We have previously shown that the phorbol ester, TPA, which activates protein kinase C, causes, in PC12 cells, a transcriptional activation of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis. The study has now been extended to examine the processes that underlie this transcriptional stimulation and, in addition, to seek whether similar mechanisms are involved in long-term trans-synaptic induction of the TH gene in adrenal medullae of rats that have been given a single injection of reserpine. In both systems, it was found that the induction of c-fos gene transcription was associated with that of the TH gene but with different kinetics. The promoter of the TH gene contains (at position -207/-200) a sequence (TGATTCA) which differs from the consensus TRE or AP-1 site (TGACTCA) by one nucleotide. Experiments were carried out to investigate whether the AP-1 protein complex which is known to contain Fos and Jun binds to the putative TRE region of the TH promoter. In the gel shift assays, the nuclear protein extracts derived from TPA-treated PC12 cells and from AM of reserpine injected rats displayed a higher magnitude of binding to a 25-mer TRE-TH oligonucleotide as compared to controls. The results showed that the behaviour of TRE-TH was atypical in that two retarded complexes (A and B) were observed, which were displaced by specific competitors. Trans-activation experiments with plasmids TRE-TH/TK/CAT and -754/-19 TH/pUC18-CAT in PC12 cells showed an increase in CAT activity in response to TPA that correlates with the previously observed increase in TH transcriptional activity by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

We have analyzed some functional aspects of the promoter of the human dopamine beta-hydroxylase (DBH) gene. A fragment of 1,247 bp directly 5' to the transcriptional start was progressively shortened, placed in front of a reporter gene, and tested in a human neuroblastoma cell line expressing DBH (SK-N-SH-TFM) and in a monkey kidney cell line (CV-1). A remarkably short region (267 bp), directly upstream from the transcription start, was sufficient to confer activity and tissue-specific expression. Furthermore, the expression of the DBH gene was shown to be inducible by cyclic AMP in SK-N-SH-TFM cells. This effect was demonstrated to occur at the transcriptional level, as shown by run-on assays, and was due to the presence of a near-consensus cyclic AMP-responsive element located in the untranscribed 5' regulatory region of the gene.

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