Dataset Information

TNF-? signals through PKC?/NF-?B to alter the tight junction complex and increase retinal endothelial cell permeability.

ABSTRACT:

Objective

Tumor necrosis factor-? (TNF-?) and interleukin-1 beta (IL-1?) 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-1? and TNF-? on retinal endothelial cell permeability were compared and the molecular mechanisms by which TNF-? 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-1? and TNF-? increased BREC permeability, and TNF-? was more potent. TNF-? 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-?-induced cell permeability through glucocorticoid receptor transactivation and nuclear factor-kappaB (NF-?B) transrepression. Preventing NF-?B activation with an inhibitor ?B kinase (IKK) chemical inhibitor or adenoviral overexpression of inhibitor ?B alpha (I?B?) reduced TNF-?-stimulated permeability. Finally, inhibiting protein kinase C zeta (PKC?) using both a peptide and a novel chemical inhibitor reduced NF-?B activation and completely prevented the alterations in the tight junction complex and cell permeability induced by TNF-? in cell culture and rat retinas.

Conclusions

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

SUBMITTER: Aveleira CA

PROVIDER: S-EPMC2963546 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Aveleira Célia A CA Lin Cheng-Mao CM Abcouwer Steven F SF Ambrósio António F AF Antonetti David A DA

Diabetes 20100806 11

<h4>Objective</h4>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 ...[more]

PMID: 20693346

Similar Datasets

Project description:How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE).A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudins were determined by real-time polymerase chain reaction, immunofluorescence, and Western blot analysis. Activation of ERK1/2 was detected by Western blot analysis with specific antiphospho protein antibodies. NF-kappaB DNA binding activity was determined by transcription factor assay. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in barrier destruction of RPE cells exposed to HIV-1 Tat.Treating cultured human retinal pigment epithelial cells with 100 nM Tat for 24 hours increased the permeability and decreased the TER of the epithelial monolayer. HIV-1 Tat also disrupted and downregulated the tight-junction proteins claudin-1, claudin-3, and claudin-4 in these cells, whereas claudin-2 was upregulated, and the expression of occludin was unaffected. HIV-1 Tat protein also induced activation of ERK1/2 and NF-kappaB. HIV-1 Tat protein induced barrier destruction, changes in expression of TJs, and activation of ERK1/2 and NF-kappaB were abrogated by inhibitor of ERK1/2 and NF-kappaB.HIV-1 Tat protein causes increases in the paracellular permeability of RPE cells in vitro concomitant with changes in expression of certain transmembrane proteins associated with the tight junction. The effects of HIV-1 Tat on barrier function of the RPE may be mediated by ERK MAPK and NF-kappaB activation, which may represent potential targets for novel therapeutic approaches for the retinopathy induced by HIV infection.