β+ SP600125 and WNT5a+ SP600125 groups were (26.24±0.28)%, (64.02±1.05)%, and (76.81±0.28)%, respectively, which were significantly lower than (90.20±0.31)% of the control group (all P<0.05).

Conclusions  The WNT5a/JNK signaling pathway, acting as a downstream target of the TGF-β signaling pathway, promotes epithelial-mesenchymal transition and extracellular matrix deposition in LECs, and enhances cell contractility.

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