Abstract
The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC-12 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 manner. Induction was attenuated by inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway (∼60%) but not by inhibition of the protein kinase A (PKA), protein kinase C, phosphoinositol kinase, or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF-responsive sequences lay within the proximal -392 base pairs (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 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 pituitary adenylyl cyclase-activating protein (PACAP), another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control seems to be another important mechanism by which neurotrophins regulate the adrenergic phenotype. NGF, PACAP, and a combination of the two stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein, but to different extents.
Footnotes
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This work was supported by the Spunk Fund Inc., the Sobel and Keller Research Support Fund, and McLean Hospital.
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ABBREVIATIONS: PNMT, phenylethanolamine N-methyltransferase; NGF, nerve growth factor; PACAP, pituitary adenylyl cyclase-activating polypeptide; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; PKA, protein kinase A; PKA-C, catalytic subunit of protein kinase A; PKC, protein kinase C; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; MEK, mitogen-activated protein kinase kinase; LY294002, 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one; U-73122, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; bp, base pair(s); PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline/Tween 20; GMSA, gel mobility shift assay; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor.
- Received May 2, 2006.
- Accepted August 22, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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