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. 2020 Jan;21(1):173-180.
doi: 10.3892/mmr.2019.10814. Epub 2019 Nov 12.

Endoplasmic reticulum stress regulates epithelial‑mesenchymal transition in human lens epithelial cells

Sheng Zhou #  1 , Jing Yang #  1 , Mingwei Wang  1 , Danying Zheng  1 , Yizhi Liu  1
Affiliations

Endoplasmic reticulum stress regulates epithelial‑mesenchymal transition in human lens epithelial cells

Sheng Zhou et al. Mol Med Rep. 2020 Jan.

Abstract

Epithelial‑to‑mesenchymal transition (EMT) of human lens epithelial cells (HLECs) serve an important role in cataract formation. The endoplasmic reticulum stress response (ER stress) has been demonstrated to regulate EMT in a number of tissues. The aim of the present study was to demonstrate the role of ER stress on EMT in HLECs. HLECs were treated with tunicamycin (TM) or thapsigargin (TG) to disturb ER homeostasis, and 4‑phenylbutyric acid (PBA) or sodium tauroursodeoxycholate (TUDCA) to restore ER homeostasis. Cell morphology was evaluated after 24 h. The long axis and aspect ratio of the cells were analyzed using ImageJ software. The results demonstrated that HLECs adopted an elongated morphology following treatment with TG, and the cellular aspect ratio increased. However, this morphological change was not observed following combination treatment with TG and PBA. Western blot analysis and immunofluorescence staining were used to measure the protein expression levels. A wound‑healing assay was performed to evaluate cell migration. Treatment with TM or TG increased the expression of the ER stress markers glucose‑regulated protein 78, phosphorylated eukaryotic initiation factor 2α, activating transcription factor (ATF)6, ATF4 and inositol‑requiring protein 1α and the EMT markers fibronectin, vimentin, α‑smooth muscle actin and neural cadherin. Furthermore, treatment with TM or TG decreased the expression of the epithelial cell marker epithelial cadherin and enhanced cell migration, which effects were inhibited following treatment with PBA or TUDCA. These results indicates that enhanced ER stress induced EMT and subsequently increased cell migration in HLECs in vitro.

Keywords: human lens epithelial cells; endoplasmic reticulum stress response; epithelial-to-mesenchymal transition; unfolded protein response.

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Figures

Figure 1.
Figure 1.
SRA01/04 cells treated with endoplasmic reticulum stress inducers exhibit a morphological change, from an epithelial- to a fibroblast-like morphology. SRA01/04 cells were treated with 0.01 µM TG or a combination of 0.01 µM TG and 0.25 mM PBA for 24 h. Untreated SRA01/04 cells served as the control group. (A) Micrographs were obtained under an inverted phase-contrast microscope (magnification, ×20). The red arrows indicate that TG-treated cells had a spindle-like appearance and an elongated long axis. (B) The cellular aspect ratio was analyzed with ImageJ software (n=3). *P<0.05 vs. control. SRA01/04 cells were treated with 0.01 µM TG, 0.01 µM TG and 0.25 mM PBA, 0.01 µM TG and 2 mM TUDCA, 0.1 µM TM, 0.1 µM TM and 0.25 mM PBA or 0.1 µM TM and 2 mM TUDCA for 24 h. (C) ER stress markers were determined by western blot analysis. (D) The expression levels of GRP78, p-IRE1α, p-eIF2α, ATF4 and ATF6 were quantified by densitometry (n=3). *P<0.05 vs. control. TG, thapsigargin; TM, tunicamycin; PBA, 4-phenylbutyric acid; TUDCA, tauroursodeoxycholate; GRP78, glucose-regulated protein 78 kDa; P-IRE1α, phosphorylated inositol-requiring protein 1α; P-eIf2α, phosphorylated eukaryotic initiation factor 2α; ATF6, activating transcription factor 6; ATF4, activating transcription factor 4; DMSO, dimethyl sulfoxide; Con, control.
Figure 2.
Figure 2.
Enhanced endoplasmic reticulum stress upregulates EMT-associated protein expression and downregulates epithelial marker expression. (A) SRA01/04 cells were treated with 0.1 µM TM, 0.1 µM TM and 0.25 mM PBA or 0.1 µM TM and 2 mM TUDCA for 24 h. (B) SRA01/04 cells were treated with 0.01 µM TG, 0.01 µM TG and 0.25 mM PBA or 0.01 µM TG and 2 mM TUDCA for 24 h. The expression levels of the EMT-associated markers fibronectin, vimentin, α-SMA, N-cadherin and E-cadherin were measured by western blot analysis (n=3). *P<0.05 vs. control. EMT, epithelial-to-mesenchymal transition; TG, thapsigargin; TM, tunicamycin; PBA, 4-phenylbutyric acid; TUDCA, tauroursodeoxycholate; α-SMA, α-smooth muscle actin. N-cadherin, neural cadherin; E-cadherin, epithelial cadherin; DMSO, dimethyl sulfoxide; Con, control.
Figure 3.
Figure 3.
Immunofluorescence analysis of endoplasmic reticulum stress-induced expression of vimentin, N-cadherin, α-SMA and fibronectin in SRA01/04 cells. SRA01/04 cells were treated with 0.01 µM TG, 0.01 µM TG and 0.25 mM PBA, 0.01 µM TG and 2 mM TUDCA, 0.1 µM TM, 0.1 µM TM and 0.25 mM PBA or 0.1 µM TM and 2 mM TUDCA for 24 h. The expression of (B) vimentin, (D) N-cadherin, (F) α-SMA and (H) fibronectin was determined by immunofluorescence analysis. The average fluorescence of (A) vimentin, (C) N-cadherin, (E) α-SMA and (G) fibronectin was quantified (n=3). *P<0.05 vs. control, magnification, ×20. α-SMA, α-smooth muscle actin; N-cadherin, neural cadherin; E-cadherin; epithelial cadherin; TG, thapsigargin; TM, tunicamycin; PBA, 4-phenylbutyric acid; TUDCA, tauroursodeoxycholate.
Figure 4.
Figure 4.
Endoplasmic reticulum stress facilitates the cell migration of SRA01/04 cells. SRA01/04 cells were treated with 0.01 µM TG, 0.1 µM TM, 0.01 µM TG and 0.25 mM or 0.1 µM TM and 0.25 mM PBA for 24 h and were subsequently subjected to a wound healing assay. Untreated and dimethyl sulfoxide-treated SRA01/04 cells served as the control groups. (A) Cells that migrated into the wounded area from the border of the wound after 24 h were visualized and images were captured under an inverted phase-contrast microscope (magnification, ×10). (B) Cell migration was used to calculate the repair rate of scarification, expressed as the percentage of the gap relative to the total area of the cell-free region, using ImageJ software (n=3). *P<0.05 vs. control. TG, thapsigargin; TM, tunicamycin; PBA, 4-phenylbutyric acid; TUDCA, tauroursodeoxycholate; α-SMA, α-smooth muscle actin; DMSO, dimethyl sulfoxide; Con, control.
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