No abstract

Tyrosine hydroxylase (TH) gene promoter activity is increased in PC12 cells that are treated with the phorbol ester, 12-Otetradecanoylphorbol 13-acetate (TPA). Mutagenesis of either the cAMP responsive element (CRE) or the activator protein-1 element (AP1) within the TH gene proximal promoter leads to a dramatic inhibition of the TPA response. The TH CRE and TH AP1 sites are also independently responsive to TPA in minimal promoter constructs. TPA treatment results in phosphorylation of cAMP responsive element binding protein (CREB) and activation of cAMP-dependent protein kinase (PKA) in PC12 cells; hence, we tested whether CREB and/or PKA are essential for the TPA response. In CREB-de®cient cells, the response of the full TH gene proximal promoter or the independent response of the TH CRE by itself to TPA is inhibited. The TPA-inducibility of TH mRNA is also blocked in CREB-de®cient cells. Expression of the PKA inhibitor protein, PKI, also inhibits the independent response of the TH CRE to TPA. Our results support the hypothesis that TPA stimulates the TH gene promoter via signaling pathways that activate either the TH AP1 or TH CRE sites. Both signaling pathways are dependent on CREB and the TH CRE-mediated pathway is dependent on PKA. Catecholamine biosynthesis is enhanced during nerve stimulation in both brain and periphery (Zigmond et al. 1989). This enhancement is primarily the result of increased activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine biosynthetic pathway. During prolonged nerve stimulation, TH enzyme protein is induced. This induction is observed in the adrenal medulla, sympathetic ganglia and the locus coeruleus in response to prolonged stress, treatment with catecholamine-depleting drugs, like reserpine, or treatment with cholinergic agonists, like carbachol or nicotine (for reviews see Kumer and Vrana 1996;Sabban et al. 1998). The increase in TH protein is usually preceded by an increase in TH-mRNA (Tank et al. 1985;Faucon Biguet et al. 1986;Fossom et al. 1991). Prolonged stress or the administration of nicotine, carbachol or reserpine induces TH-mRNA in the adrenal medulla at least partially via an initial stimulation of TH gene transcription rate (Fossom et al. 1991;Osterhout et al. 1997).The intracellular signal transduction pathways that regulate the TH gene during prolonged nerve stimulation have not been fully de®ned. Cyclic AMP, Ca 21 and phosphatidylinositol metabolites increase in the rat adrenal medulla after treatment with cholinergic agonists or after increased stimulation of the adrenal medulla transsynaptically by neurotransmitters released from the splanchnic nerve (Malhotra et al. 1989