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

AIM: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).METHODS:The cultured HCECs were exposed to media which adding sodium chloride(Na Cl) for hyperosmolar stress or adding rh-TNF-α(10 ng/m L). NF-κB inhibitor(5 μmol/L) or GM-6001(potent and broad spectrum MMP inhibitor, 20 μmol/L)was added 1 h before that treatment. The integrity of TJ proteins was determined by immunofluorescent(IF) staining. The m RNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction(RT-q PCR) and the protein expression by enzyme-linked immunosorbent assay(ELISA).RESULTS:TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The m RNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 m Os M. 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.CONCLUSION: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.

Autosomal dominant coralliform cataract related to a missense mutation of the γD-crystallin gene

Background Congenital cataract is a sight-threatening disease that affects about 1-6 cases per 10000 live births and causes 10%-30% of all blindness in children About 25% of all cases are due to genetic defects We identified autosomal dominant congenital coralliform cataracts-related genetic defect in a four-generation Chinese family Methods Complete ophthalmological examinations were performed prior to lens extraction Lens samples were then studied by electron microscopy Genomic DNA from family members were examined using whole-genomic linkage analysis, with two-point logarithm of odds (LOD) scores calculated using the Linkage program package (version 5 1) Mutation analysis of candidate genes was performed by direct sequencing Finally, a three-dimensional protein model was predicted using Swiss-Model (version 2 0) Results Eleven of the 23 examined individuals had congenital cataracts Ultrastructure studies revealed crystal deposits in the lens, and granules extensively dispersed in transformed lens fiber cells The maximum two-point LOD score, 3 5 at θ=0 1, was obtained for the marker D2S325 Mutation analysis of the γ-crystallin (CRYG) gene cluster identified a mutation (P23T) in exon 2 of γD-crystallin (CRYGD) Protein structure modeling demonstrated that the P23T mutation caused a subtle change on the surface of the γD protein Conclusions The results suggest that the coralliform cataract phenotype is due to a mutated CRYGD gene, and that this sequence change is identical to one reported by Santhiya to be related to another distinct clinical condition, lamellar cataract This study provides evidence that this same genetic defect may be associated with a different phenotype This is the first report identifying the genetic defect associated with an autosomal dominant congenital coralliform cataract