Regulation Of Tyrosine Hydroxylase Gene Expression Research Articles

Expression Levels of the Tyrosine Hydroxylase Gene and Histone Modifications Around its Promoter in the Locus Coeruleus and Ventral Tegmental Area of Rats during Forced Abstinence from Morphine

Background: Epigenetic mechanisms such as histone modifications may be involved in the structural and behavioral changes associated with addiction. We studied whether morphine-induced changes in mRNA levels of the catecholamine biosynthesis enzyme, tyrosine hydroxylase (TH), are associated with histone modifications around the promoter of this gene in the locus coeruleus (LC) and ventral tegmental area (VTA) of rats. Methods: Dependence was induced in rats by intraperitoneal injections of morphine for 11 days. The animals were killed 2 h (chronic morphine), 24 h and 7 days (spontaneous withdrawal) after the last injection of morphine. Results: Analysis of our real-time quantitative reverse transcription PCR results by 1-way ANOVA showed significant upregulation (5.13 ± 0.39 folds) of LC levels of the TH transcript 24 h after the last injection of morphine to rats, when compared with 2 h and 7 days time points. Chronic morphine and morphine abstinence failed to cause any significant changes in the levels of TH mRNA in the VTA after cessation of morphine. Consistently, chromatin immunoprecipitation real-time quantitative PCR assays revealed that 24 h after the last injection of morphine, levels of H3 acetylation were significantly increased (4.12 ± 0.38 folds) at the promoter of the TH gene in the LC but not in the VTA. Our data also showed that histone H3 trimethylation failed to change around the TH gene promoter either in the VTA or in the LC after morphine abstinence. Conclusions: Results of the present study, for the first time, demonstrate the involvement of histone H3 acetylation in the regulation of TH gene expression in the LC of rats during forced abstinence from morphine.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

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.

12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐dependent up‐regulation of dopaminergic gene expression requires Ras and neurofibromin in human IMR‐32 neuroblastoma

The dopaminergic transcriptional programme is highly regulated during development and in the adult, in response to activation of membrane receptor signalling cascades. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, is known to be regulated by receptors that act through protein kinase C (PKC) or Ras signalling. To investigate possible interactions between these two pathways before they converge on Raf activation, we evaluated whether phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA)-dependent PKC activation required Ras for regulation of TH expression in IMR-32 cells. We found that long-term treatment with TPA, which induces down-regulation of PKC-alpha, led to induction of both protein and message levels of TH by autocrine factors. This was dependent on endogenous Ras, but independent of the transcription factor Nurr1. Moreover, this mechanism of action mimicked the effects of overexpression of the Ras-GAP domain of neurofibromin, GAP-related domain (GRD) I, which is part of the upstream mechanism for regulation of Ras activation and a PKC-alpha substrate. Overexpression of Ras also led to transcriptional and translational up-regulation of TH, independent of Nurr1 induction, as well as distinct phenotypic changes consistent with cell hypertrophy and increased secretory activity shown by induction of expression of vesicular monoamine transporter 2 and synaptosomal-associated protein-25. Most interestingly, overexpression of GRDI and down-regulation of the endogenous GRDII neurofibromin led to significant increases in Nurr1 message, possibly reflecting a transcriptional hierarchy during development. Taken together, these studies suggest that PKC-alpha, neurofibromin and Ras are essential in regulation of TH gene expression in IMR-32 cells.

The tissue‐specific methylation of the human Tyrosine Hydroxylase gene reveals new regulatory elements in the first exon

The methylation status of CpG dinucleotides located in or near regulatory elements affects gene expression. The CpG-rich sequence located outside the 5' promoter region of the human Tyrosine Hydroxylase (TH) gene appears to influence the functional effect of the adjacent intronic HUMTH01 microsatellite. In order to identify new regulatory elements in this region acting on gene expression, the methylation profile of the TH CpG island was investigated using the bisulfite sequencing method. The overall methylation level of this region is correlated to TH-expressing and non-expressing status in cell lines and DNA demethylation treatment with 5-azacytidine increased TH expression. Moreover, in a homogeneous background of methylated CpGs, a single CpG in the first exon of the gene is constantly either unmethylated or methylated in, respectively, TH-expressing or non-expressing cell lines, tissues and single cells. Further analysis ascertained that this CpG is contained in a sequence characterized by putative binding sites for the AP2, Sp1 and KAISO factors. Characterization of this sequence shows that these factors specifically bind their respective sites. Finally, the binding of KAISO, a transcriptional repressor, is conditioned by the methylation of this sequence, which may, thus, participate in the regulation of TH gene expression according to its methylation pattern.

Regulation of tyrosine hydroxylase promoter activity by the von Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription factors.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen-sensitive cells of the carotid body and pheochromocytoma-derived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFalpha ubiquitination, increased accumulation of HIFalpha proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter-reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

Regulation of tyrosine-hydroxylase (TH) gene expression by hypoxia and von Hippel-Lindau tumor suppressor complex

Loss of von Hippel-Lindau (VHL) gene function leads to a familial cancer syndrome, VHL disease, characterized by CNS and retinal hemangioblastomas, renal clear cell carcinomas, and pheochromocytomas. Pheochromocytomas, tumors of the chromaffin cells of the adrenal medulla, secrete large amounts of catecholamines. This results from enhanced expression of TH, the rate-limiting enzyme in catecholamine biosynthesis. Overexpression of exogenous pVHL in PC12 cells lead to a substantial repression of TH protein and mRNA [1]. Here we examined the role of endogenous pVHL in the regulation of TH gene expression. We developed PC12 clonal cell lines that stably express VHL antisense RNA and have reduced levels of endogenous pVHL by 5–10 fold. In these cells, TH protein and mRNA were increased 2–3 fold as compared to control cells. Nuclear run-on analysis revealed an increase in the overall TH gene transcription. In addition, we measured augmented efficiency of the TH transcript elongation in the distal part of the gene. Transient co-transfections of -773 kb TH-CAT promoter-reporter constructs with the VHL antisense RNA resulted in an approximately 2–3 fold increase in reporter gene activity. TH is a hypoxia-inducible gene. The overall accumulation of THmRNA and the rate of TH gene transcription were substantially enhanced in cells with decreased levels of pVhl exposed to 1% O2 for 16 h. These results indicate that endogenous pVhl constitutively represses TH gene expression in PC12 cells. TH promoter contains a functional hypoxia-responsive element (HRE) - like sequence. Transient co-transfections of the HIF1α and HIF2α expression vectors with the wild type, but not mutated TH promoter revealed significant 3–4 fold induction of the reporter gene activity. Gel-shift analysis showed presence of the hypoxia-inducible complexes associated with the wild type but not mutated THHRE. Electroelution of protein complexes from the hypoxia-inducible complex showed presence of the HIF2α, and to lesser extent of the HIF1 subunit. pVHL is part of the E3 ubiquitin ligase complex that targets the HIF alphas for ubiquitination and degradation. Changes in the levels of the pVHL in PC12 cells by either overexpression of pVHL or expression of VHL antisense RNA resulted in respectively decreased and increased HIF-and hypoxia-mediated activation of the TH promoter. Stable changes in the pVHL levels in PC12 cells resulted in accumulation of HIF alphas during normoxia in cells with decreased amounts of pVHL, and in decreased expression of HIF alphas during hypoxia in cells overex-pressing pVHL. The changes in HIFα protein accumulation induced by pVHL resulted from ubiquitination of HIF alphas by pVHL.

Requirement for cAMP/calcium response element but not AP-1 site in fibroblast growth factor-2-elicited activation of tyrosine hydroxylase gene expression in PC12 cells

Basic fibroblast growth factor (FGF-2) mediates numerous important physiological processes, including differentiation and survival of dopaminergic neurons. FGF-2 was found to trigger elevation of tyrosine hydroxylase (TH) gene expression in PC12 cells that was sustained for 1–8 days. FGF-2 induced chloramphenicol acetyltransferase (CAT) reporter activity under control of the TH promoter, indicating that the induction is transcriptionally mediated. The transcriptional activation of TH by FGF-2 was examined using various deletions and point mutations of the 5′ flanking region controlling CAT reporter activity. In contrast to the reported mechanisms of transcriptional regulation of TH expression by NGF and phorbol esters, the AP-1 site at −205/−199 was not required for the activation by FGF-2. A construct containing only 60 nucleotides of the promoter was still inducible by FGF-2. However, a construct with a point mutation in the CRE/CaRE was not responsive to induction by FGF-2. These findings indicate that the CRE/CaRE, but not the AP-1, element is required for induction by FGF-2 and point to differences between NGF and FGF-2 in the regulation of TH gene expression.

Control of tyrosine hydroxylase gene expression in chromaffin and PC12 cells

Chromaffin and PC12 cells were used to elucidate mechanisms of stimulation of gene expression of tyrosine hydroxylase (TH), the pivotal enzyme in catecholamine biosynthesis. A plethora of treatments elevate TH gene expression. Elevated cAMP triggers increased TH gene expression, primarily by an increase in mRNA transcription. Although a variety of treatments that elevate [Ca2+]i, increase TH transcription, there are important differences among them, indicating different calcium signalling pathways in regulating TH gene expression acting at the CRE/CaRE (cyclic AMP/calcium response element). Several complexes, such as ATF-1/jun and CREB/AT-1 heterodimers bind this element. Activation of protein kinase C by phorbol esters, NGF and EGF activate TH transcription through the AP-1 site. TH transcription is also stimulated by glucocorticoids, increased cell density, hypoxia, and several other treatments. In some instances, post-transcriptional mechanisms also contribute to the intricate fine tuning of regulation of TH gene expression.

Involvement of intracellular or extracellular calcium in activation of tyrosine hydroxylase gene expression in PC12 cells.

The relationship between elevations in intracellular free Ca2+ concentration ([Ca2+]i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K+ concentration triggered rapid and sustained increases in [Ca2+]i from a basal level of approximately 50 to 110-150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP3). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP3 or inhibition of Ca(2+)-ATPase, rapidly elevated [Ca2+]i to > 200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca2+]i in each case occurred throughout the cyto-and nucleoplasm. The initial rise in [Ca2+]i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca2+]i for < 10 min, as opposed to 10-30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca2+]i, there are important differences among them in terms of the magnitude of elevated [Ca2+]i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca2+]i, indicating the involvement of different calcium signalling pathways leading to regulation of TH gene expression.

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.

A Transgenic Mouse Model to Study Transsynaptic Regulation of Tyrosine Hydroxylase Gene Expression

Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5' flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type-specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity-dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down-regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg s.c.), which up-regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X-gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine-treated transgenic animals. These data showed that the 9-kb sequence also mediates cell type-specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.

Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by age and reserpine

Tyrosine hydroxylase (TH), TH messenger RNA (TH mRNA) and dopamine (DA) were measured simultaneously in adrenal glands of individual Fischer 344 rats aged 2, 6, 13 and 23 months. Between 2 and 23 months TH activity rose 2-fold as compared to the youngest group. TH mRNA content of the adrenal gland rose 3-fold between 2 and 23 months. A 3-fold increase in adrenal DA content, the first catecholamine product of TH, provides evidence that the increases in TH gene expression are functionally significant. To determine if mechanisms that regulate gene expression are altered by aging, the effects of reserpine on induction of TH mRNA and TH activity were compared in another group of rats aged 2, 12 and 27 months. Consistent with the results of the first experiment, there were age-related increases in both TH activity and TH mRNA in the age-matched control groups. TH activity rose 2-fold and TH mRNA rose more than 6-fold between 2 and 27 months. The discrepancy in the relative magnitudes of increases in TH mRNA and TH protein suggest an uncoupling of regulation of TH mRNA and TH protein levels. Moreover, there were significant age-related differences with respect to modulation of TH gene expression by reserpine treatment. TH activity was induced by reserpine in the youngest group, but not in the two older age-groups. In contrast, reserpine caused significant induction of TH mRNA in all age groups. These results provide evidence that aging is accompanied by alterations in transcriptional and post-transcriptional mechanisms involved in regulation of TH gene expression.

Isolation and structural characterization of the bovine tyrosine hydroxylase gene

A bovine tyrosine hydroxylase (TH) cDNA probe was used to screen a charon 30 genomic library. Screening of approximately 1 million recombinant phage resulted in the identification of one clone, lambda B1, containing the entire bovine TH gene. Results derived from restriction endonuclease mapping and sequence analysis reveal that the bovine gene contains 13 exons spanning approximately 7 kb of genomic DNA. Determination of the transcription initiation site indicates that the TH gene has a 5' untranslated region of 27 bp. A TATA-box sequence is located between positions-29 and -24 from the transcription initiation site and a cyclic AMP regulatory element (CRE) between-45 and -38. Although the TH gene appears to be glucocorticoid responsive in vitro, no regions bearing identity to the consensus sequence for the glucocorticoid regulatory element (GRE) were detected within approximately 1.5 kb of 5' flanking sequence. A cross-species comparison of the 5' flanking sequences of the bovine, rat, and human TH genes reveals strong sequence and positional conservation of seven sequence elements. An analysis of the nucleotide sequence within these elements reveals similarity to the consensus sequences reported for known cis-acting regulatory elements and transcription factor binding sites, suggesting that they may play a role in the regulation of TH gene expression.

Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms.

A single human gene has been described to encode multiple tyrosine hydroxylase (TH) mRNAs. The study of this variation has been extended by S1 mapping experiments and by analysis of the 5' region of the TH gene. Four different mRNAs were found to originate solely from alternative splicing of two exons. Comparison of the 5' flanking regions of human and rat genes discloses several highly conserved segments, likely to play an important role in the regulation of TH gene expression.

Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligodeoxyribonucleotide probe.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines in neural tissues and adrenal medulla. To study the expression of the TH gene and its regulation in adult and developing neural tissues, we have synthesized an oligodeoxyribonucleotide probe (oligomer) that is specific for TH mRNA. Using Northern blot hybridization of polyadenylated RNAs from adrenal gland, brain stem, liver, and cerebral cortex with the 32P-labeled oligomer, a single TH mRNA of 1.9 kb was detected in adrenal gland and brain stem but not in liver and cerebral cortex. Using this TH-specific oligomer, TH mRNAs were localized to the chromaffin cells in the adrenal medulla and to catecholaminergic neurons in locus coeruleus and substantia nigra by in situ hybridization histochemistry. After reserpine administration, the intensity of hybridization signal was increased to threefold that of normal in sections of adrenal medulla and twofold that of normal in locus ceruleus. No difference in hybridization signal intensity was observed in the substantia nigra of normal and reserpine-treated animals. Use of this specific TH probe in in situ hybridization procedures represents a powerful approach to the study of regulation of TH gene expression at the cellular level.