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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

IL-8 expression and JNK phosphorylation levels in RPE cells. After cyclic stretch and Cytochalasin D exposure, cell lysates were prepared, and Western blotting analysis was performed on the indicated proteins. The IL-8 and JNK bands were scanned, and band densities were calculated using ImageJ software. The values are the band-density ratios compared with the baseline. 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 at 24 h. 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 at 6 h and 24 h (a-c). *P<0.01 versus baseline, n = 3 experiments
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Fig2: IL-8 expression and JNK phosphorylation levels in RPE cells. After cyclic stretch and Cytochalasin D exposure, cell lysates were prepared, and Western blotting analysis was performed on the indicated proteins. The IL-8 and JNK bands were scanned, and band densities were calculated using ImageJ software. The values are the band-density ratios compared with the baseline. 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 at 24 h. 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 at 6 h and 24 h (a-c). *P<0.01 versus baseline, n = 3 experiments

Mentions: Cyclic stretch alone did not induce a significant increase in IL-8 expression. Treatment with Cytochalasin D alone increased IL-8 expression by approximately 1.2-fold (1.18 ± 0.05; P<0.01; n = 3) at 24 h. The pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch increased IL-8 expression by approximately 1.3-fold (1.31 ± 0.02; P<0.01; n = 3) at 6 h and 1.7- fold (1.69 ± 0.06; P<0.01; n = 3) at 24 h (Fig. 2a, b).Fig. 2


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
IL-8 expression and JNK phosphorylation levels in RPE cells. After cyclic stretch and Cytochalasin D exposure, cell lysates were prepared, and Western blotting analysis was performed on the indicated proteins. The IL-8 and JNK bands were scanned, and band densities were calculated using ImageJ software. The values are the band-density ratios compared with the baseline. 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 at 24 h. 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 at 6 h and 24 h (a-c). *P<0.01 versus baseline, n = 3 experiments
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Related In: Results  -  Collection

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Fig2: IL-8 expression and JNK phosphorylation levels in RPE cells. After cyclic stretch and Cytochalasin D exposure, cell lysates were prepared, and Western blotting analysis was performed on the indicated proteins. The IL-8 and JNK bands were scanned, and band densities were calculated using ImageJ software. The values are the band-density ratios compared with the baseline. 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 at 24 h. 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 at 6 h and 24 h (a-c). *P<0.01 versus baseline, n = 3 experiments
Mentions: Cyclic stretch alone did not induce a significant increase in IL-8 expression. Treatment with Cytochalasin D alone increased IL-8 expression by approximately 1.2-fold (1.18 ± 0.05; P<0.01; n = 3) at 24 h. The pre-incubation of the RPE cells with Cytochalasin D followed by exposure to cyclic stretch increased IL-8 expression by approximately 1.3-fold (1.31 ± 0.02; P<0.01; n = 3) at 6 h and 1.7- fold (1.69 ± 0.06; P<0.01; n = 3) at 24 h (Fig. 2a, b).Fig. 2

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