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

Abstract

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...

Identification of ATF-2 as a Transcriptional Regulator for the Tyrosine Hydroxylase Gene

“…CREB, ATF-1, and CREM (2-6). Among these transcription factors, only CREB was shown to be an activator of the TH gene transcription (7)(8)(9)(10). Our data confirmed that CREB is one of the TH-CRE-binding proteins in PC12D cells (Fig.…”

“…CREB, ATF-1, and CREM were shown to recognize the cAMP-responsive element (CRE) located at position Ϫ45/Ϫ38 in the TH promoter (2)(3)(4)(5)(6). CREB was reported to mediate basal and cAMP-induced TH transcription in various cultured cells including PC12 cells by the use of dominant-negative CREB protein and antisense RNA against CREB (7)(8)(9)(10). CREB is activated by phosphorylation on Ser 133 (11,12).…”

Hypoxia Induces Phosphorylation of the Cyclic AMP Response Element-binding Protein by a Novel Signaling Mechanism

“…To test for a possible role for CREB in activation of the TH gene by hypoxia, we compared the effect of hypoxia on a TH reporter plasmid (Ϫ272TH)CAT and a similar construct in which the CRE had been mutated, (Ϫ272CRE Ϫ )CAT (26,43). Hypoxia activated wild-type TH-CAT activity by approximately 3-fold, an effect similar to that of forskolin or KCl on this construct (25,43,44). As others have reported previously (25,44), we found that mutation of the CRE resulted in a significant inhibition (ϳ3-fold) of basal levels of TH-CAT reporter activity (Fig.…”

“…Several protein kinases, including protein kinase A; calcium/calmodulin-dependent protein kinase-I, -II, and -IV; protein kinase C; RSK-2 (also termed MAPKAP kinase-1␤); and MAPKAP kinase-2, have been shown to mediate phosphorylation of CREB (14,18,(21)(22)(23)(24). Moreover, phosphorylation of CREB at Ser 133 regulates expression of the c-fos, somatostatin, and TH genes in PC12 cells (13,20,22,25). Given the role for CREB in regulating genes that mediate a multitude of cellular responses, we investigated whether physiological levels of hypoxia regulate CREB phosphorylation and function.…”

Unique Regulation of Immediate Early Gene and Tyrosine Hydroxylase Expression in the Odor-deprived Mouse Olfactory Bulb

“…The TH CRE was found to be required not only for basal activity but also for cAMPmediated up-regulation of TH promoter activity in SK-N-BE(2)C and PC12 cells (27,28). Depolarization-induced upregulation of TH promoter activity occurred through both the TH CRE and AP-1 sites in PC12 cells (29). The latter study also demonstrated that, whereas the CRE was solely responsible for cAMP-mediated regulation of TH promoter activity, both CRE and AP-1 sites were capable of mediating the effects of calcium influx on TH promoter activity.…”