A Connection Between MAPK Pathways and Circadian Clocks

Abstract

Circadian clocks and mitogen-activated protein kinase (MAPK) signaling pathways are fundamental features of eukaryotic cells. Both pathways provide mechanisms for cells to respond to environmental stimuli, and links between them are known. We recently reported that the circadian clock in Neurospora crassa regulates daily rhythms in accumulation of phosphorylated, and thus active, OS-2 MAPK, a relative of mammalian p38 MAPK, when cells are grown in constant conditions. In the absence of acute stress, rhythmically activated MAPK then signals to downstream effector molecules to regulate rhythmic expression of target genes of the pathway. Clock regulation of MAPK signaling pathways provides a mechanism to coordinately control major groups of genes such that they peak at the appropriate times of day to provide a growth and survival advantage to the organism by anticipating stresses. MAPK pathways are well known for their role in cell proliferation and tumor suppression. New evidence reveals that some mammalian clock components also function as tumor suppressors and rhythms in phospho-MAPK have been observed in higher eukaryotes. Thus, the role of the clock in regulation of the activity of MAPK pathways provides important clues into the function of the circadian clock as a tumor suppressor.

INTRODUCTION

Cells must adapt their physiology to respond to changing extracellular conditions for enhanced fitness and survival. Two complex mechanisms coexist in cells to deal with varying environmental conditions: adaptation and anticipation. When change occurs on a predictable basis, such as the rising and setting of the sun as a result of the Earth's 24 hr rotation, an internal timing mechanism called the circadian clock anticipates these changes and programs daily rhythms in the expression of target genes needed to respond to these events. ¹ For example, the circadian clock controls rhythmic expression of genes involved in photosynthesis and in UV-light protection, such that they peak in expression when the sun is up, but are at their lowest when the sun is down and their products are no longer needed.² We know that this rhythmic gene expression is anticipatory because the rhythms persist in constant conditions (e.g. constant light or constant dark) with a period of about a day. On the other hand, cellular responses to unanticipated changes, such as exposure of an organism to a high dose of damaging UV light during the day or night, uses a different mechanism that does not rely on the clock.³ Such acute changes trigger signal transduction mechanisms that lead to the activation of conserved MAPK pathways at any time of day.⁴ However, increasing evidence indicates that these two mechanisms are not independent, but instead share some remarkable linkages.⁵

MAPK PATHWAYS

MAPK pathways provide information routes by which cells sense changes in their environment and transduce this information to the inside of the cell to mount an appropriate response.⁶ The MAPK signaling module is composed of 3 consecutively activated protein kinases: MAPKKK, MAPKK, and MAPK. These individual components are often used in multiple pathways within each organism, leading to the generation of intricate networks that impact basic biological processes, including cell division and growth, morphogenesis, mating, and apoptosis.^{7, 8} Signaling through MAPK pathways begins by activation of a receptor. In lower eukaryotes the receptors include sensor histidine kinases, and the signal is typically propagated through a two-component regulatory system or phosphorelay to the MAPK pathway.^{9, 10} The situation is more complex in higher eukaryotes, with several different receptors, such as EGF, FGF, PDGF, TNFR and FAS involved in activation of different MAPK families.^{11, 12}

Four distinct MAPK family subgroups have been identified in mammals: (1) extracellular signal-regulated kinases (ERKs), (2) ERK/big MAP kinase 1 (BMK1), and the stress-activated MAPKs (SAPKs) that include, (3) c-Jun N-terminal kinase (JNKs), and (4) the p38 family of MAPKs.¹¹ Probably the best characterized MAPK signaling pathway involves the Saccharomyces cerevisiae MAPK Hog1, a relative of the p38 family of SAPKs.¹³ One major function of the HOG pathway is to sense hyperosmotic stress and respond by producing small molecules that increase the intracellular solute concentration, which in turn allows cells to adjust their osmotic pressure. However, the role of the HOG pathway in cells is obviously more complex, involving multiple inputs^{13, 14} and affecting the expression of up to 600 different genes.¹⁴ The Neurospora p38 MAPK pathway, called the osmosensing pathway (OS), is also essential for osmotic stress responses and in many respects is similar to the HOG MAPK pathway.^15-18 In mammals, SAPKs are activated by a variety of extracellular stimuli including UV light, heat shock, osmotic stress, or inflammatory cytokines, and control the expression of more than 100 different genes.¹⁹ In fungi and mammals, the direct targets of activated p38 MAPK are primarily regulatory proteins including effector kinases, transcription factors, translation factors, cell cycle regulators, and chromatin remodeling proteins, that in turn control gene expression.²⁰

THE CIRCADIAN CLOCK

Several complex biological processes in organisms display daily oscillations.¹ The endogenous timing mechanism that controls rhythmicity is called the circadian clock. The circadian clock controls rhythms in gene expression, which results in rhythms in sleep and activity, food consumption, body temperature, blood pressure, hormone activity, development, the cell cycle, and metabolism. Because of the wide variety of processes controlled by the clock, abnormalities in the circadian system affect several different aspects of human health; however, the details of the mechanisms behind clock-associated diseases are not known. Defective clocks have been linked to insomnia, epilepsy, manic depression, seasonal affective disorder, shift work disorders, cerebrovascular disease, coronary heart attacks, headaches, aging, and cancer.^21-23 Thus, considerable effort has gone into determining the molecular, biochemical, and physical properties of the circadian clock in order to understand why disease occurs when the clock is defective, and for developing new and improved therapies for treating circadian clock-associated disorders.

At the core of the circadian clock system are one or more endogenous oscillators that function to generate a free-running period that is close to 24-h when the organism is kept in constant environmental conditions (i.e. constant darkness or constant light and temperature) (Figure 1). The oscillators are composed of the products of “clock genes” that are organized in transcriptional-translational feedback loops.²⁴ Some of the clock genes encode transcription activators, while others encode negative elements that feedback to inhibit their own expression by disrupting the activity of the activators. Components of the oscillators receive environmental information through input pathways, allowing the oscillators to remain synchronized to the 24-h solar day. Time-of-day information from the oscillator(s) is then relayed through output pathways to control expression of the clock-controlled genes (ccgs) and overt rhythmicity. One mechanism by which the output pathways are predicted to be rhythmically controlled is through transcription factors or signaling molecules that are themselves components of the oscillator. These direct outputs may in turn regulate downstream ccgs in a complex web of events. For example, in mammals the positive oscillator components mCLK and BMAL1 bind to E-box elements in gene promoters and mediate rhythmic transcription of negative components of the oscillator Per1 and Per2, as well as some clock outputs including Dbp and Avp.^25-29 A similar situation exists in Neurospora, whereby the positive oscillator components WHITE COLLAR-1 (WC-1) and WC-2 dimerize through their PAS domains and function as transcription factors in the core oscillator to turn on expression of the gene encoding the negative element FREQUENCY (FRQ). In addition to the role of the WC proteins in the oscillator, they are predicted to also signal time of day information directly from the oscillator to one or more output pathways to control rhythmicity of downstream ccgs.³⁰

Figure 1. Common mechanisms regulate circadian rhythms in eukaryotes.

Circadian oscillators function to generate a free-running period that is close to 24 h when the organism is kept in constant environmental conditions. The oscillators can be reset by environmental signals, such as light and temperature, through input pathways. The oscillators send phase information through the output pathways to control the expression of clock-controlled genes (ccgs) and rhythmic processes. Eukaryotic circadian oscillators are composed of positive and negative elements that form autoregulatory feedback loops. In these networks, positive elements of the loop activate transcription of the genes that encode negative elements. As the concentrations of the negative elements rise they repress the activity of the positive elements. Phosphorylation-induced decay of the negative elements reduces their concentrations, leading to reactivation of the positive elements allowing the cycle to start again. Squiggly lines indicate rhythmicity; arrows represent activation; blocked lines indicate repression.

Research in the fungus Neurospora pioneered the isolation of ccgs and it is estimated that about 20% of the genome is under control of the clock at the level of transcript abundance.^{31, 30} The ccgs in Neurospora function in a variety of cellular processes including development, metabolism, pheromone production, and stress responses.³⁰ The expression of most of the ccgs peaks just before dawn and appears to prepare the cells for the stress, and possible opportunities, caused by daily sun exposure.³² Regulation of the ccgs is complex – in many cases, more than one pathway is involved in expression of an individual ccg. For example, several ccgs in Neurospora can be induced by light, development, and stress, independent of the circadian clock.^33-35 Interestingly, signal transduction pathways involving MAPKs control many of the same genes that are regulated by the clock, such as ccg-4 encoding a mating pheromone³⁶, ccg-9 encoding trehalose synthase³⁷, and pck-1 encoding Phosphoenolpyruvate carboxykinase^{16, 32}. These data provided the first hint that the clock might impinge on pathways that are already set up to respond to environmental signals that recur with a 24 h periodicity. This makes sense, as it would provide a simple mechanism to coordinately control large sets of genes that allow the organism to anticipate and respond to the daily occurrence of a particular event.

EMPLOYING A SIGNAL TRANSDUCTION PATHWAY: THE p38 MAPK PATHWAY AS AN OUTPUT FROM THE CLOCK

We recently demonstrated that in Neurospora, the OS pathway is used as an output pathway from the core circadian oscillator.³⁸ This oscillator, called the FRQ/WCC oscillator is constructed similarly to other eukaryotic oscillators and is composed of the negative components FRQ and FRH, and the positive components WC-1 and WC-2 that form a WC complex (WCC) (Figure 1).³⁹ Under constant environmental conditions, time-of-day information is passed from the oscillator through the MAPK signaling pathway (Figure 2), resulting in rhythms in OS-2 (p38-like) MAPK phosphorylation, with peak levels in the early subjective morning. The rhythm in phospho-OS-2 MAPK requires FRQ/WCC oscillator components and the response regulator RRG-1. These data indicate that the clock signal is received by the MAPK pathway at or upstream of RRG-1. Phospho-OS-2 MAPK then signals to transcription factors, and other effector molecules, to regulate rhythms in target gene expression. However, the FRQ/WCC oscillator was found to be unnecessary for the cells to mount an acute response, as either FRQ or WC-1 deletion strains were able to rapidly phosphorylate OS-2 in osmotic stress conditions. These data suggest that the OS pathway receives information from at least two different sources: the endogenous clock and the external environment (Figure 2).

Figure 2. Clock control of the OS MAPK pathway prepares Neurospora cells for osmotic stress.

A working model of the flow of information through the OS pathway from the environment and the clock in Neurospora. Acute osmotic shock signals through the sensor histidine kinase OS-1, and maybe other histidine kinases, to regulate the OS MAPK pathway and the levels of OS-2 phosphorylation and activity. The signal is passed from OS-1 to the histidine phosphotransferase HPT-1, and then from HPT-1 to the response regulator RRG-1. This results in activation of the MAPK pathway and phosphorylation of OS-2. Phospho-OS-2 is predicted to regulate downstream effector kinases, transcription factors, proteins involved in chromatin remodeling, and translation factors. Osmotic shock results in an acute response in the target genes, including ccg-1, at any time of the day. In constant growth conditions, the FRQ/WCC oscillator (FWO) signals to the same pathway at or before RRG-1 to regulate rhythmic phosphorylation of OS-2 and rhythmic expression of downstream genes, such as ccg-1. Some details of the flow of information through the pathway are not completely understood and arrows to and within the OS pathway are intended to represent passage of information, not activation or repression of activity.

Importantly, the daily signal from the clock to activate the OS MAPK pathway can be overridden by an acute osmotic stress at any time of day.³⁸ These data suggested that one role of the clock in control of the OS pathway may be to provide a mechanism for the organism to predict daily variations in turgor pressure. Support for this idea is that at dawn when the levels of a target ccg of the pathway, the ccg-1 gene, were already high as a result of signaling from the clock, salt exposure caused only a small increase in mRNA levels. However, at dusk, when the levels of ccg-1 were at their lowest, an osmotic shock led to an up-regulation of ccg-1 expression, resulting in levels comparable to those seen at subject dawn. Several ccgs are regulated by both the clock and acute osmotic stress, including ccg-9 and cat-1 encoding catalase (unpublished data).³⁷ Thus, the clock is likely regulating these genes to provide a mechanism for the organism to prepare for osmotic stress that might occur each day in the organism's environment, such as the desiccation that would occur during the hot midday sun. Clock control of the OS pathway is predicted to provide an advantage to the organism; rather than just responding to a change that has already happened, Neurospora can anticipate its occurrence. Altogether, our data suggests that circadian regulation of the OS-2 MAPK pathway, and possibly other MAPK pathways, provides a mechanism to coordinately control the phase of expression of target genes of the pathway.

LINKS BETWEEN MAPK PATHWAYS AND THE CLOCK IN ANIMALS

Studies in insects, amphibians, birds and mammals have also revealed links between MAPK pathways and the circadian clock. ^40-47 For example, rhythms in the phosphorylated form of ERK were observed in the mouse suprachiasmatic nuclei of the anterior hypothalamus (SCN; a small cluster of neurons that forms the site of the mammalian central pacemaker) under constant environmental conditions, with peak expression occurring during the day⁴¹. Rhythms in phospho-ERK have also been observed in the chick pineal and retina, bullfrog retina, and mammalian SCN tissue slices.^{44, 48-50} Additionally, low amplitude daily rhythms in phospho-p38 and JNK in the SCN have been reported. ^{44, 48} These observations that the clock in animals also plays a role in rhythmic control of MAPK activity provides additional support for the hypothesis that circadian oscillators have co-opted major signaling pathways to control rhythmicity in target genes of the pathways. In view of the connection between the circadian clock and in MAPK pathways, it is probably not just coincidental that defects in the clock and in MAPK signaling pathways share many commonalities in human disease.

Both MAPK and circadian clock mechanisms are associated with immune activity, heart disease, and neurodegenerative disorders, and their components function as tumor supressors. ^{23, 51, 52} It is established that deregulation of MAPK pathways is a common event in human cancer cells. However, it is not as widely appreciated that deregulation of the circadian clock is also associated with cancer. ⁵³ The clock regulates daily rhythms in cell proliferation through the rhythmic control of key cell cycle genes, including c-Myc, Wee1, and cyclinD1.^54-57 Loss of circadian control of the cell cycle can lead to transformation and cancer. For example, altered light schedules that disrupt the circadian timing mechanism in rodents leads to a significant increase in the frequency of tumors.^{58, 59} Transgenic mice with defective circadian rhythms are more susceptible to cancer.⁶⁰ Furthermore, mice deficient in either of the clock components mPer1 or mPer2 have an increased incidence of tumors, and many human tumor cells have lower levels of hPer1 and/or hPer2.^61-64 In addition, overexpression of hPer1 or hPer2 in human cancer cells blocks cell division at the G2/M phase and restricts proliferation.⁶⁴ Interestingly, malignant cells often show asynchronies in cell division and metabolism.⁵⁹ This deregulation in malignant cells is the basis of cancer chronotherapy in which chemotherapeutic drugs are given only at certain times of the day when rapidly dividing tumor cells are more susceptible, whereas non dividing normal cells are more tolerant of the drugs.^{57, 65, 66} Finally, disruption of the clock also accelerates tumor progression, and overt rhythmicity has even been used as a predictor of survival time in breast and colon cancer patients.^{67, 68} Thus, it seems likely that regulation of MAPK pathway activity by circadian oscillators is at least partly responsible for the tumor suppressor activity of the clock.

CONCLUDING REMARKS

The excitement and challenge we now face is to fully understand how the circadian clock and MAPK pathways are integrated in cells in order to begin to solve these important issues in human health. The demonstration of control of the OS MAPK pathway by the Neurospora FRQ/WCC oscillator provides a highly developed model organism to now accomplish this goal. Furthermore, links between the Neurospora clock and the cell cycle are known. It was recently shown that the clock regulates the levels of the cell cycle regulator CK-2 (first isolated as PRD-4), and following DNA damage, PRD-4 phosphorylates FRQ (to reset the clock) and other cell cycle regulators to stop the cell cycle.⁶⁹ Experiments are currently in progress using available knockout mutant strains in components of the OS MAPK pathway to determine the biochemical mechanism by which the FRQ/WCC oscillator signals to the OS MAPK pathway. Experiments are also underway to determine the mechanisms by which phospho-OS-2 globally regulates the expression of downstream ccgs. We speculate based on similarities of the OS pathway to the HOG pathway in yeast that this involves the regulation of specific transcription factors, proteins involved in chromatin remodeling, and kinases that affect translation. Lastly, rhythmic activity of the p42/44-like MAPKs involved in pheromone responses and cell wall integrity in Neurospora are currently being analyzed for rhythmic activity in constant conditions.

Elucidating the mechanism of circadian clocks is an important goal because of the ubiquity of clocks and their extensive role in the lives of most organisms. We have seen rapid advances in our understanding of the mechanisms of circadian oscillators, and we are now poised to further define circadian output pathways and the link between the clock and MAPK pathways in normal and diseased cells. Because both the clock mechanism and MAPK pathways are highly conserved from fungi to humans, unraveling the mechanisms by which circadian oscillators regulate rhythmic MAPK activity in Neurospora will likely lead to a better understanding of this link in higher eukaryotes, and may provide the information needed to develop new therapies to treat diseases common to these intertwined pathways.

Abbreviations

ccg

clock-controlled gene

SCN

suprachiasmatic nucleus

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

MEK

MAP kinase kinase

JNK

c-Jun N-terminal kinase

SAPKS

stress-activated MAPK

FRQ

FREQUENCY protein

WC

WHITE COLLAR protein

mClk

mouse Clock protein

Bmal

brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein