TNF-α Signals Through PKCζ/NF-κB to Alter the Tight Junction Complex and Increase Retinal Endothelial Cell Permeability
- Célia A. Aveleira1,2,
- Cheng-Mao Lin2,
- Steven F. Abcouwer2,3,4,
- António F. Ambrósio1,5 and
- David A. Antonetti2,4
- 1Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Research in Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal;
- 2Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, Pennsylvania;
- 3Department of Surgery, Penn State Milton S. Hershey Medical Center, Hershey, Pennsylvania;
- 4Department of Ophthalmology, Penn State Hershey Eye Center, Hershey, Pennsylvania;
- 5Centre for Neuroscience and Cell Biology, Department of Zoology, University of Coimbra, Coimbra, Portugal.
- Corresponding author: Célia A. Aveleira, .
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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- Received October 30, 2009.
- Accepted July 28, 2010.
- © 2010 by the American Diabetes Association.
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