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2.
J Clin Invest ; 129(6): 2555-2570, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31107242

RESUMO

The nuclear protein DEK is an endogenous DNA-binding chromatin factor regulating hematopoiesis. DEK is one of only 2 known secreted nuclear chromatin factors, but whether and how extracellular DEK regulates hematopoiesis is not known. We demonstrated that extracellular DEK greatly enhanced ex vivo expansion of cytokine-stimulated human and mouse hematopoietic stem cells (HSCs) and regulated HSC and hematopoietic progenitor cell (HPC) numbers in vivo and in vitro as determined both phenotypically (by flow cytometry) and functionally (through transplantation and colony formation assays). Recombinant DEK increased long-term HSC numbers and decreased HPC numbers through a mechanism mediated by the CXC chemokine receptor CXCR2 and heparan sulfate proteoglycans (HSPGs) (as determined utilizing Cxcr2-/- mice, blocking CXCR2 antibodies, and 3 different HSPG inhibitors) that was associated with enhanced phosphorylation of ERK1/2, AKT, and p38 MAPK. To determine whether extracellular DEK required nuclear function to regulate hematopoiesis, we utilized 2 mutant forms of DEK: one that lacked its nuclear translocation signal and one that lacked DNA-binding ability. Both altered HSC and HPC numbers in vivo or in vitro, suggesting the nuclear function of DEK is not required. Thus, DEK acts as a hematopoietic cytokine, with the potential for clinical applicability.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Proteínas Cromossômicas não Histona/genética , Citocinas/genética , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores de Interleucina-8B
3.
Front Immunol ; 10: 2957, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31998291

RESUMO

Sepsis results in millions of deaths every year, with acute lung injury (ALI) being one of the leading causes of mortality in septic patients. As neutrophil extracellular traps (NETs) are abundant in sepsis, neutralizing components of NETs may be a useful strategy to improve outcomes of sepsis. Citrullinated histone H3 (CitH3) has been recently shown to be involved in the NET formation. In this study, we demonstrate that CitH3 damages human umbilical vein endothelial cells (HUVECs) and potentiates NET formation through a positive feedback mechanism. We developed a novel CitH3 monoclonal antibody to target peptidylarginine deiminase (PAD) 2 and PAD 4 generated CitH3. In a mouse model of lethal lipopolysaccharide (LPS) induced shock, neutralizing CitH3 with the newly developed anti-CitH3 monoclonal antibody attenuates inflammatory responses, ameliorates ALI, and improves survival. Our study suggests that effectively blocking circulating CitH3 might be a potential therapeutic method for the treatment of endotoxemia.

4.
mBio ; 9(1)2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487235

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.


Assuntos
Encéfalo/parasitologia , Endocitose , Interações Hospedeiro-Patógeno , Listeria monocytogenes/fisiologia , Listeriose/parasitologia , Vimentina/metabolismo , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Listeriose/patologia , Camundongos , Ratos
5.
Arthritis Rheumatol ; 70(4): 594-605, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29287303

RESUMO

OBJECTIVE: The nuclear oncoprotein DEK is an autoantigen associated with juvenile idiopathic arthritis (JIA), especially the oligoarticular subtype. DEK is a secreted chemotactic factor. Abundant levels of DEK and DEK autoantibodies are found in inflamed synovium in JIA. We undertook this study to further characterize the nature of DEK autoantibodies in screening serum samples from 2 different cohorts that consisted mostly of patients with JIA. METHODS: DEK autoantibody levels were analyzed in sera from 33 JIA patients, 13 patients with other inflammatory conditions, and 11 healthy controls, as well as in 89 serum samples from JIA patients receiving anti-tumor necrosis factor (anti-TNF) therapy. Recombinant His-tagged full-length DEK protein (1-375 amino acids [aa]) and the 187-375-aa and 1-350-aa His-tagged DEK fragments made in a baculovirus system were used for enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The C-terminal 25-aa fragment of DEK was expressed in a glutathione S-transferase-tagged vector. ELISA results were calculated as area under the curve by the trapezoidal rule. RESULTS: DEK autoantibody levels were significantly higher in patients with polyarticular JIA than in those with oligoarticular JIA, and were higher in patients with polyarticular JIA who had more active disease after cessation of anti-TNF therapy. Immunoblotting against the C-terminal 25-aa fragment of DEK confirmed that this section of the DEK molecule is the most immunogenic domain. CONCLUSION: DEK autoantibody levels are higher in patients with polyarticular JIA than in those with oligoarticular JIA, and higher in patients who have disease flares after cessation of anti-TNF therapy. The C-terminal 25-aa fragment is the most immunogenic portion of DEK. These findings are significant with respect to the nature of DEK autoantibodies, their contribution to JIA pathogenesis, and their implications for JIA management.


Assuntos
Antirreumáticos/imunologia , Artrite Juvenil/sangue , Autoanticorpos/sangue , Proteínas Cromossômicas não Histona/imunologia , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adolescente , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/imunologia , Autoanticorpos/imunologia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Masculino , Exacerbação dos Sintomas , Suspensão de Tratamento
6.
Nat Commun ; 8: 14252, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28165452

RESUMO

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.


Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Artrite Juvenil/terapia , Fatores Quimiotáticos/antagonistas & inibidores , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Armadilhas Extracelulares/imunologia , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adulto , Animais , Artrite Juvenil/imunologia , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/imunologia , Fatores Quimiotáticos/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Proteínas Oncogênicas/imunologia , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/imunologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Cultura Primária de Células , Líquido Sinovial/química , Líquido Sinovial/citologia , Líquido Sinovial/imunologia , Zimosan/imunologia
7.
PLoS One ; 11(9): e0162641, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27657497

RESUMO

BACKGROUND: NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. METHODOLOGY/PRINCIPAL FINDINGS: The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. CONCLUSION/SIGNIFICANCE: Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis.

8.
Nat Microbiol ; 1: 16025, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-27572444

RESUMO

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.


Assuntos
Aderência Bacteriana , Salmonella typhimurium/fisiologia , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Vimentina/metabolismo , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/fisiologia , Animais , Antígenos de Bactérias/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Queratina-18/metabolismo , Camundongos , Ligação Proteica , Transporte Proteico
9.
Blood Cells Mol Dis ; 54(1): 123-31, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25128083

RESUMO

DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation.


Assuntos
Proteínas Cromossômicas não Histona/biossíntese , Proteínas de Ligação a DNA/biossíntese , Bases de Dados Genéticas , Regulação Leucêmica da Expressão Gênica , Hematopoese , Leucemia Mieloide Aguda/metabolismo , Proteínas Oncogênicas/biossíntese , Animais , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 9/genética , Estudos de Coortes , Proteínas de Ligação a DNA/genética , Intervalo Livre de Doença , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Estudos Multicêntricos como Assunto , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Taxa de Sobrevida , Translocação Genética
10.
Stem Cells ; 31(8): 1447-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23733396

RESUMO

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non-histone nuclear phosphoprotein initially identified as a putative proto-oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long-term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long-term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems.


Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas Oncogênicas/fisiologia , Animais , Humanos , Proteínas de Ligação a Poli-ADP-Ribose
11.
Proc Natl Acad Sci U S A ; 110(17): 6847-52, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23569252

RESUMO

DEK is a biochemically distinct, conserved nonhistone protein that is vital to global heterochromatin integrity. In addition, DEK can be secreted and function as a chemotactic, proinflammatory factor. Here we show that exogenous DEK can penetrate cells, translocate to the nucleus, and there carry out its endogenous nuclear functions. Strikingly, adjacent cells can take up DEK secreted from synovial macrophages. DEK internalization is a heparan sulfate-dependent process, and cellular uptake of DEK into DEK knockdown cells corrects global heterochromatin depletion and DNA repair deficits, the phenotypic aberrations characteristic of these cells. These findings thus unify the extracellular and intracellular activities of DEK, and suggest that this paracrine loop involving DEK plays a role in chromatin biology.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Oncogênicas/metabolismo , Comunicação Parácrina/fisiologia , Fracionamento Celular , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Proteínas de Ligação a Poli-ADP-Ribose , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética
12.
Sci Rep ; 3: 1045, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23304436

RESUMO

Vimentin, an abundant intermediate filament protein, presumably has an important role in stabilizing intracellular architecture, but its function is otherwise poorly understood. In a vimentin knockout (Vim KO) mouse model, we note that Vim KO mice challenged with intraperitoneal Escherichia coli control bacterial infection better than do wild-type (WT) mice. In vitro, Vim KO phagocytes show significantly increased capacity to mediate bacterial killing by abundant production of reactive oxygen species (ROS) and nitric oxides, likely due to interactions with the p47phox active subunit of NADPH oxidase. In acute colitis induced by dextran sodium sulfate (DSS), Vim KO mice develop significantly less gut inflammation than do WT mice. Further, Vim KO mice have markedly decreased bacterial extravasation in the setting of DSS-induced acute colitis, consistent with decreased intestinal disease. Our results suggest that vimentin impedes bacterial killing and production of ROS, thereby contributing to the pathogenesis of acute colitis.


Assuntos
Colite/metabolismo , Vimentina/metabolismo , Animais , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Escherichia coli/patogenicidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Fagocitose , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vimentina/antagonistas & inibidores , Vimentina/genética
13.
Stem Cells Dev ; 21(9): 1449-54, 2012 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21943234

RESUMO

DEK is a biochemically distinct protein that is generally found in the nucleus, where it is vital to global heterochromatin integrity. However, DEK is also secreted by cells (eg, macrophages) and influences other adjacent cells (eg, acts as a chemoattractant for certain mature blood cells). We hypothesized that DEK may modulate functions of hematopoietic stem (HSCs) and progenitor (HPCs) cells. C57Bl/6 mice were used to demonstrate that absolute numbers and cycling status of HPCs (colony forming unit-granulocyte macrophage [CFU-GM], burst forming unit-erythroid [BFU-E], and colony forming unit-granulocyte erythroid macrophage megakaryocyte [CFU-GEMM]) in bone marrow (BM) and spleen were significantly enhanced in DEK -/- as compared with wild-type (WT) control mice. Moreover, purified recombinant DEK protein inhibited colony formation in vitro by CFU-GM, BFU-E, and CFU-GEMM from WT BM cells and human cord blood (CB) cells in a dose-dependent fashion, demonstrating that DEK plays a negative role in HPC proliferation in vitro and in vivo. Suppression was direct acting as determined by inhibition of proliferation of single isolated CD34(+) CB cells in vitro. In contrast, DEK -/- BM cells significantly demonstrated reduced long term competitive and secondary mouse repopulating HSC capacity compared with WT BM cells, demonstrating that DEK positively regulates engrafting capability of self-renewing HSCs. This demonstrates that DEK has potent effects on HSCs, HPCs, and hematopoiesis, information of biological and potential clinical interest.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Transplante Heterólogo , Transplante Homólogo
14.
Arthritis Rheum ; 63(2): 556-67, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21280010

RESUMO

OBJECTIVE: DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes. Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA. METHODS: DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). RESULTS: DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS. CONCLUSION: These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.


Assuntos
Artrite Juvenil/metabolismo , Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/metabolismo , Processamento de Proteína Pós-Traducional , Membrana Sinovial/metabolismo , Acetilação , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Artrite Juvenil/imunologia , Artrite Juvenil/patologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Criança , Proteínas Cromossômicas não Histona/imunologia , Humanos , Articulações/metabolismo , Articulações/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas Oncogênicas/imunologia , Proteínas de Ligação a Poli-ADP-Ribose , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Membrana Sinovial/patologia
15.
Prostate ; 70(4): 377-89, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19866464

RESUMO

BACKGROUND: Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa. METHODS: An in vitro system designed to mimic the human prostatic microenvironment incorporating prostatic stroma (primary and immortalized prostate stromal fibroblasts), epithelium (N15C6, BPH-1, LNCaP, and PC3 cells), and inflammatory infiltrate (HL-60 cells, HH, and Molt-3 T-lymphocytes) was developed. Modified Boyden chamber assays were used to test the ability of prostate stromal and epithelial cells to attract leukocytes and to test the effect of leukocytes on prostate cellular proliferation. Antibody arrays were used to identify leukocyte-secreted cytokines mediating prostate cellular proliferation. RESULTS: Leukocytic cells migrated towards both prostate stromal and epithelial cells. CD4+ T-lymphocytes promoted the proliferation of both transformed and non-transformed prostate epithelial cell lines tested, whereas CD8+ T-lymphocytes as well as dHL-60M macrophagic and dHL-60N neutrophilic cells selectively promoted the proliferation of PCa cells. CONCLUSIONS: The results of these studies show that inflammatory cells can be attracted to the prostate tissue microenvironment and can selectively promote the proliferation of non-transformed or transformed prostate epithelial cells, and are consistent with differential role(s) for inflammatory infiltrate in the etiologies of benign and malignant proliferative disease in the prostate.


Assuntos
Linfócitos B/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/patologia , Próstata/citologia , Linfócitos T/fisiologia , Linhagem Celular Transformada , Movimento Celular/fisiologia , Citocinas/metabolismo , Células Epiteliais/citologia , Fibroblastos/citologia , Humanos , Masculino , Próstata/metabolismo , Hiperplasia Prostática/patologia , Células Estromais/citologia
16.
Chem Biol ; 14(6): 623-34, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17584610

RESUMO

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.


Assuntos
Antineoplásicos/farmacologia , Ergosterol/análogos & derivados , Vimentina/metabolismo , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Sítios de Ligação , Western Blotting , Linhagem Celular , Neovascularização da Córnea/tratamento farmacológico , Eletroforese em Gel Bidimensional , Ergosterol/química , Ergosterol/farmacologia , Ergosterol/uso terapêutico , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Vimentina/genética , Vitanolídeos
17.
Mol Cell Biol ; 26(24): 9484-96, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17030615

RESUMO

The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8+ T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.


Assuntos
Artrite Juvenil/metabolismo , Artrite Juvenil/patologia , Autoantígenos/fisiologia , Fatores Quimiotáticos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Oncogênicas/metabolismo , Artrite Juvenil/imunologia , Autoantígenos/metabolismo , Células Cultivadas , Fatores Quimiotáticos/fisiologia , Quimiotaxia de Leucócito , Criança , Proteínas Cromossômicas não Histona/fisiologia , Líquido Extracelular/imunologia , Líquido Extracelular/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Proteínas Oncogênicas/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose
18.
J Biol Chem ; 280(36): 31760-7, 2005 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-15987677

RESUMO

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.


Assuntos
Acetiltransferases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Espaço Intranuclear/metabolismo , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/fisiologia , Acetilação , Sequência de Aminoácidos , Proteína de Ligação a CREB , Linhagem Celular Tumoral , DNA/metabolismo , Genes Reporter , Histona Acetiltransferases , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Transativadores/metabolismo , Fatores de Transcrição de p300-CBP
19.
Mol Cell Biol ; 24(20): 9198-206, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15456890

RESUMO

During mammalian vascular development, endothelial cells form a complex array of vessels that differ markedly in structure and function, but the molecular basis for this vascular complexity is poorly understood. Recent insights into endothelial diversity have come from the identification of molecular markers expressed on distinct endothelial cell populations. One such marker, the PAL-E antibody, has been used for almost 20 years to distinguish blood and lymphatic vessels, but the identity of the protein recognized by PAL-E has been unknown. In the present study we have used protein purification and tandem mass spectrometry analysis of tryptic peptides to identify the PAL-E antigen as a secreted form of vimentin. Vimentin has been well characterized as an intracellular intermediate filament protein expressed broadly in mesenchymal cells. In contrast, PAL-E-reactive vimentin is secreted extracellularly, its synthesis is restricted to a distinct population of blood endothelial cells and activated macrophages, and PAL-E-reactive vimentin is found in circulating human blood. PAL-E-reactive vimentin does not arise from an endothelial cell-specific mRNA transcript but is the product of cell-specific posttranslational modification. The PAL-E antibody therefore defines secretion of vimentin as a molecular distinction among endothelial cells and exposes a novel, extracellular role for vimentin in the blood vasculature.


Assuntos
Anticorpos Monoclonais/metabolismo , Vasos Sanguíneos/metabolismo , Células Endoteliais/metabolismo , Isoformas de Proteínas/sangue , Vimentina/sangue , Sequência de Aminoácidos , Vasos Sanguíneos/citologia , Linhagem Celular , Células Endoteliais/ultraestrutura , Endotélio Vascular/citologia , Humanos , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Vimentina/genética , Vimentina/isolamento & purificação , Vimentina/metabolismo
20.
Pediatr Surg Int ; 20(6): 419-24, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15108013

RESUMO

Among factors promoting mucosal hyperplasia after bowel resection, long-chain fatty acids may have a special role. The purpose of the present study was to evaluate the effects of high-fat diet (HFD) on early intestinal adaptation in rats with short bowel syndrome (SBS). Male Sprague-Dawley rats underwent either a bowel transection with re-anastomosis (Sham rats) or 75% small bowel resection (SBS rats). Animals were randomly assigned to one of three groups: Sham rats fed normal chow (Sham-NC); SBS rats fed NC (SBS-NC); and SBS rats fed HFD (SBS-HFD). Rats were killed on days 3 or 14. Body weight and parameters of intestinal adaptation (overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth) were determined at time of killing. By day 3, SBS-HFD rats demonstrated higher duodenal and jejunal bowel and mucosal weights and ileal villus height and jejunal crypt depth vs SBS-NC rats. By day 14 SBS-HFD rats continued to demonstrate increased duodenal and jejunal bowel weight and duodenal mucosal weight vs SBS-NC animals. We conclude that early exposure to HFD both augmented and accelerated structural bowel adaptation in a rat model of SBS.


Assuntos
Adaptação Fisiológica , Gorduras na Dieta/administração & dosagem , Intestino Delgado/fisiopatologia , Síndrome do Intestino Curto/fisiopatologia , Animais , Peso Corporal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Síndrome do Intestino Curto/dietoterapia , Síndrome do Intestino Curto/patologia , Fatores de Tempo
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