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. 2019 Sep;61(7-8):410-418.
doi: 10.1111/dgd.12630. Epub 2019 Oct 13.

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Ruihua Jing  1 , Tiantian Qi  1 , Chan Wen  1 , Jiaqi Yue  1 , Guangyan Wang  1 , Cheng Pei  1 , Bo Ma  1
Affiliations

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Ruihua Jing et al. Dev Growth Differ. 2019 Sep.

Abstract

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.

Keywords: Interleukin 2; age-related macular degeneration; epithelial-mesenchymal transition; extracellular matrix synthesis; retinal pigment epithelial cells.

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Conflict of interest statement

All of the authors declare that there is no interest.

Figures

Figure 1
Figure 1
IL‐2 promoted RPE cells migration. (a) RPE cells were treated by 10 μg/L IL‐2 when cells were in 70% confluence after the insert was removed in serum‐free medium. Wound healing at 0, 24 and 48 hr. (b) The wound area was measured and analyzed with ImageJ software and there was a significance in 48 hr between the control group and IL‐2 treated group (formula image, Control; formula image, IL‐2). (c) Transwell assay between control group and 10 μg/L IL‐2 treated group after cells were seeded in the Transwell chamber 24 hr later. (d) Migrated RPE cells in control group and IL‐2 group, respectively. **p < .01, ***p < .001. Scar bar: 100 μm
Figure 2
Figure 2
IL‐2 promoted RPE cells ECM synthesis and TGF‐β2 expression. (a) RPE cells were treated by 10 μg/L IL‐2 for 0, 12, 24, 36 and 48 hr, α‐SMA, COL‐1, Fn and TGF‐β2 protein expression were detected by Western blot. (b–e) Quantification of the Western blot analysis results. IL‐2 treatment increased COL‐1, Fn and TGF‐β2 protein expression but not α‐SMA time dependently. *p < .05, **p < .01
Figure 3
Figure 3
Both JAK/STAT3 and NF‐κB signalling pathway involved in ECM synthesis and TGF‐β protein expression. (a) RPE cells were treated by 10 μg/L IL‐2 for different duration (0, 0.5, 1, 2, 4, 6 and 8 hr) and p‐IκBα, IκBα, p‐STAT3 and STAT3 expression were detected by Western blot. (b–c) Quantification of the Western blot analysis of p‐STAT3/STAT3 and p‐IκBα/IκBα. (d) RPE cells were preteated by 4 μM WP1066 for 1 hr and then 10 μg/L IL‐2 was added in serum‐free medium. (e–i) Quantification of the Western blot analysis (formula image, Control; formula image, DMSO; formula image, IL‐2 10 μg/L; formula image, IL‐2 10 μg/L+WP1066; formula image, WP1066). (j) RPE cells were preteated by 10 μM BAY for 1 hr and then 10 μg/L IL‐2 was added in serum‐free medium. (k–o) Quantification of the Western blot analysis (formula image, Control; formula image, DMSO; formula image, IL‐2 10 μg/L; formula image, IL‐2 10 μg/L+BAY; formula image, BAY). *p < .05, **p < .01
Figure 4
Figure 4
Both inhibition of JAK/STAT3 and NF‐κB signalling pathway lower migration rate. (a) RPE cells were pre‐seeded in the culture insert. When cells were 70% confluence, the insert was removed and serum‐free medium was added. Before 10 μg/L IL‐2 were loaded, WP1066 or BAY has been added into the medium for 1 hr. (b) Transwell assay of 10 μg/L IL‐2 and IL‐2 with 4 μM WP1066 or 10 μM BAY preteated for 1 hr. (c) The wound area was measured and analyzed with Image J software and there was a significance in 48 hr between the IL‐2 and IL‐2 with WP1066 or BAY group (formula image, DMSO; formula image, IL‐2; formula image, IL‐2+WP1066; formula image, IL‐2+BAY). (d) Migrated RPE cells in DMSO group and IL‐2 with P1066 or BAY group respectively. **p < .01, ***p < .001. Scar bar: 100 μm
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