Abstract
BackgroundThis study explores the role of actin cytoskeleton depolymerization induced by Cytochalasin D and mechanical stretch on the interleukin-8 (IL-8) expression and c-jun N-terminal kinase (JNK) phosphorylation levels in human retinal pigment epithelial (RPE) cells.MethodsA Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33 Hz with 20% elongation for 0 h, 6 h or 24 h. The cells were stretched alone or pre-treated with Cytochalasin D. The redistribution of the actin cytoskeleton was evaluated using phalloidin immunofluorescence staining. The protein expression levels of IL-8 and JNK in the RPE cells were determined via Western blotting.ResultsThe cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24 h, phalloidin staining revealed an unclear and irregular actin cytoskeleton. In all Cytochalasin D-treated cells, the shrinkage and disruption of the cytoskeletal structure was observed regardless of mechanical stress. The stimulation of the RPE cells with cyclic stretch alone did not induce a significant increase in IL-8 expression and JNK phosphorylation levels, which were similar to those of the control groups. After pre-treatment with Cytochalasin D alone, IL-8 expression and JNK phosphorylation levels were not significantly different at 6 h but were significantly increased by approximately 1.2-fold (1.18 ± 0.05; P<0.01) and 3.0-fold (3.01 ± 0.02; P<0.01) at 24 h, respectively. After the pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch, IL-8 expression and JNK phosphorylation levels increased by approximately 1.3-fold (1.31 ± 0.02; P<0.01) and 1.3-fold (1.31 ± 0.02; P<0.01) at 6 h, respectively, and by 1.7-fold (1.69 ± 0.06; P<0.01) and 3.2-fold (3.21 ± 0.12; P<0.01) at 24 h, respectively.ConclusionsThis study demonstrates that disruption of actin polymerization by cytochalasin D and mechanical stretch upregulates interleukin-8 expression and JNK phosphorylation levels in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in the pro-inflammatory processes in RPE cells.
Highlights
This study explores the role of actin cytoskeleton depolymerization induced by Cytochalasin D and mechanical stretch on the interleukin-8 (IL-8) expression and c-jun N-terminal kinase (JNK) phosphorylation levels in human retinal pigment epithelial (RPE) cells
Reorganization of the actin cytoskeleton induced by cyclic stretch and Cytochalasin D The effect of mechanical stretch and Cytochalasin D on the actin cytoskeleton was assessed via fluorescence confocal microscopy using phalloidin-Rhodamine staining on ARPE-19 cells
In this study, we provide evidence that the disruption of actin polymerization by Cytochalasin D and mechanical stretch enhanced IL-8 expression and JNK phosphorylation in human RPE cells, which indicates that the integrity of the actin cytoskeleton may play important roles in pro-inflammatory processes in RPE cells
Summary
This study explores the role of actin cytoskeleton depolymerization induced by Cytochalasin D and mechanical stretch on the interleukin-8 (IL-8) expression and c-jun N-terminal kinase (JNK) phosphorylation levels in human retinal pigment epithelial (RPE) cells. Published data have demonstrated that RPE cells experience chronic mechanical stress transmitted from the vitreoretinal interface and the neural retina [8, 9]. This mechanical stress may upregulate the expression levels of several pathological factors in RPE cells, such as vascular endothelial growth factor and metalloproteinases [10, 11]. Little is known about the expression of interleukins in RPE cells under mechanical stress, and the precise underlying mechanisms remain unclear
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