Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

Abstract

AIM: To evaluate the role of semaphorin 7A (Sema7A) and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells (HCEs). METHODS: Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0, 125, 250, or 500 ng/mL for 24, 48, or 72h in vitro. Transepithelial electrical resistance (TEER) as well as Dextran-fluorescein isothiocyanate (FITC) permeability assays were conducted to assess barrier function. To quantify tight junctions (TJs) such as occludin and zonula occludens-1 (ZO-1) at the mRNA level, reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed. Immunoblotting was used to examine the activity of the nuclear factor-kappa B (NF-κB) signaling pathway and the production of TJs proteins. Immunofluorescence analyses were employed to localize the TJs. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were utilized to observe changes in interleukin (IL)-1β levels. To investigate the role of NF-κB signaling activation and IL-1β in Sema7A’s anti-barrier mechanism, we employed 0.1 µmol/L IκB kinase 2 (IKK2) inhibitor IV or 500 ng/mL IL-1 receptor (IL-1R) antagonist. RESULTS: Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time- and dose-dependent manner, as well as altering the localization of TJs. Furthermore, Sema7A stimulated the activation of inhibitor of kappa B alpha (IκBα) and expression of IL-1β. The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists. CONCLUSION: Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins, as well as the expression of IL-1β. These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.