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1.
Int J Dev Biol ; 66(1-2-3): 187-197, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34881797

RESUMO

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constraints and regulations applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using two-dimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergent-free preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency, we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-ß-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs act in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.


Assuntos
Actinas , Prolactina , Actinas/metabolismo , Animais , Proliferação de Células , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas , Prolactina/farmacologia , Proteômica , Tubulina (Proteína)/metabolismo
2.
Cells ; 9(11)2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33114250

RESUMO

Mitotic spindles are highly organized, microtubule (MT)-based, transient structures that serve the fundamental function of unerring chromosome segregation during cell division and thus of genomic stability during tissue morphogenesis and homeostasis. Hence, a multitude of MT-associated proteins (MAPs) regulates the dynamic assembly of MTs in preparation for mitosis. Some tumor suppressors, normally functioning to prevent tumor development, have now emerged as significant MAPs. Among those, neurofibromin, the product of the Neurofibromatosis-1 gene (NF1), a major Ras GTPase activating protein (RasGAP) in neural cells, controls also the critical function of chromosome congression in astrocytic cellular contexts. Cell type- and development-regulated splicings may lead to the inclusion or exclusion of NF1exon51, which bears a nuclear localization sequence (NLS) for nuclear import at G2; yet the functions of the produced NLS and ΔNLS neurofibromin isoforms have not been previously addressed. By using a lentiviral shRNA system, we have generated glioblastoma SF268 cell lines with conditional knockdown of NLS or ΔNLS transcripts. In dissecting the roles of NLS or ΔNLS neurofibromins, we found that NLS-neurofibromin knockdown led to increased density of cytosolic MTs but loss of MT intersections, anastral spindles featuring large hollows and abnormal chromosome positioning, and finally abnormal chromosome segregation and increased micronuclei frequency. Therefore, we propose that NLS neurofibromin isoforms exert prominent mitotic functions.


Assuntos
Segregação de Cromossomos , Mitose/fisiologia , Neurofibromina 1/metabolismo , Fuso Acromático/metabolismo , Núcleo Celular/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Metáfase , Microtúbulos/metabolismo , Neurofibromina 1/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , Transcrição Gênica
3.
Neurochem Int ; 131: 104582, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31629778

RESUMO

The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.


Assuntos
Microdomínios da Membrana/metabolismo , Neurofibromina 1/metabolismo , Neurônios/metabolismo , Proteína Quinase C-épsilon/fisiologia , Proteína SOS1/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Fatores ras de Troca de Nucleotídeo Guanina/genética , Animais , Ácidos Araquidônicos/farmacologia , Membrana Celular/genética , Membrana Celular/metabolismo , Embrião de Galinha , Simulação por Computador , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Ácido Palmítico/metabolismo , Cultura Primária de Células , Receptor CB1 de Canabinoide/agonistas , Telencéfalo/efeitos dos fármacos , Telencéfalo/metabolismo
4.
J Neurochem ; 136(1): 78-91, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26490262

RESUMO

Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGTPase-activating protein, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here, we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to a nuclear localization signal in the C-terminal domain (CTD), by Protein Kinase C-epsilon (PKC-ε), accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. Concerned with neurofibromin's, a tumor suppressor, mechanism of action, we demonstrate in astrocytic cells that its synthesis, phosphorylation by Protein Kinase C-ε on Ser2808 (a residue adjacent to a nuclear localization sequence), and nuclear import are cell cycle-dependent, being maximal at G2. During mitosis, neurofibromin is an integral part of the spindle, while its depletion leads to aberrant chromosome congression, possibly explaining the development of chromosomal instability in Neurofibromatosis type-1. Read the Editorial Highlight for this article on page 11. Cover Image for this issue: doi: 10.1111/jnc.13300.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Cromossomos/metabolismo , Neurofibromina 1/metabolismo , Fuso Acromático/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/química , Núcleo Celular/genética , Cromossomos/genética , Células HEK293 , Humanos , Neurofibromatose 1/genética , Neurofibromatose 1/metabolismo , Neurofibromina 1/análise , Neurofibromina 1/genética , Fuso Acromático/genética
5.
PLoS One ; 8(11): e82045, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312401

RESUMO

The protein kinase C (PKC) signaling, a major regulator of chondrocytic differentiation, has been also implicated in pathological extracellular matrix remodeling, and here we investigate the mechanism of PKCε-dependent regulation of the chondrocytic phenotype in human nucleus pulposus (NP) cells derived from herniated disks. NP cells from each donor were successfully propagated for 25+ culture passages, with remarkable tolerance to repeated freeze-and-thaw cycles throughout long-term culturing. More specifically, after an initial downregulation of COL2A1, a stable chondrocytic phenotype was attested by the levels of mRNA expression for aggrecan, biglycan, fibromodulin, and lumican, while higher expression of SOX-trio and Patched-1 witnessed further differentiation potential. NP cells in culture also exhibited a stable molecular profile of PKC isoforms: throughout patient samples and passages, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas ß, γ, and θ were not. Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan. More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan. Therefore, PKCε activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKCε/ERK/CREB/AP-1-dependent transcriptional program that includes upregulation of both chondrogenic genes and microRNAs. Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.


Assuntos
Proteínas ADAM/genética , Agrecanas/genética , Regulação da Expressão Gênica , Disco Intervertebral/metabolismo , MicroRNAs/genética , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais , Proteína ADAMTS5 , Sequência de Bases , Western Blotting , Diferenciação Celular , Primers do DNA , Ativação Enzimática , Humanos , Disco Intervertebral/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Behav Brain Res ; 253: 280-9, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23911427

RESUMO

Activation of PKCɛ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCɛ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCɛ-selective peptides, the inhibitory ɛV1-2 and the activating ψɛRACK, and the novel object recognition task (NORT). Our results show that the PKCɛ-selective activator ψɛRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCɛ activation with the peptide inhibitor ɛV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCɛ activation in acutely dissected rat hippocampi, we found that ψɛRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ɛV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCɛ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCɛ-regulating peptides as memory study tools and putative therapeutic agents.


Assuntos
Memória/fisiologia , Proteína Quinase C-épsilon/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Produtos do Gene tat/farmacologia , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Wistar , Reconhecimento Psicológico/efeitos dos fármacos , Quinases raf/fisiologia , Quinases da Família src/fisiologia
7.
J Neuropathol Exp Neurol ; 72(4): 337-50, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23481708

RESUMO

Experimental studies indicate that hypoxia to the fetus, a common occurrence in many birth complications in humans, results in long-term disturbances of the central dopaminergic (DA) systems that persist in adulthood. Because dysregulation of DA systems is involved in the pathophysiology of many neurological and psychiatric disorders, we investigated the effects of perinatal hypoxia on the mesencephalic DA neurons of the human neonate using immunohistochemistry. We studied the expression of tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catecholamine synthesis, in substantia nigra, and ventral tegmental area of 18 neonates in relation to the age and severity/duration of hypoxic injury estimated by neuropathological criteria. In severe/abrupt perinatal hypoxia, intense TH staining was observed in substantia nigra, ventral tegmental area, and, surprisingly, in the nonpreganglionic Edinger-Westphal nucleus. In severe/prolonged hypoxia, there was a striking reduction or even absence of TH immunoreactivity in all the mesencephalic nuclei. These observations suggest that at early states of perinatal hypoxia, there is a massive increase in dopamine synthesis and release that is followed by feedback blockage of dopamine synthesis through inhibition of TH by the end product dopamine. Early dysregulation of DA neurotransmission could predispose infant survivors of severe perinatal hypoxia to dopamine-related neurological and/or cognitive deficits later in life.


Assuntos
Neurônios Dopaminérgicos/enzimologia , Hipóxia Fetal/enzimologia , Regulação Enzimológica da Expressão Gênica , Mesencéfalo/enzimologia , Mesencéfalo/patologia , Tirosina 3-Mono-Oxigenase/biossíntese , Neurônios Dopaminérgicos/patologia , Feminino , Hipóxia Fetal/patologia , Humanos , Recém-Nascido , Masculino , Estudos Prospectivos , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/química
8.
J Neurochem ; 116(5): 866-73, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21214560

RESUMO

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.


Assuntos
Córtex Cerebral/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/citologia , Receptor CB1 de Canabinoide/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Imunoprecipitação/métodos , Microdomínios da Membrana/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo
9.
Neurochem Int ; 58(2): 135-44, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21074588

RESUMO

Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKCɛ at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKCɛ, selectively, and that the CB1R-activated PKCɛ acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Complexos Multiproteicos/fisiologia , Neurônios/metabolismo , Proteína Quinase C-épsilon/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Quinases da Família src/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Células HEK293 , Humanos , Neurônios/enzimologia , Células PC12 , Ratos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/genética
10.
J Neurochem ; 109(2): 573-83, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19220708

RESUMO

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase-activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH-SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Surprisingly, we observed that, among other PKC-dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C-tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA-induced differentiation of SH-SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C-tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC-dependent, sustained C-tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.


Assuntos
Diferenciação Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Neurofibromina 1/metabolismo , Neurônios/metabolismo , Proteína Quinase C/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo , Linhagem Celular Tumoral , Humanos , Neurônios/citologia , Neurônios/enzimologia , Fragmentos de Peptídeos/metabolismo , Fosforilação
11.
Biochim Biophys Acta ; 1781(11-12): 731-42, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18930839

RESUMO

Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Type I PAP enzymes participate in de-novo phospholipid biosynthesis, whereas type II PAP enzymes have an established role in lipid signaling. To identify novel human type II PAPs potentially involved in de-novo phospholipid synthesis we used bioinformatics to screen for enzymes with an active site exposed to the cytosolic side of membranes. Two related enzymes, a novel lipid phosphatase related protein (LPRP-A) and a presqualene diphosphate phosphatase (PA-PSP) met this criterion. PA-PSP and LPRP-A have differential tissue and subcellular distribution, and novel yet differential roles in lipid metabolism. Specifically, PA-PSP, but not LPRP-A, was a potent Mg(2+)-independent, NEM-insensitive type II PAP. Subcellular fractionation detection indicated that both proteins were associated with membranes, while immunofluorescent deconvolution imaging revealed that these membranes were exclusively from the nuclear envelope and the endoplasmic reticulum. PA-PSP overexpression, but not LPRP-A, accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol with concomitant decrease in the rate of phosphatidylinositol synthesis. Coexpression of human CTP:phosphocholine cytidylyltransferase-alpha with PA-PSP enhanced the effect of PA-PSP on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that the eukaryotic, ER-resident PA-PSP is a bifunctional enzyme with specific type II PAP activity, and regulates, in addition to type I PAPs, the de-novo biosynthesis of phospholipids and triacylglycerols.


Assuntos
Fosfatidato Fosfatase/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatos de Poli-Isoprenil/metabolismo , Triglicerídeos/biossíntese , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Colesterol/metabolismo , Cromatografia em Camada Fina , Clonagem Molecular , Citosol/metabolismo , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Corantes de Rosanilina/química , Frações Subcelulares
12.
J Neurochem ; 97 Suppl 1: 97-103, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16635257

RESUMO

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.


Assuntos
Dopamina/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Neurofibromina 1/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tirosina 3-Mono-Oxigenase/genética , Proteínas ras/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/farmacologia , Ativação Enzimática/efeitos dos fármacos , Genes ras/genética , Humanos , Neuroblastoma , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/farmacologia
13.
J Neurosci Res ; 83(7): 1190-200, 2006 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-16555279

RESUMO

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-betaIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons.


Assuntos
Líquido Amniótico/citologia , Técnicas de Cultura de Células/métodos , Dopamina/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Líquido Amniótico/fisiologia , Animais , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Separação Celular/métodos , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Fenótipo , Gravidez , Células-Tronco/citologia
14.
Brain Res Dev Brain Res ; 143(2): 207-16, 2003 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-12855192

RESUMO

Proteoglycans have been suggested to play roles in pattern formation in the developing central nervous system. In the chick embryo, aggrecan, a chondroitin sulfate proteoglycan, has a regionally-specific and developmentally-regulated expression profile. Telencephalic neuronal cultures, when aggregated, exhibit aggrecan expression patterns comparable to those observed in vivo. The chicken mutation nanomelia produces a truncated aggrecan species that cannot be processed further and is not secreted. Neurons from normal and nanomelic chick embryo telencephalon were scored for aggregate formation and analyzed for distribution of aggrecan protein and expression of aggrecan mRNA. Distinctly different pattern formation, with respect to aggregate size (smaller) and number (fewer) were observed in poly-L-lysine plated neuronal cultures derived from nanomelic embryos when compared to those derived from normal embryos. Significantly, the nanomelic phenotype was subsequently rescued upon addition of the brain-specific form of aggrecan. Modulation of neuronal aggregate formation was mimicked by treatment with chondroitinase ABC but not other glycanases, and was rescued by addition of chondroitin 6-sulfate to the culture media. Lastly, although broad and diffuse distribution of aggrecan among the cell aggregates in the culture paradigm was observed by immunocytochemistry, mRNA in situ hybridization revealed that only a small population of cells in the center of the aggregates was responsible for the production of the secreted aggrecan found associated with neuronal aggregates. These studies suggest a function for aggrecan as a diffusible signal in CNS histomorphogenesis.


Assuntos
Proteínas da Matriz Extracelular , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Proteoglicanas/metabolismo , Telencéfalo/crescimento & desenvolvimento , Agrecanas , Animais , Agregação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Condroitina ABC Liase/metabolismo , Sulfatos de Condroitina/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lectinas Tipo C , Mutação , Proteoglicanas/genética , RNA Mensageiro/análise
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