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Keiko Yamada, Yuriko Ban, Shigeru Kinoshita; The Change of Tight Junction-Related Protein Expression in Human Telomerase Reverse Transcriptase (hTERT) Immortalized Corneal and Conjunctival Epithelial Cells by Viral Double-Strand RNA Stimulation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5511.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that polyinosinic-polycytidylic acid [Poly(I:C)] challenge strengthened the barrier function in human telomerase reverse transcriptase (hTERT) immortalized corneal and conjunctival epithelial cells. The purpose of this present study was to investigate the change of tight junction-related protein expression in corneal and conjunctival epithelial cells in response to Poly(I:C).
Conjunctival and corneal epithelial cells immortalized by preventing telomere shortening by transduction with hTERT were cultured on 12-mm Transwell® filters (Corning, Tewksbury, MA) at a density of 4 x 105 cells/cm2. The cultured epithelial cells were stimulated with 25µg/ml Poly(I:C), an analog of viral double-stranded RNA produced during viral replication. Tight junction-related proteins ZO-1, occludin, and claudin-1, -4, and -7 expressions after 3 to 48 hours of exposure to Poly(I:C) were analyzed using western blotting. Immunoreactive bands were visualized by the chemiluminescent method, and densitometry analysis was performed.
Up until 12 hours, there was no remarkable change in the tight junction-related proteins. After 24 hours, claudin-4 and occludin increased their expression in both corneal and conjunctival epithelial cells. In contrast, claudin-7 protein expression decreased after 48 hours.
Corneal and conjunctival epithelium forms a barrier that isolates the eye from the outside environment, and this barrier is created by tight junctions. Tight junctions are composed of several transmembrane and intracellular molecules. Poly(I:C) is a toll-like receptor 3 ligand. The findings of this study show that Poly(I:C) challenge, which mimics a viral infection, increased the barrier function of ocular surface epithelia. Moreover, tight junction transmembrane proteins which are expressed in corneal and conjunctival epithelia were separated into two groups; 1) proteins in which expression was increased by Poly(I:C) challenge and 2) proteins in which expression was decreased by Poly(I:C) challenge. This difference of claudin and occludin expression is owed to the strengthening of the barrier.
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