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The influence of actin depolymerization induced by Cytochalasin D and mechanical stretch on interleukin-8 expression and JNK phosphorylation levels in human retinal pigment epithelial cells

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ABSTRACT

Background: 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.

Methods: A 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.

Results: The 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.

Conclusions: This 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.

No MeSH data available.


Related in: MedlinePlus

Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments
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Fig1: Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments

Mentions: 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. The cells in the control groups displayed abundant and uniform phalloidin staining (Fig. 1a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (P<0.05; n = 3) (Fig. 1f, Fig. 1m ). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (Fig. 1g-l, Fig. 1m).Fig. 1


The influence of actin depolymerization induced by Cytochalasin D and mechanical stretch on interleukin-8 expression and JNK phosphorylation levels in human retinal pigment epithelial cells
Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384187&req=5

Fig1: Fluorescence confocal microscopic analysis of the actin cytoskeleton in the cultured human ARPE-19 cells. The cells were subjected to mechanical stretch (20%, 0.33 Hz) for 0, 6, or 24 h with or without pre-treatment with 2 μM Cytochalasin D. The structure of the actin cytoskeleton was examined with phalloidin-Rhodamine staining. The cells in the control groups displayed abundant and uniform phalloidin staining (a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (d-f, m). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (g-l, m). *P<0.05 versus baseline, n = 3 experiments
Mentions: 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. The cells in the control groups displayed abundant and uniform phalloidin staining (Fig. 1a-c). After exposure to mechanical stretch for 24 h, the phalloidin-stained actin cytoskeleton appeared unclear and irregular with a statistically significant decrease in the average fluorescence intensity of F-actin fibres (P<0.05; n = 3) (Fig. 1f, Fig. 1m ). In all the Cytochalasin D-treated cells, the disruption of the cytoskeletal structure and a decrease in the average fluorescence intensity were observed regardless of mechanical stress (Fig. 1g-l, Fig. 1m).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Methods: A Flexcell FX-5000 Tension system was used to apply cyclic stretch to cultured human RPE cells (ARPE-19) at 0.33&nbsp;Hz with 20% elongation for 0&nbsp;h, 6&nbsp;h or 24&nbsp;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.

Results: The cells in the control groups displayed abundant and uniform phalloidin staining. After exposure to mechanical stretch for 24&nbsp;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&nbsp;h but were significantly increased by approximately 1.2-fold (1.18&nbsp;&plusmn;&nbsp;0.05; P&lt;0.01) and 3.0-fold (3.01&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 24&nbsp;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&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) and 1.3-fold (1.31&nbsp;&plusmn;&nbsp;0.02; P&lt;0.01) at 6&nbsp;h, respectively, and by 1.7-fold (1.69&nbsp;&plusmn;&nbsp;0.06; P&lt;0.01) and 3.2-fold (3.21&nbsp;&plusmn;&nbsp;0.12; P&lt;0.01) at 24&nbsp;h, respectively.

Conclusions: This 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.

No MeSH data available.


Related in: MedlinePlus