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TNF? alters occludin and cerebral endothelial permeability: Role of p38MAPK.

ABSTRACT: Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNF? (10 ng/mL), IL-1? (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNF? resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1? produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNF? also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNF? was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNF? and IL-1? but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNF? also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNF? -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNF? on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNF?-induced alterations on cell morphology and permeability.

SUBMITTER: Ni Y

PROVIDER: S-EPMC5295672 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.

Ni Yawen Y Teng Tao T Li Runting R Simonyi Agnes A Sun Grace Y GY Lee James C JC

PloS one 20170207 2

Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immort ...[more]

PMID: 28170408

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Project description:BackgroundThe major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated.MethodsWe assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus.ResultsRecombinant human TNF-? increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-?. Human TNF-? did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-?. Human TNF-? decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-?-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-?, and suppressed TNF-?-augmented cytotoxicity of PIECs.ConclusionOur data suggest that human TNF-? increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-? is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.

Dataset Information

TNF? alters occludin and cerebral endothelial permeability: Role of p38MAPK.

Publications

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.

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