RESUMO
Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.
Assuntos
Dentina/metabolismo , Proteínas da Matriz Extracelular/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Dente/crescimento & desenvolvimento , Animais , Cromossomos Artificiais Bacterianos/genética , Colágeno Tipo II , Dentina/diagnóstico por imagem , Dentina/patologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/metabolismo , Incisivo/metabolismo , Incisivo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Minerais/análise , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/metabolismo , Dente/metabolismo , Microtomografia por Raio-XRESUMO
Dentin sialophosphoprotein (DSPP), which expresses and synthesizes in odontoblasts of dental pulp, is a critical protein for normal teeth mineralization. Originally, DSPP was identified as a dentin-specific protein. In 2010, DSPP was also found in femoral head cartilage, and it is still unclear what roles DSPP play in femoral head cartilage formation, growth, and maintenance. To reveal biological functions of DSPP in the femoral head cartilage, we examined Dspp null mice compared with wild-type (WT) mice to observe DSPP expression as well as localization in WT mice and to uncover differences of femoral head cartilage, bone morphology, and structure between these two kinds of mice. Expression data demonstrated that DSPP had heterogeneous fragments, expressed in each layer of femoral head cartilage and subchondral bone of WT mice. Dspp null mice exhibited a significant reduction in the thickness of femoral head cartilage, with decreases in the amount of proliferating cartilage cells and increases in apoptotic cells. In addition, the subchondral bone mineralization decreased, and the expressions of vessel markers (vascular endothelial growth factor [VEGF] and CD31), osteoblast markers (Osterix and dentin matrix protein 1 [DMP1]), osteocyte marker (sclerostin [SOST]), and osteoclast marker (tartrate-resistant acid phosphatase [TRAP]) were remarkably altered. These indicate that DSPP deletion can affect the proliferation of cartilage cells in the femoral head cartilage and endochondral ossification in subchondral bone. Our data clearly demonstrate that DSPP plays essential roles in the femoral head cartilage growth and maintenance and subchondral biomineralization.
Assuntos
Calcificação Fisiológica , Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Cabeça do Fêmur/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Cartilagem/citologia , Proliferação de Células , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/isolamento & purificação , Cabeça do Fêmur/citologia , Camundongos , Camundongos Knockout , Fosfoproteínas/deficiência , Fosfoproteínas/isolamento & purificação , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/isolamento & purificaçãoRESUMO
Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.
Assuntos
Dentina/ultraestrutura , Dentinogênese/fisiologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/ultraestrutura , Fosfoproteínas/deficiência , Fosfoproteínas/ultraestrutura , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/ultraestrutura , Calcificação de Dente/fisiologia , Animais , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , FenótipoRESUMO
Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.
Assuntos
Células-Tronco Hematopoéticas/imunologia , Inflamação/imunologia , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Sialoglicoproteínas/imunologia , Animais , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Movimento Celular/imunologia , Selectina E/metabolismo , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Inflamação/genética , Inflamação/metabolismo , Migração e Rolagem de Leucócitos/genética , Migração e Rolagem de Leucócitos/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peritonite/genética , Peritonite/imunologia , Peritonite/metabolismo , Ligação Proteica/imunologia , Receptores de Fatores de Crescimento de Fibroblastos/deficiência , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genéticaRESUMO
Recently, dentin sialophosphoprotein (DSPP) was found to be expressed in the mandibular condylar cartilage (MCC), but the possible roles of this molecule in the formation, growth, and maintenance of the cartilage are largely unclear. To analyze such roles, we examined Dspp null mice compared with wild-type mice to assess the consequences of Dspp deletion on the morphology and structure of the MCC. Our data showed that DSPP is expressed in the prechondroblastic, chondroblastic, hypertrophic layers of the MCC. Dspp null mice exhibited decreases in the amount of MCC, with reduced formation of articular and prechondroblastic layers in which progenitor cell proliferation levels were distinctly affected. The expression of extracellular matrix molecules, including biglycan and collagen II, IX, and X, was remarkably altered. The findings in this study indicate that continuous DSPP action is required for the growth and/or maintenance of the MCC.
Assuntos
Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Côndilo Mandibular/crescimento & desenvolvimento , Côndilo Mandibular/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Biglicano/metabolismo , Cartilagem/citologia , Proliferação de Células , Colágeno/metabolismo , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genética , Células-Tronco/citologiaRESUMO
TGF-ß is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-ß bioavailability by attenuating maturation of pro-TGF-ß during cartilage homeostasis. However, whether regulation of intracellular TGF-ß maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-ß/pro-TGF-ß ratio, with increased expression of TGF-ß downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-ß antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-ß signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-ß maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.
Assuntos
Remodelação Óssea , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sialoglicoproteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Anticorpos/farmacologia , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Doenças Ósseas Metabólicas/fisiopatologia , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Reabsorção Óssea/complicações , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Calcificação Fisiológica/efeitos dos fármacos , Calcificação Fisiológica/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Células Cultivadas , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fêmur/fisiopatologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , Radiografia , Receptores de Fatores de Crescimento de Fibroblastos/deficiência , Sialoglicoproteínas/deficiência , Transdução de Sinais/genéticaRESUMO
E-selectin-1 (ESL-1), also known as golgi complex-localized glycoprotein-1 (GLG1), homocysteine-rich fibroblast growth factor receptor (CGR-1), and latent transforming growth factor-ß complex protein 1 (LTCP-1), is a multifunctional protein with widespread tissue distribution. To determine the functional consequences of ESL-1 deficiency, mice were generated carrying an ESL-1 gene trap. After backcrossing to C57BL6/J for 6 generations, mice heterozygous for the gene trap (ESL-1(+/-)) were intercrossed to produce ESL-1(-/-) mice, however ESL-1(-/-) mice were not viable, even at embryonic day E10.5. To determine the effect of heterozygous ESL-1 deficiency on atherosclerosis, apolipoprotein E deficient (ApoE(-/-)), ESL-1(+/-) mice were generated and fed western diet. Compared to ApoE(-/-), ESL-1(+)(/)(+) mice, atherosclerotic lesions from ApoE(-/-), ESL-1(+/-) contained more collagen and fewer macrophages, suggesting increased plaque stability. In conclusion, heterozygous deficiency of ESL-1 is associated with features of increased atherosclerotic plaque stability while complete deficiency of ESL-1 leads to embryonic lethality.
Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Perda do Embrião/metabolismo , Haploinsuficiência , Macrófagos/metabolismo , Placa Aterosclerótica , Receptores de Fatores de Crescimento de Fibroblastos/deficiência , Sialoglicoproteínas/deficiência , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Perda do Embrião/genética , Genótipo , Idade Gestacional , Heterozigoto , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Sialoglicoproteínas/genéticaRESUMO
Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with αCaMKII and α-actinin. To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (LRRC7). Homozygous knock-out mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum disorders. At the cellular level, loss of densin results in reduced levels of α-actinin in the brain and selective reduction in the localization of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depression, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology. These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5 and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.
Assuntos
Regulação da Expressão Gênica/genética , Transtornos Mentais/genética , Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/metabolismo , Receptores de Ácido Caínico/metabolismo , Sialoglicoproteínas/deficiência , Actinas/metabolismo , Agressão/fisiologia , Animais , Comportamento Animal/fisiologia , Bicuculina/farmacologia , Peso Corporal/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Endofenótipos , Comportamento Exploratório/fisiologia , Feminino , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Genótipo , Proteína Glial Fibrilar Ácida/metabolismo , Glicina/farmacologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Técnicas In Vitro , Inibição Psicológica , Potenciação de Longa Duração/genética , Depressão Sináptica de Longo Prazo/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Memória de Curto Prazo/fisiologia , Transtornos Mentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Força Muscular/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Desempenho Psicomotor/fisiologia , Receptores de AMPA/genética , Receptores de Ácido Caínico/genética , Reconhecimento Psicológico/fisiologia , Teste de Desempenho do Rota-Rod , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.
Assuntos
Coagulação Sanguínea/genética , Deleção de Genes , Megacariócitos/metabolismo , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genética , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Contagem de Células , Cloretos/farmacologia , Compostos Férricos/farmacologia , Hemorragia/metabolismo , Hemorragia/patologia , Hemorragia/fisiopatologia , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/genética , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Trombose/induzido quimicamente , Trombose/fisiopatologiaRESUMO
Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4-/- mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4-/- mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.
Assuntos
Alérgenos/administração & dosagem , Quimiotaxia de Leucócito , Dermatite Alérgica de Contato/imunologia , Imunização , Receptores CCR4/fisiologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Administração Cutânea , Administração Oral , Transferência Adotiva , Alérgenos/toxicidade , Animais , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Toxina da Cólera/toxicidade , Dermatite Alérgica de Contato/patologia , Hipersensibilidade Alimentar/complicações , Hipersensibilidade Alimentar/imunologia , Integrinas/deficiência , Integrinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/toxicidade , Receptores CCR4/deficiência , Receptores CCR4/genética , Receptores de Fatores de Crescimento de Fibroblastos/deficiência , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Receptores de Retorno de Linfócitos/imunologia , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/fisiologia , Pele/patologia , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/transplanteRESUMO
Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.
Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Moléculas de Adesão de Célula Nervosa/metabolismo , Sialoglicoproteínas/metabolismo , Sinapses/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Proteínas do Citoesqueleto/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Moléculas de Adesão de Célula Nervosa/deficiência , Neuritos/metabolismo , Fosfoproteínas/metabolismo , Gravidez , Ácidos Siálicos/metabolismo , Sialoglicoproteínas/deficiência , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Two of the proteins found in significant quantity in the extracellular matrix (ECM) of dentin are dentin phosphoprotein (DPP) and dentin sialoprotein (DSP). DPP, the most abundant of the noncollagenous proteins (NCPs) in dentin is an unusually polyanionic protein, containing a large number of aspartic acids (Asp) and phosphoserines (Pse) in the repeating sequences of (Asp-Pse)(n). and (Asp-Pse-Pse)(n). The many negatively charged regions of DPP are thought to promote mineralization by binding calcium and presenting it to collagen fibers at the mineralization front during the formation of dentin. This purported role of DPP is supported by a sizeable pool of in vitro mineralization data showing that DPP is an important initiator and modulator for the formation and growth of hydroxyapatite (HA) crystals. Quite differently, DSP is a glycoprotein, with little or no phosphate. DPP and DSP are the cleavage products of dentin sialophosphoprotein (DSPP). Human and mouse genetic studies have demonstrated that mutations in, or knockout of, the Dspp gene result in mineralization defects in dentin and/or bone. The discoveries in the past 40 years with regard to DPP, DSP, and DSPP have greatly enhanced our understanding of biomineralization and set a new stage for future studies. In this review, we summarize the important and new developments made in the past four decades regarding the structure and regulation of the Dspp gene, the biochemical characteristics of DSPP, DPP, and DSP as well as the cell/tissue localizations and functions of these molecules.
Assuntos
Calcificação Fisiológica/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Calcificação Fisiológica/genética , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Mutação/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genéticaRESUMO
Bone healing is a complex multi-step process, which depends on the position and size of the lesion, and on the mechanical stability of the wounded area. To address more specifically the mechanisms involved in cortical bone healing, we created drill-hole defects in the cortex of mouse femur, a lesion that triggers intramembranous repair, and compared the roles of bone sialoprotein (BSP) and osteopontin (OPN), two proteins of the extracellular matrix, in the repair process. Bone regeneration was analyzed by ex vivo microcomputerized X-ray tomography and histomorphometry of bones of BSP-deficient, OPN-deficient and wild-type mice. In all mouse strains, the cortical gap was bridged with woven bone within 2 weeks and no mineralized tissue was observed in the marrow. Within 3 weeks, lamellar cortical bone filled the gap. The amount and degree of mineralization of the woven bone was not affected by OPN deficiency, but cortical bone healing was delayed in BSP-deficient mice due to delayed mineralization. Gene expression studies showed a higher amount of BSP transcripts in the repair bone of OPN-deficient mice, suggesting a possible compensation of OPN function by BSP in OPN-null mice. Our data suggest that BSP, but not OPN, plays a role in primary bone formation and mineralization of newly formed bone during the process of cortical bone healing.
Assuntos
Regeneração Óssea/fisiologia , Calcificação Fisiológica/fisiologia , Fêmur/patologia , Osteopontina/deficiência , Sialoglicoproteínas/deficiência , Cicatrização/fisiologia , Animais , Diáfises/diagnóstico por imagem , Diáfises/patologia , Diáfises/fisiopatologia , Fêmur/diagnóstico por imagem , Fêmur/fisiopatologia , Fêmur/cirurgia , Regulação da Expressão Gênica , Sialoproteína de Ligação à Integrina , Camundongos , Osteogênese/fisiologia , Osteopontina/genética , Osteopontina/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Cicatrização/genética , Microtomografia por Raio-XRESUMO
Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP-/-) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP-/- and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by microCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP-/- mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP-/- mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.
Assuntos
Osso e Ossos/patologia , Sialoglicoproteínas/deficiência , Cicatrização , Animais , Remodelação Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/cirurgia , Sialoproteína de Ligação à Integrina , Imageamento por Ressonância Magnética , Camundongos , Sialoglicoproteínas/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND: Osteopontin (OPN), a secreted glycoprotein that promotes TH1 immune responses, is involved in several inflammatory conditions. Recently, OPN plasma levels have been demonstrated to be elevated in patients with Crohn's disease. From this evidence, we investigated in the present study whether OPN deficiency protects mice against dextran sodium sulfate (DSS)-induced colitis. MATERIALS AND METHODS: Colitis was induced in OPN -/- mice and matched wild-type Black Swiss control mice by adding 3.5% DSS to their drinking water. Disease progression was evaluated for 10 days by measuring body weight, stool consistency, rectal bleeding, colon lengths, histology, and immunohistochemistry. Levels of the acute-phase protein serum amyloid A, O PN, the proinflammatory cytokines interleukin (IL)-6 and IL-12, and the anti-inflammatory cytokine IL-10 were measured in the serum and, in the case of IL-10 and IL-12, in supernatants from colonic explants at the end of treatment. RESULTS: After DSS treatment, OPN -/- mice exhibited significantly decreased disease activity compared with wild-type mice, as evidenced by reduced rectal bleeding, weight loss, and histological intestinal injury (P < 0.002). Furthermore, serum levels of serum amyloid A and IL-6 increased to a lesser extent (P < 0.001), which also was the case for the release of IL-12 by colonic explants (P < 0.01). The release of IL-10 by colonic explants, however, was increased (P < 0.01). Serum levels of IL-10 and IL-12 were not affected by DSS treatment in both wild-type and OPN-/- mice. Macrophage infiltration into inflamed colonic tissue also was markedly attenuated in DSS-treated OPN -/- mice compared with wild-type mice. CONCLUSIONS: This study shows that OPN deficiency significantly protected mice from colitis by attenuating the TH1 response and macrophage chemotaxis. OPN may represent a novel attractive target for pharmacological treatment of inflammatory bowel disease.
Assuntos
Colite/prevenção & controle , Citocinas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Sialoglicoproteínas/deficiência , Administração Oral , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colo/patologia , Citocinas/sangue , Sulfato de Dextrana , Camundongos , Camundongos Transgênicos , OsteopontinaRESUMO
Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN-/- mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN-/- mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-kappaB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN-/- mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.
Assuntos
Reabsorção Óssea/induzido quimicamente , Expressão Gênica , Elevação dos Membros Posteriores/métodos , Subunidade p50 de NF-kappa B/metabolismo , Sialoglicoproteínas/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação para Baixo/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Subunidade p50 de NF-kappa B/genética , Osteopontina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/deficiência , Estresse Mecânico , Regulação para Cima/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Osteopontin is a secreted, adhesive glycoprotein, whose expression is markedly elevated in several types of cancer and premalignant lesions, implicating its association with carcinogenesis. To test the hypothesis that induced osteopontin is involved in tumor promotion in vivo, osteopontin-null and wild-type (WT) mice were subjected to a two-stage skin chemical carcinogenesis protocol. Mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) applied on to the dorsal skin followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 27 weeks. Osteopontin-null mice showed a marked decrease both in tumor/papilloma incidence and multiplicity compared with WT mice. Osteopontin is minimally expressed in normal epidermis, but on treatment with TPA its expression is highly induced. To determine the possible mechanism(s) by which osteopontin regulates tumor development, we examined cell proliferation and cell survival. Epidermis from osteopontin-null and WT mice treated with TPA thrice or with DMBA followed by TPA for 11 weeks showed a similar increase in epidermal hyperplasia, suggesting that osteopontin does not mediate TPA-induced cell proliferation. Bromodeoxyuridine staining of papillomas and adjacent epidermis showed no difference in cell proliferation between groups. However, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analyses indicated a greater number of apoptotic cells in DMBA-treated skin and papillomas from osteopontin-null versus WT mice. These studies are the first to show that induction of the matricellular protein osteopontin facilitates DMBA/TPA-induced cutaneous carcinogenesis most likely through prevention of apoptosis.
Assuntos
Apoptose/fisiologia , Papiloma/patologia , Sialoglicoproteínas/deficiência , Neoplasias Cutâneas/patologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Apoptose/efeitos dos fármacos , Carcinógenos , Processos de Crescimento Celular/fisiologia , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Feminino , Masculino , Camundongos , Osteopontina , Papiloma/induzido quimicamente , Papiloma/metabolismo , Sialoglicoproteínas/biossíntese , Pele/efeitos dos fármacos , Pele/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Acetato de TetradecanoilforbolRESUMO
Osteopontin (OPN), a pro-inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN-null and wild-type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN-null mice. Although peripheral blood neutrophil numbers were lower in DSS-treated OPN-null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS-treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS-treated OPN-null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8alpha+) lymphocytes. Whereas pro-inflammatory cytokines, including G-CSF, RANTES, MIP1alpha, and TNF-alpha, were increased < 10-fold in DSS-treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS-treated OPN-null splenocytes as well as gut tissues. The suppressed TNF-alpha response in OPN-null mice was reflected in a marked increase in non-apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis.
Assuntos
Colite/genética , Colite/patologia , Sulfato de Dextrana/farmacologia , Sialoglicoproteínas/deficiência , Fator de Necrose Tumoral alfa/genética , Animais , Peso Corporal , Morte Celular , Colo/patologia , Regulação da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Osteopontina , Sialoglicoproteínas/genética , Baço/patologiaRESUMO
OBJECTIVE: To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T cell-depleted, nonmyeloablative bone marrow transplantation (BMT) on chronic inflammatory arthritis and autoimmunity in mice deficient in interleukin-1 receptor antagonist (IL-1Ra). METHODS: IL-1Ra(-/-) mice (H-2K(d)) were treated with antibody to asialoganglioside G(M1) (anti-natural killer cell), total body irradiation (500 cGy), and T cell-depleted, nonmyeloablative BMT derived from C57BL/6 mice (H-2K(b)). Engraftment and chimerism were evaluated in peripheral blood, lymph nodes, and spleen by multicolor flow cytometry. The severity of arthritis was evaluated by clinical scoring and histopathologic assessment. Levels of IgG1 and IgG2a subtypes of anti-type II collagen (anti-CII) antibodies were measured in serum samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T cell proliferative responses and levels of cytokine production (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha], interleukin-10 [IL-10], and IL-17) were assayed in culture supernatants. RESULTS: All IL-1Ra(-/-) mice receiving BMT showed marked improvement in arthritis within 3 weeks, as well as successful induction of mixed chimerism. These mice showed higher levels of IgG1, and lower levels of IgG2a anti-CII antibodies and weaker T cell proliferative responses than did mice in the control groups (either no treatment or conditioning alone without bone marrow rescue). In mixed chimeras, the levels of IFNgamma, TNFalpha, and IL-17 produced from CII-stimulated T cells were significantly suppressed and IL-10 production was significantly higher as compared with controls. CONCLUSION: The introduction of allogeneic mixed chimerism showed a strong immunoregulatory potential to correct established chronic inflammatory arthritis and autoimmunity originating from a dysregulated proinflammatory cytokine network.
Assuntos
Artrite/imunologia , Autoimunidade/imunologia , Transplante de Medula Óssea/imunologia , Quimerismo , Sialoglicoproteínas/deficiência , Animais , Artrite/patologia , Artrite/terapia , Autoanticorpos/sangue , Doença Crônica , Colágeno Tipo II/imunologia , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-17/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Linfócitos T/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Although the anti-inflammatory effect of interleukin-1 (IL-1) receptor antagonist (IL-1Ra) has been described, the contribution of this cytokine to non-alcoholic steatohepatitis (NASH) remains unclear. Our aim was to ascertain whether deficiency of IL-1Ra leads to hepatic inflammation and fibrosis upon consumption of an atherogenic diet. IL-1Ra deficient mice (IL-1Ra(-/-)) showed severe steatosis and pericellular fibrosis containing many inflammatory cells following 20 weeks of an atherogenic diet when compared with wild type (WT) mice. The hepatic histology of IL-1Ra(-/-) mice is similar to that of NASH.