Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to +27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained within 773 bases.The rate of biosynthesis of specific neurotransmitters can be modulated by a variety of environmental, neuronal, and hormonal stimuli. In catecholamine biosynthesis, the rate of flow through the pathway is largely dependent on the activity of the initial enzyme, tyrosine hydroxylase (TH), which is expressed in the adrenal medulla, sympathetic ganglia, and certain defined nuclei of the brain. Activity of tyrosine hydroxylase can be modulated both by changes in the synthetic rate of new tyrosine hydroxylase polypeptide and by posttranslational modification of preexisting enzyme molecules.The synthesis of TH is influenced by a variety of factors; in vivo, TH is induced in response to environmental stimuli, such as stress (1), and this induction can be mimicked pharmacologically by treatment of animals with agents that deplete cellular catecholamine stores, such as reserpine (2). In cultures of adrenal chromaffin or pheochromocytoma cells, tyrosine hydroxylase activity is induced by a number of effectors, including glucocorticoid (3, 4), cyclic AMP (5, 6), epidermal growth factor (7), and nerve growth factor (6,8,9).Whether these effectors act through similar mechanisms and whether changes are modulated transcriptionally or posttranscriptionally is not known. Using a cloned cDNA probe for TH, we have previously shown increases in the mRNA for tyrosine hydroxylase following treatment of clonal rat pheochromocytoma cells from an adrenal medullary tumor with analogs of cyclic AMP and glucocorticoid (10). The TH RNA is also increased in adrenal glands and superior cervical ganglia in vivo when animals are subjected to cold stress or reserpine treatment (11-14). In this communication, we have extended those studies by demonstrating that both glucocorticoid and cyclic AMP stimulate the transcriptional activity of the TH gene. In addition, we report that the 5...

In order to characterize the specificity of expression of the neurotransmitter biosynthetic gene dopamine beta-hydroxylase (DBH), the identification of proteins that interact with the DB1 enhancer was initiated. A homeobox-containing cDNA was isolated from a PC12 expression cDNA library screened with the DB1 enhancer. The homeodomain is a member of the paired-like class, and is encoded by several nonidentical cDNAs. The cDNAs contain the same sequence in the homeodomain and 3' coding and noncoding sequences, but diverge in sequence 5' to the homeodomain. This family of homeobox-containing cDNAs is named Arix. Arix mRNA transcripts are found only in noradrenergic, DBH-positive tissues, and in cell lines derived from those tissue. The DB1 enhancer contains two binding sites for the Arix homeodomain, and both sites contribute to basal activity of the DBH promoter. When introduced into tissue culture, Arix regulates the transcriptional activity from the DBH promoter, and also from the promoter of the tyrosine hydroxylase gene, encoding the initial enzyme of the catecholamine biosynthetic pathway. The pattern of expression of the Arix transcripts, the presence of the homeodomain, and the transcriptional regulatory properties suggest that this family of proteins may be involved in the specificity of expression of the catecholamine biosynthetic genes.

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine ␤-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to each other, between bases ؊180 and ؊150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in nonneuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element, and antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissuespecific expression of neuroendocrine genes.The phenotypic expression of genes encoding neurotransmitter biosynthetic enzymes is influenced by local environmental signals such as growth factors and neurohormones. Expression of genes encoding the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) 1 and dopamine ␤-hydroxylase (DBH) has been shown to be modulated by extracellular signals, including glucocorticoid hormones (1-4), the neuropeptide vasoactive intestinal peptide (5, 6), nicotine (7,8), and the neurotrophic factors insulin-like growth factor I (9) and acidic fibroblast growth factor (10). In addition, physiological neural stimuli, such as cold or immobilization stress, can modulate expression of these genes (11)(12)(13)(14).These extracellular signals are transduced from the cell surface through second messenger cascades to the nucleus, where they influence transcriptional control mechanisms. The widely studied second messenger systems initiated through elevations of cyclic AMP and diacylglycerols and mediated through protein kinases A and C stimulate transcription from both the TH and DBH promoters (1, 4, 15-17, 19, 20). In the TH gene, separate regulatory elements mediate these responses: elements at Ϫ45 and Ϫ102 are essential for the response to cyclic AMP (19,(22)(23)(24), whereas an AP1 site at Ϫ205 represents the region responsive to phorbol esters (25). The cyclic AMP regulatory element (CRE) at Ϫ45 also mediates the cellular response to calcium (26) and nicotine (8), and contributes to the cell-type specificity of TH expression (19,28). In the DBH gene, a single region, spanning from Ϫ180 to Ϫ150 of the rat gene, (Ϫ189 to Ϫ176 of the human gene), mediates the response to both...

The homeodomain transcription factors Arix/Phox2a and NBPhox/Phox2b play a role in the specification of the noradrenergic phenotype of central and peripheral neurons. To better understand the functions of these two factors, we have compared the genetic organization, chromosomal location, and transcriptional regulatory properties of Arix and NBPhox. The gene structure is very similar, with each gene containing three exons and two introns, extending a total of approximately 5 kb. Arix and NBPhox are unlinked in human and mouse genomes. NBPhox is located on human Chromosome 4p12 and mouse Chromosome 5, while Arix is located on human Chromosome 11q13 and mouse Chromosome 7. Both proteins bind to three sites in the promoter proximal region of the rat dopamine beta-hydroxylase gene (DBH). In vitro, Arix and NBPhox form DNA-independent multimers and exhibit cooperative binding to the DB1 regulatory element, which contains two homeodomain recognition sites. Both proteins regulate transcription from the rat DBH promoter, and transcription is synergistically increased in the presence of the protein kinase A catalytic subunit (PKA) plus either Arix or NBPhox. The transcription factors exhibit similar concentration-dependent efficacies, and when they are coexpressed, transcription is stimulated to a value approximately equal to that seen with either factor alone. The N-terminal segment of Arix is essential for transcriptional regulatory activity, and this region bears 50% identity with NBPhox, suggesting a similar mechanism of transcriptional activation of the DBH gene. We conclude from this study that Arix and NBPhox exhibit indistinguishable and independent transcriptional regulatory properties on the DBH promoter.

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