Hyperosmolarity disrupts tight junction via TNF-α/MMP pathway in primary human corneal epithelial cells.

Abstract

To investigate the mechanism of the tight junction (TJ) disruption and the association between tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMPs) under hyperosmotic condition in primary human corneal epithelial cells (HCECs). The cultured HCECs were exposed to media which adding sodium chloride (NaCl) for hyperosmolar stress or adding rh-TNF-α (10 ng/mL). NF-κB inhibitor (5 µmol/L) or GM-6001 (potent and broad spectrum MMP inhibitor, 20 µmol/L) was added 1h before that treatment. The integrity of TJ proteins was determined by immunofluorescent (IF) staining. The mRNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the protein expression by enzyme-linked immunosorbent assay (ELISA). TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The mRNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 mOsM. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity. TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor (GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.