DEMONSTRATION OF A 2ND 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 dea th in neurons. However, the therapeutic potential of NGF is limited by metabolic instability and poor CNS penetration. These limitations mig ht 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 differe ntial analysis based on semi-quantitative reverse transcription polyme rase chain reaction (RT-PCR). Mouse L929 cells were serum deprived and NGF mRNA was induced by treatment with phorbol 12-myristate 13-acetat e (PMA), 1,25-dihydroxy-vitamin D-3 (calcitriol) or horse serum. An in crease in transcripts initiating at exon 1 was noted in cDNA from cell s induced with all three agents. In addition, we also observed an incr ease in cDNA transcripts that initiate at exon 3 and do not include ex ons 1 and 2 (4.38 +/- 0.42, 2.56 +/- 0.05 and 3.04 +/- 0.03 fold incre ase over control for PMA, calcitriol and serum, respectively). Each of these increases was completely inhibited in the presence of actinomyc in D, indicating that the increased levels of mRNA were due to increas es in transcription and not mRNA stabilization. These results confirm the previous demonstration of a promoter for NGF near exon 1 and estab lish a pharmacologically inducible promoter in the NGF gene near exon 3 that could be targeted for therapeutic intervention.