RESUMO
Recent studies of Schwann cell differentiation in vivo and in vitro have provided new insights into determinative signal transduction events both at the cell surface and in the nucleus. Several polypeptide growth factors and their receptors, most notably the neuregulins and receptors of the ErbB family, have been implicated in the specification of cell fate, the control of precursor cell proliferation, and the regulation of programmed cell death during both early and late Schwann cell differentiation. Our understanding of the transcriptional control of Schwann cell development, particularly by the POU protein SCIP and the zinc-finger protein Krox-20, has been advanced by transgenic, knockout, and expression studies.
Assuntos
Células de Schwann/citologia , Animais , Diferenciação Celular/fisiologiaRESUMO
Receptor tyrosine kinases and their ligands mediate cell-cell communication and interaction in many organ systems, but have not been known to act in this capacity in the mature immune system. We now provide genetic evidence that three closely related receptor tyrosine kinases, Tyro 3, Axl, and Mer, play an essential immunoregulatory role. Mutant mice that lack these receptors develop a severe lymphoproliferative disorder accompanied by broad-spectrum autoimmunity. These phenotypes are cell nonautonomous with respect to lymphocytes and result from the hyperactivation of antigen-presenting cells in which the three receptors are normally expressed.
Assuntos
Sistema Imunitário/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Moléculas de Adesão de Célula Nervosa/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Diferenciação Celular , Transplante de Células , Células Dendríticas/imunologia , Feminino , Homeostase , Ativação Linfocitária , Linfócitos/citologia , Linfócitos/imunologia , Tecido Linfoide/crescimento & desenvolvimento , Tecido Linfoide/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Mutação , Moléculas de Adesão de Célula Nervosa/genética , Proteínas Oncogênicas/genética , Biossíntese de Proteínas , Proteína S/biossíntese , Proteína S/genética , Proteínas/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Baço/citologia , Baço/imunologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
POU proteins have been shown to transcriptionally active cell-specific genes and to participate in the determination of cell fate. It is therefore thought that these proteins function in development through the stable activation of genes that define specific developmental pathways. Evidence is provided here for an alternative mode of action. The primary structure of SCIP, a POU protein expressed by developing Schwann cells of the peripheral nervous system, was deduced and SCIP activity was studied. Both in normal development and in response to nerve transection, SCIP expression was transiently activated only during the period of rapid cell division that separates the premyelinating and myelinating phases of Schwann cell differentiation. In cotransfection assays, SCIP acted as a transcriptional repressor of myelin-specific genes.
Assuntos
Genes Homeobox/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas Repressoras/fisiologia , Células de Schwann/citologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Clonagem Molecular , AMP Cíclico/fisiologia , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Homeobox/genética , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Fator 6 de Transcrição de Octâmero , Ratos , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
We have used PCR to identify 13 novel protein-tyrosine kinase genes (tyro-1 to -13), six of which (tyro-1 to -6) are preferentially expressed in the developing vertebrate nervous system. The tyro-2 and tyro-9 genes encode kinase domains that exhibit strong amino acid sequence similarity to the equivalent regions of the receptors for EGF and FGF, respectively, and may encode novel receptors for these or related polypeptide ligands. The tyro-1 to -6 genes are all expressed during central nervous system neurogenesis and exhibit distinct and highly regionalized patterns of expression in the adult brain. Together with recent studies in invertebrates, these data are consistent with the hypothesis that protein-tyrosine kinases play a central role in neural development.
Assuntos
Expressão Gênica , Sistema Nervoso/metabolismo , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Encéfalo/metabolismo , Células Cultivadas , Receptores ErbB/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Sistema Nervoso/embriologia , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/biossíntese , Sondas RNA , RNA Mensageiro , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
We have isolated the gene encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. In rats and mice, this gene is split into six exons distributed over 7 kb of DNA. The segregation of these exons is consistent with the functional segregation of the P0 protein into extracellular, membrane-spanning, and cytoplasmic domains. We find that the P0 extracellular domain is similar in structure to a single immunoglobulin variable region domain. In contrast to prototypical immunoglobulin domains, however, this P0 domain is encoded by two exons, the partitioning of which provides genetic evidence for the evolution of immunoglobulin-related domains from an ancestral half-domain. We also describe procedures for transfection of cultures of nontransformed rat Schwann cells and use these procedures to show that the Schwann cell-specific expression of the P0 gene is controlled by cis-acting elements localized upstream of exon I.
Assuntos
Regulação da Expressão Gênica , Proteínas da Mielina/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Proteína P0 da Mielina , Ratos , TransfecçãoRESUMO
We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.
Assuntos
AMP Cíclico/genética , Regulação da Expressão Gênica , Genes , Neuroglia/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , AMP Cíclico/farmacologia , Cicloeximida/farmacologia , Cinética , Dados de Sequência Molecular , Bainha de Mielina/fisiologia , RatosRESUMO
We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.
Assuntos
Regulação da Expressão Gênica , Proteínas da Mielina/genética , Regiões Promotoras Genéticas , Células de Schwann/fisiologia , Fatores Etários , Animais , Divisão Celular , Doenças Desmielinizantes/patologia , Toxina Diftérica/genética , Hormônio do Crescimento/genética , Camundongos , Camundongos Transgênicos , Proteína P0 da Mielina , Nervos Periféricos/patologia , Doenças do Sistema Nervoso Periférico/patologia , Fenótipo , RNA Mensageiro/genéticaRESUMO
SCIP is a POU domain transcription factor expressed by glial progenitor cells in the peripheral and central nervous systems (dividing Schwann cells and O-2A cells, respectively), where it appears to act as a repressor of myelin-specific genes. We have isolated genomic clones encoding the rat SCIP gene. Comparison of the structure of these clones with genomic Southern blots and SCIP cDNAs demonstrates that SCIP is encoded in a single-copy, intronless gene that has the general features of an expressed retroposon. This gene contributes to an extended CpG island. It is transcribed to produce a 3.1-kb mRNA that encodes a 451-amino-acid protein with a predicted molecular mass of 45 kDa. Immunopurified SCIP antibodies specifically recognize a nuclear protein of this size in cultured proliferating Schwann cells, and gel shift analyses demonstrate that this protein is the predominant octamer-binding protein in these cells.
Assuntos
Elementos de DNA Transponíveis , Fatores de Transcrição/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Clonagem Molecular , DNA , Expressão Gênica , Dados de Sequência Molecular , Fator 6 de Transcrição de Octâmero , Ratos , Mapeamento por Restrição , Retroviridae , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The past year has seen significant progress in the analysis of transcriptional regulation as it relates to neural development. Highlights include the identification and analysis of new homeobox genes that delimit developmental boundaries in the vertebrate forebrain, the study of upstream regulators of homeobox genes, the analysis of Pax genes that may contribute to specification of the vertebrate dorso-ventral neuraxis, and the functional analysis of transcription factors that are likely to specify particular neural cell types in both vertebrate and invertebrate nervous systems.
Assuntos
Neuroglia/fisiologia , Neurônios/fisiologia , Transcrição Gênica , Animais , Senescência Celular/fisiologia , Humanos , Fenômenos Fisiológicos do Sistema Nervoso , Fenótipo , Fatores de Transcrição/fisiologia , Vertebrados/fisiologiaRESUMO
The neuregulins are a family of closely related proteins that play important roles in neural and cardiac development, as well as in mammary carcinogenesis. The pleiotropic activities of these molecules are transduced by a set of receptor protein tyrosine kinases that exhibit structural similarity to the receptor for epidermal growth factor. Recent results have demonstrated essential roles for the neuregulins and their receptors in regulating cell number, determining cell fate, and establishing pattern in the developing central and peripheral nervous systems.
Assuntos
Glicoproteínas/fisiologia , Sistema Nervoso/embriologia , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Diferenciação Celular , Desenvolvimento Embrionário e Fetal , Crista Neural/citologia , Neurregulinas , Receptor ErbB-2 , Células de SchwannRESUMO
We have isolated cDNA clones encoding Tyro 10, a novel receptor protein-tyrosine kinase (PTK) whose catalytic domain exhibits significant similarity to the Trk family of neurotrophin receptors (Lai & Lemke, 1991). We find that the Tyro 10 gene is widely expressed, both within and outside the nervous system, and in both developing and mature neural tissue. The primary structure of Tyro 10, deduced from cDNA sequence, defines a new sub-family of receptor PTKs. Although the Tyro 10 kinase domain is more closely related to the equivalent domains of Trk, TrkB and TrkC than to the catalytic domains of other receptor PTKs, it is less closely related to these Trk domains than they are to each other. More significantly, the Tyro 10 extracellular (ligand binding) domain is not structurally related to the extracellular domains of the Trk receptors, but instead bears homology to cell surface mediators of protein-protein interactions, including blood coagulation Factors V and VIII, and the neuronal recognition protein A5. These appear to be structural features of a distinct receptor PTK sub-family, in that they are also found in the recently-described discoidin domain receptor (DDR).
Assuntos
Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Receptores com Domínio Discoidina , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ratos , Homologia de Sequência de AminoácidosRESUMO
We have isolated mouse cDNA clones encoding Tyro 3, a receptor protein-tyrosine kinase (PTK) of the mammalin central nervous system (CNS). Expression of the Tyro 3 gene is strongly up-regulated in neurons of the mouse neocortex, cerebellum, and hippocampus after the day of birth, during periods of active synaptogenesis, and high expression is maintained in the adult CNS. The sequence of Tyro 3 cDNAs predicts a glycoprotein receptor with similarity to neural cell recognition and adhesion molecules--the extracellular (ligand binding) region of this receptor is composed of two immunoglobulin-related domains followed by two fibronectin type III repeats. Immunoblot and immunoprecipitation analyses with anti-Tyro 3 antibodies indicate that the 125 kD Tyro 3 protein is abundantly expressed in CNS synaptosomes, and immunohistochemical analysis of cultured hippocampal cells demonstrates that Tyro 3 is a product of neurons. Rat-2 fibroblasts stably transfected with a Tyro 3 expression construct acquire the ability to grow in soft agar, suggesting that Tyro 3 is potentially oncogenic.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Receptores Proteína Tirosina Quinases/química , Sequência de Aminoácidos , Animais , Química Encefálica , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Cultivadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/toxicidadeRESUMO
SCIP is a POU domain transcription factor expressed by Schwann cells, the myelin-forming glial cells of the peripheral nervous system. In this study, we investigate SCIP regulation of the gene encoding P0, the major structural protein of peripheral myelin. We find that SCIP represses transcription of this gene through the joint action of the SCIP POU domain and an amino terminal domain that acts cell specifically. Maximal repression is DNA-binding-dependent, and analysis of the P0 promoter reveals the presence of multiple SCIP binding sites. Surprisingly, none of these sites in their native positions dramatically affect P0 promoter activity or its repression by SCIP, although they mediate repression when moved closer to the P0 transcription start site. We propose that repression occurs through a quenching mechanism mediated by the SCIP POU and amino terminal domains acting in concert with other nuclear proteins, including a Schwann cell-specific adapter.
Assuntos
Proteínas da Mielina/genética , Proteínas Repressoras/fisiologia , Células de Schwann/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação/fisiologia , Camundongos , Camundongos Transgênicos , Proteína P0 da Mielina , Fator 6 de Transcrição de Octâmero , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Transativadores , TransfecçãoRESUMO
We have studied the transcription factors SCIP and Krox-20 in differentiating Schwann cells-during normal development, in experimentally induced degenerating and regenerating peripheral nerves, and in cell culture-and have compared the expression of these regulators to a battery of genes that mark distinct stages in Schwann cell differentiation. In the myelinating Schwann cell lineage, we find that SCIP is initially induced by contact with axons and first appears near the last round of cell division in immature cells. This expression is transient-it is maximal in "promyelinating" cells and is then extinguished as Schwann cells overtly differentiate and myelinate axons. In contrast, Krox-20 appears in cells 24-36 h after they become SCIP+ and continues to be expressed in mature myelinating cells. These differences in regulation are seen in normal development, in regenerating nerves following nerve crush, and in cultured Schwann cells stimulated to adopt a myelination phenotype by elevation of intracellular cyclic AMP. Importantly, transient SCIP expression is also observed in the nonmyelinating Schwann cell lineage, but Krox-20 expression is not. Together with the myelination phenotypes exhibited by SCIP and Krox-20 mutant mice, these results suggest that SCIP preferentially acts during the predifferentiated phases of Schwann cell development, while in contrast, Krox-20 is associated with the later commitment to myelination and may therefore function as a direct transactivator of myelination genes.
RESUMO
Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Citometria de Fluxo , Humanos , Integrina alfaV/metabolismo , Macrófagos/metabolismo , Microscopia Eletrônica de Varredura , Fagocitose/genética , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina QuinaseRESUMO
Partial cDNAs of the human TYRO3 gene, encoding a putative receptor tyrosine kinase, and its processed pseudogene (TYRO3P) were cloned from human teratocarcinoma cell, bone marrow and melanocyte cDNA libraries. The tyrosine kinase homologous domains of TYRO3 and TYRO3P were sequenced and compared with each other and with the mouse TYRO3 gene. Abundant levels of the 4.2-kb TYRO3 mRNA were detected in human brain, and lower levels in other human tissues. TYRO3 and TYRO3P were both assigned to human chromosome 15q14-q25 by analysis of DNAs from somatic cell hybrids.
Assuntos
Cromossomos Humanos Par 15 , Pseudogenes , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , DNA , Humanos , Camundongos , Dados de Sequência Molecular , Receptores Proteína Tirosina Quinases/biossínteseRESUMO
Following peripheral nerve crush or transection, B-50 mRNA expression increased dramatically in the distal nerve stump. This increase has been fully attributed to an up-regulation of B-50 synthesis in reactive Schwann cells. Here we describe that B-50 mRNA expression in primary Schwann cell cultures is strongly down-regulated by cyclic AMP. Treatment of neonatal Schwann cell cultures with as low as 20 nM forskolin decreased B-50 mRNA expression. We show that B-50 promoter P2, but not P1, is active in Schwann cells and that the activity of P2 is inhibited 2.5 fold by forskolin. P2 does not contain a consensus sequence of a known cyclic AMP responsive element suggesting that the effect of forskolin is indirect.
Assuntos
AMP Cíclico/fisiologia , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/metabolismo , Células de Schwann/metabolismo , Animais , Animais Recém-Nascidos , Vírus do Sarcoma Aviário/genética , Células Cultivadas , Clonagem Molecular , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Proteína GAP-43 , Luciferases/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Oligodendrocytes, the myelinating cells of the central nervous system, arise from a profilerating pool of motile progenitor cells. The proliferation and survival of these cells is dependent on signal transduction via several protein tyrosine kinases (PTKs) including receptors for fibroblast growth factor -2, the platelet-derived growth factor receptors and the neurotrophin receptor, trkC. We hypothesized that additional PTKs could also influence oligodendroglial development. Utilizing RTPCR, we amplified from post-natal day 6 rat oligodendroglia 17 distinct kinase domain sequences, 14 of which were not previously known to be expressed by oligodendroglia. Amongst the sequences identified were the c-met and Fak genes, whose protein products regulate the motility of other epithelial cell types. Utilizing immunohistochemistry, we confirmed that both c-met and Fak are expressed by cultured oligodendroglia, suggesting that these proteins could also be implicated in regulating the motility of these cells.