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. Keywords SCN; peripheral oscillators; Period genes; valproic acid; real time bioluminescence monitoringDeveloped through evolution to adjust to environmental conditions, self-sustained circadian oscillators generate daily fluctuations in behavior and physiology with a period of approximately 24 h. The study of the mammalian circadian clock at molecular levels has progressed dramatically over the past decade (Fukuhara and Tosini, 2003;Schibler et al., 2003;Gachon et al., 2004). In mammals, the central circadian clock is localized to the suprachiasmatic nucleus (SCN) of the hypothalamus, which synchronizes circadian events within the body. Peripheral circadian clocks have been found in many types of cells and tissues, e.g. fibroblasts, pineal gland, heart, liver, kidney, lung, and so on. Sustained circadian rhythm can be generated not only in a SCN neuron, but also in a fibroblast and other tissues and organs. Numerous studies of the molecular clock mechanism have suggested that these circadian pacemakers consist of transcriptional/translational autoregulatory feedback loops of several clock gene products (Hastings and Herzog, 2004;Ko and Takahashi, 2006). The stability of clock proteins, regulated through phosphorylation and dephosphorylation via protein kinases and phosphatases, is ...

Selective serotonin reuptake inhibitors and raft inhibitors shorten the period of Period1-driven circadian bioluminescence rhythms in rat-1 fibroblasts

“…In a previous study, we have shown that SP600125, an inhibitor of one of the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK), similarly extends period in fibroblasts and the mouse SCN (Chansard et al, 2007). In addition, valproic acid shortens the period in rat-1 fibroblasts (Chansard et al, 2007). These results further suggest that peripheral cell models have a potential to allow us to study the effects of chemicals on circadian rhythm generation.…”

“…Detailed procedures are described in the previous study (Chansard et al, 2007). In brief, 3 days before starting experiments, 10 6 rat-1 fibroblasts, stably transfected with Period1-luciferase reporter gene ( [Izumo et al, 2003], generous gift from Carl Johnson, Vanderbilt University, Nashville, TN, USA), were seeded in a 35 mm Petri dish.…”

“…For example, lithium lengthens period of SCN neuronal rhythms and behavioral rhythms in many organisms (Klemfuss, 1992). In a previous study, we have shown that SP600125, an inhibitor of one of the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK), similarly extends period in fibroblasts and the mouse SCN (Chansard et al, 2007). In addition, valproic acid shortens the period in rat-1 fibroblasts (Chansard et al, 2007).…”

“…These results further suggest that peripheral cell models have a potential to allow us to study the effects of chemicals on circadian rhythm generation. Indeed, the fibroblast model has been used to record circadian rhythms in rodents and even humans (Balsalobre et al, 1998;Brown et al, 2005;Chansard et al, 2007). This model also enables us to reduce the number of animals to be used.…”

“…In the present study, we used real-time monitoring of bioluminescence in rat-1 fibroblasts, stably transfected with Period1-luciferase (Period1 promoter; one of circadian clock genes) reporter gene (Chansard et al, 2007) as well as Period1-luciferase transgenic mouse SCN explants to test any effects that SSRIs might have on the circadian rhythm generation. Since SSRI inhibits serotonin reuptake by inhibiting the activity of serotonin transporter (SERT), we first confirmed that SERT mRNA is expressed in rat-1 fibroblasts.…”