Received for publication May 3, 2006.
Revised July 21, 2006.
Accepted for publication August 22, 2006.

NGF regulates adrenergic expression

Abstract

The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC12 cells transfected with a rat phenylethanolamine N-methyl-transferase (PNMT) promoter-luciferase reporter gene construct pGL3RP893. NGF treatment increased PNMT promoter-driven luciferase activity in a dose- and time-dependent fashion. Induction was attenuated by inhibition of the extracellular-signal-regulated-kinase (ERK) mitogen-activated protein kinase (MAPK) pathway (~60%) but not by inhibition of the protein kinase A (PKA), protein kinase C (PKC), phosphoinositol kinase (PI3K) or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF responsive sequences lay within the proximal -392 bp of PNMT promoter, wherein binding elements for Egr-1 (-165 bp) and Sp1 (-48 bp) reside. Western analysis further showed that NGF increased nuclear levels of Egr-1, but not Sp1 or PKA-C, the catalytic subunit of PKA. Gel mobility shift assays showed increased potential for Egr-1, but not Sp1, protein-DNA binding complex formation. Mutation of either the Egr-1 or Sp1 binding sites in the PNMT promoter attenuated NGF activation. NGF, combined with PACAP, another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control appears another important mechanism by which neurotrophins regulate the adrenergic phenotype. Independently NGF and PACAP and the combination stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein but to a different extents.

Key words: Adrenergic, Promoter analysis, Regulation of gene expression