Ultraviolet B Radiation Stimulates the Interaction between Nuclear Factor of Activated T Cells 5 (NFAT5) and Nuclear Factor-Kappa B (NF-κB) in Human Lens Epithelial Cells

ABSTRACT

Purpose: Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells.

Methods: Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm² and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining.

Results: At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm²) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased.

Conclusions: Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.

KEYWORDS:

• Cataract
• HLE-B3
• NFAT5
• nuclear factor-kappa B
• ultraviolet B

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A1A02037702) and Biochemical Research Institute funds (GNUHBRIF-2014-0003) from the Gyeongsang National University Hospital.