Redox-mediated Synchronization of the Circadian Clock and Clock-regulated MiRNAs and Antioxidant Genes in ARPE-19 Cells

Abstract

Purpose: : In most organisms, including mammals, light is thought to be the major entraining stimulus for the circadian clock. However, most of the mammalian cell lines cultured in vitro are light insensitive. In ARPE-19 cells, serum-induced H₂O₂ can synchronize the circadian clock and the clock output pathways: antioxidant defense mechanism (ADS) and the clock-regulated miRNAs predicted to target the clock and antioxidant genes.

Methods: : To investigate circadian rhythm of clock and clock-regulated genes, total RNA from both serum-shocked (50% serum) and control ARPE-19 cells were extracted at times 0, 3, 6 h after treatment and then every 6 h for 72 h. To test if H₂O₂ functions as a signaling mediator for the expression of Per2, cells were treated with an H₂O₂ scavenger N-acetylcysteine (NAC, 20mM) for 1h and then stimulated with H₂O₂ (50µM) for 6 h. To determine whether serum induction of NAD(P)H oxidase results in H₂O₂ production, cells were treated with NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI, 10µM). To examine if H₂O₂ signaling relies on the MAPK pathway, cells were treated with MAPK inhibitor U0126 (40µM) for 1 h, and then stimulated with H₂O₂ (50µM) for 2 h before harvesting RNA samples.

Results: : After serum-shock, ARPE-19 cells showed a circadian rhythm in the expression of Bmal1, Per2, catalase, Gpx-s, and miRNAs (miR-183/96, miR-494, and miR-30b), with a period length of 24 h. The per2 and Bmal1 showed high amplitude oscillation and their Per2 and Bmal1 mRNA levels displayed peaks at 3-6 h and 12 h after serum shock, respectively, and trough levels at the end of the 24 h cycle, and this trend continued in the following cycles. Pretreatment of cells with NAC and DPI attenuated serum- and H₂O₂-induced expression of Per2 (p<0.05). H₂O₂-induced Per2 expression was inhibited by U0126 ((p<0.05), indicative of the involvement of MAPK in H₂O₂ signaling as a mediator for clock gene expression.

Conclusions: : H₂O₂ functions as a signaling mediator for the circadian clock. Serum induces production of H₂O₂ in RPE cells probably by activating NADPH oxidases, which in turn stimulate the MAPK signaling pathway and induce transactivation of Per2 gene. The redox-mediated synchronization of circadian clock probably regulates the circadian rhythmicity of the ADS and miRNAs.

Keywords: retinal pigment epithelium • antioxidants • signal transduction