Cis-urocanic acid inhibits SAPK/JNK signaling pathway in UV-B exposed human corneal epithelial cells in vitro

Abstract

Purpose: The cornea is sensitive to ultraviolet B (UV-B) radiation-induced oxidative stress and inflammation. Its clinical manifestations are photokeratitis and climatic droplet keratopathy. Urocanic acid (UCA) is a major endogenous UV-absorbing chromophore in the epidermis and it is also an efficacious immunosuppressant. We have previously shown that cis-UCA can suppress UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal epithelium (HCE) cells. In the current study, we further wanted to investigate the effects of cis-UCA on UV-B-induced inflammatory and apoptotic responses in HCE-2 cells, focusing on the nuclear factor kappa B (NF-κB) and AP-1 (subunits c-Fos and c-Jun) signaling pathways.

Methods: After exposing HCE-2 cells to UV-B and cis-UCA, DNA binding of c-Fos, c-Jun and NF-κB was measured with ELISA. In addition, the endogenous levels of phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (phospho-SAPK/JNK) and phospho-c-Jun were determined. The proliferative capacity of HCE-2 cells was also quantified, and the cytotoxicity of the cis-UCA and UV-B treatments was monitored by measuring the release of lactate dehydrogenase enzyme in the culture medium.

Results: UV-B irradiation induced the binding of transcription factors c-Jun, c-Fos, and NF-κB to DNA. Cis-UCA inhibited the binding of c-Jun and c-Fos but not that of NF-κB. Moreover, UV-B increased the levels of phospho-c-Jun and phospho-JNK, and the expression of both was attenuated by cis-UCA. Cis-UCA also alleviated the UV-B-induced apoptosis and proliferative decline in human corneal cells.

Conclusions: The results from this study suggest that cis-UCA suppresses JNK signaling pathway, which provides potential for treating UV-B-induced inflammatory defects in human corneal cells.

Figure 1

DNA binding of c-Fos and c-Jun subunits of the transcription factor AP-1 heterodimer. Binding of c-Fos (A) and c-Jun (B) to DNA. Results are presented as mean optical density (OD) ± SEM cis-UCA concentration was 100 µg/ml. Seven parallel samples were measured in control and cis-UCA, and nine parallel samples in UV and cis-UCA + UV treatments. *p<0.05; **p<0.01 (Mann–Whitney).

Figure 2

Binding of NF-κB (p65) to DNA 6 h after stimulation. Results are presented as mean counts per second (CPS) ±SEM cis-UCA concentration was 100 µg/ml. Five parallel samples were measured in control and cis-UCA, and seven parallel samples in UV and cis-UCA + UV treatments.

Figure 3

Phosphorylation of c-Jun (Ser63) 6 h after stimulation. Results are presented as mean optical density (OD) ±SEM cis-UCA concentration was 100 µg/ml. Seven parallel samples were measured in control and cis-UCA, and nine parallel samples in UV and cis-UCA + UV treatments. ***p<0.001 (Mann–Whitney).

Figure 4

Phosphorylation of SAPK/JNK (Thr183/Tyr185) 6 h after stimulation. Results are presented as mean optical density (OD) ±SEM cis-UCA concentration was 100 µg/ml. Seven parallel samples were measured in control and cis-UCA, and nine parallel samples in UV and cis-UCA + UV treatments. *p<0.05 (Mann–Whitney).

Figure 5

Proliferation of HCE-2 cells. Results are presented as mean cell numbers ±SEM cis-UCA concentration was 100 µg/ml. Eight parallel samples were measured in all groups. **p<0.01; ***p<0.001 (Mann–Whitney).

Figure 6

Release of lactate dehydrogenase (LDH). Results are presented as mean optical density (OD) ±SEM cis-UCA concentration was 100 µg/ml. Six parallel samples were measured in all groups. **p<0.01 (Mann–Whitney).