Cholinergic stimulation of early growth response-1 DNA binding activity requires protein kinase C and mitogen-activated protein kinase kinase activation and is inhibited by sodium valproate in SH-SY5Y cells

J Neurochem. 1999 Oct;73(4):1384-92. doi: 10.1046/j.1471-4159.1999.0731384.x.


Activation of muscarinic receptors in human neuroblastoma SH-SY5Y cells with carbachol stimulated a rapid and large increase in early growth response-1 (Egr-1, also called zif268 and NGF1-A) protein levels and DNA binding activity. Egr-1 DNA binding activity was stimulated within 15 min of treatment with carbachol and maintained a maximum 20-fold increase over basal between 1 and 2 h after treatment, and the EC50 was approximately 1 microM carbachol. Carbachol-stimulated Egr-1 DNA binding activity was dependent on protein kinase C, as it was potently inhibited by GF109203X (IC50 approximately 0.1 microM) and was reduced by 85 +/- 5% by down-regulation of protein kinase C. Inhibitors of increases in intracellular calcium levels reduced carbachol-induced Egr-1 DNA binding activity by 25-35%. Carbachol-stimulated activation of Egr-1 was reduced 35% by genistein, a tyrosine kinase inhibitor, and 60% by PD098059, an inhibitor of mitogen-activated protein kinase kinases 1/2 (MEK1/2) that activates extracellular-regulated kinases 1/2 (ERK1/2). A novel inhibitory action was caused by chronic (7-day) administration of sodium valproate but not by two other bipolar disorder therapeutic agents, lithium and carbamazepine. Valproate treatment reduced carbachol-stimulated Egr-1 DNA binding activity by 60% but did not alter carbachol-induced activation of ERK1/2 or p38 or increases in Egr-1 protein levels. These results reveal that muscarinic receptors activate Egr-1 through a signaling cascade primarily encompassing protein kinase C, MEK1/2, and ERK1/2 and that valproate substantially inhibits Egr-1 DNA binding activity stimulated by carbachol or protein kinase C.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Carbachol / pharmacology
  • DNA, Neoplasm / metabolism
  • DNA-Binding Proteins / metabolism*
  • Early Growth Response Protein 1
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • Genistein / pharmacology
  • Humans
  • Immediate-Early Proteins / metabolism
  • Indoles / pharmacology
  • Kinetics
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • Maleimides / pharmacology
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinase Kinases
  • Mitogen-Activated Protein Kinases*
  • Neuroblastoma
  • Protein Kinase C / metabolism*
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Tyrosine Kinases / metabolism
  • Transcription Factors / metabolism*
  • Tumor Cells, Cultured
  • Valproic Acid / pharmacology*


  • DNA, Neoplasm
  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Enzyme Inhibitors
  • Flavonoids
  • Immediate-Early Proteins
  • Indoles
  • Maleimides
  • Transcription Factors
  • Valproic Acid
  • Carbachol
  • Genistein
  • Protein Kinases
  • MAP2K2 protein, human
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Protein Kinase C
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • MAP2K1 protein, human
  • Mitogen-Activated Protein Kinase Kinases
  • bisindolylmaleimide I
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one