Changes in transepithelial electrical resistance and intracellular ion concentration in TGF-β-induced epithelial-mesenchymal transition of retinal pigment epithelial cells

Abstract

Objective: This study aimed to investigate the changes in transepithelial electrical resistance (TEER) and ion concentrations, and their relationship in TGF-β-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells.

Methods: RPE cell line ARPE-19 was employed and treated with 10 ng/ml TGF-β1 and TGF-β2 to establish the EMT model in vitro. The EMT markers fibronectin, N-cadherin, occludin, zona occludens 1(ZO-1) and claudin-19 were investigated by western blot and immunofluorescence. CellZscope system was used to monitor the TEER values. Fluorescent probe, flow cytometry and automatic microplate reader were employed to detect the changes of Ca²⁺, Mg²⁺, Zn²⁺, Na⁺ and K⁺ in ARPE-19 cells.

Results: The TGF-β1-induced EMT of ARPE-19 cells was marked by the disruption of the distribution of occludin, ZO-1, and claudin-19. The development of TEER was significantly disturbed in both TGF-β1 and TGF-β2 treatment groups. Also, the time course of the maximum slope indicated that the fastest decrease in TEER values occurred after 36 hours. The concentrations of Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ increased in TGF-β1- and TGF-β2-treated ARPE-19 cells, while the concentration of Na⁺ decreased. Significant inverse correlations were detected between the concentrations of Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ and TEER values in ARPE-19 cells treated with TGF-β1. The Na⁺ concentration and TEER values showed a positive correlation. Similar results were observed in the TGF-β2 treatment group. The time-effect analysis showed that the concentrations of Ca²⁺, Mg²⁺, Zn²⁺ and K⁺ increased and peaked after 72, 72, 48, and 72 h, respectively, with the extension of TGF-β1 treatment time. In the TGF-β2 treatment group, the Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ concentrations were also upregulated and reached their highest after 72, 72, 72, and 36 h, respectively. In contrast, the concentration of Na⁺ decreased and reached the lowest after 48 h in the TGF-β1 treatment group and after 72 h in the TGF-β2 treatment group.

Conclusion: TGF-β1 and TGF-β2 disrupted the ARPE-19 cell monolayer, disturbed TJs integrity, downregulated TEER values, and changed intracellular ion permeability. These findings might help further understand the EMT of RPE cells during PVR.

Keywords: Epithelial-mesenchymal transition; ion; retinal pigment epithelial cell; tight junction proteins; transepithelial electrical resistance.

Figure 1

TGF-β1 destroyed the distribution of TJ proteins in ARPE-19 cells. The distribution of TJ proteins occludin, ZO-1, and claudin-19 were detected by immunofluorescence. Representative images of occludin (green), ZO-1 (green), and claudin-19 (green) expression in TGF-β1-treated ARPE-19 cells after 36 h and normal control. DAPI (blue) was used as a nuclear marker. Magnification, 40 ×. Scale bar, 20 μm.

Figure 2

Measurement of TEER. A. Schematic structure and measurement method of the CellZscope. B. When three groups (2.5 × 10⁴ cells/ mL, 5.0 × 10⁴ cells/ mL, and 1.0 × 10⁵ cells/mL) of ARPE-19 cells were cultured for 2 weeks, the CellZscope was used to record TEER, C_CL and Rmed values. C. TEER values in the three groups (2.5 × 10⁴ cells/mL, 5.0 × 10⁴ cells/mL and 1.0 × 10⁵ cells/mL) decreased using TGF-β1 and TGF-β2.

Figure 3

Intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, Na⁺, and K⁺ were determined using fluorescent probes by flow cytometry and automatic microplate reader. A-J. Measurement of intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, and Na⁺ using the fluorescent probe by flow cytometry. Left, representative flow cytometric histogram of fluorescent levels. Right, quantification of intracellular ions levels from three independent experiments. I and J. Quantification of the K⁺ concentrations in TGF-β1-treated and TGF-β2-treated groups using an automatic microplate reader after fluorescent probe staining. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Correlation analysis between intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, Na⁺ and TEER values. A-E. Correlation analysis between intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, Na⁺ and TEER changes in the TGF-β1 group. F-J. Correlation analysis between intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, Na⁺ and TEER values in the TGF-β2 group.

Figure 5

Time-effect analysis of intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, and Na⁺ after treatment with TGF-β1 and TGF-β2. A-E. Time-effect analysis of intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, and Na⁺ in the TGF-β1 group. F-J. Time-effect analysis between intracellular concentrations of Ca²⁺, Mg²⁺, Zn²⁺, K⁺, and Na⁺ in the TGF-β2 group.