c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms

Abstract

Circadian rhythms are endogenous cycles with periods close to, but not exactly equal to, 24 h. In mammals, circadian rhythms are generated in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as several peripheral cell types, such as fibroblasts. Protein kinases are key regulators of the circadian molecular machinery. We investigated the role of the c-Jun N-terminal kinases (JNK), which belong to the mitogen-activated protein kinases family, in the regulation of circadian rhythms. In rat-1 fibroblasts, the p46 kDa, but not the p54 kDa, isoforms of JNK expressed circadian rhythms in phosphorylation. The JNK-inhibitor SP600125 dose-dependently extended the period of Period1-luciferase rhythms in rat-1 fibroblasts from 24.23+/-0.17-31.48+/-0.07 h. This treatment also dose-dependently delayed the onset of the bioluminescence rhythms. The effects of SP600125 on explant cultures from Period1-luciferase transgenic mice and Period2(Luciferase) knockin mice appeared tissue-specific. SP600125 lengthened the period in SCN, pineal gland, and lung explants in Period1-luciferase and Period2(Luciferase) mice. However, in the kidneys circadian rhythms were abolished in Period1-luciferase, while circadian rhythms were not affected by SP600125 treatment in Period2(Luciferase) mice. Valproic acid, already known to affect period length, enhanced JNK phosphorylation and, as predicted, shortened the period of the Period1-bioluminescence rhythms in rat-1 fibroblasts. In conclusion, our results showed that SP600125 treatment, as well as valproic acid, alters JNK phosphorylation levels, and modulates the period length in various tissues. We conclude that JNK phosphorylation levels may help to set the period length of mammalian circadian rhythms.

Fig. 1. Phosphorylation of JNK isoforms in synchronized rat-1 fibroblasts

Twenty-four hours after synchronization, cells were harvested at 4-h intervals on a circadian cycle. (A) Phosphorylation of p46 kDa isoforms showed a rhythmic pattern (open square, P<0.05) over the circadian cycle with a peak of phosphorylation reached 44 h after synchronization. Total amount of p46 kDa was unchanged (filled circle, P>0.1). (B) p54 kDa isoform phosphorylation (open square) as well as total protein amounts (filled circle) did not show significant variations (both cases, P>0.1). Period1-luciferase rhythmic bioluminescence expression on the same time window is represented (A and B). Values are means±S.E.M. n=4–5 For each time point. *P<0.05. For symbols without standard error bars, the bars are smaller than the size of symbols.

Fig. 2. The effects of SP600125 on the period of Period1-luciferase bioluminescence rhythms in rat-1 fibroblasts

Fibroblasts were synchronized by medium change and Period1-driven bioluminescence was recorded for several days. SP600125 (30 μM) lengthened the period of the bioluminescence rhythms. SP600125 negative control (30 μM) showed no significant effect. The left panels show the representative raw bioluminescence recordings and the right panels the baseline-corrected bioluminescence data. The columns in the lower panel show the estimated period length in each experimental condition. Values are means±S.E.M. *** P<0.005; ns, not significant. In all figures a number shown in a column indicates sample size.

Fig. 3. The effects of SP600125 on the acute and chronic activation of JNK in rat-1 fibroblasts

(A) Fibroblasts were synchronized by medium change with or without SP600125. The acute phosphorylation of p46 kDa and p54 kDa isoforms, measured 20 min after medium change was inhibited by SP600125. The lower panel shows quantitative data. Control (0 min) levels were normalized as 100. See detailed description in Fig. 1. (B) Cells were harvested on a full circadian cycle at 5-h intervals starting 30 h after medium change. p46 kDa and p54 kDa isoforms (open square), normalized toward the respective peak time level of expression in the control condition (shown in Fig. 1), did not show significant rhythmicity (both cases, P>0.01). Total amounts of both isoforms remained unvaried (both cases, P>0.1). Values are means±S.E.M. n=3 For each time point. Period1-luciferase rhythmic expression on the same time window is represented. * P<0.05; ** P<0.005; *** P<0.0005.

Fig. 4. Characteristics of SP600125 in the regulation of the Period1-driven bioluminescence rhythm in rat-1 fibroblasts

(A) Circadian rhythmic expression of Period1-bioluminescence in fibroblasts were synchronized and recorded for 3 days in the presence of 30 μM SP600125 (filled bar, 30.73±0.16 h). Medium was then replaced by assay medium without SP600125. After washout, the period was significantly shortened (24.44±0.10 h, P<0.0005). The graph is representative of three independent experiments. n=7. (B) SP600125 showed dose-dependent effects on the period lengthening. Data represent means±S.E.M. n=3–8 For each dose. Comparisons with the control condition (“0”): * P<0.05; *** P<0.005. Comparisons among SP600125 conditions: ^a P<0.05; ^c P<0.005. (C) SP600125 dose-dependently delayed the time of the onset. Data shown represent means±S.E.M. Comparisons with the control condition: * P<0.05; *** P<0.0005. Comparisons among SP600125 conditions: ^a P<0.05; ^b P<0.0005.

Fig. 5. The effects of SP600125 treatment on the period of Period1- and Period2-driven bioluminescence rhythms in SCN and pineal gland of transgenic/knockin mice

SCN slices from Period1-luciferase transgenic mice (left panels) and Period2^Luciferase knockin mice (right panels) showed a significant period lengthening in the presence of SP600125 (A). A similar effect of SP600125 was found in the pineal gland (B). Bioluminescence values for each corresponding daily time are plotted on the chart with time 0 as midnight (00:00 h) on the starting day of the cultures. For each tissue, the left panel represents the raw bioluminescence record and the right panel the baseline-corrected bioluminescence data. The lower panel shows the estimated period length. Each graph is representative of three to four independent experiments. Values are means±S.E.M. ** P<0.005; *** P<0.0005.

Fig. 6. The effects of SP600125 treatment on the period of Period1- and Period2-driven bioluminescence rhythms in peripheral tissues of transgenic/knockin mice

Lung explants from Period1-luciferase transgenic mice (left panels) and Period2^Luciferase knockin mice (right panels) showed a significant period lengthening in the presence of SP600125 (A). However, in the kidneys, circadian rhythms were abolished in Period1-luciferase mice, while circadian rhythms were not affected by SP600125 treatment in Period2^Luciferase mice (B). Bioluminescence values for each corresponding daily time are plotted on the chart with time 0 as midnight (00:00 h) on the starting day of the cultures. For each tissue, the left panel represents the raw bioluminescence record and the right panel the baseline-corrected bioluminescence data. The lower panel shows the estimated period length. For Period1-luciferase kidneys, only control samples are shown because no significant circadian rhythms or period was obtained in the presence of SP600125. Each graph is representative of four to seven independent experiments. Values are means ±S.E.M. ns, Not significant; * P<0.05; *** P<0.0005.

Fig. 7. The effects of valproic acid treatment on the period of Period1-bioluminescence rhythms and the phosphorylation level of JNK in rat-1 fibroblasts

(A) Rat-1 fibroblasts were synchronized and treated with 1 mM valproic acid. Period was significantly shortened in the presence of valproic acid. Values are means±S.E.M. In each graph, a number shown in a column indicates sample size. * P<0.05. Student’s t-test. (B) Rat-1 fibroblasts were cultured and treated for 12 h with either 30 μM SP600125 or 1 mM valproic acid. SP600125 (SP) did not significantly affect the phosphorylation levels of p46 kDa and p54 kDa, while valproic acid (VA) hyper-phosphorylated both isoforms. Total JNK levels were not affected by SP or VA treatment. Values are means±S.E.M. n=3–4 For each treatment. ** P<0.005; *** P<0.0005; ns, not significant.