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Nerve growth factor regulates adrenergic expression.

T C Tai1, David C Wong-Faull, Robert Claycomb

  • 1Laboratory of Molecular and Developmental Neurobiology, Department of Psychiatry, McLean Hospital, Harvard Medical School, 115 Mill St., MRC 116, Belmont, MA 02478, USA.

Molecular Pharmacology
|August 24, 2006
PubMed
Summary
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Nerve growth factor (NGF) regulates adrenergic expression in PC-12 cells by activating the extracellular signal-regulated kinase MAPK pathway and influencing Egr-1 binding sites. This study also highlights post-transcriptional regulation by neurotrophins.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Cell Biology

Background:

  • Nerve growth factor (NGF) plays a crucial role in neuronal development and function.
  • Understanding the molecular mechanisms of NGF in regulating gene expression is vital for neurobiology.
  • The phenylethanolamine N-methyl-transferase (PNMT) gene is a key marker for adrenergic differentiation.

Purpose of the Study:

  • To elucidate the molecular mechanisms by which NGF regulates PNMT gene expression in PC-12 cells.
  • To identify the specific signaling pathways and DNA elements involved in NGF-mediated PNMT induction.
  • To investigate the role of post-transcriptional regulation in NGF's effect on adrenergic phenotype.

Main Methods:

  • PC-12 cells were transfected with a rat PNMT promoter-luciferase reporter construct.

Related Experiment Videos

  • Reporter gene assays were used to measure PNMT promoter activity.
  • Inhibition of signaling pathways (MAPK, PKA, PKC, PI3K) was employed.
  • Deletion constructs and site-directed mutagenesis identified key promoter regions and transcription factors (Egr-1, Sp1).
  • Western blot and gel mobility shift assays assessed protein levels and DNA binding.
  • mRNA and protein levels were quantified to evaluate post-transcriptional effects.

    • Main Results:

    • NGF dose- and time-dependently increased PNMT promoter activity.
    • The extracellular signal-regulated kinase (ERK) MAPK pathway significantly mediated NGF's effect.
    • NGF-responsive elements were localized to the proximal -392 bp of the PNMT promoter, involving Egr-1 and Sp1 binding sites.
    • NGF increased nuclear Egr-1 levels and its DNA binding activity.
    • Mutation of Egr-1 or Sp1 sites attenuated NGF-induced activation.
    • NGF and pituitary adenylyl cyclase-activating protein (PACAP) cooperatively stimulated PNMT promoter activity.
    • NGF and PACAP also modulated PNMT mRNA and protein levels, indicating post-transcriptional regulation.

    Conclusions:

    • NGF regulates adrenergic expression via the ERK MAPK pathway and interaction with Egr-1 and Sp1 binding sites in the PNMT promoter.
    • Both transcriptional and post-transcriptional mechanisms contribute to NGF's regulation of the adrenergic phenotype.
    • The findings provide insights into the complex molecular control of neuronal differentiation by neurotrophins.