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1.
Mod Rheumatol ; 27(6): 1098-1100, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25867226

RESUMO

Chronic infantile neurological, cutaneous, and articular (CINCA) syndrome is a systemic autoinflammatory disease caused by increased production of interleukin (IL)-1ß. We present a case of CINCA syndrome followed up to skeletal maturity. Joint contracture and valgus deformity of the knee had developed before diagnosis. Surgical interventions by soft tissue release and hemiepiphysiodesis improved the contracture and the deformity, and IL-1 receptor antagonist dramatically controlled systemic inflammation, and the patient lives without any disabilities.


Assuntos
Contratura/diagnóstico , Síndromes Periódicas Associadas à Criopirina/diagnóstico , Antirreumáticos/uso terapêutico , Contratura/tratamento farmacológico , Contratura/cirurgia , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Síndromes Periódicas Associadas à Criopirina/cirurgia , Humanos , Lactente , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Articulação do Joelho/patologia , Articulação do Joelho/cirurgia , Masculino
2.
J Biol Chem ; 285(39): 29842-50, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20663886

RESUMO

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.


Assuntos
Cartilagem , Regulação para Baixo/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Membrana/biossíntese , Células-Tronco Mesenquimais/metabolismo , Fator de Transcrição YY1/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Células Cultivadas , Metilação de DNA/fisiologia , Histona Desacetilases/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Especificidade de Órgãos , Proteínas Repressoras/metabolismo , Elementos de Resposta/fisiologia , Fator de Transcrição Sp3/metabolismo
3.
Stem Cell Reports ; 16(3): 610-625, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33636111

RESUMO

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.


Assuntos
Cartilagem/fisiologia , Condrócitos/fisiologia , Colágeno Tipo X/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Animais , Osso e Ossos/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Condrócitos/citologia , Condrogênese , Colágeno Tipo X/genética , Estresse do Retículo Endoplasmático , Matriz Extracelular/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Homeostase , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Proteínas Matrilinas/genética , Proteínas Matrilinas/metabolismo , Camundongos , Modelos Biológicos , Mutação , Osteocondrodisplasias/patologia , Fenótipo , Resposta a Proteínas não Dobradas
4.
Nat Biomed Eng ; 3(7): 558-570, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182836

RESUMO

The recapitulation of bone formation via the in vitro generation of bone-like nodules is frequently used to understand bone development. However, current bone-induction techniques are slow and difficult to reproduce. Here, we report the formation of bone-like nodules within ten days, via the use of retinoic acid (RA) to induce the osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) into osteoblast-like and osteocyte-like cells that create human bone tissue when implanted in calvarial defects in mice. We also show that the induction of bone formation depends on cell signalling through the RA receptors RARα and RARß, which simultaneously activate the BMP (bone morphogenetic protein) and Wnt signalling pathways. Moreover, by using patient-derived hiPSCs, the bone-like nodules recapitulated the osteogenesis-imperfecta phenotype, which was rescued via the correction of disease-causing mutations and partially by an mTOR (mechanistic target of rapamycin) inhibitor. The method of inducing bone nodules may serve as a fast and reproducible model for the study of the formation of both healthy and pathological bone.


Assuntos
Osso e Ossos/patologia , Osso e Ossos/fisiologia , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Osteogênese/fisiologia , Animais , Proteínas Morfogenéticas Ósseas , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Mutação , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , Receptores do Ácido Retinoico/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacos , Transplante , Tretinoína/farmacologia , Via de Sinalização Wnt
5.
Biochem Biophys Res Commun ; 365(3): 406-12, 2008 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-17983593

RESUMO

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.


Assuntos
Adipogenia , Células da Medula Óssea/citologia , Osteogênese , Molécula 1 de Adesão de Célula Vascular/metabolismo , Antígenos CD/análise , Antígenos CD/metabolismo , Células da Medula Óssea/química , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem Celular , Humanos , Células Estromais/química , Células Estromais/citologia , Células Estromais/metabolismo , Molécula 1 de Adesão de Célula Vascular/análise
6.
Biochem Biophys Res Commun ; 365(1): 124-30, 2008 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17980151

RESUMO

The expression of the chondromodulin-I (ChM-I) gene, a cartilage-specific gene, is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma (OS) cells. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by histone deacetylase 2 (HDAC2) in three ChM-I-negative OS cell lines. Treatment with an HDAC inhibitor induced the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and, surprisingly, the demethylation of the core promoter region. The demethylation was transient, and gradually replaced by methylation after a rapid recovery of histone deacetylaion. These results represent an example of the plasticity of differentiation being regulated by the cell-specific plasticity of epigenetic regulation.


Assuntos
Epigênese Genética , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Acetilação , Sítios de Ligação , Linhagem Celular Tumoral , Metilação de DNA , Replicação do DNA , Histona Desacetilase 2 , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp3/metabolismo
7.
Stem Cells ; 25(9): 2371-82, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17569790

RESUMO

The precise biological characteristics of human mesenchymal stem cells (hMSCs), including growth regulatory mechanisms, have not yet been defined. Using 29 strains of hMSCs isolated from bone marrow, we have performed extensive analyses of the growth profiles of hMSCs in vitro. All 29 strains stopped proliferating with a mean population doubling (PD) of 28, although there was a considerable difference among strains. The mean telomere restriction fragment length of the cells passaged twice correlated well with the final number of PDs in each strain, suggesting the value of this measurement to be predictive of the growth potential of hMSCs. The expression level of the p16INK4A gene was associated closely with the PD number of each strain (p = .00000001). Most of the p16INK4A-positive cells were Ki67-negative and senescence associated beta-galactosidase-positive, and the suppression of p16INK4A gene expression by small interfering RNA in senescent hMSCs reduced the number of senescent cells and endowed them with the ability to proliferate. Twenty-five of the 29 strains showed a steady gradual increase in the expression of p16INK4A. The remaining four strains (13.8%) showed different profiles, in which DNA methylation in the promoter region occurred in vitro. One of the four strains continued to proliferate for much longer than the others and showed chromosomal aberrations in the later stages. These results indicated p16INK4A to be a key factor in the regulation of hMSC growth, and, most importantly, careful monitoring of DNA methylation should be considered during the culture of hMSCs, particularly when a prolonged and extended propagation is required.


Assuntos
Proliferação de Células , Senescência Celular/genética , Metilação de DNA , Inativação Gênica/fisiologia , Genes p16/fisiologia , Células-Tronco Mesenquimais/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Criança , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fatores de Tempo , Translocação Genética
8.
J Pediatr Orthop B ; 26(6): 519-525, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28107267

RESUMO

The aim of this study is to evaluate the complications of humeral lengthening and their associated factors. Fifty-four achondroplastic patients were treated by bilateral humeral lengthening. Our original shoulder sling was sufficient to prevent shoulder dislocation. Pre-existing radial head dislocation was observed in 18 patients. Lengthening was accomplished in all cases without a decrease in the elbow function. Seven humeri fractured after the fixator removal. The risk factors for postoperative fracture were a waiting period of less than 5 days, a healing index less than 25, and the concave shape of the callus. There was no radial nerve palsy.


Assuntos
Acondroplasia/cirurgia , Alongamento Ósseo/efeitos adversos , Úmero/cirurgia , Complicações Pós-Operatórias/etiologia , Adolescente , Alongamento Ósseo/métodos , Criança , Fixadores Externos , Feminino , Humanos , Fraturas do Úmero/etiologia , Fraturas do Úmero/prevenção & controle , Masculino , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fatores de Risco , Luxação do Ombro/etiologia , Luxação do Ombro/prevenção & controle , Resultado do Tratamento
9.
J Child Orthop ; 10(3): 193-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27075984

RESUMO

PURPOSE: The aim of this study was to first develop and use 1.0 s ultrafast magnetic resonance imaging (MRI) to confirm the location of the femoral head in non-sedated infants with developmental dysplasia of the hip (DDH) after reduction with spica cast application in clinical settings. METHODS: The ultrafast acquisition was achieved by employing a balanced steady-state free precession sequence and immobilizing the patient with dedicated sandbags. On completion of the ultrafast MRI study, all infants were sedated for conventional MRI scanning. Two orthopaedic surgeons retrospectively evaluated the image quality, result of the reduction and total MRI study time (including patient immobilization, coil setup, and scanning) in 14 DDHs of 13 infants (one with bilateral DDHs). RESULTS: Both reviewers stated that there were no motion artefacts for non-sedated infants during the ultrafast MRI and that the quality of both the ultrafast and conventional MRI images were acceptable to assess the femoral head location. Assessment of the reduction procedure resulted in two hips being categorized as 'incomplete reduction' requiring a re-reduction procedure. The total study time of ultrafast and conventional MRI was 6 ± 1 min and 14 ± 3 min, respectively (P < 0.001). No complications due to sedation, such as hypoxia, were reported. The average sedation waiting time was 1 h 25 min ± 34 min. CONCLUSION: The ultrafast MRI procedure reported here can be readily employed to confirm the location of the femoral head in infants with DDHs, without the use of any sedation.

10.
J Pediatr Orthop B ; 24(4): 286-90, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25812029

RESUMO

The aims of this study were to quantify the femoral head volume (FHV) in developmental dysplasia of the hip (DDH) and to estimate its relation with the severity of the disease. Fifty-one patients (age range 2-11 months) with unilateral DDH were evaluated using three-dimensional MRI. The relation among FHV, age, severity, and displacement was investigated. The affected FHV gradually decreased according to severity. Cephalad displacement of the femoral head correlated negatively with FHV. This new approach showed severity-dependent growth disturbance of the femoral head. This quantification is a promising technique for understanding the pathology of DDH.


Assuntos
Cabeça do Fêmur/patologia , Luxação Congênita de Quadril/diagnóstico , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Índice de Gravidade de Doença , Feminino , Cabeça do Fêmur/metabolismo , Luxação Congênita de Quadril/metabolismo , Humanos , Lactente , Masculino
11.
J Orthop Trauma ; 16(6): 431-3, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12142834

RESUMO

We describe a patient with bilateral pubic ramus nonunions who was treated successfully with a modification of the retrograde medullary screw technique, in which the screw orientation was altered so that it engaged the cancellous bone in the inferior part of the anterior column and the anterior-inferior cortex of the fossa acetabuli. The modification should be one option when the original technique is judged to be difficult to perform.


Assuntos
Parafusos Ósseos , Fixação Intramedular de Fraturas/instrumentação , Fraturas não Consolidadas/cirurgia , Osso Púbico/lesões , Adulto , Desenho de Equipamento , Humanos , Masculino
12.
J Pediatr Orthop B ; 23(2): 107-11, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24335579

RESUMO

Developmental dysplasia of the hip (DDH) includes various abnormalities such as instability, subluxation, and dislocation. In selecting the appropriate treatment method, it is important to distinguish these abnormalities from each other. We developed a novel approach for diagnosing DDH using three-dimensional MRI, which are used to visualize the spatial relation between the dislocated femoral head and the acetabulum and to clarify the changes during hip joint movement. The three-dimensional MRI are useful for confirming the diagnosis of DDH and for evaluating the reducibility of the affected hip.


Assuntos
Luxação Congênita de Quadril/diagnóstico , Articulação do Quadril/patologia , Imageamento Tridimensional , Imageamento por Ressonância Magnética/métodos , Feminino , Humanos , Lactente , Masculino , Reprodutibilidade dos Testes , Estudos Retrospectivos
13.
Tissue Eng Part C Methods ; 16(1): 81-91, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19364273

RESUMO

Bone marrow stromal cells (BMSCs) include cells with multidirectional differentiation potential described as mesenchymal stem cells. For clinical use, it is important to develop a way to isolate BMSCs from bone marrow in a closed system without centrifugation. After screening 200 biomaterials, we developed a device containing a nonwoven fabric filter composed of rayon and polyethylene. The filter selectively traps BMSCs among mononuclear cells in bone marrow based on affinity, not cell size. The cells are then recovered by the retrograde flow. Using canine and human bone marrow cells, the biological properties of BMSCs isolated by the device were compared with those obtained by conventional methods using centrifugation. The total number isolated by the device was larger, as was the number of CD106(+)/STRO-1(+) double-positive cells. The cells showed osteogenic, chondrogenic, and adipogenic differentiation potential in vitro. Finally, the direct transplantation of cells isolated by the device without in vitro cultivation accelerated bone regeneration in a canine model of osteonecrosis in vivo. The proposed method is rapid and efficient, does not require a biological clean area, and will be useful for the clinical application of mesenchymal stem cells in bone marrow.


Assuntos
Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Animais , Antígenos de Superfície/biossíntese , Regeneração Óssea , Diferenciação Celular , Celulose/química , Condrócitos/citologia , Cães , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura/métodos , Osteonecrose , Polietileno/química , Molécula 1 de Adesão de Célula Vascular/biossíntese
14.
Biochem Biophys Res Commun ; 353(1): 60-6, 2007 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-17173860

RESUMO

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.


Assuntos
Técnicas de Cultura de Células/métodos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Proteínas ras/metabolismo , Transformação Celular Neoplásica/genética , Células Cultivadas , Genes ras/genética , Humanos , Sarcoma/genética , Proteínas ras/genética
15.
J Biol Chem ; 281(50): 38941-50, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17060315

RESUMO

Synovial sarcoma, a soft tissue sarcoma that develops in adults, is pathologically subclassified into monophasic spindle synovial sarcoma and biphasic synovial sarcoma with epithelial components. The molecular mechanism building the epithelial components in biphasic synovial sarcoma is totally unknown. Here we investigated claudins, critical molecules in the tight junction, in biphasic synovial sarcoma. Expression profiles of 21 claudins in 17 synovial sarcoma tumor samples, including 9 biphasic tumors, identified claudin4, claudin7, and claudin10 as biphasic tumor-related claudins, and immunohistochemical analyses demonstrated the localization of these claudins in the epithelial component in biphasic tumors, with claudin7 the most closely associated with the epithelial component. The mRNA expression and protein localization of claudin7 coincided with those of the ELF3, an epithelia-specific member of the Ets family of transcription factors. Luciferase reporter assays demonstrated that the presence of the Ets-binding site at -150 in the promoter region of the claudin7 gene was critical for the transcriptional activity, and gel shift and chromatin immunoprecipitation assays confirmed the binding of ELF3 to the Ets site at -150. Inhibition of ELF3 expression by small interfering RNA simultaneously down-regulated the mRNA expression of the claudin7 gene, and the introduction of ELF3 expression in claudin7-negative cell lines induced mRNA expression of the claudin7 gene. Therefore, the induction of claudin7 expression by ELF3 appears critical to the formation of the epithelial structures in biphasic synovial sarcoma.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sarcoma Sinovial/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Claudinas , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-ets , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma Sinovial/patologia
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