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

Yawen Ni,Agnes Simonyi,James C Lee,Runting Li,Tao Teng,Grace Y Sun,Donghui Zhu

doi:10.1371/journal.pone.0170346

Yawen Ni, Agnes Simonyi + Show 5 more

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https://doi.org/10.1371/journal.pone.0170346

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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 TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β 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 TNFα 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 TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.

Highlights

Blood–brain barrier (BBB) is a highly selective permeability barrier for protecting the brain from harmful substances circulating in the bloodstream [1]
Since tight junction (TJ) are important for endothelial cell functions, the goal for this study is to investigate whether cytokines and endotoxins alter occludin expression and function in the hCMEC/D3 cells
In subsequent experiments, 7.5% gel was used for blotting occludin and ERK1/2, and 10% gel was used for blotting p38MAPK

Summary

Introduction

Blood–brain barrier (BBB) is a highly selective permeability barrier for protecting the brain from harmful substances circulating in the bloodstream [1]. ECs are unique as they have continuous intercellular Tight Junction proteins (TJs). TNFα on occludin in endothelial cells and ability to resist immune cells to pass through the BBB and enter into the central nervous system [2]. Many neurodegenerative diseases, such as multiple sclerosis, epilepsy, Alzheimer’s disease, or diabetes, show abnormality of TJs function. Occludin is a major component of the TJ, and is a transmembrane protein present in the plasma membranes of ECs. Its extracellular domains can join one another directly. Immunoblotting and immunocytochemistry show distribution of occludin continuously at cell-cell contacts in brain ECs [3]

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