Tyrosine Hydroxylase Promoter Activity Research Articles

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cβ3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors.

The transcription factor REST up-regulates tyrosine hydroxylase and antiapoptotic genes and protects dopaminergic neurons against manganese toxicity

Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein-binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1β (IL-1β), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2-associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration.

G-Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity.

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (“TH–Vglut2 Class1”) also expressed the dopamine transporter (DAT) gene while one did not (“TH–Vglut2 Class2”), and the remaining population did not express TH at all (“Vglut2-only”). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-014-0778-9) contains supplementary material, which is available to authorized users.

The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1‐ and FAAH‐independent mechanisms

Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between 'on demand' generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. Anandamide decreased TH promoter activity in N1E115 cells through CB₁ receptor activation. Unexpectedly, URB597 reduced TH expression (pEC₅₀ = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB₁ receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB₁ and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when interpreting the data.

Tyrosine hydroxylase assay: a bioassay for aryl hydrocarbon receptor-active compounds based on tyrosine hydroxylase promoter activation

Tyrosine hydroxylase (TH) assay is a novel bioassay for screening aryl hydrocarbon receptor (AhR)-active compounds with diverse toxicities. The TH assay employs reporter genes to evaluate TH promoter activity, which is linked to dopamine synthesis and regulated by the AhR-aryl hydrocarbon receptor responsive element-III (AHRE-III)-mediated pathway. The toxic equivalency factors for higher chlorinated dioxin congeners in the TH assay (TH-TEF) exhibit the same tendency as those for the WHO-TEF, indicating that the activity of the TH assay is consistent with that of existing methods. Furthermore, other chemicals, such as lower chlorinated dioxin congeners that were minimally investigated in the past could be detected preferentially by this assay.

A neuroprotective role of the human uncoupling protein 2 (hUCP2) in a Drosophila Parkinson's Disease model

Parkinson's disease (PD), caused by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta, is the most common movement disorder. While its etiology remains unknown, mitochondrial dysfunction is recognized as one of the major cellular defects contributing to PD pathogenesis. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in neuroprotection in several neuronal injury models. Here we show that hucp2 expression in Drosophila DA neurons under the control of the tyrosine hydroxylase (TH) promoter protects those flies against the mitochondrial toxin rotenone-induced DA neuron death, head dopamine depletion, impaired locomotor activity and energy deficiency. Under normal conditions, hUCP2 flies maintain an enhanced locomotor activity and have higher steady-state ATP levels suggesting improved energy homeostasis. We show that while no increased mitochondrial DNA content or volume fraction is measured in hUCP2 flies, augmented mitochondrial complex I activity is detected. Those results suggest that it is increased mitochondrial function but not mitochondrial biogenesis that appears responsible for higher ATP levels in hUCP2 flies. Consistent with this notion, an up-regulation of Spargel, the Drosophila peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) homologue is detected in hUCP2 flies. Furthermore, a Spargel target gene Tfam, the mitochondrial transcription factor A is up-regulated in hUCP2 flies. Taken together, our results demonstrate a neuroprotective effect of hUCP2 in DA neurons in a Drosophila sporadic PD model. Moreover, as the TH promoter activity is present in both DA neurons and epidermis, our results reveal that hucp2 expression in those tissues may act as a stress signal to trigger Spargel activation resulting in enhanced mitochondrial function and increased energy metabolism.

Differential and Interactive Effects of Ligand-Bound Progesterone Receptor A and B Isoforms on Tyrosine Hydroxylase Promoter Activity

The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurones. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurones. The present study aimed to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0 kb promoter activity in PR-B expressing cells, although only a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the -1403 to -1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated that multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposing effects on PR-B-mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene, and also that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.

Interference of alpha-synuclein with cAMP/PKA-dependent CREB signaling for tyrosine hydroxylase gene expression in SK-N-BE(2)C cells

Alpha-synuclein (α-SYN) is a small and highly conserved presynaptic protein that is implicated in both normal brain function and the pathogenesis of neurodegenerative diseases. Although the physiological function of α-SYN has not been fully evaluated, several lines of evidence suggest that it plays an important role in the regulation of dopamine homeostasis in the brain. Early studies have demonstrated that interaction of α-SYN and tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine synthesis, is functionally significant for dopaminergic neurotransmission and the pathophysiology of Parkinson's disease. In the present study, we would like to evaluate whether overexpression of wild-type or mutant α-SYN might affect cAMP/PKA-dependent TH activation in DA-producing SK-N-BE(2)C cells. Here we show that wild-type and mutant A30P and A53T α-SYN attenuate forskolin-induced TH up-regulation, but do not suppress TH basal expression in SK-N-BE(2)C cells. Forskolin-induced increase in TH promoter activity and CRE-dependent transcription are significantly suppressed in α-SYN-overexpressing cells. Alpha-SYN enters the nucleus, but does not bind to CREB or interfere with forskolin-induced CREB phosphorylation. These data indicate that elevated levels of α-SYN due to a specific disease or the normal aging process could be associated with dopaminergic neuronal dysfunction through interference with TH regulation.

Retinoid X receptor α acts as a negative regulator in Nurr1-induced dopaminergic differentiation in rat neural precursor cells

To investigate the role of retinoid X receptor (RXRα)–Nurr1 heterodimers in tyrosine hydroxylase (TH) expression, we observed retrovirus-induced RXRα–Nurr1 heterodimer interactions with, and transactivation of, the TH promoter region in cultured rat embryonic neural precursor cells. Interestingly, forced expression of RXRα with Nurr1 remarkably reduced Nurr1 activity in TH+ dopaminergic neuron generation and significantly down-regulated TH promoter activity. These regulatory activities were altered in both Nurr1dim- and RXRαdim- that disrupted dimeric binding, verifying that the Nurr1–RXRα heterodimer represses TH promoter activity. Therefore, a plausible explanation for the inhibitory role of RXRα in Nurr1-induced TH expression is that RXRα differentially affects an inhibitory element of the TH promoter.

Activation of tyrosine hydroxylase (TH) gene transcription induced by brain-derived neurotrophic factor (BDNF) and its selective inhibition through Ca 2+ signals evoked via the N-methyl- d-aspartate (NMDA) receptor

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamine but its transcriptional regulation is not fully understood. Using a reporter assay with cultured rat cortical neurons, we demonstrated that the TH gene promoter was activated by brain-derived neurotrophic factor (BDNF), through its specific receptor TrkB and the ERK/MAP kinase pathway. Using a series of mutant TH gene promoters, we found that the cAMP-response element (CRE) plays a crucial role in the TH promoter activity and the Egr-1-responsive element (ERE), at least in part, is responsible for the BDNF-induced activation. Notably, the influx of Ca 2+ evoked via the N-methyl- d-aspartate receptor (NMDA-R) but not via the L-type voltage-dependent Ca 2+ channel (L-VDCC) selectively antagonized the activation of the gene promoter, suggesting a new link between the catecholaminergic and glutamatergic systems. The Ca 2+ signals evoked via NMDA-R did not affect the phosphorylation of ERK1/2 induced by BDNF. These results suggest that the TH gene's transcription is positively regulated by BDNF, through the CRE and ERE of the promoter, but selectively antagonized by the Ca 2+ signals evoked via NMDA-R without disturbing the ERK/MAP kinase pathway, the regulation by which may underlie the development of the catecholaminergic system in the brain.

Urocortin 2 Lowers Blood Pressure and Reduces Plasma Catecholamine Levels in Mice with Hyperadrenergic Activity

Exaggerated adrenergic activity is associated with human hypertension. The peptide urocortin 2 (Ucn 2) inhibits catecholamine synthesis and secretion from adrenal chromaffin cells in vitro and administration to mammals lowers blood pressure (BP). The chromogranin A-null mouse (Chga-/-) manifests systemic hypertension because of excessive catecholamine secretion from the adrenal and decreased catecholamine storage. In the present study, we investigated whether systemic administration of Ucn 2 could reduce BP and adrenal and plasma levels of catecholamines in vivo. Ucn 2 peptide was administered to freely moving, conscious Chga-/- and wild-type control mice. Telemetry and HPLC measured changes in BP and catecholamine levels, respectively. In both groups of mice, Ucn 2 dose-dependently decreased BP, and this effect was mediated by corticotropin factor-receptor type 2. However, in Chga-/- mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in wild-type mice (P=0.04). In Chga-/- mice only, Ucn 2 decreased adrenal and plasma levels of catecholamines as well as adrenal levels of tyrosine hydroxylase protein and phosphorylation. In vitro mechanistic studies demonstrated that Ucn 2 reduces both catecholamine secretion and tyrosine hydroxylase promoter activity, suggesting that the exaggerated action of Ucn 2 to reduce BP in the Chga-/- mouse is mediated through inhibition of both catecholamine synthesis and secretion. The data suggest that Ucn 2 may be therapeutically useful in regulating the exaggerated sympathoadrenal function of hyperadrenergic hypertension.

Membrane‐initiated estradiol signaling increases tyrosine hydroxylase promoter activity with ERα in PC12 cells

Tyrosine hydroxylase (TH) promoter activity is induced by 17beta-estradiol (E(2)) in PC12 cells expressing estradiol receptor-alpha (ERalpha) requiring a cAMP/calcium response element (CRE/CaRE) at -45. To examine whether membrane-initiated estradiol signaling is underlying this induction, cells co-transfected with TH reporter construct and ERalpha expression vector were exposed to membrane-impermeant estradiol conjugate (beta-estradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin, E(2)BSA). TH promoter activity was elevated by E(2)BSA in dose- and time-dependent manner. E(2)BSA also elicited rapid phosphorylation of CRE binding protein (CREB) and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E(2)BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E(2) dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E(2) or E(2)BSA. Blocking S-palmitoylation of ERalpha with C451A mutation and/or pre-treatment with 2-Bromopalmitate did not prevent but instead enhanced E(2) or E(2)BSA-elicited induction of TH promoter activity. These findings reveal, for the first time, that estradiol induction of TH gene transcription with ERalpha in PC12 cells involves membrane-initiated estradiol signaling, rapid activation of dual PKA/MEK signaling pathways, leading to CREB phosphorylation, acting at CRE/CaRE. The data demonstrate possible mechanism whereby estradiol affects catecholaminergic systems in vivo.

Divergent effects of estradiol on gene expression of catecholamine biosynthetic enzymes

Within the catecholaminergic systems, there are contradictory findings regarding ability of estradiol to regulate expression of genes related to catecholamine biosynthesis. Several parameters important for effects of estradiol on the catecholamine (CA) related enzyme gene expression were examined in two CA regions. Ovariectomized (OVX) female rats were given prolonged estradiol treatments, either in a pulsatile fashion by injections or continuously by pellets. The mode affected the response of tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH) mRNAs differentially in the nucleus of solitary tract (NTS) and the locus coeruleus (LC). In rostral–medial NTS, TH mRNA levels were increased with injections, but declined in rats administered estradiol by pellets. In LC, a significant change was only observed in GTPCH with injections. These differences may reflect activation of different estrogen receptors (ER). The response to estradiol in the presence of ERα and ER β was examined in PC12 cell culture. Estradiol directly regulated promoter activity of TH, GTPCH and dopamine β-hydroxylase (DBH) genes. With ERα, 17 β-estradiol elevated TH promoter activity, while there was a decline with ERβ. In contrast, both DBH and GTPCH promoters were enhanced by 17 β-estradiol over a wide range of concentrations with either ER subtype. Thus, mode of administration, location examined and ER subtype expressed are important considerations in the overall response of catecholamine related enzymes to estradiol.

Mechanisms for Differential Effects of Estrogen on Tyrosine Hydroxylase (TH) Gene Transcription

There are conflicting findings regarding the ability of estrogen to regulate TH gene expression in vivo, with some reports of elevation, reduction or no change. The basis for these discrepancies is unclear and may result from differences in dose, duration, mode of administration, or estradiol receptor (ER) subtype expressed. We found that in the nucleus of the solitary tract of OVX female rats, the response of the TH gene depends on the mode of estrogen administration. Daily injections of estradiol benzoate (EB) at 25 μg/kg for 16 days led to a greater than 2‐fold rise in TH mRNA, while administration of 17‐β estradiol (E2) (0.1 mg, 21 day release) for a similar time by pellets, triggered a 90% reduction in TH mRNA compared to vehicle treated controls. Another important parameter is the ER subtype. Many catecholaminergic cells express both ERα and ERβ. To study the mechanism and effect of ER subtypes, we co‐transfected PC12 cells with expression vectors for ERα or ERβ and TH reporter constructs. E2 elevated TH promoter driven reporter activity with ERα, while it triggered a reduction with ERβ. Further studies found that in the presence of ERα, the membrane impermeable E2‐BSA triggered a similar elevation of TH promoter activity requiring the CRE/CaRE motif. CREB and its phosphorylation at Ser133 were required for the response to E2 or E2‐BSA. The findings reveal several ways whereby estrogen can regulate TH transcription.

Adrenocorticotropic hormone elevates gene expression for catecholamine biosynthesis in rat superior cervical ganglia and locus coeruleus by an adrenal independent mechanism

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague–Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

Up-regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Elk-1 and Egr-1

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signaling molecules, including EGF, NGF, and glucocorticoids. We have analyzed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in noradrenergic locus coeruleus-like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Elk-1, a key transcriptional regulator of serum response elementdriven gene transcription. Likewise, TPA strongly upregulated the biosynthesis of the transcription factor Egr-1 via distal serum response elements within the Egr-1 5'-flanking region. Subsequently, Egr-1 responsive transcriptional activation was observed. Overexpression of Egr-1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr-1. Expression of dominant-negative mutants of Elk-1 or Egr-1 impaired TPA-induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant-negative mutants of the transcription factors ATF2, ATF4, CREB, c-Jun and C/EBP did not change TPAinduced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA-mediated signaling cascade directed towards the tyrosine hydroxylase gene. from Annual Meeting of the Study Group Neurochemistry. International Conference of the Gesellschaft fur Biochemie und Molekularbiologie 2006 (GBM 2006): Molecular pathways in health and disease of the nervous system Witten, Germany. 28–30 September 2006

Regulation of human tyrosine hydroxylase gene by neuron-restrictive silencer factor

Tyrosine hydroxylase (TH), the biosynthetic enzyme of catecholamine, is synthesized specifically in catecholaminergic neurons. Thus, it is possible that neuronal cell type-specific expression of this gene is coordinately regulated. One of the neuron-specific transcription regulators, neuron-restrictive silencer factor (NRSF)/repressor element 1 (RE1) silencing transcription factor (REST), represses the expression of neuronal genes in non-neuronal cells. To elucidate the molecular mechanisms that control catecholaminergic neuronal expression of human TH, we initially characterized the 5′ regulatory region. Previous studies have shown that a 3174 bp fragment of the human TH promoter confers specific expression to the reporter gene in dopaminergic neuron-like cell lines. Within this 5′ regulatory region, three putative neuron-restrictive silencer elements (NRSE)/RE1 were identified, which bound NRSF/REST in a sequence-specific manner, as confirmed using EMSA and ChIP assays. In transient transfection assays, deletion or mutation of NRSE/RE1 elements led to a 7-fold increase in activity of the 3.2 kb TH promoter in human neural stem cells (NSCs), but had no major effects on differentiated neuron-like cells. Suppression of NRSF/REST functions with either the histone deacetylase inhibitor, trichostatin, or DN-NRSF induced TH promoter activity. Our data strongly suggest that NRSF/REST functions as a repressor of TH transcription in NSCs via a mechanism dependent on the TH NRSE/RE1 sites.

Prolonged Activation of cAMP-response Element-binding Protein and ATF-2 Needed for Nicotine-triggered Elevation of Tyrosine Hydroxylase Gene Transcription in PC12 Cells

Phosphorylation (P-) of cAMP-response element-binding protein (CREB) by protein kinase A or mitogen-activated protein kinases was implicated in mediating the increased tyrosine hydroxylase (TH) gene expression after prolonged exposure to nicotine in vivo and in cell culture. We examined the time course and signaling pathways for phosphorylation of CREB and possible involvement of ATF-2. Treatment of PC12 cells with 200 microm nicotine triggered rapid but transient elevation of P-CREB followed by a second sustained rise after 2-5 h of continuous nicotine. In contrast, ERK1/2 was only phosphorylated with short term nicotine exposure. MEK inhibitor U0126 abolished nicotine-induced rise in P-ERK1/2, but not P-CREB, nor did it inhibit nicotine-evoked elevation in TH promoter activity, indicating that ERK1/2 was not needed for induction of TH gene expression by nicotine. In contrast, protein kinase A inhibitor H-89 or Ca(2+)/calmodulin-activated protein kinase inhibitor KN-93 reduced the nicotine-triggered rise in P-CREB and TH promoter activity. There was a delayed elevation of P-ATF-2 after 1 h of nicotine treatment, accompanied by increased ATF-2 protein. Upstream kinase JNK, but not p38, was phosphorylated especially after 5 min to 2 h of nicotine exposure. To examine the requirement for CREB and ATF-2, cells were transfected with dominant negative forms of ATF-2 or CREB. Both reduced the basal TH promoter activity and the response to nicotine. Knockdown of ATF-2 or CREB with siRNA did not alter basal TH promoter activity or mRNA but greatly attenuated the response to nicotine. The results suggest that both ATF-2 and CREB mediate activation of TH gene transcription by nicotine.

Up‐regulation of tyrosine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated by the transcription factors Ets‐like protein‐1 (Elk‐1) and Egr‐1

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signalling molecules, including epidermal growth factor (EGF), nerve growth factor (NGF) and glucocorticoids. We have analysed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in noradrenergic locus coeruleus-like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription. Likewise, TPA strongly up-regulated the biosynthesis of the transcription factor Egr-1 via distal serum response elements within the Egr-1 5'-flanking region. Subsequently, enhancement of the transcriptional activation potential of Egr-1 was observed. Overexpression of Egr-1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr-1. Expression of dominant-negative mutants of Elk-1 or Egr-1 impaired TPA-induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant-negative mutants of the transcription factors activating transcription factor (ATF)-2, ATF4, cAMP response element-binding protein, c-Jun and CCAAT/enhancer binding protein (C/EBP) did not change TPA-induced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA-mediated signalling cascade directed towards the tyrosine hydroxylase gene.