MAP kinase additively activates the mouse Per1 gene promoter with CaM kinase II

Abstract

In a previous study, we showed that the Ca²⁺/calmodulin-dependent protein kinase IIδ (CaMKIIδ) activates the mouse Per1 (mPer1) promoter through a 5′-GAGGGG-3′ motif near exon1B. Here we use luciferase reporter gene assays to document additive activation of the mPer1 promoter by CaMKIIδ and mitogen-activated protein kinase (MAPK) pathways. Transfection of constitutively active MEKK markedly increased mPer1 promoter activity in NB2A cells. Experiments using MAPK inhibitors and dominant-negative c-Jun NH2-terminal kinase 1 (JNK1) showed that extracellular signal-regulated kinase (ERK) accounts for MEKK-induced mPer1 gene activation. We next defined the ERK-responsive region in the mPer1 promoter. A region from − 1735 to − 1721 was required for ERK-induced promoter activation. We also identified a CaMKII-responsive element near exon 1B. Although mutation of the CaMKII-responsive element has no effect on the ERK responsiveness, elimination of a GC-rich sequence downstream of the CaMKII-responsive region totally abolished ERK responsiveness. Finally, ERK-induced promoter activation was additively potentiated by co-transfection with active CaMKIIδ. These results suggest that additive activation by ERK and CaMKII, most likely as a result of photic stimulation in the suprachiasmatic nucleus, plays a critical role in activating the mPer1 gene promoter.

Introduction

In mammals, the suprachiasmatic nucleus (SCN) in the hypothalamus functions as a biological clock center, in which circadian oscillation of clock genes including period and cryptochrome underlies coordination of circadian rhythmicity approximately every 24 h (Albrecht et al., 1997, Dunlap, 1999, Sherman et al., 1997, Sun et al., 1997, Tei et al., 1997, Takumi et al., 1998a, Takumi et al., 1998b, Zylka et al., 1998). Other clock genes, Clock and Bmal1, with basic helix–loop–helix and PAS domains activate transcription of the three mammalian period genes (Per1, Per2 and Per3) and two cryptochrome genes (Cry1 and Cry2) by binding to E-boxes on their promoters. The three Per proteins interact with themselves and with the two Cry proteins, and heteromultimers of these proteins function in a negative feedback loop to repress transcription of Per genes by attenuating the activation potential of Clock/Bmal1.

Light exposure during the subjective night induces rapid mPer1 and mPer2 mRNA expression, which in turn resets the biological clock in the SCN (Sherman et al., 1997, Shigeyoshi et al., 1997, Takumi et al., 1998a). However, the signaling cascade mediating Per gene expression following photic stimulation is not fully understood. Both cAMP and calcium are intracellular second messengers that mediate induction of the Per1 gene and phase resetting in the SCN. Accumulation of intracellular cAMP leads to phosphorylation of the cAMP-response element binding protein (CREB) via activation of protein kinase A (PKA). By contrast, elevation of intracellular calcium through NMDA activation is believed to activate both the extracellular signal-regulated kinase (ERK) and CaMKII in the SCN. ERK also accounts for CREB phosphorylation in the SCN, thereby inducing Per1 gene. Previous reports using luciferase reporter assays indicate that ERK and CaMKII markedly increase mPer1 gene promoter activity (Nomura et al., 2003). Light-induced activation of CRE-dependent transcription is also disrupted by blocking ERK pathway activation (Dziema et al., 2003). However, we previously documented that CaMKII inhibitors but not ERK inhibitors inhibited light-induced Per1 gene expression and phase delay in the hamster SCN (Yokota et al., 2001). Thus, mechanisms underlying Per1 induction through ERK and CaMKII signaling pathways and their crosstalk are still controversial.

Here, we define an ERK-responsive element in the Per1 promoter and assess crosstalk between ERK and CaMKII signaling pathways in mPer1 gene expression using a luciferase reporter assay. ERK but not JNK or p38MAPK functioned in MEKK-induced mPer1 promoter activation. We also found an additive stimulatory effect of ERK with CaMKIIδ on mPer1 promoter activity. Taken together, additive activation of mPer1 promoter by ERK and CaMKII likely mediates photic entrainment of circadian rhythm in the suprachiasmatic nucleus.