Noradrenaline Induces Clock Gene Per1 mRNA Expression in C6 Glioma Cells Through β 2 -Adrenergic Receptor Coupled With Protein Kinase A – cAMP Response Element Binding Protein (PKA–CREB) and Src-Tyrosine Kinase – Glycogen Synthase

Astrocytes in the hypothalamic suprachiasmatic nucleus, site of the master circadian pacemaker, play an essential role in the regulation of systemic circadian rhythms. To evaluate involvement of noradrenergic systems in regulation of circadian variation of clock-genes in astrocytes, we investigated effects of noradrenaline (NA) on expression of several clock genes in C6 glioma cells by using real-time PCR analysis. Treatment with NA (10 μ M) induced transient expression of Per1 mRNA, but not of Per2 , Bmal1 , Clock , Cry1 , or Cry2 mRNA, through activation of β 2 adrenoceptors. Action of NA was partially blocked by H-89 [protein kinase A (PKA) inhibitor] or KG-501 [inhibitor of cAMP response element binding protein (CREB)]. We found that pretreatment with genistein or PP2 (general or Src tyrosine kinase inhibitors, respectively) or LiCl [inhibitor of glycogen synthase kinase-3 β (GSK-3 β )] significantly inhibited NA-induced Per1 mRNA expression. In addition, treatment with H-89 and either genistein or LiCl completely blocked NA stimulatory effects. NA markedly induced tyrosine phosphorylation of Src and GSK3 β via activation of β 2 adrenoceptors. Phosphorylation of GSK-3 β by NA was completely eliminated by genistein or PP2. These results primarily suggest that two distinct NA-mediating pathways, PKA–CREB and Src–GSK-3 β , play crucial roles in regulation of Per1 expression in astroglial cells.