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TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.

Aveleira CA, Lin CM, Abcouwer SF, Ambrósio AF, Antonetti DA - Diabetes (2010)

Bottom Line: TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins.Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability.These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Research in Light and Image, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. caveleira@ibili.uc.pt

ABSTRACT

Objective: Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated.

Research design and methods: Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization.

Results: IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas.

Conclusions: These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

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Related in: MedlinePlus

TNF-α alters tight junction proteins content and cell localization. BRECs were treated with 5 ng/ml TNF-α for 0.5 and 6 h. Whole-cell extracts were assayed for ZO-1 (A), claudin-5 (B), and occludin (C) immunoreactivity by Western blotting. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least five independent experiments and are expressed as the relative amount compared with control (Ctrl). *P < 0.05, **P < 0.01, significantly different from control as determined by ANOVA followed by Dunnett post hoc test. Total RNA was isolated after 6 h of TNF-α treatment, and the transcript levels of ZO-1 (D), claudin-5 (E), and occludin (F) were analyzed by qPCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Results represent the mean ± SEM of eight independent experiments and are expressed as the relative amount compared with control conditions. *P < 0.05, ***P < 0.001, significantly different from control as determined by Student t test. G: Cells were immunolabeled for ZO-1, claudin-5, and occludin 6 h after TNF-α treatment, and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. Arrows indicate continuous staining at cell borders. These results are representative of four independent experiments. Scale bar, 25 μm.
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Figure 2: TNF-α alters tight junction proteins content and cell localization. BRECs were treated with 5 ng/ml TNF-α for 0.5 and 6 h. Whole-cell extracts were assayed for ZO-1 (A), claudin-5 (B), and occludin (C) immunoreactivity by Western blotting. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least five independent experiments and are expressed as the relative amount compared with control (Ctrl). *P < 0.05, **P < 0.01, significantly different from control as determined by ANOVA followed by Dunnett post hoc test. Total RNA was isolated after 6 h of TNF-α treatment, and the transcript levels of ZO-1 (D), claudin-5 (E), and occludin (F) were analyzed by qPCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Results represent the mean ± SEM of eight independent experiments and are expressed as the relative amount compared with control conditions. *P < 0.05, ***P < 0.001, significantly different from control as determined by Student t test. G: Cells were immunolabeled for ZO-1, claudin-5, and occludin 6 h after TNF-α treatment, and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. Arrows indicate continuous staining at cell borders. These results are representative of four independent experiments. Scale bar, 25 μm.

Mentions: To determine the effect of TNF-α on expression of specific tight junction proteins, BRECs were exposed to 5 ng/ml TNF-α for 0.5 and 6 h and the protein contents of ZO-1, claudin-5, and occludin were determined by Western blotting. TNF-α significantly decreased ZO-1 content (58.2 ± 6.5% of control) after 6 h of exposure (Fig. 2A). Claudin-5 content was rapidly reduced after 0.5 h of treatment (70.8 ± 7.0% of control, Fig. 2B), and 6 h of TNF-α exposure further downregulated claudin-5 content (57.6 ± 8.7% of control). In contrast, TNF-α increased occludin content (130.6 ± 6.6% of control; Fig. 2C). To determine whether alterations in protein content were due to changes in mRNA expression, total mRNA content was evaluated by qPCR 6 h after TNF-α exposure. TNF-α significantly decreased ZO-1 (74.6 ± 3.3% of control; Fig. 2D) and claudin-5 mRNA content (80.9 ± 5.7% of control; Fig. 2E) but induced a twofold increase in occludin mRNA (Fig. 2F). To investigate whether TNF-α alters the tight junction complex at the cell membrane, the cellular localization of the tight junction proteins were evaluated by immunocytochemistry and confocal microscopy. In control conditions, ZO-1, claudin-5, and occludin immunoreactivity appeared as a near continuous staining at the cell border (Fig. 2G and supplementary Fig. 1, available in an online appendix). Upon TNF-α treatment, a loss of both ZO-1 and claudin-5 immunostaining was observed, leading to a fragmented border staining, although the effect on ZO-1 was more pronounced. Also, a diffuse cytoplasmic distribution of claudin-5 and occludin was observed in TNF-α–treated cells. After TNF-α treatment, occludin staining increased, and it was irregularly distributed at the cell border (Fig. 2G and supplementary Fig. 1).


TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability.

Aveleira CA, Lin CM, Abcouwer SF, Ambrósio AF, Antonetti DA - Diabetes (2010)

TNF-α alters tight junction proteins content and cell localization. BRECs were treated with 5 ng/ml TNF-α for 0.5 and 6 h. Whole-cell extracts were assayed for ZO-1 (A), claudin-5 (B), and occludin (C) immunoreactivity by Western blotting. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least five independent experiments and are expressed as the relative amount compared with control (Ctrl). *P < 0.05, **P < 0.01, significantly different from control as determined by ANOVA followed by Dunnett post hoc test. Total RNA was isolated after 6 h of TNF-α treatment, and the transcript levels of ZO-1 (D), claudin-5 (E), and occludin (F) were analyzed by qPCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Results represent the mean ± SEM of eight independent experiments and are expressed as the relative amount compared with control conditions. *P < 0.05, ***P < 0.001, significantly different from control as determined by Student t test. G: Cells were immunolabeled for ZO-1, claudin-5, and occludin 6 h after TNF-α treatment, and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. Arrows indicate continuous staining at cell borders. These results are representative of four independent experiments. Scale bar, 25 μm.
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Figure 2: TNF-α alters tight junction proteins content and cell localization. BRECs were treated with 5 ng/ml TNF-α for 0.5 and 6 h. Whole-cell extracts were assayed for ZO-1 (A), claudin-5 (B), and occludin (C) immunoreactivity by Western blotting. Representative Western blots for each tight junction protein and β-actin (loading control) are presented above each respective graph. The results are normalized to β-actin and represent the mean ± SEM of at least five independent experiments and are expressed as the relative amount compared with control (Ctrl). *P < 0.05, **P < 0.01, significantly different from control as determined by ANOVA followed by Dunnett post hoc test. Total RNA was isolated after 6 h of TNF-α treatment, and the transcript levels of ZO-1 (D), claudin-5 (E), and occludin (F) were analyzed by qPCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Results represent the mean ± SEM of eight independent experiments and are expressed as the relative amount compared with control conditions. *P < 0.05, ***P < 0.001, significantly different from control as determined by Student t test. G: Cells were immunolabeled for ZO-1, claudin-5, and occludin 6 h after TNF-α treatment, and 10 confocal Z-stacks were taken through 2.56 μm and projected into one image. Arrows indicate continuous staining at cell borders. These results are representative of four independent experiments. Scale bar, 25 μm.
Mentions: To determine the effect of TNF-α on expression of specific tight junction proteins, BRECs were exposed to 5 ng/ml TNF-α for 0.5 and 6 h and the protein contents of ZO-1, claudin-5, and occludin were determined by Western blotting. TNF-α significantly decreased ZO-1 content (58.2 ± 6.5% of control) after 6 h of exposure (Fig. 2A). Claudin-5 content was rapidly reduced after 0.5 h of treatment (70.8 ± 7.0% of control, Fig. 2B), and 6 h of TNF-α exposure further downregulated claudin-5 content (57.6 ± 8.7% of control). In contrast, TNF-α increased occludin content (130.6 ± 6.6% of control; Fig. 2C). To determine whether alterations in protein content were due to changes in mRNA expression, total mRNA content was evaluated by qPCR 6 h after TNF-α exposure. TNF-α significantly decreased ZO-1 (74.6 ± 3.3% of control; Fig. 2D) and claudin-5 mRNA content (80.9 ± 5.7% of control; Fig. 2E) but induced a twofold increase in occludin mRNA (Fig. 2F). To investigate whether TNF-α alters the tight junction complex at the cell membrane, the cellular localization of the tight junction proteins were evaluated by immunocytochemistry and confocal microscopy. In control conditions, ZO-1, claudin-5, and occludin immunoreactivity appeared as a near continuous staining at the cell border (Fig. 2G and supplementary Fig. 1, available in an online appendix). Upon TNF-α treatment, a loss of both ZO-1 and claudin-5 immunostaining was observed, leading to a fragmented border staining, although the effect on ZO-1 was more pronounced. Also, a diffuse cytoplasmic distribution of claudin-5 and occludin was observed in TNF-α–treated cells. After TNF-α treatment, occludin staining increased, and it was irregularly distributed at the cell border (Fig. 2G and supplementary Fig. 1).

Bottom Line: TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins.Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability.These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Research in Light and Image, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. caveleira@ibili.uc.pt

ABSTRACT

Objective: Tumor necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1β) are elevated in the vitreous of diabetic patients and in retinas of diabetic rats associated with increased retinal vascular permeability. However, the molecular mechanisms underlying retinal vascular permeability induced by these cytokines are poorly understood. In this study, the effects of IL-1β and TNF-α on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-α increases cell permeability were elucidated.

Research design and methods: Cytokine-induced retinal vascular permeability was measured in bovine retinal endothelial cells (BRECs) and rat retinas. Western blotting, quantitative real-time PCR, and immunocytochemistry were performed to determine tight junction protein expression and localization.

Results: IL-1β and TNF-α increased BREC permeability, and TNF-α was more potent. TNF-α decreased the protein and mRNA content of the tight junction proteins ZO-1 and claudin-5 and altered the cellular localization of these tight junction proteins. Dexamethasone prevented TNF-α-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-κB) transrepression. Preventing NF-κB activation with an inhibitor κB kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor κB alpha (IκBα) reduced TNF-α-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKCζ) using both a peptide and a novel chemical inhibitor reduced NF-κB activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-α in cell culture and rat retinas.

Conclusions: These results suggest that PKCζ may provide a specific therapeutic target for the prevention of vascular permeability in retinal diseases characterized by elevated TNF-α, including diabetic retinopathy.

Show MeSH
Related in: MedlinePlus