title: Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation.
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 Ca2+. 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 Ca2+ is primarily dependent on both the TH cAMP-responsive 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 Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ 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.
authors.affiliation: Department of Pharmacology and Physiology, and the Neuroscience Program, University of Rochester Medical Center, Rochester, New York 14642, USA.
authors.forename: K
authors.initials: K
authors.lastname: Nagamoto-Combs
authors.forename: K M
authors.initials: KM
authors.lastname: Piech
authors.forename: J A
authors.initials: JA
authors.lastname: Best
authors.forename: B
authors.initials: B
authors.lastname: Sun
authors.forename: A W
authors.initials: AW
authors.lastname: Tank
mesh_terms: D033961:Adaptor Protein Complex 1; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
chemical_list: D033961:Adaptor Protein Complex 1; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D017362:Cyclic AMP Response Element-Binding Protein; D008565:Membrane Proteins; D009687:Nuclear Proteins; D000001:Calcimycin; D014446:Tyrosine 3-Monooxygenase; D002118:Calcium