RESUMO
Articular cartilage is crucial for joint function but its avascularity limits intrinsic repair, leading to conditions like osteoarthritis (OA). Chondromodulin-I (Cnmd) has emerged as a key molecule in cartilage biology, with potential implications for OA therapy. Cnmd is primarily expressed in cartilage and plays an important role in chondrocyte proliferation, cartilage homeostasis, and the blocking of angiogenesis. In vivo and in vitro studies on Cnmd, also suggest an involvement in bone repair and in delaying OA progression. Its downregulation correlates with OA severity, indicating its potential as a therapeutic target. Further research is needed to fully understand the mode of action of Cnmd and its beneficial implications for managing OA. This comprehensive review aims to elucidate the molecular characteristics of Cnmd, from its expression pattern, role in cartilage maintenance, callus formation during bone repair and association with OA.
Assuntos
Cartilagem Articular , Peptídeos e Proteínas de Sinalização Intercelular , Osteoartrite , Animais , Humanos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , AdultoRESUMO
When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.
Assuntos
Lesões do Ligamento Cruzado Anterior , Reconstrução do Ligamento Cruzado Anterior , Humanos , Adulto , Ratos , Animais , Camundongos , Ligamento Cruzado Anterior/cirurgia , Tendões/cirurgia , Lesões do Ligamento Cruzado Anterior/cirurgia , Articulação do Joelho/cirurgiaRESUMO
Chondromodulin (Cnmd) is a glycoprotein known to stimulate chondrocyte growth. We examined in this study the expression and functional role of Cnmd during distraction osteogenesis that is modulated by mechanical forces. The right tibiae of the mice were separated by osteotomy and subjected to slow progressive distraction using an external fixator. In situ hybridization and immunohistochemical analyses of the lengthened segment revealed that Cnmd mRNA and its protein in wild-type mice were localized in the cartilage callus, which was initially generated in the lag phase and was lengthened gradually during the distraction phase. In Cnmd null (Cnmd-/-) mice, less cartilage callus was observed, and the distraction gap was filled by fibrous tissues. Additionally, radiological and histological investigations demonstrated delayed bone consolidation and remodeling of the lengthened segment in Cnmd-/- mice. Eventually, Cnmd deficiency caused a one-week delay in the peak expression of VEGF, MMP2, and MMP9 genes and the subsequent angiogenesis and osteoclastogenesis. We conclude that Cnmd is necessary for cartilage callus distraction.
Assuntos
Calo Ósseo , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana , Osteogênese por Distração , Animais , Camundongos , Cartilagem , Fixadores Externos , Osteogênese/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genéticaRESUMO
Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/patologia , Proteínas de Membrana/metabolismo , Esclerose/patologia , Transdução de Sinais , Proteínas Smad/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Mutantes , Mutação/genética , Osteoblastos/patologia , Linhagem , Fosforilação , Crânio/patologia , Adulto JovemRESUMO
Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.
Assuntos
Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Fibroblastos/metabolismo , Hibridização In Situ , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Gravidez , RNA-Seq , Esterno/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti-angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament-specific marker and anti-angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age-dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd-/- ) mice exhibited more rapid progression of age-related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic-like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd-/- Chm1-/- ) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF-derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age-related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age-related IVD degeneration.
Assuntos
Envelhecimento/metabolismo , Progressão da Doença , Degeneração do Disco Intervertebral/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Animais , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Células Cultivadas , Condrócitos/metabolismo , Técnicas de Cocultura , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Fatores de Risco , Adulto JovemRESUMO
In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.
Assuntos
Proteínas ADAM/metabolismo , Crista Neural/metabolismo , Transdução de Sinais , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 6/metabolismo , Cartilagem/crescimento & desenvolvimento , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular , Condrogênese , Embrião de Mamíferos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Miocárdio/citologia , Miocárdio/metabolismo , Crista Neural/citologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Regulação para CimaRESUMO
BACKGROUND: The effects of fibroblast growth factor 2 (FGF-2) on healing after surgical repair of chronic rotator cuff (RC) tears remain unclear. HYPOTHESIS: FGF-2 enhances tenogenic healing response, leading to biomechanical and histological improvement of repaired chronic RC tears in rats. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male Sprague-Dawley rats (n = 117) underwent unilateral surgery to refix the supraspinatus tendon to its insertion site 3 weeks after detachment. Animals were assigned to either the FGF-2 group or a control group. The effects of FGF-2 were assessed via biomechanical tests at 3 weeks after detachment and at 6 and 12 weeks postoperatively and were assessed histologically and immunohistochemically for proliferating cell nuclear antigen and mesenchymal stem cell (MSC)-related markers at 2, 6, and 12 weeks postoperatively. The expression of tendon/enthesis-related markers, including SRY-box 9 (Sox9), scleraxis (Scx), and tenomodulin (Tnmd), were assessed by real-time reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The effect of FGF-2 on comprehensive gene expressions at the healing site was evaluated by microarray analysis. RESULTS: The FGF-2 group showed a significant increase in mechanical strength at 6 and 12 weeks compared with control; the FGF-2 group also showed significantly higher histological scores at 12 weeks than control, indicating the presence of more mature tendon-like tissue. At 12 weeks, Scx and Tnmd expression increased significantly in the FGF-2 group, whereas no significant differences in Sox9 were found between groups over time. At 2 weeks, the percentage of positive cells expressing MSC-related markers increased in the FGF-2 group. Microarray analysis at 2 weeks after surgery showed that the expression of several growth factor genes and extracellular matrix-related genes was influenced by FGF-2 treatment. CONCLUSION: FGF-2 enhanced the formation of tough tendon-like tissues including an increase in Scx- or Tnmd-expressing cells at 12 weeks after surgical repair of chronic RC tears. The increase in mesenchymal progenitors and the changes in gene expression upon FGF-2 treatment in the early phase of healing appear to be related to a certain favorable microenvironment for tenogenic healing response of chronic RC tears. CLINICAL RELEVANCE: These findings may provide advantages in therapeutic strategies for patients with RC tears.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Lesões do Manguito Rotador/cirurgia , Manguito Rotador/cirurgia , Animais , Fenômenos Biomecânicos , Osso e Ossos/cirurgia , Matriz Extracelular/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Tendões/cirurgia , Cicatrização/fisiologiaRESUMO
The immune system evolved to efficiently eradicate invading bacteria and terminate inflammation through balancing inflammatory and regulatory T-cell responses. In autoimmune arthritis, pathogenic TH17 cells induce bone destruction and autoimmune inflammation. However, whether a beneficial function of T-cell-induced bone damage exists is unclear. Here, we show that bone-damaging T cells have a critical function in the eradication of bacteria in a mouse model of periodontitis, which is the most common infectious disease. Bacterial invasion leads to the generation of specialized TH17 cells that protect against bacteria by evoking mucosal immune responses as well as inducing bone damage, the latter of which also inhibits infection by removing the tooth. Thus, bone-damaging T cells, which may have developed to stop local infection by inducing tooth loss, function as a double-edged sword by protecting against pathogens while also inducing skeletal tissue degradation.
Assuntos
Perda do Osso Alveolar/imunologia , Bacteriemia/microbiologia , Periodontite/imunologia , Células Th17/fisiologia , Perda de Dente/imunologia , Animais , Modelos Animais de Doenças , Feminino , Interleucina-6/metabolismo , Camundongos Endogâmicos C57BL , Microbiota , Boca/microbiologia , Periodontite/complicações , Periodontite/metabolismo , Periodontite/microbiologia , Ligante RANK/metabolismoRESUMO
BACKGROUND: Transforming growth factor ß1 (TGF-ß1) positively regulates the tenogenic marker genes scleraxis ( Scx) and tenomodulin ( Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-ß1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. HYPOTHESIS: TGF-ß1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-ß1 (100 ng) or phosphate-buffered saline. The effects of TGF-ß1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [ Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro-computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell-related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. RESULTS: The TGF-ß1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell-related marker immunoreactivity were not affected by exogenously administrated TGF-ß1 at all time points. In the TGF-ß1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased, whereas the expression of MMP-9 and MMP-13 significantly decreased at 2 and 4 weeks postoperatively. CONCLUSION: TGF-ß1 enhances formation of tough fibrous tissues at the healing site by inhibiting MMP-9 and MMP-13 expression to increase collagen accumulation but without the growth of tenogenic lineage cells. CLINICAL RELEVANCE: These findings suggest that TGF-ß1 could be used for enhancing biomechanical strength after RC surgical repair.
Assuntos
Matriz Extracelular/química , Lesões do Manguito Rotador/metabolismo , Manguito Rotador/metabolismo , Tendões/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fenômenos Biomecânicos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Manguito Rotador/diagnóstico por imagem , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/diagnóstico por imagem , Lesões do Manguito Rotador/genética , Tendões/citologia , Tendões/cirurgia , Microtomografia por Raio-XRESUMO
Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel ScxCre knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of ScxCre/Cre KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of ScxCre/Cre KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Desenvolvimento Musculoesquelético/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/patologia , Cartilagem/anormalidades , Cartilagem/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Marcação de Genes , Genes Reporter , Loci Gênicos , Recombinação Homóloga , Ligamentos/anormalidades , Ligamentos/crescimento & desenvolvimento , Ligamentos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Fatores de Transcrição SOX9/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Tendões/anormalidades , Tendões/crescimento & desenvolvimento , Tendões/metabolismoRESUMO
Tenomodulin (TNMD, Tnmd) is a gene highly expressed in tendon known to be important for tendon maturation with key implications for the residing tendon stem/progenitor cells as well as for the regulation of endothelial cell migration in chordae tendineae cordis in the heart and in experimental tumour models. This review aims at providing an encompassing overview of this gene and its protein. In addition, its known expression pattern as well as putative signalling pathways will be described. A chronological overview of the discovered functions of this gene in tendon and other tissues and cells is provided as well as its use as a tendon and ligament lineage marker is assessed in detail and discussed. Last, information about the possible connections between TNMD genomic mutations and mRNA expression to various diseases is delivered. Taken together this review offers a solid synopsis on the up-to-date information available about TNMD and aids at directing and focusing the future research to fully uncover the roles and implications of this interesting gene.
Assuntos
Proteínas de Membrana/metabolismo , Células-Tronco/metabolismo , Tendões/metabolismo , Animais , Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Transdução de Sinais , Tendões/citologiaRESUMO
OASIS/CREB3L1, an endoplasmic reticulum (ER)-resident transcription factor, plays important roles in osteoblast differentiation. In this study, we identified new crosstalk between OASIS and the hypoxia signaling pathway, which regulates vascularization during bone development. RT-PCR and real-time PCR analyses revealed significant decreases in the expression levels of hypoxia-inducible factor-1α (HIF-1α) target genes such as vascular endothelial growth factor A (VEGFA) in OASIS-deficient (Oasis(-/-)) mouse embryonic fibroblasts. In coimmunoprecipitation experiments, the N-terminal fragment of OASIS (OASIS-N; activated form of OASIS) bound to HIF-1α through the bZIP domain. Luciferase assays showed that OASIS-N promoted the transcription activities of a reporter gene via a hypoxia-response element (HRE). Furthermore, the expression levels of an angiogenic factor Vegfa was decreased in Oasis(-/-) osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of Oasis(-/-) mice. These results suggest that OASIS affects the expression of HIF-1α target genes through the protein interaction with HIF-1α, and that OASIS-HIF-1α complexes may play essential roles in angiogenesis during bone development.
Assuntos
Osso e Ossos/irrigação sanguínea , Osso e Ossos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipóxia/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Osteogênese/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Elementos de Resposta , Transcrição GênicaRESUMO
BACKGROUND: Fibroblast growth factor (FGF)-2 has the potential to enhance tendon-to-bone healing after rotator cuff (RC) injury. HYPOTHESIS: FGF-2 stimulates tenogenic differentiation of progenitors to improve the biomechanical strength and histological appearance of repaired RCs in rats. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male Sprague-Dawley rats (N = 156) underwent unilateral surgery to repair the supraspinatus tendon to insertion sites. The FGF-2-treated group (gelatin hydrogel containing 5 µg of FGF-2) and a control group (gelatin hydrogel only) were compared to investigate the effects of FGF-2 at 2, 4, 6, 8, and 12 weeks postoperatively. Biomechanical testing was performed at 6 and 12 weeks. Semiquantitative histological analysis and immunohistochemical analysis for the proliferating cell nuclear antigen (PCNA) were performed, and the expression of tendon-related markers, including Scleraxis (Scx) and Tenomodulin (Tnmd), was monitored by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. SRY-box containing gene 9 (Sox9) expression was monitored by RT-PCR and immunohistochemical analysis. At 2 and 4 weeks, immunohistochemical analysis for mesenchymal stem cell (MSC) markers was also performed. RESULTS: The FGF-2-treated group demonstrated a significant improvement in mechanical strength at 6 and 12 weeks and significantly higher histological scores than the control group at ≥4 weeks. The average incidence of PCNA-positive cells was significantly higher at 2 and 4 weeks, and more cells expressing MSC markers were detected at the insertion site in the FGF-2-treated group. The expression level of Scx increased significantly in the FGF-2-treated group from 4 to 8 weeks, while the Tnmd level increased significantly from 4 to 12 weeks postoperatively. The localization of Tnmd overlapped with the locations of reparative tissues accompanying collagen fibers with an aligned orientation. Sox9 expression was significantly upregulated at 4 weeks in the FGF-2-treated group. CONCLUSION: FGF-2 promotes growth of the tenogenic progenitor cells, which participate in tendon-to-bone healing, resulting in biomechanical and histological improvement of the repaired RC. CLINICAL RELEVANCE: These findings provide clues regarding the clinical development of regenerative repair strategies for RC injury.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas de Membrana/metabolismo , Procedimentos Ortopédicos/métodos , Lesões do Manguito Rotador , Células-Tronco/efeitos dos fármacos , Traumatismos dos Tendões/cirurgia , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Manguito Rotador/patologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/patologiaRESUMO
Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development.
RESUMO
Sox9 is a high-mobility group box-containing transcription factor that functions as a key regulator of chondrogenesis. We here report that Sox9 mediates the direct conversion of tenocytes to chondrocytes through an intermediate state in which both differentiation programs are active. Sox9 is abundantly expressed in cartilage but is undetectable in limb tendons that express Scleraxis (Scx) and Tenomodulin (Tnmd), tendon-specific early and late molecular markers, respectively. Upon forced expression of Sox9 in the chick forelimb, ectopic cartilage formation is preferentially observed in fibrous tissues including the tendons, ligaments, perichondrium/periosteum, dermis, and muscle connective tissues. Tnmd expression in tenocytes isolated from leg tendons was markedly upregulated by forced expression of basic helix-loop-helix (b-HLH) activators including Scx, Paraxis, Twist1 and Twist2. In contrast, the overexpression of Sox9 in monolayer tenocytes resulted in the downregulation of Tnmd and Scx expressions during passaging in culture, and the induction of cartilage molecular markers such as type II collagen (Col2a1) and Chondromodulin-I (ChM-I). This Sox9-driven switching from a tenocytic to a chondrocytic gene expression profile was associated with a dramatic change from a spindle to a polygonal cellular morphology. The extracellular accumulation of cartilage-characteristic proteoglycans was also observed. These data suggest that tenocytes have a strong potential for conversion into chondrocytes through the activities of Sox9 both in vitro and in vivo.
Assuntos
Proteínas Aviárias/metabolismo , Transdiferenciação Celular/fisiologia , Condrócitos/citologia , Condrócitos/metabolismo , Fatores de Transcrição SOX9/metabolismo , Tendões/citologia , Tendões/metabolismo , Animais , Proteínas Aviárias/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Transdiferenciação Celular/genética , Células Cultivadas , Embrião de Galinha , Condrogênese/genética , Condrogênese/fisiologia , Coristoma/genética , Coristoma/metabolismo , Coristoma/patologia , Primers do DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/citologia , Mioblastos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição SOX9/genéticaRESUMO
Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein in cartilage matrix that inhibits angiogenesis. It contains two distinctive structural domains: the N-terminal third of the molecule is a hydrophilic domain that contains O-linked and N-linked oligosaccharide chains, and the C-terminal two-thirds is a hydrophobic domain that contains all of the cysteine residues. In the present study, we have attempted to further uncover the structural requirements for ChM-I to exert anti-angiogenic activity by monitoring its inhibition of the vascular endothelial growth factor (VEGF)-A-induced migration of HUVEC in vitro. Site-directed mutagenesis experiments revealed that the cyclic structure formed by the disulfide bridge between Cys(83) and Cys(99) in human ChM-I is indispensable for its anti-angiogenic function. Moreover, the C-terminal hydrophobic tail (from Trp(111) to Val(120) ) was found to play an important role in ensuring the effectiveness of ChM-I activity on HUVEC. A synthetic cyclic peptide corresponding to the ChM-I region between Ile(82) to Arg(100) also inhibited the migration of HUVEC, while replacing the Cys(83) and Cys(99) residues in this peptide with Ser completely negated this inhibitory activity. An additional synthetic cyclic peptide harboring the hydrophobic C-terminal tail of ChM-I clearly mimicked the inhibitory action of this protein on the migration of HUVEC and successfully inhibited tumor angiogenesis and growth in a xenograft mouse model of human chondrosarcoma.
Assuntos
Inibidores da Angiogênese/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteínas de Membrana/farmacologia , Peptídeos Cíclicos/farmacologia , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Animais , Bovinos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Condrossarcoma/irrigação sanguínea , Condrossarcoma/tratamento farmacológico , Condrossarcoma/patologia , Cisteína/química , Cisteína/genética , Dissulfetos/química , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Neovascularização Patológica/prevenção & controle , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Proteínas Recombinantes/química , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Tendões/citologia , Adipogenia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular , Expressão Gênica , Humanos , OsteogêneseRESUMO
BACKGROUND: Chondromodulin-I (ChM-I) is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro. RESULTS: Northern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9)-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I) markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation. CONCLUSIONS: This study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/análise , Proteínas de Membrana/análise , Trofoblastos/metabolismo , Animais , Células CHO , Linhagem Celular , Movimento Celular , Cricetinae , Cricetulus , Decídua/metabolismo , Feminino , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Trofoblastos/citologiaRESUMO
Chondromodulin-I (ChM-I) is a 25-kDa glycoprotein that specifically localizes in the extracellular matrix of cartilage and negatively regulates angiogenesis. ChM-I comprises two domains: an N-terminal hydrophilic domain (domain 1) containing an N-linked glycosylation site and a C-terminal hydrophobic domain (domain 2) with all four disulfide bonds that are present in this protein. We generated a nonglycosylated recombinant human ChM-I (NG-hChM-I) and compared its bioactivity with that of the glycosylated form of human ChM-I (G-hChM-I) expressed in Chinese hamster ovary cells in vitro. NG-hChM-I exhibited the growth factor/inhibitor activity in the cultures of chondrocytes and vascular endothelial cells but required markedly higher doses. Although domain 1 is predicted to be hydrophilic per se on the basis of its amino acid sequence, NG-hChM-I remains insoluble in aqueous solution as much as ΔN-hChM-I that lacks the N-terminal 37 amino acids containing an N-glycosylation site. Circular dichroism measurements revealed that the content of α-helix was calculated to be 34% in G-hChM-I, whereas the content of the characteristic secondary structures in NG-hChM-I was distinctly lower than those in G-hChM-I. These results indicate that glycosylation in domain 1 is critical for the structural integrity for biological functions of ChM-I in vitro.