Circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. Little is known about the output pathways. In this study, we show that the Neurospora crassa osmosensing MAPK pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. Rhythmic activation of the highly conserved stress-activated p38-type MAPK [Osmotically Sensitive-2 (OS-2)] by the N. crassa circadian clock allows anticipation and preparation for hyperosmotic stress and desiccation that begin at sunrise. These results suggest a conserved role for MAPK pathways in circadian rhythmicity.circadian output pathway ͉ circadian rhythm ͉ Neurospora crassa ͉ osmotic stress phosphorelay

The mitogen-activated protein kinase (MAPK) family of genes aids cells in sensing both extracellular and intracellular stimuli, and emerging data indicate that MAPKs have fundamental, yet diverse, roles in the circadian biological clock. In the mammalian sup-rachiasmatic nucleus (SCN), MAPK pathways can function as inputs allowing the endogenous clock to entrain to 24 h environmental cycles. MAPKs can also interact physically and/or genetically with components of the molecular circadian oscillator, implying that MAPKs can affect the cycling of the clock. Finally, circadian rhythms in MAPK pathway activation exist in many different tissue types and in model organisms, providing a mechanism to coordinately control the expression tissue-specific target genes at the proper time of day. As such, it should probably not come as a surprise that MAPK signaling pathways and circadian clocks affect similar biological processes and defects in either pathway lead to many of the same types of human diseases, highlighting the need to better define the mechanisms that link these two fundamental pathways together.

MAPK signal transduction pathways are important regulators of stress responses, cellular growth, and differentiation. In Neurospora, the circadian clock controls rhythms in phosphorylation of the p38-like MAPK (OS-2); however, the mechanism for this regulation is not known. We show that the WCC, a transcription factor and clock component, binds to the os-4 MAPKKK promoter in response to light and rhythmically in constant darkness, peaking in the subjective morning. Deletion of the WCC binding sites in the os-4 promoter disrupts both os-4 mRNA and OS-2 phosphorylation rhythms. The clock also indirectly regulates rhythmic expression of the histidyl-phosphotransferase gene, hpt-1, which peaks in the evening. Anti-phase expression of positive (OS-4) and negative (HPT-1) MAPK pathway regulators likely coordinate to enhance rhythmic MAPK activation to prepare cells to respond to osmotic stress during the day in the natural environment. Consistent with this idea, we show that wild type cells have a clock-dependent morning kinetic advantage in glycerol accumulation after salt stress as compared to evening treatment. Thus, circadian transcriptional control of MAPK pathway components leads to striking time-of-day-specific effects on the signaling status and physiological response of the pathway.

BackgroundThe circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro.MethodsThe levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests.ResultsWe demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness.ConclusionsGlioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3896-y) contains supplementary material, which is available to authorized users.

No abstract

In the filamentous fungus Neuropora crassa, the Osmotically Sensitive (OS) signal transduction pathway is rhythmically activated and functions as an output pathway from the circadian clock. This conserved fungal pathway is composed of a sensory phosphorelay coupled to a mitogen activated protein kinase (MAPK) cascade. Our goal is to define the mechanism utilized by the FREQUENCY/WHITE COLLAR (FRQ/WCC) circadian oscillator to mediate rhythmicity of OS pathway activity that subsequently affects transcription of clock controlled genes. We show that expression of two components in the OS pathway, the histidine phosphotransferase encoded by hpt‐1 and the downstream MAPK kinase kinase encoded by os‐4, is regulated by the FRQ/WCC oscillator. We also demonstrate that the promoter of os‐4 is rhythmically bound by the White Collar Complex (WCC), a transcription factor and core component of the FRQ/WCC oscillator, with a period and phase that correlate with the rhythmic accumulation of os‐4 transcript. Furthermore, deletion of the promoter region bound by the WCC results in arrhythmic expression of os‐4 indicating that direct clock interaction is required for rhythmicity of os‐4. Interestingly, hpt‐1 expression rhythms peak in an opposite phase relative to os‐4 expression, and the hpt‐1 promoter is not bound directly by the WCC. Our research tests how clock regulation of OS pathway components affects the activation state of the pathway and expression of downstream genes. Our working model predicts that the activity of HPT‐1 inhibits pathway activation, while the activity of OS‐4 promotes pathway activation; therefore, by expressing these two components in opposite phases, the clock coordinates their activity to set the activation state of the pathway. By variably expressing the levels of these two components, we hypothesize that the clock can tune the basal level of pathway activation resulting in rhythmicity. This work is funded by a grant from the NIH.