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1.
J Orthop Res ; 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34783068

RESUMO

Tendons and ligaments are jointed to bones via an enthesis that is essential to the proper function of the muscular and skeletal structures. The aim of the study is to investigate the effect of mechanical stress on the enthesis. We used ex vivo models in organ cultures of rat Achilles tendons with calcaneus including the enthesis. The organ was attached to a mechanical stretching apparatus that can conduct cyclic tensile strain. We made the models of 1-mm elongation (0.5 Hz, 3% elongation), 2-mm elongation (0.5 Hz, 5% elongation), and no stress. Histological evaluation by Safranin O staining and Toluidin Blue and Picro Sirius red staining was conducted. Expression of sex-determining region Y-box 9 (Sox9), scleraxis (Scx), Runt-related transcription factor 2 (Runx2), and matrix metalloproteinase 13 (Mmp13) were examined by real-time polymerase chain reaction and immunocytochemistry. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling and live/dead staining and was conducted for evaluation of the apoptosis and cell viability. The structure of the enthesis was most maintained in the model of 1-mm elongation. The electronic microscope showed that the enthesis of the no stress model had ill-defined borders between fibrocartilage and mineralized fibrocartilage, and that calcification of mineralized fibrocartilage occurred in the model of 2-mm elongation. Sox9 and Scx was upregulated by 1-mm elongation, whereas Runx2 and Mmp13 were upregulated by 2-mm elongation. Apoptosis was inhibited by low stress. The results of this study suggested that 1-mm elongation can maintain the structure of the enthesis, while 2-mm elongation promotes degenerative changes.

2.
Nat Commun ; 12(1): 4148, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230481

RESUMO

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.


Assuntos
Cartilagem/metabolismo , Homeostase , Hipóxia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Osteoartrite/genética , Fatores de Transcrição SOX9
3.
J Bone Miner Metab ; 39(5): 780-786, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33988755

RESUMO

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.


Assuntos
Osteoclastos , Técnicas de Movimentação Dentária , Animais , Remodelação Óssea , Masculino , Ligamento Periodontal , Ratos , Ratos Wistar , Fosfatase Ácida Resistente a Tartarato
4.
Bone ; 143: 115609, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32829041

RESUMO

Tendons transmit power from muscles to bones, and ligaments maintain the stability of joints, thus producing smooth and flexible movements of articular joints. However, tendons have poor self-healing ability upon damage due to injuries, diseases, or aging. To maintain homeostasis or promote regeneration of the tendon/ligament, it is critical to understand the mechanism responsible for the coordination of tendon/ligament-specific gene expression and subsequent cell differentiation. In this review, we have discussed the core molecular mechanisms involved in the development and homeostasis of tendons and ligaments, with particular focus on transcription factors, signaling, and mechanical stress.


Assuntos
Ligamentos , Tendões , Diferenciação Celular , Músculos , Estresse Mecânico
5.
Methods Mol Biol ; 2245: 151-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315201

RESUMO

MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a critical role in various biological processes including musculoskeletal formation and arthritis pathogenesis via regulating target gene expressions, raising the potentially substantial effects on gene expression networks. Over 2000 miRNAs are encoded in the human genome and a single miRNA potentially targets hundreds of genes. To examine the expression and function of miRNAs in chondrocytes and arthritis pathogenesis, we describe the protocols for the current miRNA related experiments including miRNA expression profiling by (1) Next Generation Sequencing and by TaqMan Array system, (2) miRNA target prediction by TargetScan, (3) miRNA target screening by cell-based reporter library assay, and (4) miRNA and its target interaction by HITS-CLIP (high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation) in cartilage and chondrocyte research.


Assuntos
Condrócitos/metabolismo , Perfilação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , RNA Mensageiro/genética , Transcriptoma , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
6.
Front Cell Dev Biol ; 8: 307, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32671057

RESUMO

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

7.
Bone ; 137: 115434, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32422296

RESUMO

Endochondral ossification is a critical event in bone formation, particularly in long shaft bones. Many cellular differentiation processes work in concert to facilitate the generation of cartilage primordium to formation of trabecular structures, all of which occur within the growth plate. Previous studies have revealed that the growth plate is tightly regulated by various transcription factors, epigenetic systems, and microRNAs. Hence, understanding these mechanisms that regulate the growth plate is crucial to furthering the current understanding on skeletal diseases, and in formulating effective treatment strategies. In this review, we focus on describing the function and mechanisms of the transcription factors, epigenetic systems, and microRNAs known to regulate the growth plate.


Assuntos
Epigênese Genética , Lâmina de Crescimento , MicroRNAs , Animais , Cartilagem , Condrócitos , Condrogênese , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MicroRNAs/genética , Osteogênese
8.
JOR Spine ; 3(1): e1081, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32211592

RESUMO

Damage to the intervertebral discs (IVDs) occurs due to aging or excessive mechanical stress, causing a series of IVD-related degenerative diseases, such as spinal disc herniation and spondylosis. These IVD-related diseases are difficult to cure, partially because the regeneration ability of IVDs is not sufficient. As a novel strategy for treatment of IVD-related diseases, mesenchymal stem cell transplantation to the damaged discs has been reported in animal studies. To further develop and improve this approach, it is necessary to gain a better understanding of the molecular network regulating IVD development by critical transcription factors. Recent findings reveal that during IVD development, nucleus pulposus and annuls fibrosus differentiation is coordinated by a series of transcription factors, such as Mkx, Pax1, 9, Shh, Foxa1, 2, T-Brachyury, and Sox5, 6, 9. The combination of mesenchymal stem cell transplantation with the regulation of these molecules may provide a novel strategy for treatment of degenerative disc diseases.

9.
Nat Commun ; 10(1): 2429, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160553

RESUMO

The WW domain-containing protein 2 (Wwp2) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.


Assuntos
Proteína ADAMTS5/metabolismo , Cartilagem Articular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoartrite/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Cartilagem Articular/diagnóstico por imagem , Modelos Animais de Doenças , Humanos , Articulação do Joelho/diagnóstico por imagem , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/metabolismo , RNA Mensageiro/farmacologia , Transdução de Sinais , Crânio/diagnóstico por imagem , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Microtomografia por Raio-X , Adulto Jovem
10.
Clin Calcium ; 28(6): 809-816, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29848827

RESUMO

Ligaments of joint have an essential role of proper mobilization and stabilization between bone and bone. Damage to ligaments caused by ageing, injury, and arthritis induce a disability of musculoskeletal system and has a problem to reduce our quality of life. To aim for the regeneration of ligaments, we have researched from the point of view of the developmet, found out that the transcription factor Mohawk has been important for the development and homeostasis of tendons and ligaments, and analyzed its function. Furthermore, we have also attempted to induce stem cells to tendon and ligament cells to produce type Ⅰ collagen fibers. In this article, we outline the mechanism of the development that has been reported including our approaches.


Assuntos
Ligamentos/crescimento & desenvolvimento , Osteoartrite/terapia , Regeneração , Cartilagem Articular , Condrócitos , Colágeno Tipo I , Humanos , Qualidade de Vida , Células-Tronco/citologia , Tendões/crescimento & desenvolvimento
11.
Mod Rheumatol ; 28(6): 933-940, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29667905

RESUMO

Tendons and ligaments play essential roles in connecting muscle and bone and stabilizing the connections between bones. The damage to tendons and ligaments caused by aging, injury, and arthritis induces the dysfunction of the musculoskeletal system and reduces the quality of life. Current therapy for damaged tendons and ligaments depends on self-repair; however, it is difficult to reconstruct normal tissue. Regeneration therapy for tendons and ligaments has not been achieved, partly because the mechanism, cell biology, and pathophysiology of tendon and ligament development remain unclear. This review summarizes the role of the transcription factor, Mohawk, which controls tendon and ligament cell differentiation, in the maintenance of cell homeostasis, as well as its function in disease and the possibility of new therapeutic approaches.


Assuntos
Proteínas de Homeodomínio/metabolismo , Disco Intervertebral/metabolismo , Ligamento Periodontal/metabolismo , Tenócitos/metabolismo , Animais , Diferenciação Celular , Proteínas de Homeodomínio/genética , Homeostase , Humanos , Disco Intervertebral/citologia , Disco Intervertebral/patologia , Ligamento Periodontal/citologia , Ligamento Periodontal/patologia , Tenócitos/citologia
12.
Development ; 144(2): 313-320, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27993989

RESUMO

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx-/- mice, age-dependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx-/- mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx+/+ mice. PDL cells lost their shape in Mkx-/- mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx-/- PDL revealed an increase in osteogenic gene expression and no change in PDL- and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.


Assuntos
Proteínas de Homeodomínio/fisiologia , Homeostase/genética , Ligamento Periodontal/fisiologia , Perda do Osso Alveolar/genética , Perda do Osso Alveolar/patologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/genética
13.
Nat Commun ; 7: 12503, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27527664

RESUMO

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx(-/-) mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.


Assuntos
Anel Fibroso/fisiologia , Proteínas de Homeodomínio/metabolismo , Disco Intervertebral/fisiologia , Regeneração , Adulto , Animais , Anel Fibroso/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Disco Intervertebral/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Adulto Jovem
14.
Proc Natl Acad Sci U S A ; 113(28): 7840-5, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27370800

RESUMO

Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)(-/-) rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx(-/-) mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx(-/-) mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx(-/-) TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx(+/+) TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9 Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.


Assuntos
Proteínas de Homeodomínio/fisiologia , Ossificação Heterotópica/etiologia , Tendão do Calcâneo/patologia , Adipogenia , Animais , Condrogênese , Técnicas de Inativação de Genes , Masculino , Ossificação Heterotópica/patologia , Osteogênese , Ratos Wistar , Estresse Mecânico
15.
Mol Cell Biol ; 36(8): 1297-309, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26884464

RESUMO

Mechanoforces experienced by an organ are translated into biological information for cellular sensing and response. In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover. We show that Mohawk (Mkx), a tendon-specific transcription factor, is essential in mechanoresponsive tenogenesis through regulation of its downstream ECM genes such as type I collagens and proteoglycans such as fibromodulin both in vivo and in vitro Wild-type (WT) mice demonstrated an increase in collagen fiber diameter and density in response to physical treadmill exercise, whereas in Mkx(-/-) mice, tendons failed to respond to the same mechanical stimulation. Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation. Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Tendões/fisiologia , Transativadores/metabolismo , Animais , Células Cultivadas , Deleção de Genes , Proteínas de Homeodomínio/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Nucleares/genética , Condicionamento Físico Animal , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Tendões/citologia , Transativadores/genética , Ativação Transcricional , Regulação para Cima , Suporte de Carga
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