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1.
Genet Med ; 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31316167

RESUMO

PURPOSE: Congenital contractural arachnodactyly (CCA) is an autosomal dominant connective tissue disorder manifesting joint contractures, arachnodactyly, crumpled ears, and kyphoscoliosis as main features. Due to its rarity, rather aspecific clinical presentation, and overlap with other conditions including Marfan syndrome, the diagnosis is challenging, but important for prognosis and clinical management. CCA is caused by pathogenic variants in FBN2, encoding fibrillin-2, but locus heterogeneity has been suggested. We designed a clinical scoring system and diagnostic criteria to support the diagnostic process and guide molecular genetic testing. METHODS: In this retrospective study, we assessed 167 probands referred for FBN2 analysis and classified them into a FBN2-positive (n = 44) and FBN2-negative group (n = 123) following molecular analysis. We developed a 20-point weighted clinical scoring system based on the prevalence of ten main clinical characteristics of CCA in both groups. RESULTS: The total score was significantly different between the groups (P < 0.001) and was indicative for classifying patients into unlikely CCA (total score <7) and likely CCA (total score ≥7) groups. CONCLUSIONS: Our clinical score is helpful for clinical guidance for patients suspected to have CCA, and provides a quantitative tool for phenotyping in research settings.

2.
Neurology ; 92(23): e2679-e2690, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31068484

RESUMO

OBJECTIVE: We took advantage of a large multinational recruitment to delineate genotype-phenotype correlations in a large, trans-European multicenter cohort of patients with spastic paraplegia gene 7 (SPG7). METHODS: We analyzed clinical and genetic data from 241 patients with SPG7, integrating neurologic follow-up data. One case was examined neuropathologically. RESULTS: Patients with SPG7 had a mean age of 35.5 ± 14.3 years (n = 233) at onset and presented with spasticity (n = 89), ataxia (n = 74), or both (n = 45). At the first visit, patients with a longer disease duration (>20 years, n = 62) showed more cerebellar dysarthria (p < 0.05), deep sensory loss (p < 0.01), muscle wasting (p < 0.01), ophthalmoplegia (p < 0.05), and sphincter dysfunction (p < 0.05) than those with a shorter duration (<10 years, n = 93). Progression, measured by Scale for the Assessment and Rating of Ataxia evaluations, showed a mean annual increase of 1.0 ± 1.4 points in a subgroup of 30 patients. Patients homozygous for loss of function (LOF) variants (n = 65) presented significantly more often with pyramidal signs (p < 0.05), diminished visual acuity due to optic atrophy (p < 0.0001), and deep sensory loss (p < 0.0001) than those with at least 1 missense variant (n = 176). Patients with at least 1 Ala510Val variant (58%) were older (age 37.6 ± 13.7 vs 32.8 ± 14.6 years, p < 0.05) and showed ataxia at onset (p < 0.05). Neuropathologic examination revealed reduction of the pyramidal tract in the medulla oblongata and moderate loss of Purkinje cells and substantia nigra neurons. CONCLUSIONS: This is the largest SPG7 cohort study to date and shows a spasticity-predominant phenotype of LOF variants and more frequent cerebellar ataxia and later onset in patients carrying at least 1 Ala510Val variant.

3.
Elife ; 82019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30950395

RESUMO

In mouse embryo gastrulation, epiblast cells delaminate at the primitive streak to form mesoderm and definitive endoderm, through an epithelial-mesenchymal transition. Mosaic expression of a membrane reporter in nascent mesoderm enabled recording cell shape and trajectory through live imaging. Upon leaving the streak, cells changed shape and extended protrusions of distinct size and abundance depending on the neighboring germ layer, as well as the region of the embryo. Embryonic trajectories were meandrous but directional, while extra-embryonic mesoderm cells showed little net displacement. Embryonic and extra-embryonic mesoderm transcriptomes highlighted distinct guidance, cytoskeleton, adhesion, and extracellular matrix signatures. Specifically, intermediate filaments were highly expressed in extra-embryonic mesoderm, while live imaging for F-actin showed abundance of actin filaments in embryonic mesoderm only. Accordingly, Rhoa or Rac1 conditional deletion in mesoderm inhibited embryonic, but not extra-embryonic mesoderm migration. Overall, this indicates separate cytoskeleton regulation coordinating the morphology and migration of mesoderm subpopulations.

4.
Development ; 141(11): 2349-59, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24821988

RESUMO

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.


Assuntos
Eletroporação/instrumentação , Animais , Movimento Celular , Simulação por Computador , Eletroporação/métodos , Embrião de Mamíferos/metabolismo , Endoderma/metabolismo , Desenho de Equipamento , Feminino , Análise de Elementos Finitos , Corantes Fluorescentes/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Miniaturização , Modelos Teóricos
5.
J Med Genet ; 50(9): 585-92, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23812909

RESUMO

BACKGROUND: Harstfield syndrome is the rare and unique association of holoprosencephaly (HPE) and ectrodactyly, with or without cleft lip and palate, and variable additional features. All the reported cases occurred sporadically. Although several causal genes of HPE and ectrodactyly have been identified, the genetic cause of Hartsfield syndrome remains unknown. We hypothesised that a single key developmental gene may underlie the co-occurrence of HPE and ectrodactyly. METHODS: We used whole exome sequencing in four isolated cases including one case-parents trio, and direct Sanger sequencing of three additional cases, to investigate the causative variants in Hartsfield syndrome. RESULTS: We identified a novel FGFR1 mutation in six out of seven patients. Affected residues are highly conserved and are located in the extracellular binding domain of the receptor (two homozygous mutations) or the intracellular tyrosine kinase domain (four heterozygous de novo variants). Strikingly, among the six novel mutations, three are located in close proximity to the ATP's phosphates or the coordinating magnesium, with one position required for kinase activity, and three are adjacent to known mutations involved in Kallmann syndrome plus other developmental anomalies. CONCLUSIONS: Dominant or recessive FGFR1 mutations are responsible for Hartsfield syndrome, consistent with the known roles of FGFR1 in vertebrate ontogeny and conditional Fgfr1-deficient mice. Our study shows that, in humans, lack of accurate FGFR1 activation can disrupt both brain and hand/foot midline development, and that FGFR1 loss-of-function mutations are responsible for a wider spectrum of clinical anomalies than previously thought, ranging in severity from seemingly isolated hypogonadotropic hypogonadism, through Kallmann syndrome with or without additional features, to Hartsfield syndrome at its most severe end.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Dedos/anormalidades , Deformidades Congênitas da Mão/genética , Holoprosencefalia/genética , Mutação INDEL/genética , Deficiência Intelectual/genética , Deformidades Congênitas dos Membros/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Sequência de Bases , Sítios de Ligação , Fenda Labial/enzimologia , Fissura Palatina/enzimologia , Exoma , Feminino , Genômica , Deformidades Congênitas da Mão/enzimologia , Holoprosencefalia/enzimologia , Humanos , Deficiência Intelectual/enzimologia , Deformidades Congênitas dos Membros/enzimologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Análise de Sequência de DNA
6.
Dev Biol ; 364(2): 192-201, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22342906

RESUMO

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.


Assuntos
Padronização Corporal , Movimento Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , PTEN Fosfo-Hidrolase/fisiologia , Animais , Endoderma/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , PTEN Fosfo-Hidrolase/genética , Gravidez
7.
Development ; 138(14): 3011-20, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21693517

RESUMO

The establishment of the mammalian body plan depends on signal-regulated cell migration and adhesion, processes that are controlled by the Rho family of GTPases. Here we use a conditional allele of Rac1, the only Rac gene expressed early in development, to define its roles in the gastrulating mouse embryo. Embryos that lack Rac1 in the epiblast (Rac1Δepi) initiate development normally: the signaling pathways required for gastrulation are active, definitive endoderm and all classes of mesoderm are specified, and the neural plate is formed. After the initiation of gastrulation, Rac1Δepi embryos have an enlarged primitive streak, make only a small amount of paraxial mesoderm, and the lateral anlage of the heart do not fuse at the midline. Because these phenotypes are also seen in Nap1 mutants, we conclude that Rac1 acts upstream of the Nap1/WAVE complex to promote migration of the nascent mesoderm. In addition to migration phenotypes, Rac1Δepi cells fail to adhere to matrix, which leads to extensive cell death. Cell death is largely rescued in Rac1Δepi mutants that are heterozygous for a null mutation in Pten, providing evidence that Rac1 is required to link signals from the basement membrane to activation of the PI3K-Akt pathway in vivo. Surprisingly, the frequency of apoptosis is greater in the anterior half of the embryo, suggesting that cell survival can be promoted either by matrix adhesion or by signals from the posterior primitive streak. Rac1 also has essential roles in morphogenesis of the posterior notochordal plate (the node) and the midline.


Assuntos
Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Gástrula/fisiologia , Mesoderma/embriologia , Morfogênese/fisiologia , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Apoptose/fisiologia , Movimento Celular/genética , Indóis , Camundongos , Transdução de Sinais/fisiologia , Estatísticas não Paramétricas , Proteínas rac1 de Ligação ao GTP
8.
J Immunol ; 187(3): 1475-85, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21709160

RESUMO

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.


Assuntos
Proteínas de Transporte/metabolismo , Catepsina D/fisiologia , Fatores Quimiotáticos/agonistas , Hemeproteínas/metabolismo , Peptídeos/agonistas , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Receptores de Formil Peptídeo/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Proteínas de Transporte/biossíntese , Catepsina D/deficiência , Células Cultivadas , Fatores Quimiotáticos/biossíntese , Fatores Quimiotáticos/metabolismo , Cricetinae , Cricetulus , Hemeproteínas/biossíntese , Humanos , Ligantes , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos/metabolismo , Ligação Proteica/imunologia , Precursores de Proteínas/biossíntese , Receptores de Formil Peptídeo/biossíntese
9.
Proc Natl Acad Sci U S A ; 108(17): 7022-7, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21482783

RESUMO

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/embriologia , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos/embriologia , Coração/embriologia , Complexos Multiproteicos/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
10.
PLoS Biol ; 8(8): e1000442, 2010 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-20689803

RESUMO

Cell migration and cell rearrangements are critical for establishment of the body plan of vertebrate embryos. The first step in organization of the body plan of the mouse embryo, specification of the anterior-posterior body axis, depends on migration of the anterior visceral endoderm from the distal tip of the embryo to a more proximal region overlying the future head. The anterior visceral endoderm (AVE) is a cluster of extra-embryonic cells that secretes inhibitors of the Wnt and Nodal pathways to inhibit posterior development. Because Rac proteins are crucial regulators of cell migration and mouse Rac1 mutants die early in development, we tested whether Rac1 plays a role in AVE migration. Here we show that Rac1 mutant embryos fail to specify an anterior-posterior axis and, instead, express posterior markers in a ring around the embryonic circumference. Cells that express the molecular markers of the AVE are properly specified in Rac1 mutants but remain at the distal tip of the embryo at the time when migration should take place. Using tissue specific deletions, we show that Rac1 acts autonomously within the visceral endoderm to promote cell migration. High-resolution imaging shows that the leading wild-type AVE cells extend long lamellar protrusions that span several cell diameters and are polarized in the direction of cell movement. These projections are tipped by filopodia-like structures that appear to sample the environment. Wild-type AVE cells display hallmarks of collective cell migration: they retain tight and adherens junctions as they migrate and exchange neighbors within the plane of the visceral endoderm epithelium. Analysis of mutant embryos shows that Rac1 is not required for intercellular signaling, survival, proliferation, or adhesion in the visceral endoderm but is necessary for the ability of visceral endoderm cells to extend projections, change shape, and exchange neighbors. The data show that Rac1-mediated epithelial migration of the AVE is a crucial step in the establishment of the mammalian body plan and suggest that Rac1 is essential for collective migration in mammalian tissues.


Assuntos
Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Embrião de Mamíferos/fisiologia , Endoderma/citologia , Neuropeptídeos/metabolismo , Vísceras/embriologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Indução Embrionária , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Neuropeptídeos/genética , Vísceras/citologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP
11.
Dev Biol ; 346(2): 237-46, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20678497

RESUMO

The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.


Assuntos
Padronização Corporal/genética , Embrião de Mamíferos/metabolismo , Proteínas de Membrana/genética , Morfogênese/genética , Animais , Proteínas do Citoesqueleto , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL
12.
J Immunol ; 178(3): 1450-6, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17237393

RESUMO

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.


Assuntos
Fatores Quimiotáticos/fisiologia , Neutrófilos/fisiologia , Receptores de Formil Peptídeo/metabolismo , Animais , Cálcio , Proteínas de Transporte , Quimiotaxia , Hemeproteínas , Humanos , Camundongos , Peptídeos
13.
Cytokine Growth Factor Rev ; 17(6): 501-19, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17084101

RESUMO

The formyl peptide receptor (FPR) family is involved in host defence against pathogens, but also in sensing internal molecules that may constitute signals of cellular dysfunction. It includes three subtypes in human and other primates. FPR responds to formyl peptides derived from bacterial and mitochondrial proteins. FPRL1 displays a large array of exogenous and endogenous ligands, including the chemokine variant sCKbeta8-1, the neuroprotective peptide humanin, and lipoxin A4. Two high affinity agonists (F2L and humanin) were recently described for FPRL2. In mouse, eight FPR-related receptors have been described. Fpr1 is the ortholog of human FPR, while fpr2 appears to share many ligands with human FPRL1. Altogether, the physiological role of the FPR family is still incompletely understood, due in part to the large variety of ligands, the redundancy with other chemoattractant agents, and the lack of clear orthologs between human and mouse receptors. Newly developed tools will allow to study further this family of receptors.


Assuntos
Receptores de Formil Peptídeo/imunologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Filogenia , Receptores de Formil Peptídeo/classificação , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/imunologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual
14.
J Exp Med ; 201(1): 83-93, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15623572

RESUMO

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein-coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases through the G(i) class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.


Assuntos
Cálcio/metabolismo , Fatores Quimiotáticos/genética , Quimiotaxia/imunologia , Células Dendríticas/imunologia , Receptores de Formil Peptídeo/metabolismo , Transdução de Sinais/genética , Sequência de Aminoácidos , Anticorpos Monoclonais , Proteínas de Transporte/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia/genética , Primers do DNA , Células Dendríticas/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Hemeproteínas/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos , Receptores de Formil Peptídeo/agonistas , Receptores de Lipoxinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
15.
J Exp Med ; 198(7): 977-85, 2003 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-14530373

RESUMO

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein-coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42-p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Quimiocinas/fisiologia , Receptores de Quimiocinas/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Movimento Celular , Quimiocinas/química , Quimiocinas/genética , Quimiocinas/isolamento & purificação , Células Dendríticas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular
16.
Eur J Immunol ; 32(2): 494-501, 2002 02.
Artigo em Inglês | MEDLINE | ID: mdl-11828366

RESUMO

Human chemokine receptor (HCR) is a putative chemokine receptor sharing high similarity with CCR1, CCR2, CCR3 and CCR5. Its gene is located within the main cluster of CC-chemokine receptor genes, in the 3p21 region of the human genome. We generated monoclonal antibodies directed at human HCR, and studied its distribution in human leukocyte populations and cell lines, and its regulation following maturation or activation of these populations. In peripheral blood leukocytes, HCR is expressed on CD4+ and CD8+ T lymphocytes, including most memory and part of naive cells, but is absent from B cells. Expression of HCR was enhanced following stimulation of T cells by OKT3 and IL-2. HCR is present on monocytes and macrophages. Monocyte-derived dendritic cells harbored HCR, and expression was enhanced following stimulation by lipopolysaccharides, poly (I:C), IFN-gamma or CD40L. Neutrophils strongly expressed HCR. A similar distribution was found in bone marrow,and HCR was also expressed in CD34+ precursors. Expression of HCR and its regulation were confirmed by real-time PCR. In a panel of human tissues, we found abundant HCR transcripts in thymus, spleen, lymph nodes and lung. This large distribution across leukocyte populations, and the up-regulation during DC maturation, represent a new profile among chemokine receptors. We speculate that HCR responds to inflammatory chemokines, and might be involved in the interaction between antigen presenting and T cells, and in hematopoiesis.


Assuntos
Leucócitos/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Animais , Anticorpos Monoclonais , Sequência de Bases , Diferenciação Celular , DNA Complementar/genética , Regulação da Expressão Gênica , Humanos , Leucócitos/citologia , Ativação Linfocitária , Camundongos , Distribuição Tecidual
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