Abstract:
Objective To explore the role of c-jun N-terminal kinase (JNK)/activated protein-1 (AP-1) signaling pathway in paraquat (PQ)-induced epithelial-mesenchymal transition (EMT) of rat type Ⅱ alveolar epithelial cells.
Methods RLE-6TN cells were incubated with designed concentrations of PQ (0, 25, 50, 100, 200, 300, and 400 μmol/L) for 24 h or 200 μmol/L PQ for 0, 12, 24, 36, 48, 60, and 72 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined by MTT assay to evaluate dose/time-effect relationship. Cell apoptosis was detected by Annexin V-FITC/PI. The migration and invasion abilities of cells were detected by wound healing and Transwell assays. The protein expressions of epithelial phenotype markers (E-cad and ZO-1) and mesenchymal phenotype markers (FSP-1 and α-SMA) were determined by immunofluorescence and Western blot respectively. The protein expressions of JNK/p-JNK, c-jun/p-c-jun, c-fos/p-c-fos were detected by Western blot. The activity of AP-1 was measured by luciferase reporter assay to evaluate the activation of JNK signaling pathway by PQ.
Results An elongated shape and a lack of tight cell-cell adhesions of mesenchymal cells were induced by 200 μmol/L PQ treatment in a time-dependent manner. The cell viability decreased in the cells treated with PQ at 100, 200, 300, and 400 μmol/L (P<0.05) or with 200 μmol/L PQ for 24, 36, 48, 60, and 72 h (P<0.05). After 200 μmol/L PQ treatment for 12, 24, and 36 h, the percentage of apoptosis cells increased with more exposure time, especially in the 36h treatment group as compared with the control group (P<0.05); cell migration index and invasive cells increased; the protein expressions of epithelial phenotype markers (E-cad and ZO-1) significantly decreased while those of mesenchymal phenotype markers (α-SMA and FSP-1) dramatically increased in a time-dependent manner after PQ exposure (P<0.05). Compared with the control group, after exposure to PQ (100, 200, and 300 μmol/L) for 24 h, the protein expressions of p-JNK, p-c-jun, and p-c-fos significantly increased (P<0.05). The PQ treatment at 200 and 300 μmol/L induced a significant increase of AP-1 activity (P<0.05).
Conclusion PQ affects cell viability of epithelial cells in a dose-and time-dependent manner. PQ can induce EMT in rat alveolar epithelial cells, in which JNK/AP-1 signaling pathway is involved.
下载:
