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Background: Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli beta-galactosidase or enhanced green fluorescence protein (EGFP) using PEI.Results: Optimal transfection efficiency was observed with 1 microg/ml of plasmid DNA and 5 microg/ml PEI. Expression of beta-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with approximately 9% of the neurons expressing beta-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector.Conclusions: These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures. [ABSTRACT FROM AUTHOR]

Schizophrenia is a neurodevelopmental disorder featuring complex aberrations in the structure, wiring, and chemistry of multiple neuronal systems. The abnormal developmental trajectory of the brain appears to be established during gestation, long before clinical symptoms of the disease appear in early adult life. Many genes are associated with schizophrenia, however, altered expression of no one gene has been shown to be present in a majority of schizophrenia patients. How does altered expression of such a variety of genes lead to the complex set of abnormalities observed in the schizophrenic brain? We hypothesize that the protein products of these genes converge on common neurodevelopmental pathways that affect the development of multiple neural circuits and neurotransmitter systems. One such neurodevelopmental pathway is Integrative Nuclear FGFR1 Signaling (INFS). INFS integrates diverse neurogenic signals that direct the postmitotic development of embryonic stem cells, neural progenitors and immature neurons, by direct gene reprogramming. Additionally, FGFR1 and its partner proteins link multiple upstream pathways in which schizophrenia-linked genes are known to function and interact directly with those genes. A th-fgfr1(tk-) transgenic mouse with impaired FGF receptor signaling establishes a number of important characteristics that mimic human schizophrenia - a neurodevelopmental origin, anatomical abnormalities at birth, a delayed onset of behavioral symptoms, deficits across multiple domains of the disorder and symptom improvement with typical and atypical antipsychotics, 5-HT antagonists, and nicotinic receptor agonists. Our research suggests that altered FGF receptor signaling plays a central role in the developmental abnormalities underlying schizophrenia and that nicotinic agonists are an effective class of compounds for the treatment of schizophrenia. [ABSTRACT FROM AUTHOR]

Genetic and post mortem evidence has implicated the α7 neuronal nicotinic receptor (NNR) in the etiology of schizophrenia and related disorders. In schizophrenia, enhanced subcortical dopamine (DA) correlates with positive and cognitive of the disease, including impairments in sensorimotor gating. We measured the levels of extracellular DA and DA metabolites during an acoustic test session of prepulse inhibition (PPI) of the startle response, a measure of sensorimotor gating, by microdialysis and HPLC-EC in a transgenic mouse model of schizophrenia. In th-fgfr1(tk-) mice, blockade of fibroblast growth factor receptor 1 (FGFR1) signaling during development in catecholaminergic neurons results in reduced size and density of midbrain DA neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These mice displayed reduced PPI and enhanced startle response relative to control mice as well as a potentiation of DA release in the dorsal striatum during a 30 minute PPI test session. Acute administration of a partial α7 NNR agonist TC-7020 (1.0 mg/kg) normalized PPI and startle deficits and attenuated increases of DA release during acoustic PPI testing. These results provide direct evidence of elevated striatal dopaminergic transmission with impaired sensorimotor gating that may underlie cognitive and positive symptoms and motor deficits in schizophrenia and related disorders. Also, systemic targeting of alpha7 NNRs may ameliorate these deficits by functionally suppressing striatal DA activity. [ABSTRACT FROM AUTHOR]

The watershed-hypothesis of schizophrenia asserts that over 200 different mutations dysregulate distinct pathways that converge on an unspecified common mechanism(s) that controls disease ontogeny. Consistent with this hypothesis, our RNA-sequencing of neuron committed cells (NCCs) differentiated from established iPSCs of 4 schizophrenia patients and 4 control subjects uncovered a dysregulated transcriptome of 1349 mRNAs common to all patients. Data reveals a global dysregulation of developmental genome, deconstruction of coordinated mRNA networks, and the formation of aberrant, new coordinated mRNA networks indicating a concerted action of the responsible factor(s). Sequencing of miRNA transcriptomes demonstrated an overexpression of 16 miRNAs and deconstruction of interactive miRNA-mRNA networks in schizophrenia NCCs. ChiPseq revealed that the nuclear (n) form of FGFR1, a pan-ontogenic regulator, is overexpressed in schizophrenia NCCs and overtargets dysregulated mRNA and miRNA genes. The nFGFR1 targeted 54% of all human gene promoters and 84.4% of schizophrenia dysregulated genes. The upregulated genes reside within major developmental pathways that control neurogenesis and neuron formation, whereas downregulated genes are involved in oligodendrogenesis. Our results indicate (i) an early (preneuronal) genomic etiology of schizophrenia, (ii) dysregulated genes and new coordinated gene networks are common to unrelated cases of schizophrenia, (iii) gene dysregulations are accompanied by increased nFGFR1-genome interactions, and (iv) modeling of increased nFGFR1 by an overexpression of a nFGFR1 lead to up or downregulation of selected genes as observed in schizophrenia NCCs. Together our results designate nFGFR1 signaling as a potential common dysregulated mechanism in investigated patients and potential therapeutic target in schizophrenia., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Studies have shown the presence of expanded polyQ containing proteins in brain cells related to Huntington disease (HD) and other poly-glutamine disorders. We report the use of organically modified silica (ORMOSIL) nanoparticles as an efficient non-viral gene carrier in an effort to model brain pathology associated with those disorders induced by expanded polyQ peptides. In experiment 1, plasmids expressing Hemaglutinin-tagged polypeptides with 20 glutamine repeats (Q20) or with extended 127-glutamine repeats (Q127) were complexed with ORMOSIL nanoparticles and injected twice (2 weeks apart) into the lateral ventricle of the mouse brain. Fourteen days post-injection of Q127, immunocytochemistry revealed the presence of the characteristic nuclear and cytoplasmic Q127 aggregates in numerous striatal, septal and neocortical neuronal cells as well as ubiquitin-containing aggregates indicative of the neuronal pathology. The mice receiving Q127 showed a marked increase in the reactive GFAP (+) astrocytes in striatum, septum and brain cortex, further indicating the neurodegenerative changes, accompanied by motor impairments. In experiment 2, plasmids Q20 or Q127 were complexed with ORMOSIL and were injected into the brain lateral ventricle or directly into the striatum of adult rats. In both routes of transfection, Q127 induced the appearance of reactive GFAP (+) astrocytes and activated ED1 antigen expressing microglia. An increase in the size of the lateral ventricle was also observed in rats receiving Q127. In transgenic mouse polyQ models, extensive pathologies occur outside the nervous system and the observed brain pathologies could reflect developmental effects of the toxic polyQ proteins. Our experiments show that the nervous tissue restricted expression of poly Q-extended peptides in adult brain is sufficient to evoke neuropathologies associated with HD and other polyQ disorders. Thus, nanotechnology can be employed to model pathological and behavioral aspects of genetic brain diseases in mice as well as in other species, providing a novel research tool for in vivo testing of single or multi-gene therapies.

This study shows that an ICP4-replication-deficient herpes simplex virus containing the Moloney murine leukaemia virus LTR fused with the coding sequence for the beta-galactosidase gene can be used as a very effective vector for delivering the beta-galactosidase reporter gene into the rat brain septum. F344 rats received bilateral stereotaxic injections into the nucleus of the diagonal band and into the medial septum. The X-gal stain was used to detect the activity of the expressed beta-galactosidase enzyme. The delivered reporter gene was expressed successfully not only in the neuronal cells of the injected areas but also in cells that project to the injection area such as cortex cells about 6 mm away from the injection sites. Expression was visible at 1, 3 and 9 weeks following injection. We conclude that this vector can effectively deliver genes into different regions of the mature mammalian brain and also to areas distant from the injection site.

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Parkin is an ubiquitin-protein ligase molecule abundantly expressed in mammalian brains. Deletional mutations of Parkin protein produce a disease-related parkinsonian phenotype which is inherited with an autosomal recessive mode of transmission. To gain a greater insight into the evolutionary trajectory of the protein among vertebrate species, we describe here the (i) distribution pattern, (ii) sizing of specific fragments and (iii) embryonic development of Parkin in Xenopus laevis utilizing two antibodies to the N- and C-terminal sequence of the human Parkin protein. Parkin immunoreactivity was distributed in a heterogeneous fashion throughout the adult frog brain. The telencephalon, including the olfactory bulb, striatum and nucleus accumbens, harbored high numbers of Parkin-containing cells. High numbers of immunoreactive neurons were also present in discrete regions of the thalamus and hypothalamus. Relatively moderate expression of Parkin protein was noted in the nucleus anterodorsalis tegmenti, nucleus reticularis medius and torus semicircularis. The substantia nigra exhibited a distinctive heterogeneous pattern of Parkin-immunoreactivity, especially within presumptive dopamine neurons. The cerebellum also showed high expression of Parkin-positive material. Characterization of the subcellular distribution of the protein indicated both a cytoplasmic and nuclear integration of Parkin-immunoreactivity. This pattern of subcellular localization was similar to that observed in human brain material, perhaps reflecting distinct structural phosphorylation sites of the Parkin protein. Western blot analysis identified three specific bands with molecular weights varying from 50 to 65 kDa in adult Xenopus brain. However, studies on the temporal expression of Parkin during development showed a complete absence of cellular immunoreactivity which was especially conspicuous during late premetamorphic stages of frog development. These results suggest that the ubiquitination activity of Parkin is limited or non-existent during embryogenesis, but appears to assume a more functional role during adulthood as reflected by the high distribution pattern of the protein within major circuits of the amphibian brain.

Manipulation of gene expression in developing or in mature central nervous systems (CNS) holds a promise for the resolution of many compelling neurobiological questions, including the feasibility of gene therapy to treat diseases of the brain. In this context, a number of viral vectors have been used in recent years to introduce and express genes into the CNS. This article discusses a gene transfer system based on the Herpes Simplex Virus-1 (HSV-1). We describe here the use of non-replicating, non-toxic HSV-1 vector, 8117/43, in a series of studies carried in our joint program. This vector proves further the utility of HSV-1 as a delivery vehicle to a number of distinct sites within the CNS.

Mutations within the amino acid sequence of Parkin, the encoded protein of the parkin gene, appear to trigger the degeneration of dopaminergic neurons in the substantia nigra. Here, the presence and anatomical distribution of Parkin within the rat was examined. Immunoblot analysis of tissue homogenates showed two major bands at 50 and 44kDa. Within the brain, Parkin-containing neurons were identified in the basal ganglia, including the substantia nigra and caudate-putamen. Parkin was visualized in the raphe nucleus, which as in the substantia nigra, was closely localized to monoaminergic-encoding neurons. In addition, Parkin was detected in laminar structures such as the cortex and hippocampus; a substantial number of Parkin-immunoreactive neurons was seen in the cerebellum as well. Parkin therefore is widely distributed in brain pathways implicated in the pathology of Parkinson's disease.

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

FGF-2, a mitogenic/neurotrophic protein, controls the development and plasticity of many types of neural cells. In neural crest-derived adrenal pheochromatocytes, induction of FGF-2 coincides with the establishment of functional innervation and is reproduced in vitro by stimulating acetylcholine receptors (AChR). The mechanisms by which AChR activate the FGF-2 gene were examined in cultured bovine adrenal medullary chromaffin (BAMC) cells in which AChR induce expression and nuclear accumulation of growth-promoting FGF-2 and FGF-2 receptors. Carbachol or nicotine increased expression of transfected FGF-2 gene promoter-luciferase constructs and were more potent than the muscarinic agonist ABMCB. Deletion analysis has identified a unique -555/-512 bp element that confers AChR stimulation and basal activity to the downstream FGF-2 promoter, and a separate protein kinase C/cAMP-responsive sequence (-625/-555 bp). Stimulation of AChR increased in vitro formation of protein complexes with the AChR-responsive element which were not displaced by target oligonucleotides for common trans-activators. Southwestern analysis identified 50-55, 125, 140 and 170 kDa proteins that interact with the AChR-responsive element in a manner stimulated by AChR. Nicotine increased tyrosine phosphorylation of cytoplasmic and nuclear proteins, including 50-55 kDa promoter-binding factors. Activation of the FGF-2 promoter was reduced by genistein. Thus, nicotinic AChR activate the FGF-2 gene via a new signaling mechanism separate from the cAMP/PKC pathways. It utilizes tyrosine phosphorylation and interaction of trans-activating factors with a novel cis-acting element. It offers a new pathway through which trans-synaptic signals may control neural development and plasticity., (Copyright 1998 Elsevier Science B. V.)

The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bone adrenal medullary cells. Angiotensin II-responsive elements are located within -54/+25-bp and -269/-55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12-O-tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.

Fractionation of human astrocytes revealed the presence of 103, 118, and 145 kDa forms of FGF receptor 1 (FGFR1) in isolated nuclei. Only trace amounts of FGFR1 proteins were detected in the cell membrane or cytoplasmic fractions. Nuclear FGFR1 is found in the nucleoplasm and nuclear matrix but not in chromatin. Immuno-confocal microscopy further demonstrates the intranuclear presence of FGFR1 and its colocalization with FGF-2. Nuclear FGFR1 binds to FGF-2 and has tyrosine kinase activity. Translocation of functional growth factor receptors into the cell nucleus offers a novel mechanism for growth factor action.

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.

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 Ca2+ 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, Met5-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.

bFGF is a neurotrophic protein expressed in various regions of the adult peripheral and central nervous system. The present study was undertaken to examine the role of bFGF in multihormonal, catecholaminergic and enkephalinergic cells of the adrenal medulla (AM). Western blot analysis revealed the presence of at least three bFGF isoforms (18, 22/23, and 24 kDa) in cultured bovine AM cells. Incubation of AM cells with the exogenous 18 kDa bFGF produced time-dependent increases in tyrosine hydroxylase (TH) and proenkephalin (PEK) mRNA, with maximal changes occurring at 12 h (TH) or 24 h (PEK) of bFGF exposure. Effects of bFGF on TH and PEK mRNA were non-additive with increases induced by exposure of AM cells to nicotine, the depolarizing agent veratridine, or the adenylate cyclase activator forskolin. These data indicate that bFGF effects may occur through intracellular pathways accessed during transsynaptic induction of TH and PEK genes. The increases in PEK mRNA induced by nicotine or bFGF were inhibited by the calcium antagonist TMB-8. TMB-8 also inhibited bFGF-induced increases in TH mRNA as well. However, treatment with TMB-8 increased basal levels of TH mRNA. The addition of bFGF increased endogenous levels of c-fos mRNA, c-Fos and c-Fos-related proteins, suggesting that bFGF may activate TH and PEK gene expression through a calcium-AP1 transcriptional regulatory pathway. Immunohistochemical analysis revealed the presence of bFGF-immunoreactivity (bFGF-IR) in the cytoplasm and in the nucleus of AM cells. Incubation of cells with exogenous bFGF produced time-dependent increases of nuclear bFGF-IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells and is transcriptionally activated via a protein kinase C (PKC)-dependent pathway. To identify regulatory regions of the TH gene, transfected neural crest-derived catecholaminergic cells [human neuroblastoma SH-SY5Y and bovine adrenal medullary (BAM) cells] or glia-derived SF-763 cells were used to analyze expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5'-flanking sequences of the bovine TH gene. Plasmid pTH(-245/+21)CAT was constructed by fusing the -245 to +21 region of the TH gene to CAT sequences in the promoterless pCAT basic plasmid. Cells transfected with pTH(-245/+21)CAT expressed CAT activity at levels 8- to 100-fold higher than those of cells transfected with pCAT basic. In SH-SY5Y or BAM cells, pTH(-245/+21)CAT supported the expression of CAT at levels similar to or higher than that supported by the tissue nonspecific viral SV40 promoter/enhancer. In glia-like cells, CAT expression from pTH(-245/+2 1)CAT was about 13-fold lower than that under the control of the SV40 promoter. Incubation of neuroblastoma cells with the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), increased expression of CAT from pTH(-245/+2 1)CAT over 6-fold and was accompanied by induction of c-fos and c-jun mRNAs and proteins. The regulation of TH promoter function by c-Fos/c-Jun was corroborated by transactivation of the TH promoter in SH-SY5Y cells engineered to overexpress exogenous c-Fos and c-Jun. TH gene sequences essential for c-Fos/c-Jun transactivation were located in the -245 to -52 region, upstream from a CRE-like element. Gel mobility assays demonstrated binding of c-Fos-antigen(s) to the region of the TH promoter that supports activation by PMA and c-Fos/c-Jun. Temporal patterns of nicotinic agonist-stimulated induction of c-fos and TH mRNA are also consistent with a role for c-Fos in TH gene transcription. Our results demonstrate that the 5'-flanking region of the bovine TH gene operates as an authentic promoter capable (i) of directing cell-specific expression of a bacterial CAT gene and (ii) of conferring responsivity to PKC-stimulation. The results also suggest that the nuclear proteins c-Fos and c-Jun contribute to PKC pathway-mediated activation of the TH gene via direct interaction with the TH gene promoter. The activation of TH gene expression by PKC/c-Fos/c-Jun may serve as an additional or alternative mechanism to activation by the cAMP-CRE pathway.

The purpose of this study was to examine the effects of angiotensin on the release of enkephalin peptides and the expression of the proenkephalin (pEK) gene. Incubation of cultured bovine adrenal medullary (AM) cells in serum-free medium resulted in calcium- and time-dependent accumulation of [Met(5)]-enkephalin (MEK) in the medium. Fifteen minutes to three hours of incubation with 2 nM [Sar(1)]-angiotensin II (s(1)-AII) did not affect basal secretion of MEK; however, longer incubations (24 h) resulted in four- to fivefold increases. Northern and dot blot analyses with bovine pEK cDNA demonstrated increases in the relative abundance of pEK mRNA in angiotensin-treated cells, suggesting that the long-term increases in MEK release may reflect increased expression of pEK gene and MEK synthesis. Stimulation of MEK release and induction of pEK mRNA were concentration dependent (ED(50) approximately 1 nM. Changes in pEK mRNA levels were not observed until 12 h of incubation with s(1)-AII and continued to increase during an additional 12 h of incubation. Addition of an angiotensin antagonist, saralasin, at 0-16 h, but not at 18-20 h, to cells incubated continuously for 24 h with s(1)-AII inhibited changes in pEK mRNA and MEK release. These observations demonstrate the absence of apparent desensitization of angiotensin receptor function and indicate that long-term receptor-ligand interactions are required to induce changes in gene expression and MEK release. Induction of pEK mRNA and stimulation of MEK release were additive to the effects of veratridine and forskolin, respectively, indicating that the effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Angiotensin-induced increases in pEK mRNA were partially inhibited by nifedipine and also by dantrolene and TMB-8, drugs that inhibit voltage-dependent calcium channels and mobilization of calcium from intracellular stores, respectively. s(1)-AII-induced changes in pEK mRNA were inhibited with calmidazolium, suggesting involvement of calmodulin. The participation of protein kinase C in the induction of pEK gene and long-term stimulation of MEK release was indicated by inhibition of the s(1)-AII effects by pretreatment of cells with protein kinase C inhibitor sphingosine. Effects of s(1)-AII on induction of pEK mRNA by angiotensin and by nicotine were prevented by the translational inhibitor cycloheximide. In conclusion, angiotensin receptors were found to control expression of the pEK gene and secretion of MEK. Unlike nicotinic receptors, which may control secretion of enkephalin peptides directly by stimulating exocytosis and indirectly by controlling peptide synthesis, the effects of angiotensin appear to be mediated indirectly at the level of pEK gene expression.

Previous studies from our laboratory suggest that protein kinase C (PKC) is involved in the angiotensin II (AII)-induced increase in the expression of genes encoding proenkephalin and catecholamine biosynthesizing enzymes in primary cultured bovine adrenal medullary (BAM) cells. The purpose of this study was to examine the effects of [Sar1]-AII (S1-AII), an AII agonist, on PKC activity in BAM cells. Thirty-minute incubation with S1-AII produced a dose-dependent activation of PKC. The particulate PKC activity was significantly increased by 2 nM S1-AII after both 30 min and 12 h of incubation. A high concentration of S1-AII (200 nM) caused an increase in particulate PKC activity after 30 min of incubation and this increase was still observed after 18 h of continuous incubation. [Sar1, Thr8]-angiotensin II (S1, T8-AII) (100 microM), an AII antagonist, inhibited the effect of S1-AII (20 nM) on PKC activity, suggesting a specific AII receptor-mediated effect. An increase in BAM cell particulate PKC immunoreactivity after 18 h of S1-AII treatment was observed in Western blot analysis of PKC-immunoreactive protein (82 kDa). The persistent activation of PKC seen in this study is consistent with our hypothesis that PKC may mediate the S1-AII-induced increase in the expression of genes encoding proenkephalin and catecholamine synthesizing enzymes in BAM cells.

Previous studies identified nicotine and angiotensin receptors as major trans-synaptic and hormonal inputs controlling activities and mRNA levels of the catecholamine biosynthetic enzymes in adrenal medullary (AM) cells. The purpose of this study was to further explore the molecular mechanisms underlying these modes of regulations. Incubation of cultured bovine AM cells with nicotine or a stable analog of angiotensin II, sar1-angiotensin II, increased synthesis of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNAs (2- to 3-fold), suggesting transcriptional activation of these genes. No effects of nicotine or sar1-angiotensin II on TH and PNMT mRNA levels could be demonstrated in cells treated with cycloheximide or puromycin, suggesting regulation by inducible proteins. However, translational inhibitors produced small increases in basal TH mRNA levels (1.5- to 1.9-fold), indicating that repressor-like mechanisms participate in the regulation of the TH gene. Sequence analysis of the bovine TH promoter (1.6 kb) revealed the presence of several putative regulatory elements including four nonidentical AP1-like sites, potential targets for c-Fos/c-Jun/AP1 regulation. Formation of sequence-specific complexes between nuclear proteins and five fragments of the TH promoter (200-400 nt) was demonstrated by gel mobility shift assay (one to three complexes per fragment). Binding of proteins to three promoter fragments containing single AP1-like elements was antagonized by an oligodeoxynucleotide-containing AP1 consensus sequence (oligo-AP1), but not by other unrelated DNA sequences. Oligo-AP1 inhibited the formation of one or two complexes with individual DNA fragments. These observations suggest formation of different AP1-like complexes on individual promoter fragments. c-Fos antibody inhibited the formation of three oligo-AP1-displaceable complexes. The formation of the majority of retarded bands, including all AP1-like complexes, was increased by incubation of cells with sar1-angiotensin II or nicotine. Western analysis demonstrated the presence of c-Fos (p54), c-Jun (p36-38), and several c-Fos- and c-Jun-related antigens in control and stimulated AM cells. Sar1-angiotensin II and nicotine increased steady-state levels of c-Fos (1.5- to 2-fold) and c-fos mRNA (10- to 14-fold) and the levels of several c-Fos-related antigens (FRA) (2- to 7-fold). Some of FRA showed differential regulation by angiotensin II and nicotinic receptors. The levels of p36-38 c-Jun and c-jun mRNA were increased by sar1-angiotensin II (2- to 5-fold) and were less affected by nicotine. In conclusion, our results suggest transcriptional regulation of the TH gene by angiotensin II and nicotinic receptors, through partially different nuclear pathways. The regulation may involve multiple target sites in the TH promoter, inducible proteins, AP1-like factors, c-Fos, and c-Fos-related antigens.

Primary cultures of bovine adrenal medullary cells (AM) in a chemically defined media were used to examine the role of neural and hormonal factors in the expression of proenkephalin A (pEK), phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) genes. Acetylcholine or nicotine reduced cellular content of catecholamines by 30% and increased the relative abundance of pEK, TH, and PNMT mRNAs. The increases produced by acetylcholine were +129%, +147%, and +43% for pEK, TH, and PNMT mRNA, respectively. The kinetics of increases produced by nicotine were different for the 3 mRNAs, with pEK and TH showing enhanced levels over 48 h incubation, while PNMT showed increase during the initial 18 h (+90%) followed by decline to control levels at 48 h. 8-Br cAMP and forskolin elicited a similar pattern of changes as nicotine, suggesting that cyclic AMP may be involved in the mediation of the nicotinic effects. To examine the role of depletion of cellular catecholamines in the regulation of mRNA levels, cells were exposed to tetrabenazine or reserpine. Decreases in cellular catecholamine contents were accompanied by increases in TH and pEK mRNA levels, while the expression of PNMT gene exhibited a transient 4-fold increase and then profound inhibition (60-95%) over a 48-h period. The tetrabenazine effect on TH and pEK mRNA was reduced by alpha-amanitin, suggesting transcriptionally-mediated regulation. Inductions of pEK but not TH or PNMT mRNAs were inhibited by cycloheximide. Hormonal regulation of TH, PNMT, and pEK mRNAs was examined by incubation of cells with dexamethasone. Low concentrations of dexamethasone (0.1, 10 nM) were effective to increase PNMT (+35%, +90%) and pEK (+27%, 45%) mRNA levels. TH mRNA was not affected by similar concentrations of dexamethasone, however, there was a 45% increase at 1 microM. Dexamethasone-elicited increases in PNMT mRNA levels were observed at 48 h and persisted up to 7 days, suggesting that hormonal mechanisms may be distinct from those mediating effects of nicotine, cAMP or tetrabenazine. Taken together, these results indicate that (1) the level of TH, PNMT, and pEK mRNAs are regulated by direct neural (acetylcholine) and hormonal (glucocorticoid) inputs to adrenal medullary cells; (2) effects of acetylcholine could be mediated by cyclic AMP and alterations in catecholamine content; and (3) expression of individual genes is regulated differentially. Such differential regulation of TH, PNMT, and pEK mRNAs may contribute to the long-term selective control of hormonal output from adrenomedullary cells.

The nuclear proto-oncogene, c-fos, has been implicated in the coordinated regulation of gene expression during cell proliferation and differentiation. In this study, we have demonstrated the induction of the c-fos gene products in differentiated cells of the adrenal medulla by non-mitogenic signals. Activation of adrenal medullary cells in vivo by insulin-induced hypoglycemia, and in vitro by nicotine or angiotensin resulted in the rapid and transient elevation of c-fos mRNA levels. Induction of the c-fos mRNA by angiotensin and nicotine were accompanied by the appearance of the c-fos protein. The increase in c-fos protein occurred initially in the cytoplasm and, later, in the nucleus, and it was co-localized with tyrosine hydroxylase. Nuclear expression of the c-fos protein was also induced by veratridine, forskolin and the calcium ionophore A231287. The role of calcium in the regulation of the c-fos gene by angiotensin with nifedipine and inhibition of the effects of angiotensin with nifedipine and sphingosine, a protein kinase C inhibitor. Activation of the c-fos gene may play a role in the coordinated induction of genes involved in the long-term adaptation of adrenal medullary cells to increased functional demands.