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1.
J Exp Med ; 203(4): 1067-80, 2006 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-16606672

RESUMO

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that has multiple ligands and is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. However, the role of RAGE in normal physiology is largely undefined. Here, we present evidence for a role of RAGE in osteoclast maturation and function, which has consequences for bone remodeling. Mice lacking RAGE had increased bone mass and bone mineral density and decreased bone resorptive activity in vivo. In vitro-differentiated RAGE-deficient osteoclasts exhibited disrupted actin ring and sealing zone structures, impaired maturation, and reduced bone resorptive activity. Impaired signaling downstream of alphavbeta3 integrin was observed in RAGE(-/-) bone marrow macrophages and precursors of OCs. These results demonstrate a role for RAGE in osteoclast actin cytoskeletal reorganization, adhesion, and function, and suggest that the osteosclerotic-like phenotype observed in RAGE knockout mice is due to a defect in osteoclast function.


Assuntos
Osso e Ossos/fisiologia , Osteoclastos/fisiologia , Receptores Imunológicos/fisiologia , Actinas/fisiologia , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Regulação para Baixo/fisiologia , Produtos Finais de Glicação Avançada , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/fisiologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética
2.
Blood ; 115(15): 3136-45, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20065295

RESUMO

Neogenin, a deleted in colorectal cancer (DCC) family member, has been identified as a receptor for the neuronal axon guidance cues netrins and repulsive guidance molecules repulsive guidance molecules (RGM). RGMc, also called hemojuvelin (HJV), is essential for iron homeostasis. Here we provide evidence that neogenin plays a critical role in iron homeostasis by regulation of HJV secretion and bone morphogenetic protein (BMP) signaling. Livers of neogenin mutant mice exhibit iron overload, low levels of hepcidin, and reduced BMP signaling. Mutant hepatocytes in vitro show impaired BMP2 induction of Smad1/5/8 phosphorylation and hepcidin expression. Neogenin is expressed in liver cells in a reciprocal pattern to that of hepcidin, suggesting that neogenin functions in a cell nonautonomous manner. Further studies demonstrate that neogenin may stabilize HJV, a glycosylphosphatidylinositol-anchored protein that interacts with neogenin and suppresses its secretion. Taken together, our results lead the hypothesis that neogenin regulates iron homeostasis via inhibiting secretion of HJV, an inhibitor of BMP signaling, to enhance BMP signaling and hepcidin expression. These results reveal a novel mechanism underlying neogenin regulation of HJV-BMP signaling.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Homeostase/efeitos dos fármacos , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Transporte de Cátions/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína da Hemocromatose , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepcidinas , Humanos , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
3.
Nat Cell Biol ; 7(11): 1124-32, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16244667

RESUMO

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.


Assuntos
Embrião de Galinha/citologia , Fatores de Crescimento Neural/fisiologia , Neuritos/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Células Cultivadas , Embrião de Galinha/metabolismo , Receptor DCC , Humanos , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Netrina-1 , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra
4.
J Leukoc Biol ; 87(3): 415-23, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19955209

RESUMO

PYK2, a major cell adhesion-activated tyrosine kinase, is highly expressed in macrophages and implicated in macrophage activation and inflammatory response. However, mechanisms by which PYK2 regulates inflammatory response are beginning to be understood. In this study, we demonstrate that PYK2 interacts with MyD88, a crucial signaling adaptor protein in LPS and PGN-induced NF-kappaB activation, in vitro and in macrophages. This interaction, increased in macrophages, stimulated by LPS, requires the death domain of MyD88. PYK2-deficient macrophages exhibit reduced phosphorylation and degradation of IkappaB, an inhibitor of NF-kappaB nuclear translocation, and decreased NF-kappaB activation and IL-1beta expression by LPS. These results suggest that via interaction with MyD88, PYK2 is involved in modulating cytokine (e.g., LPS) stimulation of NF-kappaB activity and signaling, providing a mechanism underlying PYK2 regulation of an inflammatory response.


Assuntos
Quinase 2 de Adesão Focal/metabolismo , Macrófagos/enzimologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/genética , Peptidoglicano/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
J Bone Miner Res ; 23(7): 1084-96, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18302500

RESUMO

High-mobility group box 1 (HMGB1), a nonhistone nuclear protein, is released by macrophages into the extracellular milieu consequent to cellular activation. Extracellular HMGB1 has properties of a pro-inflammatory cytokine through its interaction with receptor for advanced glycation endproducts (RAGE) and/or toll-like receptors (TLR2 and TLR4). Although HMGB1 is highly expressed in macrophages and differentiating osteoclasts, its role in osteoclastogenesis remains largely unknown. In this report, we present evidence for a function of HMGB1 in this event. HMGB1 is released from macrophages in response to RANKL stimulation and is required for RANKL-induced osteoclastogenesis in vitro and in vivo. In addition, HMGB1, like other osteoclastogenic cytokines (e.g., TNFalpha), enhances RANKL-induced osteoclastogenesis in vivo and in vitro at subthreshold concentrations of RANKL, which alone would be insufficient. The role of HMGB1 in osteoclastogenesis is mediated, in large part, by its interaction with RAGE, an immunoglobin domain containing family receptor that plays an important role in osteoclast terminal differentiation and activation. HMGB1-RAGE signaling seems to be important in regulating actin cytoskeleton reorganization, thereby participating in RANKL-induced and integrin-dependent osteoclastogenesis. Taken together, these observations show a novel function of HMGB1 in osteoclastogenesis and provide a new link between inflammatory mechanisms and bone resorption.


Assuntos
Divisão Celular/fisiologia , Proteína HMGB1/fisiologia , Osteoclastos/citologia , Ligante RANK/fisiologia , Receptores Imunológicos/fisiologia , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Imunofluorescência , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 14(1): 123-7, 2006 Feb.
Artigo em Zh | MEDLINE | ID: mdl-16584607

RESUMO

This study was aimed to establish downstream purification procedure by which the protein of interest can be purified to higher purity rapidly and efficiently. The different combinations of various purification strategies, methods and conditions were compared, including reversed phase chromatography, metal chelating chromatography, anion exchange chromatography, blue dye affinity chromatography, filtration chromatography and so on. The results showed that in reversed phase chromatography, isolated protein of interest was denatured and precipitated immediately after chromatography because methanol or acetonitrile were adopted as the organic phase. In blue dye affinity chromatography expecting to purify the protein of interest in one step, protein of interest was difficultly differentiated from mixed protein as much proteins bound to the chromatography media by non-specific affinity. While there is a translation-enhancing sequence T7-g10 in the PRSETA-B7-2-PE40KDEL expression vector, so it adds 6 histidines to the N terminus of the protein of interest, this allows to purify the protein of interest by metal chelating chromatography. Based on this characteristic, a three-step chromatography line including metal chelating chromatography, anion exchange chromatography and filtration chromatography was finally established after repeated experiments. By this way the purity of protein of interest reached 95% and the total recovery rate was 8%. The result of Western blot indicated that the expressed and purified recombinant B7-2-PE40KDEL could specifically bind with mAb against human B7-2 and multiple antibody against PEA. The cytotoxicity of the recombinant toxin tested by MTT method showed that the B7-2-PE40KDEL could selectively kill Jurkat cell line expressing CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor. It is concluded that a high efficient and speedy three-step purification procedure for the purifying recombinant protein B7-2-PE40KDEL was established, and this procedure possess selective killing activity on CD28 positive T lymphocytes.


Assuntos
Antígeno B7-2/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Antígeno B7-2/genética , Proteínas de Bactérias/genética , Antígenos CD28/imunologia , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
7.
Zhonghua Xue Ye Xue Za Zhi ; 26(1): 19-22, 2005 Jan.
Artigo em Zh | MEDLINE | ID: mdl-15946503

RESUMO

OBJECTIVE: To study the expression of recombinant human SCF-TPO fusion protein and its biological function. METHODS: Four primers were designed according to known sequences of TPO and SCF. The functional amino acid domains of TPO and SCF were amplified by RT-PCR from fetus hepatocytes, respectively. The expression plasmid pET32a/SCF-TPO was constructed by VOE gene fusion technique and expressed in E. coli BL21(DE3) plysS as inclusion body after isopropyl-beta-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein was tested by SDS-PAGE and Western blot. The biological functions of SCF-TPO fusion protein in MO7e cells was investigated by MTT method after purification with metal chelating chromatography. RESULTS: The high expression SCF-TPO fusion protein was obtained, reaching up to 30% of the total cellular protein. Western blot verified the correct fusion expression and MTT results showed the growth promoting effect of the SCF-TPO fusion protein on MO7e cells, with a higher promoting activity at 100 ng/ml. CONCLUSIONS: Expressed SCF-TPO fusion protein after renaturation has biological activity in promoting the proliferation of MO7e cells.


Assuntos
Proliferação de Células , Proteínas Recombinantes de Fusão/genética , Fator de Células-Tronco/genética , Trombopoetina/genética , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Humanos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/fisiologia , Trombopoetina/metabolismo , Trombopoetina/fisiologia
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