Light stimulates MSK1 activation in the suprachiasmatic nucleus via a PACAP-ERK/MAP kinase-dependent mechanism

Abstract

Signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been shown to be a key intracellular signaling event that couples light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Because many of the physiological effects of the MAPK pathway are mediated by extracellular signal-regulated kinase (ERK)-regulated kinases, it was of interest to identify kinase targets of ERK in the SCN. In this study, we examined whether mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of ERK in the SCN and whether it couples to clock gene expression. Here we show that photic stimulation during the subjective night stimulates MSK1 phosphorylation at serine 360, an event required for robust kinase activation. Activated ERK and MSK1 were colocalized in SCN cell nuclei after photic stimulation. The in vivo administration of the MAP kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene] attenuated MSK1 phosphorylation. MSK1 phosphorylation was more responsive to late-night than early-night photic stimulation, indicating that MSK1 may differentially contribute to light-induced phase advancing and phase delaying of the clock. The potential connection between pituitary adenylate cyclase-activating polypeptide (PACAP) (a regulator of clock entrainment) and MSK1 phosphorylation was examined. PACAP infusion stimulated MSK1 phosphorylation, whereas PACAP receptor antagonist infusion attenuated light-induced MSK1 phosphorylation in the SCN. In reporter gene assays, MSK1 was shown to couple to mPeriod1 via a cAMP response element-binding protein-dependent mechanism. Together, these data identify MSK1 as both a downstream target of the MAPK cascade within the SCN and a regulator of clock gene expression.

Figure 1.

Light induces MSK1 phosphorylation. A, Mice were exposed to light during the subjective day (CT 6) or subjective night (CT 15 and CT 22). Representative images of SCN-containing coronal brain sections immunohistochemically labeled for pMSK1 are shown. Boxed regions are enlarged below each respective image. Box, 100 μm². 3V, Third ventricle; OC, optic chiasm. B, Mean number of pMSK1-positive cells per SCN section. Light exposure during the subjective day (CT6) did not elicit pMSK1 (p > 0.1). Photic treatment at either night time point (CT 15 or CT 22) produced a significant increase in the number of cells containing pMSK1 relative to control animals killed at the same time points. Light treatment at CT 22 was also found to produce a significant increase in the number of pMSK1-positive cells compared with light exposure at CT 15. ^*p < 0.05. Numbers below each bar represent the number of animals used for each condition. C, RT-PCR analysis of MSK1 and CYCα mRNA expression in the SCN. D, Densitometric analysis of data presented in C revealed no significant variation in total MSK1 mRNA expression as a function of circadian time. Results are presented as the ratio of the MSK1 to cyclophilin. Data were averaged from triplicate determinations for each time point.

Figure 2.

Light triggers the colocalized expression of pMSK1 and pERK in neuronal nuclei. Compared with controls (No Light, A), photic stimulation (Light, B) elicited an increase in the number of pMSK1-positive and pERK-positive cells. Merging of the pERK and pMSK1 signals revealed that the expression of the activated forms of the two kinases was colocalized. Scale bar, 100 μm. The boxed region is magnified in the inset. C, A representative image of ventral SCN tissue from a light-treated animal labeled with antibodies directed against pMSK1 (red) and NeuN (green). The colocalized expression of pMSK1 and NeuN indicates that activated MSK1 is expressed in neuronal nuclei. Scale bar, 15 μm.

Figure 3.

The MAPK and p38/MAPK pathways couple light to MSK1 activation. Cannulated animals were infused with 3 μl of DMSO (vehicle), U0126, SB203508, or a combination of U0126 and SB203508 45 min before light exposure (15 min, 100 lux) at CT 22. A, Representative coronal tissue sections immunolabeled for phosphorylated MSK1. Boxed regions are magnified below each image. B, Average number of pMSK1-positive cells per SCN. Disruption of the MAPK pathway significantly reduced the number of positive cells relative to DMSO infusion. SB203508 attenuated light-induced MSK1 activation; however, this effect failed to reach significance relative to DMSO infused, light-treated animals (p > 0.06). The combined administration of U0126 and SB203508 blocked MSK1 phosphorylation. ^*p < 0.001, relative to the DMSO-infused light-treated condition. Numbers below each bar represent the number of animals used for each condition.

Figure 4.

PACAP stimulates MSK1 phosphorylation. A, Representative pMSK1 immunostaining from saline (vehicle)- and PACAP (200 μm)-infused mice. Mice were infused via the lateral ventricle at CT 22 and killed 45 min later. Relative to vehicle infusion, PACAP elicited a marked increase in pMSK1 expression in the periventricular regions, including the SCN. B, PACAP also elicited ERK activation. Representative sections immunolabeled for pERK are from the same animals as in A. C, Primary neuronal cultures were stimulated (10 min) with PACAP (200 nm) or glutamate (10 μm). Confocal images of cells double immunolabeled for phosphorylated MSK1 (red) and for the neuronal-specific structural protein MAP2 (green) are shown. Arrows indicate the locations of neuronal nuclei expressing activated MSK1. D, Quantified cells counts. Relative to mock-stimulated neurons (control), both glutamate and PACAP elicited a significant increase in the number of pMSK1-positive cells (^*p < 0.001). Data are expressed as fold stimulation relative to control levels of pMSK, which were normalized to a value of 1. Numbers below bars indicate the number of cells examined for each condition.

Figure 5.

Inhibition of the PAC1 receptor significantly attenuates light-induced MSK1 phosphorylation. Animals were infused with the PAC1-specific antagonist PACAP 6-38 or saline (vehicle) 15 min before a light treatment (15 min, 100 lux) at CT 15 and CT 22. A, Representative pMSK1 immunolabeled sections from each of the four treatment groups at CT 22. B, Compared with saline infusion, the infusion of PACAP 6-38 significantly reduced the capacity of light to elicit pMSK1 expression (^*p < 0.001). Although PACAP 6-38 attenuated the number of pMSK-expressing cells, a modest but significant residual pMSK1 signal was observed compared with controls (^**p < 0.05). Numbers below bars denote the number of animals used for that condition. C, Histological examination of cannula placement within the dorsal third ventricle (3V). Black arrow denotes the location of the cannula tip.

Figure 6.

MSK1 stimulates mPer1-dependent transcription. A, HEK 293 cells were transfected with an mPer1-luciferase reporter construct (200 ng/well) and one or a combination of the following constructs: DN-MSK1 (800 ng/well), CA-MSK1 (200 ng/well), or A-CREB (800 ng/well). pcDNA3.1 (empty vector) was cotransfected as needed to bring the total to 1 μg/well. Relative to mock-stimulated cells, TPA (200 ng/ml) elicited a marked increase in mPer1-luciferase expression. In contrast, TPA-induced mPer1 expression was attenuated by cotransfection with DN-MSK1 and by pretreatment (30 min) with U0126 (10 μm). CA-MSK1 (black bars) stimulated robust mPer1-luciferase expression. Cotransfection with A-CREB significantly attenuated the effects of CA-MSK1, indicating that MSK couples to mPer1-dependent transcription via a CREB-dependent mechanism. Data are expressed as absolute intensity units. Experiments were performed four times, and representative data were averaged from quadruplicate determinations. ^*p < 0.001. B, HEK 293 cells were immunolabeled for FLAG-tagged DN-MSK1 (red) and the cotransfection marker protein GFP (green) and stained with Hoechst 33258 (blue). Note the nuclear-specific expression of DN-MSK1. Scale bar, 25 μm.