Promotive effect of the TGF-β/WNT5a/JNK signaling pathway on the epithelial-mesenchymal transition in human lens epithelial cells
10.3760/cma.j.cn115989-20230425-00147
- VernacularTitle:TGF-β/WNT5a/JNK信号通路对人晶状体上皮细胞上皮－间充质转化的促进作用
- Author:
Yu SONG
1
;
Xiuli BAO
Author Information
1. 内蒙古医科大学第一临床医学院，呼和浩特 010050
- Publication Type:Journal Article
- Keywords:
Lens epithelial cells;
c-Jun N-terminal kinase;
Transforming growth factor-β;
Wingless 5a;
Epithelial mesenchymal transition;
Fibrosis
- From:
Chinese Journal of Experimental Ophthalmology
2025;43(3):219-226
- CountryChina
- Language:Chinese
-
Abstract:
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 ( Ftime=71.599, P<0.001) and among different groups ( Fgroup=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.
