Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca 2؉ . In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5 -flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca 2؉ is primarily dependent on both the TH cAMPresponsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca 2؉ -mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca 2؉ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.Biosynthesis of the catecholamines is tightly regulated by the activity of the rate-limiting enzyme, tyrosine hydroxylase (TH 1 ; EC 1.14.16.2). Experimental manipulations that lead to long-term stimulation of catecholaminergic cells in sympathetic ganglia and adrenal medulla are associated with increases in TH gene expression (1-8). The mechanisms responsible for regulation of the TH gene are complex and have not been fully elucidated. Cultured rat pheochromocytoma cells have been used extensively to study the underlying mechanisms for TH gene regulation. Using this model system, a number of laboratories have shown that cAMP analogs (9 -11), active phorbol esters (12), and glucocorticoids (8 -10, 13) induce TH mRNA, stimulate TH gene transcription rate, and/or activate TH gene promoter activity. These results are consistent with the hypothesis that protein kinase A, protein kinase C, and glucocorticoid receptors participate in signaling pathways that regulate the TH gene.Treatments that lead to increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) also increase expression of the TH gene. Studie...

Tyrosine hydroxylase (TH) gene transcription rate is stimulated by cyclic AMP in cultured rat pheochromocytoma cells. This effect is at least partially due to the interaction of transcription factors with the canonical cyclic AMP‐response element (CRE) at position −45 to −38 within the TH gene promoter. In this study we test whether a region of the TH gene promoter, which is adjacent to and upstream of the canonical TH CRE, also participates in the response of the promoter to cyclic AMP. Using electrophoretic mobility shift assays, we demonstrate that nuclear proteins from rat pheochromocytoma cell lines bind to the region of the TH gene from −102 to −73. A comparison of promoter sequences indicates that sequences within this region of the TH gene are highly homologous to proenkephalin promoter sequences (between −110 and −80) designated ENKCRE‐1 and ENKCRE‐2. We designated the TH gene sequence homologous to ENKCRE‐1 as TH E1 and the sequence homologous to ENKCRE‐2 as TH E2. Competition displacement binding assays suggest that protein binding to the −102/−73 region of the TH gene is critically dependent on the TH E1 sequence. Transient transfection assays using minimal promoter constructs demonstrate that this region acts as a cyclic AMP‐responsive element. Mutagenesis of the TH E1 sequence within the normal context of the TH gene proximal promoter leads to a 50% decrease in the cyclic AMP inducibility of the promoter. These results support the hypothesis that the full response of the TH gene to cyclic AMP requires both the canonical TH CRE and this newly discovered element, which we term TH CRE2.

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