Demonstration of a second pharmacologically active promoter region in the NGF gene that induces transcription at exon 3.
Nerve growth factor (NGF) has been demonstrated to facilitate neurite outgrowth, rescue neurons from injury, and prevent programmed cell death in neurons. However, the therapeutic potential of NGF is limited by metabolic instability and poor CNS penetration. These limitations might be circumvented by identifying compounds which increase endogenous production of NGF in the brain. We sought to determine the site of all pharmacologically inducible promoters in the NGF gene using a differential analysis based on semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Mouse L929 cells were serum deprived and NGF mRNA was induced by treatment with phorbol 12-myristate 13-acetate (PMA), 1,25-dihydroxy-vitamin D3 (calcitriol) or horse serum. An increase in transcripts initiating at exon 1 was noted in cDNA from cells induced with all three agents. In addition, we also observed an increase in cDNA transcripts that initiate at exon 3 and do not include exons 1 and 2 (4.38 +/- 0.42, 2.56 +/- 0.05 and 3.04 +/- 0.03 fold increase over control for PMA, calcitriol and serum, respectively). Each of these increases was completely inhibited in the presence of actinomycin D, indicating that the increased levels of mRNA were due to increases in transcription and not mRNA stabilization. These results confirm the previous demonstration of a promoter for NGF near exon 1 and establish a pharmacologically inducible promoter in the NGF gene near exon 3 that could be targeted for therapeutic intervention.
Racke
M M
MM
Hoechst Marion Roussel, Cincinnati, OH 45215-6300, USA.
Mason
P J
PJ
Johnson
M P
MP
Brankamp
R G
RG
Linnik
M D
MD
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006736:Horses / Q000097:blood; D007739:L Cells / Q000187:drug effects; D051379:Mice; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate