Unique and cell-type-specific tyrosine hydroxylase gene expression.
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Activating Transcription Factors↗
Biomedical subjects
T H Joh
Publications and source records attributed to T H Joh.
Structure/function relationship of the cAMP response element in tyrosine hydroxylase gene transcription.
Expression of tyrosine hydroxylase (TH) is limited to catecholamine-producing neurons and neuroendocrine cells in a cell type-specific manner and is inducible by the cAMP-regulated signaling pathway. Previous results indicated that the cAMP response element (CRE) residing at -45 to -38 base pairs upstream of the transcription initiation site is essential for both basal and cAMP-inducible promoter activity of the 2.4-kilobase or shorter upstream sequence of the TH gene (Kim, K. S., Lee, M. K., Carroll, J. , and Joh, T. H. (1993) J. Biol. Chem. 268, 15689-15695; Lazaroff, M. , Patankar, S., Yoon, S. O., and Chikaraishi, D. M. (1995) J. Biol. Chem. 270, 21579-21589). Here, we further report that the CRE is critical for the promoter activity of the 5.6- or 9.0-kilobase upstream sequences of the rat TH gene, which had been shown to direct the cell-specific TH expression in vivo. To define the structure/function relationship of the CRE in transcriptional activation of the TH gene, we performed saturated mutational analyses of 12 nucleotides encompassing the CRE. Mutation of any nucleotide within the octamer motif results in a significant decrease of both basal and cAMP-inducible transcriptional activity of the TH reporter gene construct. Among the four nucleotides adjacent to the CRE (two 5' and two 3'), only the G residue at the immediate 3' position is important for full transcriptional activity. DNase I footprint analysis indicates a positive correlation between in vivo promoter activity and in vitro interaction between the CRE motif and its cognate protein factor(s). Reconstruction experiments using a TH promoter in which the native CRE was rendered inactive show that the CRE can transactivate transcription in either orientation through a window of approximately 200 base pairs upstream of the transcription initiation site, suggesting that CRE supports transcriptional activation of the TH gene in a distance-dependent manner. Finally, when the distance between the CRE and TATA box was changed by inserting an additional 5 or 10 bases, it was observed that both insertional mutations increased activity by approximately 3-fold. The cAMP inducibility was as intact as the wild type construct. Together, these results are consistent with a model in which transcriptional activation of the TH gene by the CRE requires that it be located within a certain proximity of the CAP site but does not depend on a stringent stereospecific alignment in relationship to the TATA element.
Animals↗
Monitoring catecholamine differentiation in the embryonic brain and peripheral neurons using E. coli lacZ as a reporter gene.
An X-gal based histochemical assay was used to detect catecholamine (CA) cells in transgenic mouse embryos, in which the expression of the lacZ reporter was driven by the tissue-specific promoter of the rat tyrosine hydroxylase (TH) gene. As the first enzyme in the biosynthetic pathway for CA neurotransmitters, TH is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals. During embryogenesis, TH expression appears permanently within CA-producing cells, and transiently within several other cell types. In this study we were able to monitor TH expression in transgenic mouse embryos by following the expression of the lacZ reporter in substantia nigral dopaminergic neurons in the central nervous system, the trigeminal (V) sensory ganglia, and dorsal root ganglia in the periphery. Our results demonstrate that the rat TH promoter-lacZ transgene provides an important experimental tool for monitoring catecholaminergic lineage cells during embryogenesis.
Animals↗
Melatonin administration protects CA1 hippocampal neurons after transient forebrain ischemia in rats.
Melatonin administered at the beginning of cerebral reperfusion protected CA1 neurons against 10, 20 and 30 min of transient forebrain ischemia. Intraperitoneal injections of saline or melatonin (10 mg/kg) were given after 0, 2 and 6 h, or 1, 2 and 6 h of cerebral reperfusion, or 30 min prior to ischemia. One week later, quantitative histological analysis demonstrated that CA1 neuronal density was significantly increased in the melatonin groups that were treated at 0, 2, 6 h compared to the saline-treated controls. Ischemic protection of CA1 was lost in the animals in which the melatonin treatment was delayed by 1 h, or given 30 min prior to the ischemia.
Analysis of Variance↗
Induction of interferon-gamma inducing factor in the adrenal cortex.
Interferon-gamma inducing factor (IGIF) is a recently identified cytokine also called interleukin-1gamma (IL-1gamma) or interleukin-18 (IL-18). Its biological activity is pleiotropic, and, so far, it has been shown to induce interferon-gamma production in Th1 cells, to augment the production of granulocyte-macrophage-CSF, and to decrease that of interleukin-10 (IL-10). We first detected newly synthesized IGIF mRNA by differential display in the adrenal gland of reserpine-treated rats and then isolated two transcripts by reverse transcription polymerase chain reaction. They were identified as rat IGIF on the basis of the high homology with mouse: 91% at both the nucleotide and the amino acid level. Subsequently, we investigated the effects of stress on IGIF mRNA levels and found that acute cold stress strongly induced IGIF gene expression. In situ hybridization analysis showed that IGIF is synthesized in the adrenal cortex, specifically in the zona reticularis and fasciculata that produce glucocorticoids. The presence of IGIF mRNA was also detected in the neurohypophysis although induction by stress was not significant. Our results call for more attention to the role of the adrenal gland as a potential effector of immunomodulation and suggest that IGIF itself might be a secreted neuroimmunomodulator and play an important role in orchestrating the immune system following a stressful experience.
Adrenal Cortex↗
Localization of monoamine oxidase A and B mRNA in the rat brain by in situ hybridization.
Monoamine oxidases A and B (MAOA and MAOB) are the major catabolic isoenzymes of catecholamines and serotonin in the mammalian brain. Although the distribution of the monoamine oxidase protein has been mapped by ligand binding and immunohistochemistry, the sites of MAOA and MAOB synthesis have not been precisely determined. In this study, we used in situ hybridization to visualize MAOA and MAOB mRNA in the rat brain by using specific cDNA and oligonucleotide probes. MAOA mRNA was localized in major monoaminergic cell groups, such as the dorsal vagal complex, the C1/A1 groups, the locus ceruleus, the raphe nuclei, the substantia nigra, and the ventral tegmental area. MAOA mRNA was also found in forebrain structures, such as the cortex, the hippocampus, the thalamus, and the hypothalamus. In contrast to the distribution of MAOA mRNA, high levels of MAOB mRNA were present in only three brain regions: the area postrema, the subfornical organ, and the dorsal raphe. The in situ visualization of MAO mRNA demonstrates that MAOA mRNA synthesis is wide spread in many catecholaminergic and serotonergic cell groups, whereas MAOB mRNA synthesis is far more discrete and limited. The different expression patterns of MAOA and MAOB suggests that may also have different physiological functions.
Animals↗
Expression of catecholamine-synthesizing enzymes, peptidylglycine alpha-amidating monooxygenase, and neuropeptide Y mRNA in the rat adrenal medulla after acute systemic nicotine.
The expression of catecholamine-synthesizing enzymes in the adrenal medulla is upregulated in parallel by stress and pharmacological treatments. In this study we examined whether a neuropeptide and its processing enzyme are regulated in parallel with catecholamine enzyme genes after drug treatment. Because the main effect of stress on the adrenal medulla is via splanchnic nerve stimulation of nicotinic receptors, we used nicotine to stimulate the medulla and visualized expression of catecholamine enzyme genes, the medullary peptide neuropeptide Y (NPY), and the neuropeptide-processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) by in situ hybridization quantified by image analysis of autoradiographic images. Rats received a single injection of nicotine (0, 1, or 5 mg/kg sc). Six hours later, rats were transcardially perfused. Free-floating adrenal gland sections were hybridized with 35S-labeled cDNA probes for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), PAM, and NPY. Nicotine treatment upregulated the expression of TH, PNMT, and NPY genes in a dose-dependent fashion. Small but nonsignificant increases were observed in DBH and PAM mRNA levels. These results suggest that common transcriptional activation mechanisms may upregulate both catecholamine and neuropeptide synthesis in the adrenal medulla after nicotinic stimulation.
Adrenal Medulla↗
Stimulation of human DBH gene expression by prostaglandin E2 in human neuroblastoma SK-N-BE(2)C cells.
Prostaglandin E2 (PGE2) enhances transcription of the human dopamine beta-hydroxylase (DBH) gene in human neuroblastoma SK-N-BE(2)C cells. To identify a PGE2-responsive cis-acting element in the human DBH gene, serial deletion constructs of the human DBH 5'-upstream region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into SK-N-BE(2)C cells. Treatment of the transformed cells with PGE2 increased CAT expression two- to threefold in all constructs except where the promoter region was shortened beyond position -114 bp. There are several cis-regulatory elements in the region between -262 and -114 bp from the transcription initiation site that include a cyclic AMP response element (CRE) and a putative AP1 sequence. We presupposed that the CRE and AP1 might be candidates for PGE2 stimulation, and therefore, used site-directed mutagenesis to change the CRE and AP1 motives and test which of the two elements mediated the transcriptional enhancement. Only a specific mutation within the CRE sequence abolished the PGE2 effect. In addition, cotransfection with an expression vector expressing PKA inhibitor resulted in the specific blockage of the PGE2 effect on DBH gene expression. Northern blot analysis revealed that the increase in DBH gene transcription caused by PGE2 results in elevated DBH mRNA levels. Gel-retardation and competition assays confirmed that the binding of nuclear factors to the CRE site is sequence specific. Our data, therefore, indicate that PGE2 enhances the transcription of the human DBH gene. The effect is mediated by the CRE motif through activation of PKA.
Cyclic AMP Response Element-Binding Protein↗
Genetically engineered neural transmission.
Neural transmission is a communication between neurons and target cells, resulting in behavioral and physiological changes. Defective or altered neural transmission is thought to occur in neuropsychiatric and neurodegenerative illnesses. To probe the biological consequences of defective or altered neural transmission, various genetically engineered transgenic mouse models have been developed, together with conventional pharmacological manipulation. Via genetic manipulation, we are able to engineer specific neurotransmitters, receptors, inactivation of neurotransmitters or neural innervation density. Moreover, recently developed molecular genetic techniques make it possible to induce either a gene knock out event or transgene expression at a discrete time point in a specific neuronal population in both embryos and adult animals. In conjunction with pharmacological manipulation, these sophisticated genetic manipulations of neural transmission will provide new tools to control neural transmission in both normal and pathophysiological conditions.
Animals↗
Two distinct P2 purinergic receptors, P2Y and P2U, are coupled to phospholipase C in mouse pineal gland tumor cells.
We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-beta) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5'-O-(3-thiotriphosphate) > or = UTP > 2-chloro-ATP > 3'-O-(4-benzoyl)benzoyl ATP, GTP > or = 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) > CTP. AMP, adenosine, alpha,beta-methyleneadenosine 5'-triphosphate, beta,gamma-methyleneadenosine 5'-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP- and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4- and UTP4- concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADP beta S induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of approximately 100 microM. In sequential stimulation, UTP and ADP beta S do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADP beta S results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45-55%, whereas it has no effect on the ADP beta S response. Treatment with 1 microM phorbol 12-myristate 13-acetate inhibits the ADP beta S-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-beta cells and that both receptors are linked to phospholipase C.
Adenosine Diphosphate↗
Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector.
A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac). The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control. LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis. Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site. These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry. Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection. In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN. However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression. These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport. In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively. Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector. Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors.
Defective Viruses↗
Inducible cAMP early repressor can modulate tyrosine hydroxylase gene expression after stimulation of cAMP synthesis.
Members of the CREB/CREM/ATF family of transcription factors either enhance or repress transcription after binding to the cAMP response elements (CREs) of numerous genes. The rat gene for tyrosine hydroxylase (TH) bears a canonical CRE, at base pairs -38 through -45 from the transcription initiation site, that is essential for basal and cAMP-stimulated transcription (Kim, K.-S., Lee, M. K., Carroll, J., and Joh, T. H. (1993) J. Biol. Chem. 268, 15689-15695; Lazaroff, M., Patankar, S., Yoon, S. O., and Chikaraishi, D. M. (1995) J. Biol. Chem. 270, 21579-21589). The current study identifies CRE-binding proteins induced in pharmacological paradigms characterized by TH activation. PC12- and rat adrenal gland-derived nuclear proteins retarded a TH-CRE oligonucleotide in gel mobility shift assays with virtually identical patterns. These differed substantially from patterns exhibited by extracts from locus ceruleus or from neuroblastoma (SK-N-BE()C) and locus ceruleus-derived (CATH.a) cell lines. Forskolin stimulation of PC12 cells and reserpine treatment of rats increased, in nuclear extracts derived from cells and adrenal glands, respectively, the amount of a fast moving CRE/protein complex that was supershifted by an anti-CREM antibody. Subsequent Western, Northern, and polymerase chain reaction analyses indicated that a specific member of the CREM family, the inducible cAMP early repressor (ICER), was strongly induced in both systems. Cotransfection of PC12 cells with TH2400CAT plasmid and the expression vector pCMV-ICER-Ib demonstrated that ICER efficiently represses the transcriptional activity of the TH gene promoter. In addition, PKA-stimulated transcriptional activity of the promoter was effectively suppressed by ICER. These results suggest that ICER can modulate cAMP-stimulated transcription of the TH gene and provide a model accounting for rapid reversal of increased TH transcription following elevations in cAMP.
Activating Transcription Factors↗
Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.
Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development.
Animals↗
Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene.
As the first and rate limiting enzyme in the biosynthetic pathway for catecholamine (CA) neurotransmitters, tyrosine hydroxylase (TH) is a specific phenotypic marker for CA cells in the central and peripheral nervous systems of adult animals. During embryogenesis, TH expression appears permanently within cells destined to be CA-secreting during adult life, and transiently in several cell types that will not express TH in adulthood. In this study, we examined the early ontogeny of TH expression in transgenic mouse embryos by following the expression of a lacZ reporter, driven by the tissue-specific promoter of the rat TH gene. The lacZ reporter product, beta-galactosidase (beta-gal), visualized by X-gal staining, first became apparent in primordia of sensory ganglia serving the glossopharyngeal (IX) and vagal (X) cranial nerves at embryonic day (E)9.0. Between E9.5 and E10.5, beta-gal expression extended to the remaining cranial sensory ganglia serving the trigeminal (V) and facial (VII) nerves, dorsal root ganglia, ventrolateral neural tube and sympathetic ganglion primordia. During that same period, the first beta-gal expression in the embryonic brain also appeared within distinct regions, such as the ventral prosencephalon, the ventral and dorsolateral mesencephalon and the rostral and caudal rhombencephalon. The level of beta-gal expression in all these tissues decreased at E13.5, but a distinct adult pattern of beta-gal expression started to emerge in the substantia nigra and ventral tegmental area in the central nervous system and the adrenal medulla in the periphery. Our findings indicate that the proximal 9.0 kb of the 5' promoter region of the rat TH gene encodes sufficient information to direct development of the appropriate catecholaminergic lineage cells in the central and most peripheral nervous systems during embryogenesis.
Animals↗
Immortalization of neuroendocrine pinealocytes from transgenic mice by targeted tumorigenesis using the tryptophan hydroxylase promoter.
Tryptophan hydroxylase (TPH) is the first enzyme in both serotonin and melatonin biosynthesis in neuroendocrine cells of the pineal gland. The lack of immortalized neuroendocrine pineal cell lines has been a major obstacle to the study of the tissue-specific and circadian regulation of TPH gene expression in the pineal gland. Previously, we demonstrated that a 6.1 kb 5' upstream region of the mouse TPH gene directs the restricted expression of a lacZ reporter gene to the pineal gland and the raphe nuclei of transgenic mice. Therefore, to develop TPH-expressing pineal cell lines we first established transgenic mice carrying a construct consisting of 6.1 kb of 5' flanking region fused to the SV40 T-antigen. These animals developed highly invasive pineal tumors and died at 12-15 weeks of age. The pineal tumors obtained from the transgenic mice were utilized to establish the immortalized pinealocyte-derived cell lines. These cells express two marker enzymes, TPH and serotonin N-acetyltransferase (NAT). In pineal gland TPH and NAT expressions have been known to be regulated during circadian cycle. The two established cell lines therefore promise to be a valuable in vitro model system for the study of the rhythmic nature of the pineal function at molecular level in mammal.
Animals↗
c-Fos induction in the rat nucleus of the solitary tract by intraoral quinine infusion depends on prior contingent pairing of quinine and lithium chloride.
Intraoral infusions of sucrose or saccharin induce c-Fos-like immunoreactivity (c-FLI) in the intermediate nucleus of the solitary tract (iNTS) of rats after acquisition of a conditioned taste aversion (CTA). The induction of c-FLI in the iNTS may be a consequence of the shift in behavioral response from ingestive to aversive behaviors that characterize acquisition and expression of a CTA. To test this hypothesis, rats were intraorally infused with 0.3 mM quinine sulfate, an aversive taste, 1. prior to conditioning, 2. after 3 noncontingent (unpaired) infusions of quinine and toxic lithium chloride (LiCl) injections, 3. after conditioning with 3 contingent pairings of quinine and LiCl, and 4. after extinction with repeated unpaired infusions of quinine. Intraoral infusions of quinine induced c-FLI in the iNTS only after acquisition of a CTA against quinine; quinine failed to induce c-FLI in the iNTS of unconditioned, noncontingently treated, or extinguished rats. The pattern of c-FLI in the iNTS induced by expression of a CTA against quinine was quantitatively and anatomically similar to that elicited by sucrose in rats expressing a CTA against sucrose. We conclude that aversive responses per se are not sufficient to induce c-FLI in the iNTS. Furthermore, contingent pairing of quinine and LiCl does not cause a shift in behavioral response from palatable, ingestive behaviors to aversive behaviors as in acquisition of a CTA against sucrose. Thus, we also conclude that a shift in behavior from ingestive to aversive responses is not required for increased c-FLI expression in the iNTS during CTA expression. Therefore, the induction of c-FLI in the iNTS during expression of a CTA may be correlated with neuronal processes specific to acquisition and expression of a CTA.
Animals↗
Alternate promoters in the rat aromatic L-amino acid decarboxylase gene for neuronal and nonneuronal expression: an in situ hybridization study.
Aromatic L-amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue-specific expression of the rat AADC gene.
Adrenal Gland Neoplasms↗
AP1-mediated transcriptional enhancement of the rat tyrosine hydroxylase gene by muscarinic stimulation.
We investigated transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by muscarinic stimulation in human neuroblastoma SK-N-BE(2)M17 cells. Carbachol treatment increased the levels of intracellular Ca2+ and inositol 1,4,5-trisphosphate (IP3) and enhanced transcription of the TH gene. The muscarinic receptor antagonist atropine completely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 microM 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) to chelate intracellular Ca2+, carbachol still raised intracellular IP3 level and enhanced TH gene expression. Transient transfection analysis of the 5' upstream region of TH gene revealed that the AP1 cis-acting element at -205 to -199 bp was responsible for carbachol stimulation. But carbachol did not enhance TH gene expression in protein kinase C (PKC)-activated or down-regulated cells that had been induced by 5-min or 24-h exposure to phorbol 12-myristate 13-acetate (PMA), respectively. Thus, Ca(2+)-independent PKC may play a role in carbachol-induced TH gene expression. We demonstrated by gel retardation and competition assays that a DNA sequence containing the wild-type AP1 site formed the specific DNA-protein complex. However, treatment with carbachol or PMA did not change the amount of the specific DNA-protein complex. Our results indicate that stimulation of phospholipase C-linked muscarinic receptors leads to elevated TH gene expression via AP1-mediated enhancement in a PKC-dependent pathway.
Base Sequence↗