Glucocorticoids (GCs) generally stimulate gene transcription via consensus glucocorticoid response elements (GREs) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9-kilobase (kb) TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by mifepristone (RU486). Serial deletion mutations revealed a Dex-responsive 7-base pair (bp) sequence, TGACTAA, located at Ϫ5734 to Ϫ5728. Deletion of just these seven nucleotides from the 9-kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester but not to forskolin. The Dex response was fully retained in a construct in which most of the 9-kb promoter was deleted, except for 100 bp around the Ϫ5.7-kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal cAMPresponse element (Ϫ45/Ϫ38) or activator protein-1 (AP-1) (Ϫ207/Ϫ201) sites in the 9-kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25-bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against the glucocorticoid receptors and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a chromatin immunoprecipitation assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene.

The purpose of the work reported here was to determine whether the tyrosine hydroxylase glucocorticoid-responsive element (TH-GRE) interacts with the cyclic AMP pathway and the CRE in regulating mouse TH promoter activity, and whether an additional, previously identi®ed downstream GRElike element also participates in the function of the TH-GRE and CRE. To determine the role of the cAMP pathway on TH-GRE function, we compared the effects of forskolin and dexamethasone on TH mRNA, TH gene transcription and TH promoter activity in a mutant PC12 cell line (A126±1B2) de®cient in cAMP-dependent protein kinase A (PKA) with their effects in the wild-type parental strain. Forskolin treatment increased TH mRNA content, transcriptional activity and the activity of a chimeric gene with 3.6 kb of the TH promoter in wild-type cells, but not in PKA-de®cient cells. In contrast, dexamethasone treatment stimulated equivalent increases in TH mRNA, TH gene transcription and TH promoter activity in each cell type. Mutation of the CRE in chimeric constructs containing 3.6 kb of the 5H¯a nking sequence of the mouse TH gene or coexpression of a dominant-negative mutant of CREB prevented the stimulation of TH promoter activity by forskolin. However, neither the mutation of the CRE nor inhibition of CREB in¯uenced basal or dexamethasone-stimulated promoter activity. Site-directed mutagenesis of the TH-GRE eliminated the response of the promoter to dexamethasone. However, the mutagenesis of a more proximal 15-bp region with a GRE-like sequence had no demonstrable effect on the ability of dexamethasone to stimulate TH promoter activity. Neither mutagenesis of the TH-GRE or the downstream GRElike sequence had an effect on the ability of forskolin to activate this chimeric gene. Taken together, these results provide evidence that a single GRE is suf®cient for maximal induction of transcriptional activity by glucocorticoids and that the CRE is not required for either partial or full activity of this upstream GRE sequence.

Abbreviations used: cAMP, cyclic AMP; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A, the cyclic AMP-dependent protein kinase; PKC, protein kinase C; TH, tyrosine hydroxylase; VIP, vasoactive intestinal polypeptide.

No abstract