Induction Of Tyrosine Hydroxylase mRNA Research Articles

CAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3'-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3'-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a approximately 44-kDa protein as a major TH mRNA 3'-UTR binding factor, and cAMP induces the 40- to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3'-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3'-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons.

Induction of tyrosine hydroxylase mRNA by nicotine in rat midbrain is inhibited by mifepristone

Repeated nicotine administration induces tyrosine hydroxylase (TH) mRNA in rat midbrain. In this study we investigate the mechanisms responsible for this response using two models of midbrain dopamine neurons, rat ventral midbrain slice explant cultures and mouse MN9D cells. In both models nicotine stimulates TH gene transcription rate in a dose-dependent manner. However, this stimulation is short-lived, lasting for 1 h, but less than 3 h, and is not sufficient to induce TH mRNA or TH protein. Nicotine elevates circulating glucocorticoids, which induce TH expression in some model systems. We tested the hypothesis that the effect of nicotine on midbrain TH mRNA is mediated by the glucocorticoid receptor. When rats are administered the glucocorticoid receptor antagonist mifepristone, the induction of TH mRNA by nicotine in both substantia nigra and ventral tegmentum is inhibited. Furthermore, the glucocorticoid receptor agonist dexamethasone stimulates TH gene transcription for sustained periods of time in both midbrain slices and MN9D cells, leading to induction of TH mRNA and TH protein. Our results are consistent with the hypothesis that nicotine induces TH mRNA in midbrain by elevating glucocorticoids, which then act on glucocorticoid receptors in dopamine neurons leading to transcriptional activation of the TH gene.

Activation of Tyrosine Hydroxylase mRNA Translation by cAMP in Midbrain Dopaminergic Neurons

During prolonged stress or chronic treatment with neurotoxins, robust compensatory mechanisms occur that maintain sufficient levels of catecholamine neurotransmitters in terminal regions. One of these mechanisms is the up-regulation of tyrosine hydroxylase (TH), the enzyme that controls catecholamine biosynthesis. In neurons of the periphery and locus coeruleus, this up-regulation is associated with an initial induction of TH mRNA. In contrast, this induction either does not occur or it is nominal in mesencephalic dopamine neurons. The reasons for this lack of compensatory TH mRNA induction remain obscure, because so little is known about the regulation of TH expression in these neurons. In this study, we test whether activation of the cAMP signaling pathway regulates TH gene expression in two rodent models of midbrain dopamine neurons, ventral midbrain organotypic slice cultures and MN9D cells. Our results demonstrate that elevation of cAMP leads to induction of TH protein and TH activity in both model systems; however, TH mRNA levels are not up-regulated by cAMP. The induction of TH protein is the result of a novel post-transcriptional mechanism that activates TH mRNA translation. This translational activation is mediated by sequences within the 3' untranslated region (UTR) of TH mRNA. Our results support a model in which cAMP induces or activates trans-factors that interact with the TH mRNA 3'UTR to increase TH protein synthesis. An understanding of this novel regulatory mechanism may help to explain the control of TH gene expression and consequently dopamine biosynthesis in midbrain neurons under different physiological and pathological conditions.

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.

Nicotinic and muscarinic acetylcholine receptors are essential for the long-term response of tyrosine hydroxylase gene expression to chronic nicotine treatment in rat adrenal medulla

Nicotine induces tyrosine hydroxylase (TH) mRNA by interacting with nicotinic acetylcholine receptors (nAChRs) in cultured adrenal medullary cell systems; however, the mechanisms responsible for the induction of adrenal TH in response to systemically administered nicotine under in vivo conditions are more complex. In the present study, we tested whether nAChRs and muscarinic acetylcholine receptors (mAChRs) participate in the induction of adrenal TH observed after long-term treatment with nicotine. Chronic nicotine treatment (1.6 mg/kg, two daily injections spaced 12 h apart for 7 days) induced TH mRNA, TH protein and TH activity in rat adrenal medulla. This induction of TH gene expression was totally blocked when an antagonist of either nAChRs or mAChRs was administered prior to each nicotine injection. Repeated injections of the mAChR agonist bethanechol (5 mg/kg injected twice per day for 7 days) also produced increases in TH mRNA levels; however, TH protein levels and TH activity did not increase in response to bethanechol. In denervated adrenal glands chronic nicotine treatment did not lead to induction of either TH mRNA, TH protein or TH activity, whereas chronic bethanechol treatment led to induction of TH mRNA, but not TH protein or activity. These results suggest that agonist occupation of both nAChRs and mAChRs are essential for the complete response of TH gene expression to chronic nicotine treatment in rat adrenal medulla, but that stimulation of either cholinergic receptor by itself is not sufficient to elicit a full response. The results also suggest that both transcriptional and post-transcriptional mechanisms may potentially need to be regulated to induce TH protein in response to some stimuli.

Ras/MEK pathway is required for NGF-induced expression of tyrosine hydroxylase gene

Neurotrophins are essential for the development and survival of catecholaminergic neurons. However, the critical pathway for expression of the tyrosine hydroxylase (TH) gene induced by neurotrophin is still unclear. Here we found that Ras/MEK pathway is required for NGF-induced expression of the TH gene in PC12D cells. Induction of TH mRNA by NGF was abolished by pretreatment of the cells with U0126, an inhibitor for MEK1/2, but not with inhibitors for p38 MAPK, PI3K, and PKA. U0126 inhibited TH promoter activity at the same concentration as it acted on ERK1/2 phosphorylation. A dominant-negative form of Ras suppressed the NGF-induced activation of the TH reporter gene, and transient transfection of cells with wild-type Ras and an active form of MEK1 increased the TH promoter activity. The reporter assay also demonstrated that the Ras/MEK pathway acted on both the AP-1-binding motif and the cAMP-responsive element in the TH promoter.

Glial cell line-derived neurotrophic factor up-regulates the expression of tyrosine hydroxylase gene in human neuroblastoma cell lines.

The role of glial cell line-derived neurotrophic factor (GDNF) in the survival of dopaminergic neurons has been well documented, but its effect on dopamine biosynthesis remains to be elucidated. In this study, the effect of GDNF on the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis, was investigated. We found that GDNF elevated the expression of the TH gene at both mRNA and protein levels in TGW cells, a human neuroblastoma cell line. GDNF significantly enhances the transcription rate of the TH gene as actinomycin D prevented the induction of TH mRNA and GDNF increased the activity of the TH promoter. In addition, GDNF exerts a relatively weak but significant effect on the stability of TH mRNA, because GDNF delayed the degradation of TH mRNA and strengthened a special TH mRNA/protein interaction known to be relevant with TH mRNA stability. By comparing several human neurogenic cell lines, we found that GDNF-induced TH expression was only observed in the cells possessing Ret protein and coincided with the expression levels. Taken together, these results indicate that GDNF up-regulates the expression of the TH gene by promoting the transcription of the TH gene and the stability of TH mRNA with the Ret receptor dependency in some neuroblastoma cell lines. Thus, GDNF exerts its neurotrophic role not only in promoting cells survival, but also in affecting dopamine biosynthesis.

Induction of tyrosine hydroxylase and neuropeptide Y by carbachol: modulation with age.

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of dopamine beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH) mRNA levels and TH enzyme activity in the adrenal medulla of senescent compared with younger animals. Cold exposure induces TH and DbetaH mRNA, and the cholinergic pathway is believed to be involved in the cold-stimulated increase in TH expression in the adrenal medulla. However, TH gene expression in the senescent rat is resistant to stimulation by cold exposure, suggesting that the cholinergic pathway may be impaired with age in the adrenal medulla. To investigate this possibility, we administered carbachol (0.5 mg/kg i.p., every 12 hours for 3 consecutive days), a mixed nicotinic-muscarinic agonist, to young (4-month-old) and senescent (24-month-old) male F-344 rats. We examined the induction of TH mRNA, TH immunoreactivity, and TH enzyme activity in the adrenal medulla in young and old rats. In addition DbetaH and NPY mRNA levels were determined in the adrenal medulla with or without carbachol administration. Basal levels of TH mRNA, TH immunoreactivity, and TH activity as well as DbetaH and neuropeptide Y (NPY) mRNA were 1.5- to 4-fold greater in the adrenal medullae of old rats compared with young rats. Carbachol administration increased TH mRNA, TH immunoreactivity, and TH activity as well as DbetaH and NPY mRNA to the same or a greater extent in the senescent compared with the young rats. The present study indicates that the cholinergic induction of TH or DbetaH are not impaired with age, and that senescent rats retain the capacity to respond to carbachol stimulation. The present findings cannot explain why the adrenal medullae from senescent rats are resistant to the cold-induced elevation of TH mRNA and TH activity observed in young rats.

Regulation of tyrosine hydroxylase gene expression by muscarinic agonists in rat adrenal medulla.

Tyrosine hydroxylase (TH) gene expression in the adrenal medulla is regulated by numerous stimuli via transsynaptic mechanisms. The adrenal chromaffin cell receptors that mediate this transsynaptic response remain unidentified. In this report we demonstrate that the muscarinic acetylcholine receptor agonist bethanechol stimulates the TH gene transcription rate in both innervated and denervated adrenal glands. Hence, this muscarinic response is not dependent on transsynaptic influences, suggesting that agonist occupation of adrenal chromaffin cell muscarinic receptors is sufficient to activate intracellular signaling pathways that stimulate the TH gene. When bethanechol is administered repeatedly over a 3-h interval (four injections spaced 1 h apart), TH mRNA levels are increased two- to threefold at 6 and 12 h after the initial injection of drug. It is surprising that this induction of TH mRNA does not lead to increases in TH activity or TH protein level. These results are consistent with the hypothesis that both transcriptional and posttranscriptional mechanisms must be regulated to induce TH protein and that muscarinic agonists activate only a subset of these mechanisms.

Role of H2O2 and heme-containing O2 sensors in hypoxic regulation of tyrosine hydroxylase gene expression.

In the current study, we investigated links between O2-regulated H2O2 formation and the hypoxic induction of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in O2-sensitive PC-12 cells. During exposure of PC-12 cells to 5% O2, H2O2 concentration decreased by 40% as measured with 2',7'-dichlorofluorescein (DCF). Treatment with H2O2 reduced TH mRNA during normoxia and prevented the induction of TH mRNA during hypoxia. Treatment with catalase or N-(2-mercaptopropionyl)-glycine, a reducing antioxidant agent that decreases H2O2 concentration, also induced TH mRNA. Deferoxamine (DF), an iron chelator, failed to affect H2O2 formation but induced TH mRNA in normoxia and hypoxia. CoCl2 led to a decrease in H2O2 at 20 h of treatment but induced TH mRNA during normoxia and hypoxia before it affected H2O2. In conclusion, TH gene expression correlates inversely with H2O2 formation. DF and CO2+ seem to affect TH gene expression in the mechanism downstream from the H2O2 formation rather than by interfering with the H2O2-generating activity of the O2 sensor.

Induction of tyrosine hydroxylase by forskolin: modulation with age

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of tyrosine hydroxylase messenger RNA (mRNA) levels and tyrosine hydroxylase enzyme activity in the adrenal medulla of senescent compared with younger animals. In addition, tyrosine hydroxylase gene expression in the senescent rat is resistant to further stimulation by cold exposure as compared with younger animals. Collectively, these observations suggest either that tyrosine hydroxylase expression is already maximally stimulated in senescent rats or that tyrosine hydroxylase gene induction pathways are impaired with senescence. To help distinguish between these possibilities, we examined the induction of tyrosine hydroxylase mRNA, tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity in the adrenal medulla following forskolin administration to young and old F-344 rats. Forskolin at doses of 1.8 and 3.5 mg/kg increased tyrosine hydroxylase mRNA levels 2.5-fold in adrenal medulla from young rats but did not increase either tyrosine hydroxylase immunoreactivity or tyrosine hydroxylase enzyme activity 5 h after administration. Prolonged treatment with forskolin (3 doses, 12 h apart) increased tyrosine hydroxylase mRNA levels and tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity. In senescent rats, the baseline level of tyrosine hydroxylase mRNA was more than 2-fold higher compared with young rats. A single injection of the lower dose of forskolin increased tyrosine hydroxylase mRNA levels by the same increment in senescent as compared with young rats. These data indicate that the tyrosine hydroxylase gene in the adrenal medulla from senescent rats is still capable of further stimulation. © 1997 Elsevier Science B.V. All rights reserved.

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

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.

The effect of chronic ethanol treatement and cold stress on catecholaminergic enzyme activity and mRNA in rat brain and adrenals

Sprague-Dawley male rats (150 g) were chronically treated with 5 v/v % ethanol admixed with nutritionally complete liquid diet and fed ad libitum for 3 weeks. One half of each group was exposed to cold stress at 4°C either for 24 h (for determination of mRNA by in situ hybridization) or for 48 h (for determination of enzyme activity). Chronic ethanol treatment (ethanol) did not affect tyrosine hydroxylase(TH) mRNA level in locus coeruleus(LC) of brain and adrenal medulla(AM) compared to controls. Cold stress showed strong increase of TH mRNA level in LC and AM compared to controls. Pretreated ethanol reduced the increased TH mRNA level by cold stress in LC and AM. Ethanol did not affect TH activity in LC and adrenal glands(adrenals). Cold stress increased TH activity in LC but not in adrenals. Pretreated ethanol did not reduce the increased TH activity by cold stress in LC but this result was not shown in adrenals. Phenylethanolamine-N-methyltransferase(PNMT) activity in C1C2 and adrenals increased only in ethanol treated group. These results suggest that ethanol does not affect TH mRNA level and activity in LC and adrenals, but increases PNMT activity in C1C2 and adrenals in normal rat. It is also suggested that pretreated ethanol reduces the magnitude of cold stress response, that is induction of TH mRNA in LC and AM, and does not reduce the protein activation of TH that is also cold stress response in LC.

Regulation of tyrosine hydroxylase gene expression by the m1 muscarinic acetylcholine receptor in rat pheochromocytoma cells.

Tyrosine hydroxylase (TH) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca2+]i; these increases are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5'-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca2+/calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca2+/calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.

Regulation of tyrosine hydroxylase gene expression by the m1 muscarinic acetylcholine receptor in rat pheochromocytoma cells

Tyrosine hydroxylase ( TH ) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca 2+ ] i ; these increase are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5′-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca 2+ /calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca 2+ /calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells.

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.

Differential effects of the intraventricular administration of 6-hydroxydopamine on the induction of type II beta-tubulin and tyrosine hydroxylase mRNA in the locus coeruleus of the aging Fischer 344 rat.

Noradrenergic neurons of the locus coeruleus have been shown to respond to injury by increasing the synthesis of neurotransmitter (via the activation and induction of tyrosine hydroxylase, the rate-limiting catalyst in the production of catecholamines) and initiating compensatory axonal sprouting. However, this laboratory has recently described a significant deficit in the activation of tyrosine hydroxylase in the aged Fischer 344 rat, in contrast to the young and mature rat, following partial damage to cortical and hippocampal noradrenergic terminals induced by the neurotoxin 6-hydroxydopamine. To extend these observations, we measured changes in the relative levels of neuron-specific type II beta-tubulin and tyrosine hydroxylase mRNA in locus coeruleus neurons of 2, 12, and 24-month-old Fischer 344 rats following intraventricular infusions of 6-hydroxydopamine by using in situ hybridization histochemistry. These measures were used as markers of the responsiveness of these neurons to injury. 6-Hydroxydopamine treatment induced a persistent increase (at least 10 days) in the expression of type II beta-tubulin mRNA only in 2-month-old animals; this marker decreased in the 12 and 24-month-old animals. Relative levels of tyrosine hydroxylase mRNA increased in 2 and 12-month-old lesioned animals both 3 and 10 days post-treatment. In contrast, the induction of tyrosine hydroxylase mRNA in 24-month-old animals, seen three days post-treatment, was attenuated by 10 days. These data indicate that the capacity of locus coeruleus neurons to compensate for injury by either initiating a potential sprouting response or increasing their capacity to synthesize neurotransmitter is reduced in older animals.

Regulation of tyrosine hydroxylase gene expression in depolarized non-transformed bovine adrenal medullary cells: second messenger systems and promoter mechanisms

Activation of the tyrosine hydroxylase (TH) gene in the adrenal medulla during stress is mediated by trans-synaptic mechanisms and may involve cholinergic receptors. Stimulation of nicotinic receptors in adrenal medullary cells induces cell depolarization, influx of Ca 2+ ions and increases levels of cAMP. We have shown that both cAMP and membrane depolarization produce an increase in the expression of the TH gene in cultured bovine adrenal medullary cells (BAMC). Others have proposed that transcriptional activation of the TH gene by cAMP is mediated through the sequence homologous to a cAMP responsive element (CRE) located in the proximal region of the TH gene promoter. In the present study we have examined the mechanisms by which membrane depolarization increases the TH gene activity. Treatment of serum-free BAMC cultures with the depolarizing agent, veratridine, increased the extracellular concentration of catecholamines, Met 5-enkephalin, and the relative abundance of TH mRNA. Veratridine treatment also increased the levels of mRNAs for the catecholamine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT), and proenkephalin A (PEK). Treatment for longer than 3 h was required to increase TH mRNA levels. By contrast, our previous studies indicated that cAMP stimulation for 2 h produces a maximal increase in TH mRNA levels in BAMC. The effects of veratridine and forskolin on TH mRNA levels were additive, further indicating that depolarization and cAMP activate TH gene expression via different pathways. Calmidazolium, an antagonist of calmodulin, had no effect on the veratridine-induced increase in TH mRNA levels. Similarly sphingosine treatment or preincubation with PMA, which reduce protein kinase C (PKC) activity and attenuate the induction of TH mRNA by PMA or the hormone, angiotensin II, did not affect the induction by veratridine. To identify promoter mechanisms of TH gene activation in depolarized cells we transfected BAMC with a plasmid pTHgoodLuc and treated with veratridine for 24 h. pTHgoodLUC contains a luciferase reporter gene linked to a −428 +21 bp fragment of the bovine TH gene promoter (relative to the transcription start site). Veratridine increased the expression of luciferase from the TH promoter 2.5-fold. Deletion of the −194 −54 bp promoter region containing SP-1 and POU/Oct sites reduced veratridine stimulation by 40%. Additional deletion of the −269 to −190 bp promoter segment, including an AP-1 element, further reduced veratridine stimulation to a statistically non-significant level. In conclusion, activation of TH gene expression upon depolarization is not mediated by calmodulin and PKC. Promoter sequences involved in this activation are located upstream from the CRE. Depolarization may activate TH gene transcription by acting on more than one regulatory region.

Regulation of gene expression by hypoxia.

The present study was undertaken to determine if gene expression for tyrosine hydroxylase (TH), the rate limiting enzyme in the biosynthesis of catecholamines, is regulated in the carotid body, sympathetic ganglia and adrenal medulla by hypoxia. We found that a reduction in oxygen tension from 21% to 10% caused a substantial increase (200% at 1 hour and 500% at 6 hours exposure) in the concentration of TH mRNA in carotid body type I cells but not in either the sympathetic ganglia or adrenal gland. In addition, we found that hypercapnia, another natural stimulus of carotid body activity, failed to enhance TH mRNA in type I cells. Removal of the sensory and sympathetic innervation of the carotid body failed to prevent the induction of TH mRNA by hypoxia in type I cells. Our results show that TH gene expression is regulated by hypoxia in the carotid body but not in other peripheral catecholamine synthesizing tissue and that the regulatory mechanism is intrinsic to type I cells.

The neuropeptide VIP regulates the expression of the tyrosine hydroxylase gene in cultured avian sympathetic neurons

The neuropeptide vasoactive intestinal polypeptide (VIP) increases the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in cultured chicken sympathetic neurons. We report here that VIP acts by increasing TH mRNA levels in these cells. Induction of TH mRNA is transient and reaches maximal values 6–8 h after the addition of the peptide to the cultures. TH mRNA levels return to control values after 1–2 days. The quail cDNA probe detects a single mRNA species of approximately 9 kb in RNA extracted both from embryonic chicken sympathetic neurons and adult quail adrenal medulla.