Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.6 (CAD)
4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.
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PMID:Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. 900 67

Recently, a tyrosine hydroxylase (TH)-expressing CNS-derived cell line, CAD, was obtained that is capable of undergoing reversible morphological differentiation. The isolation of the CAD line allowed us to ask whether different DNA regulatory elements direct TH transcription when cells are growing and undifferentiated versus postmitotic and differentiated. To this end, we compared expression of a transiently transfected bacterial chloramphenicol acetyltransferase reporter gene under the transcriptional control of TH 5' flanking DNA in CAD cells grown in the presence and absence of serum. Mutational analysis indicates that CAD cells differently regulate TH transcription depending on their state of differentiation. In both states, the cyclic AMP response element and AP1 site each activate transcription. However, in undifferentiated cells, the dyad/E-box element represses expression by approximately 2.7-fold, whereas it modestly activates transcription in differentiated cells. The role of the dyad/ E-box as a repressor correlates well with the two- to threefold lower amount of endogenous TH protein present in the undifferentiated CAD cells. This study demonstrates the differential use of TH DNA regulatory elements in proliferating, undifferentiated and nonproliferating, differentiated immortalized neuronal cells.
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PMID:Differentiation of a catecholaminergic CNS cell line modifies tyrosine hydroxylase transcriptional regulation. 964 50

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.
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PMID:Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation. 1111 Aug 18

The CAD cell line originates from catecholaminergic neurons in the central nervous system (CNS) of a simian virus large T antigen transgenic mouse. In the present study, we have immunohistochemically characterized the cell line after differentiation in serum-free medium, using immunofluorescence in combination with confocal laser scanning microscopy (CLSM), immunoblot, and ribonuclease protection assay (RPA). Tyrosine hydroxylase (TH)-, phenylethanolamine-N-methyltransferase (PNMT)-, neuropeptide Y (NPY)-, vesicular monoamine transporter subtype 2-, vasoactive intestinal peptide (VIP)-, somatostatin (SS)-, synaptophysin-, synaptic vesicle protein 2 (SV2)-, and growth-associated protein of 43 (GAP-43)-immunoreactivities (IRs) were present in the cells but not choline acetyltransferase and vesicular acetylcholine transporter. The immunoreactive substances were present in cell bodies in serum-containing medium (SCM), but after serum withdrawal (protein-free medium, PFM) these proteins and peptides were partially shifted into the long process and their varicosities. A few cells cultured in PFM were occasionally found with extremely high TH-immunoreactivity (IR) in cell bodies and processes. Growth-associated protein of 43-immunoreactivity was weak in SCM but was up-regulated (verified with immunoblot) in PFM and concentrated in varicosities along the processes and the distal tips of neurites. The somatostatin receptor subtype 2a (SSR(2(a))) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR(2(a)) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR(2(a)).
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PMID:Adrenergic differentiation and SSR2a receptor expression in CAD-cells cultured in serum-free medium. 1244 Nov 63

CAD cells are a murine CNS catecholaminergic (tyrosine hydroxylase-positive; TH+) neuronal cell line that undergoes morphological differentiation to resemble CNS catecholaminergic neurons upon serum deprivation. We show here that CAD cells also express neuronal nitric oxide synthase (nNOS) mRNA and protein and produce readily measurable levels of NO. Since both NO and catecholamines (L-DOPA; dopamine; norepinephrine) are redox active molecules, their production within the same cell may affect the cell's vulnerability to insult. Thus, we examined the regulation of NO production by CAD cells and the effect of NO on cell survival. NO is generated in a dose-dependent fashion by treatment with agents (ionomycin; A23817; KCl) known to increase calcium entry across the cell membrane. The NO level can be increased further by pretreatment with sepiapterin, a membrane permeable precursor for BH4 synthesis, suggesting that the BH4 levels or access required for nNOS activation is limited in CAD cells. Reducing mitochondrial Ca2+ uptake using ruthenium red (RuR) increased ionomycin-mediated NO production over ionomycin alone and indicates a critical role for mitochondria in nNOS regulation. Cell death was significantly increased by ionomycin treatment alone or in conjunction with reduced mitochondrial Ca2+ uptake. However, NO was not the primary mediator of cell death since NOS inhibitors rescued only less than 10% of the cells. These data suggest that endogenous NO production by nNOS is not a major factor in CAD cell death under these conditions.
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PMID:Nitric oxide production and regulation of neuronal NOS in tyrosine hydroxylase containing neurons. 1524 34

In neural crest (NC) cultures cAMP signaling is an instructive signal in catecholaminergic, sympathoadrenal cell development. However, the extracellular signals activating the cAMP pathway during NC cell development have not been identified. We demonstrate that in avian NC cultures, evidenced by tyrosine hydroxylase expression and catecholamine biosynthesis, adenosine and not adrenergic signaling, together with BMP2, promotes sympathoadrenal cell development. In NC cultures, addition of the adenosine receptor agonist NECA in the presence of BMP2 promotes sympathoadrenal cell development, whereas the antagonist CGS 15943 or the adenosine degrading enzyme adenosine deaminase (ADA) suppresses TH expression. Importantly, NC cells express A2A and A2B receptors which couple with Gsalpha increasing intracellular cAMP. Employing the CNS-derived catecholaminergic CAD cell line, we also demonstrate that neuronal differentiation mediated by serum withdrawal is further enhanced by treatment with IBMX, a cAMP-elevating agent, or the adenosine receptor agonist NECA, acting via cAMP. By contrast, the adenosine receptor antagonist CGS 15943 or the adenosine degrading enzyme ADA inhibits CAD cell neuronal differentiation mediated by serum withdrawal. These results support that adenosine is a physiological signal in neuronal differentiation of the CNS-derived catecholaminergic CAD cell line and suggest that adenosine signaling is involved in NC cell development in vivo.
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PMID:Adenosine signaling promotes neuronal, catecholaminergic differentiation of primary neural crest cells and CNS-derived CAD cells. 1588 17

Ceramide is a lipid second-messenger generated in response to stimuli associated with neurodegeneration that induces apoptosis, a mechanism underlying neuronal death in Parkinson's disease. We tested the hypothesis that insulin-like growth factor-1 (IGF-1) could mediate a metabolic response in CAD cells, a dopaminergic cell line of mesencephalic origin that differentiate into a neuronal-like phenotype upon serum removal, extend processes resembling neurites, synthesize abundant dopamine and noradrenaline and express the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase, and that this process was phosphatidylinositol 3-kinase (PI 3-K)-Akt-dependent and could be inhibited by C(2)-ceramide. The metabolic response was evaluated as real-time changes in extracellular acidification rate (ECAR) using microphysiometry. The IGF-1-induced ECAR response was associated with increased glycolysis, determined by increased NAD(P)H reduction, elevated hexokinase activity and Akt phosphorylation. C(2)-ceramide inhibited all these changes in a dose-dependent manner, and was specific, as it was not induced by the inactive C(2)-ceramide analogue C(2)-dihydroceramide. Inhibition of the upstream kinase, PI 3-K, also inhibited Akt phosphorylation and the metabolic response to IGF-1, similar to C(2)-ceramide. Decreased mitochondrial membrane potential occurred after loss of Akt phosphorylation. These results show that IGF-1 can rapidly modulate neuronal metabolism through PI 3-K-Akt and that early metabolic inhibition induced by C(2)-ceramide involves blockade of the PI 3-K-Akt pathway, and may compromise the first step of glycolysis. This may represent a new early event in the C(2)-ceramide-induced cell death pathway that could coordinate subsequent changes in mitochondria and commitment of neurons to apoptosis.
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PMID:Insulin-like growth factor-1-dependent maintenance of neuronal metabolism through the phosphatidylinositol 3-kinase-Akt pathway is inhibited by C2-ceramide in CAD cells. 1756 16

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.
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PMID:Differential and interactive effects of ligand-bound progesterone receptor A and B isoforms on tyrosine hydroxylase promoter activity. 2181 51

The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are induced by amino acid starvation and are required to dissociate TSC2, re-tether and activate MTORC1 on endolysosomal membranes. This study characterizes TFEB-mediated endocytosis as a critical process leading to activation of MTORC1 and autophagic function, thus identifying the importance of the dynamic endolysosomal system in cellular clearance. Abbreviations: CAD: central adrenergic tyrosine hydroxylase-expressing-a-differentiated; ChIP-seq: chromosome immunoprecipitation sequencing; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; EEA1: early endosomal antigen 1; EGF: epidermal growth factor; FBS: fetal bovine serum; GFP: green fluorescent protein; GTPase: guanosine triphosphatase; HEK293T: human embryonic kidney 293 cells expressing a temperature-sensitive mutant of the SV40 large T antigen; LAMP: lysosomal-associated membrane protein; LYNUS: lysosomal nutrient-sensing complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha/beta; MTOR: mechanistic target of rapamycin kinase; MTORC: mechanistic target of rapamycin kinase complex; OE: overexpression; PH: pleckstrin homology; PtdIns(3,4,5)P₃: phosphatidylinositol 3,4,5-trisphosphate; RRAGD: Ras related GTPase binding D; RHEB: Ras homolog enriched in brain; SLC38A9: solute carrier family 38 member 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TSC2: tuberous sclerosis 2; TMR: tetramethylrhodamine; ULK1: unc-51 like kinase 1; WT: wild type.
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PMID:TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy. 3014 26

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