Identification of an Activator Protein-1-Like Sequence as the Glucocorticoid Response Element in the Rat Tyrosine Hydroxylase Gene

Abstract

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.

Induction of Tyrosine Hydroxylase Gene Expression by Glucocorticoids in the Perinatal Rat Brain is Age-Dependent

“…Hormone-activated GRs are able to enhance gene expression when AP-1 consists of the Jun/ Jun homodimer, but don't do it when AP-1 appears as the Jun/Fos heterodimer [53]. Furthermore, as has been shown recently, the GRE/AP-1 composite site is the major site of interaction of glucocorticoids with the TH gene in the pheochromocytoma cell line [58]. Ontogenetic variation in the expression of Fos and Jun family proteins, which affects their ratio [59,60], can be one of the reasons for the TH gene regulation by glucocorticoids in fetuses but not in neonates.…”

“…Ontogenetic variation in the expression of Fos and Jun family proteins, which affects their ratio [59,60], can be one of the reasons for the TH gene regulation by glucocorticoids in fetuses but not in neonates. However, to date this hypothesis is supported only by in vitro data [58], and existence of such mechanism in vivo conditions need to be explored in further studies.…”

A Novel Model of Dexamethasone-Induced Hypertension: Use in Investigating the Role of Tyrosine Hydroxylase

“…For example, activator protein-1 has a lower binding to the TH promoter motif upon immobilization in the SCG than in the adrenal medulla (Sabban et al, 2004). The genomic effect of DEX on TH via the atypical glucocorticoid response element might not occur at the SCG, and was less likely to be of stimulatory nature that we saw in vitro (Rani et al, 2009;Sheela Rani et al, 2013). Consistent with this, systemic a-MPT administration generates a greater decrease in blood pressure than guanethidine, an inhibitor of the norepinephrine uptake transporter (Chiou-Tan et al, 1994).…”

“…At the molecular level, our laboratory identified an atypical glucocorticoid response element in the promoter region of rat and human TH gene that is stimulated by DEX (Rani et al, 2009;Sheela Rani et al, 2013). In the present study, we hypothesized that an underlying mechanism for DEX-induced hypertension involves increases in TH gene expression in the adrenal medulla, and perhaps in other sympathetic terminals, upon chronic DEX treatment; this in turn leads to a sustained increase in catecholamine synthesis, higher enzyme activity, and finally to hypertension.…”

Effect of dexamethasone on the expression of immediate early genes c-fos and c-jun in different regions of the neonatal brain

“…The regulation of gene expression by glucocorticoids can be mediated either by the canonical mechanism that involves the interaction of the glucocorticoid receptor (GR), a transcription factor activated by the hormone, with glucocorticoid response element (GRE) of gene promoter (GRE), or by the so-called noncanonical mechanism that involves the interaction of GR with the AP-1 complex [2]. This mechanism enables the regulation of gene expression by glucocorticoids in the absence of GRE from the respective gene promoter so that the hormone can modulate the expression of these genes as well [3,4]. In this case, the expression of target transcripts can be either enhanced or suppressed depending on the protein composition of the AP-1 complex.…”