Differential effect of membrane depolarization on levels of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs in PC12 pheochromocytoma cells

104

125

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...

mentioning

confidence: 61%

“…Studies by Sabban and co-workers (14,15) have shown that membrane depolarization or agonist occupation of nicotinic cholinergic receptors leads to the induction of TH mRNA in PC12 cells. Both of these treatments lead to Ca 2ϩ influx through voltage-sensitive Ca 2ϩ channels.…”

mentioning

confidence: 99%

Tyrosine Hydroxylase Gene Promoter Activity Is Regulated by Both Cyclic AMP-responsive Element and AP1 Sites following Calcium Influx

Nagamoto-Combs¹,

Piech²,

Best³

et al. 1997

104

125

“…Levels and activity of the enzyme are finely and differentially regulated in these cells by a large variety of pharmacological and physiological stimuli. Modulation of TH activity occurs at the protein level via phosphorylation of the enzyme (40) and at the gene expression level via transcriptional changes (41). In vitro experiments with reporter gene constructs have shown that very proximal cisacting elements play important roles in functional regulation of TH gene transcription (42)(43)(44).…”

Section: Induction Of a Subclinical Parkinson's-like Disease Inmentioning

confidence: 99%

HIV-1 Tat-mediated Inhibition of the Tyrosine Hydroxylase Gene Expression in Dopaminergic Neuronal Cells

Zauli¹,

Secchiero²,

Rodella³

et al. 2000

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH ؉ neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

“…Levels and activity of the enzyme are finely and differentially regulated in these cells by a large variety of pharmacological and physiological stimuli (33)(34)(35)(36)(37)(38)(39). Modulation of TH activity occurs at the protein level via phosphorylation of the enzyme catalyzed by PKA and possibly other kinases (40), and at the gene expression level via transcriptional changes (14,(41)(42)(43)(44). Transgenic models were developed to identify the minimal promotor regions necessary to drive tissue specificity and stimulus-associated induction in vivo (45)(46)(47).…”

mentioning

confidence: 99%

Inducible cAMP Early Repressor Can Modulate Tyrosine Hydroxylase Gene Expression after Stimulation of cAMP Synthesis

Tinti

Conti

Cubells

et al. 1996