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)/metabolismoRESUMO
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 GênicaRESUMO
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/metabolismoRESUMO
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 ReversaRESUMO
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/farmacologiaRESUMO
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/citologiaRESUMO
During papillomavirus infection, the E5 protein localizes in the cell Golgi apparatus and other endomembrane compartments. Cells transformed by E5 do not express major histocompatibility class I complex (MHC I) on the cell surface, while cells transformed by the other transforming proteins E6 and E7 do. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Here we show that expression of bovine papillomavirus E5 causes the retention of MHC I in the Golgi apparatus, thus preventing its transport to the cell surface. We ascribe this effect to a failure of acidification of the Golgi apparatus, as similar effects are observed in control cells treated with the ionophore monensin. Treatment of E5-transformed cells with either beta- or gamma-interferon increases the synthesis of MHC I, showing that inhibition of MHC I expression by E5 is not irreversible. However, even after interferon treatment, MHC I, although increased in quantity, is not transported to the cell surface. E5 therefore affects MHC I at several levels, but prevention of MHC I transport to the cell surface appears to be the dominant effect. Lack of surface MHC I would have profound consequences for presentation of viral peptides to the immune system.
Assuntos
Papillomavirus Bovino 1/fisiologia , Complexo de Golgi/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Transporte Proteico/fisiologia , Animais , Antivirais/farmacologia , Papillomavirus Bovino 1/efeitos dos fármacos , Bovinos , Transformação Celular Viral , Regulação da Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I , Genes Reporter , Proteínas de Fluorescência Verde , Concentração de Íons de Hidrogênio , Interferon beta/farmacologia , Interferon gama/farmacologia , Ionóforos/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Monensin/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/fisiologiaRESUMO
The human papillomavirus (HPV) transcription/replication factor E2 is essential for the life cycle of HPVs. E2 protein binds to DNA target sequences in the viral long control regions to regulate transcription of the viral genome. It also enhances viral DNA replication by interacting with the viral replication factor E1 and recruiting it to the origin of replication and may also play a more direct role in replication. The cellular proteins with which E2 interacts to carry out these functions are largely unknown. To identify these proteins a yeast two-hybrid screen was carried out with the transcription/replication domain of HPV16 E2. This screen identified several candidate interacting partners for E2 including TopBP1 (topoisomerase II beta-binding protein 1). TopBP1 has eight BRCA1 carboxyl-terminal domains that are found in proteins regulating the DNA damage response, transcription, and replication. Here we demonstrate that HPV16 E2 and TopBP1 interact in vitro and in vivo and that TopBP1 can enhance the ability of E2 to activate transcription and replication. This is the first time that TopBP1 has been shown to function as a transcriptional coactivator and that E2 interacts with TopBP1. Removal of the amino-terminal domain of TopBP1 abolishes coactivation of transcription and replication. This interaction may have functional consequences upon the viral life cycle.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Western Blotting , Cromatina/metabolismo , Deleção de Genes , Glutationa Transferase/metabolismo , Humanos , Proteínas Nucleares , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Ativação Transcricional , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
The papillomavirus E5 protein is localized in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of the host cell. Transformed bovine fibroblasts expressing bovine papillomavirus (BPV) E5 are highly vacuolated and have a much enlarged, distorted and fragmented GA. Major histocompatibility complex class I (MHC I) is processed and transported to the cell surface through the GA. Given the cellular localization of E5 in the GA and the morphologically abnormal GA, we investigated the expression of MHC I in cells transformed by E5 from BPV-1 and BPV-4. Two cell lines were used: bovine cells that also express E6, E7 and activated ras, and NIH3T3 cells that express only E5. In addition, PalF cells acutely infected with a recombinant retrovirus expressing E5 were also examined. In contrast to non-transformed normal cells, or transformed cells expressing other papillomavirus proteins, cells expressing E5 do not express MHC I on their surface, but retain it intracellularly, independently of the presence of other viral or cellular oncogenes, or of whether the cells are long-term transformants or acutely infected. We conclude that expression of E5 prevents expression of MHC I to the cell surface and causes its retention within the cell. In addition, lower amounts of total MHC I heavy chain and of heavy chain RNA are detected in E5-transformed cells than in control cells. As surface expression of another glycosylated membrane protein, the transferrin receptor, is not affected, it appears that E5 targets MHC I with at least a degree of specificity. In papillomavirus lesions this effect would have important implications for antigen presentation by, and immunosurveillance of, virally infected cells.