Circadian control by the reduction/oxidation pathway: catalase represses light-dependent clock gene expression in the zebrafish

Abstract

Light is the key entraining stimulus for the circadian clock, but several features of the signaling pathways that convert the photic signal to clock entrainment remain to be deciphered. Here, we show that light induces the production of hydrogen peroxide (H(2)O(2)) that acts as the second messenger coupling photoreception to the zebrafish circadian clock. Treatment of light-responsive Z3 cells with H(2)O(2) triggers the induction of zCry1a and zPer2 genes and the subsequent circadian oscillation of zPer1. Remarkably, the induction kinetics and oscillation profile in response to H(2)O(2) are identical to those initiated by light. Catalase (Cat), an antioxidant enzyme degrading H(2)O(2), shows an oscillating pattern of expression and activity, antiphasic to zCry1a and zPer2. Interestingly, overexpression of zCAT results in a reduced light-dependent zCry1a and zPer2 gene induction. In contrast, inhibition of zCAT function enhances light-mediated inducibility of these clock genes. These findings implicate the enzymatic function of zCAT enzyme in the negative regulation of light-dependent clock gene transcriptional activation. Our findings provide an attractive link between the regulation of the cellular reduction/oxidation (redox) state and the photic signaling pathways implicated in circadian control.

Fig. 1.

Light produces H₂O₂ in Z3 cells. Quantitative analysis of DCF fluorescence. Z3 cells were loaded with CM-H₂DCFDA and then were exposed to light. CM-H₂DCFDA fluorescence was detected by spectrofluorimetry. Values are means ± SE of three independent experiments. In each experiment, the CM-H₂DCFDA fluorescence of the nontransduced cells kept in the dark was set as 1. Where indicated, the cells were transduced with zCat or luciferase genes by retroviral vector (shown by zCAT and LUC, respectively).

Fig. 2.

H₂O₂ induces zCry1a and zPer2 expression in Z3 cells and this induction is mediated by MAPK pathway. (A) A time course of circadian gene induction upon H₂O₂ treatment of confluent Z3 cells was examined by RNA protection assay. Z3 cells permanently cultured in the dark were treated with H₂O₂ (100 μM). At indicated time points after the treatment, cells were harvested for RNA preparation. tRNA serves as our negative control reaction (t). Relative amounts of total RNA used for each sample are displayed (RNA). (B) Circadian gene induction by different concentrations of H₂O_2. Z3 cells kept in the dark and stimulated with H₂O₂ at the concentrations of 10⁻³, 10⁻², 10⁻¹, 1, 10, 50, and 100 μM. After 100 min, cells were harvested for RNA preparation. As the negative control (0 μM H₂O₂ concentration), we added water to the culture media. (C) Z3 cells kept in dark and stimulated with light (L), H₂O₂ (H) (100 μM), UV (U) (10mJ/m², 30 seconds), or MNU (M) (2 mM). After 100 min, cells were harvested for RNA preparation. (D) Confluent Z3 cells were treated with the indicated inhibitors or with the vehicle (DMSO, 0 M). The concentrations of U0126 and SB203580 inhibitors used were 40 μM and 10 μM, respectively. After 1 h, cells were stimulated with light (L) or H₂O₂ (100 μM) (H), or were left in the dark (D). After 100 min, cells were harvested, and RNA was prepared. (E) Confluent Z3 cells were treated with the indicated inhibitors or with the vehicle (DMSO, 0 M). The concentrations of U0126 and SB203580 inhibitors used were 40 μM and 10 μM, respectively. After 1 h, cells were stimulated with light (L) or were left in the dark (D). After 7 h, cells were harvested, and RNA was prepared.

Fig. 3.

H₂O₂ induces circadian gene oscillation in Z3 cells. Z3 cells maintained in constant darkness were stimulated with H₂O₂ (100 μM), and RNA was harvested at each time point indicated after the treatment. zCry1a, zPer1, zPer2, and zCat gene expression was examined by RPA, as shown in Fig. 2A.

Fig. 4.

Oscillation of zCat and circadian clock genes under LD and DD cycles in Z3 cells. Z3 cells maintained in constant darkness were exposed to 12:12 LD cycles for 2 days and then transferred to constant dark condition for another 2 days. RNA was harvested at each time point indicated after light onset. zCry1a, zPer1, zPer2, zCat, and zAox gene expression was examined by RPA (A) or RT-PCR analysis (B), as shown in Fig. 2. In RT-PCR analysis, the value from the cells kept in dark was set as 1 for each gene, and zebrafish actin gene was used for normalization. The bar above the blots indicates light (white) and dark (black) periods.

Fig. 5.

zCat gene expression and catalase activity in Z3 cells cycle in antiphase to zPer2 and zCry1a mRNAs. (A) Z3 cells maintained in constant darkness were exposed to light, and RNA was harvested at each time point indicated after light onset. zCry1a, zPer2, and zCat gene expression was examined by RT-PCR analysis as shown in Fig. 4B. (B) Z3 cells maintained in constant darkness were exposed to a 12:12 LD cycle. Cells were harvested at each time point indicated after light onset for catalase activity assay. The catalase activity of cells at time 0 was taken as 1. Values are means ± SE of three independent experiments. The bar above the graph indicates light (white) and dark (black) periods. (C) Catalase activity was assayed at each time point indicated, as shown in B.

Fig. 6.

zCAT is a key negative regulator for light-dependent activation of circadian genes. (A and B) Light induction of zCry1a and zPer2 expression in non- (Ctr, black line), zCat- (zCAT, red line), and luciferase- (LUC, green line) infected Z3 cells was examined by RPA (A) or RT-PCR analysis (B), as shown in Fig. 2A and 4B, respectively. (C) Confluent Z3 cells were treated with catalase inhibitor sodium azide (350 μM) or with the vehicle (DMSO, 0 M). After 2 h, cells were stimulated with light, and RNA was harvested at each time point indicated after light onset. zCry1a, zPer2, and zAox gene expression was examined by RPA, as shown in Fig. 2D. (D) A model depicting the molecular mechanism underlying light-dependent transcription in zebrafish circadian clock.