Egr together with catecholamine biosynthetic
Egr1, together with catecholamine biosynthetic enzymes, such as tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT), is induced in adrenal medulla by different types of stress (Liu et al., 2005, Morita et al., 1996, Nankova et al., 1993, Papanikolaou and Sabban, 1999, Papanikolaou and Sabban, 2000, Sabban and Kvetnansky, 2001).
A functional Egr1 binding site on proximal TH promoter was previously identified and shown to interact with an AP1 motif in the induction of TH promoter activity (Nakashima et al., 2003, Papanikolaou and Sabban, 2000). Egr1 alone, or together with other factors such as glucocorticoids, also regulates PNMT, the final enzyme in epinephrine biosynthesis (Ebert et al., 1994, Morita et al., 1995, Tai et al., 2001). The role of Egr1 in transcriptional activation of the DBH gene has not been previously studied.
DBH (EC 126.96.36.199) catalyzes the synthesis of norepinephrine (NE) from dopamine (DA). Modulation of DBH activity or expression is implicated in several neuropsychiatric disorders (reviewed by (Cubells and Zabetian, 2004)). For example, an elevated DA/NE ratio resulting from decreased DBH expression or activity is associated with increased vulnerability to psychotic depression (Meyers et al., 1999, Schatzberg and Rothschild, 1992). Polymorphisms of the human DBH gene have been linked to reactive schizophrenia, psychotic depression, alcoholism, attention deficit hyperactivity disorders, modulation of smoking and other reward-seeking behavior, and susceptibility to Parkinson\'s disease (reviewed by (Anney et al., 2004, Cubells and Zabetian, 2004, Healy et al., 2004)).
DBH gene expression is regulated in response to several pharmacological and physiological stimuli (Afar et al., 1996, Ishiguro et al., 1998, Kilbourne et al., 1992, McMahon and Sabban, 1992, Shaskus et al., 1992). On the DBH promoter, there are several functional motifs that are homologous to known cis-acting regulatory elements including Sp1, three homeodomains, an overlapping cAMP/AP1 response cystic fibrosis chloride channel [(CRE)/AP1], and AP2 motifs (Kim et al., 2001, Kim et al., 1998, Swanson et al., 1997). The three homeobox motifs can bind Phox2a/Arix, which is important for cell-specific DBH gene expression. The overlapping CRE/AP1 motif can bind CREB and also AP1 family members (Seo et al., 1996, Swanson et al., 2000, Swanson et al., 1998).
In this study, we examined whether Egr1 can regulate DBH gene expression. Our results show that Egr1 suppressed DBH promoter activity, and that the region −227/−224 of the promoter is responsible for this effect. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) indicated that this involves specific binding of Egr1 to the DBH promoter. Our results suggest, for the first time, that Egr1 may play a role in the physiological regulation of transcription of the DBH gene.
Discussion This is the first study to characterize the effect of Egr1 on DBH gene expression. We found that Egr1 can play an inhibitory role on DBH promoter activity; it also can reduce endogenous DBH mRNA. Mutation of the promoter indicated that the site at -227/−224 is involved in this inhibition. Both in vitro and in vivo binding studies confirmed the presence of an Egr1 binding site in this region of the proximal DBH promoter. The Egr1 gene is one of the early response genes dramatically and rapidly induced upon stimulation with many environmental signals including growth factors, hormones, and neurotransmitters (Gupta et al., 1991, Kaufmann et al., 2001, Liu et al., 2005, Sato et al., 1999). Egr1 functions as a convergence point for many signaling cascades and is thought to couple extracellular signals with long-term responses by altering gene expression of Egr1 target genes (Silverman and Collins, 1999). The suppression of DBH promoter activity by Egr1 was evident with several lengths of the promoter, with the proximal 247 nucleotides being sufficient. With the longest construct tested [p5′DBH/Luc (−1625/+21)], Egr1 also reduced DBH promoter activity, but not as much as with shorter constructs. In this regard, Afar et al. (Afar et al., 1996) also observed that elevation of reporter activity with 1 kb DBH promoter in response to cAMP was not as great as the responses with the shorter promoter constructs (−210, −190, and −173). It is possible that there is a more dominant regulatory element in the distal region of the first kb of the promoter. Alternatively, the conformation of the promoter may restrict access to Egr1 and other regulatory elements in the longer promoter, leading to an attenuated response.