RESUMO
The actions of the retinoic acid nuclear receptor gamma (RARγ) agonist, palovarotene, on pre-existing osteochondromas were investigated using a mouse multiple osteochondroma model. This approach was based on the knowledge that patients often present to the clinic after realizing the existence of osteochondroma masses, and the findings from preclinical investigations are the effects of drugs on the initial formation of osteochondromas. Systemic administration of palovarotene, with increased doses (from 1.76 to 4.0 mg/kg) over time, fully inhibited tumor growth, keeping the tumor size (0.31 ± 0.049 mm3) similar to the initial size (0.27 ± 0.031 mm3, p = 0.66) while the control group tumor grew (1.03 ± 0.23 mm3, p = 0.023 to the drug-treated group). Nanoparticle (NP)-based local delivery of the RARγ agonist also inhibited the growth of osteochondromas at an early stage (Control: 0.52 ± 0.11 mm3; NP: 0.26 ± 0.10, p = 0.008). Transcriptome analysis revealed that the osteoarthritis pathway was activated in cultured chondrocytes treated with palovarotene (Z-score = 2.29), with the upregulation of matrix catabolic genes and the downregulation of matrix anabolic genes, consistent with the histology of palovarotene-treated osteochondromas. A reporter assay performed in cultured chondrocytes demonstrated that the Stat3 pathway, but not the Stat1/2 pathway, was stimulated by RARγ agonists. The activation of Stat3 by palovarotene was confirmed using immunoblotting and immunohistochemistry. These findings suggest that palovarotene treatment is effective against pre-existing osteochondromas and that the Stat3 pathway is involved in the antitumor actions of palovarotene.
Assuntos
Condrócitos , Modelos Animais de Doenças , Osteocondroma , Receptores do Ácido Retinoico , Receptor gama de Ácido Retinoico , Animais , Camundongos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Osteocondroma/tratamento farmacológico , Osteocondroma/patologia , Osteocondroma/metabolismo , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Fator de Transcrição STAT3/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , MasculinoRESUMO
Osteochondromas are cartilage-capped tumors that arise near growing physes and are the most common benign bone tumor in children. Osteochondromas can lead to skeletal deformity, pain, loss of motion, and neurovascular compression. Currently, surgery is the only available treatment for symptomatic osteochondromas. Osteochondroma mouse models have been developed to understand the pathology and the origin of osteochondromas and develop therapeutic drugs. Several cartilage regulatory pathways have been implicated in the development of osteochondromas, such as bone morphogenetic protein, hedgehog, and WNT/ß-catenin signaling. Retinoic acid receptor-γ is an important regulator of endochondral bone formation. Selective agonists for retinoic acid receptor-γ, such as palovarotene, have been investigated as drugs for inhibition of ectopic endochondral ossification, including osteochondromas. This review discusses the signaling pathways involved in osteochondroma pathogenesis and their possible interactions with the retinoid pathway.
Assuntos
Neoplasias Ósseas/etiologia , Osteocondroma/etiologia , Retinoides/metabolismo , Animais , Neoplasias Ósseas/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Osteocondroma/patologia , Transdução de Sinais/fisiologiaRESUMO
Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.
Assuntos
Desenvolvimento Ósseo/genética , Diferenciação Celular/genética , Células-Tronco Mesenquimais , Osteogênese/genética , Animais , Cartilagem/crescimento & desenvolvimento , Condrócitos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Camundongos , Transdução de Sinais/genética , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Osteochondromas are cartilage-capped growths located proximate to the physis that can cause skeletal deformities, pain, limited motion, and neurovascular impingement. Previous studies have demonstrated retinoic acid receptor gamma (RARγ) agonists to inhibit ectopic endochondral ossification, therefore we hypothesize that RARγ agonists can target on established osteochondromas. The purpose of this study was to examine the action of RARγ agonist in human osteochondromas. Osteochondroma specimens were obtained during surgery, subjected to explant culture and were treated with RARγ agonists or vehicles. Gene expression analysis confirmed the up-regulation of RARγ target genes in the explants treated with NRX 204647 and Palovarotene and revealed strong inhibition of cartilage matrix and increased extracellular matrix proteases gene expression. In addition, immunohistochemical staining for the neoepitope of protease-cleaved aggrecan indicated that RARγ agonist treatment stimulated cartilage matrix degradation. Interestingly, cell survival studies demonstrated that RARγ agonist treatment stimulated cell death. Moreover, RNA sequencing analysis indicates changes in multiple molecular pathways due to RARγ agonists treatment, showing similarly to human growth plate chondrocytes. Together, these findings suggest that RARγ agonist may exert anti-tumor function on osteochondromas by inhibiting matrix synthesis, promoting cartilage matrix degradation and stimulating cell death.
Assuntos
Neoplasias Ósseas/metabolismo , Osteocondroma/metabolismo , Receptores do Ácido Retinoico/agonistas , Animais , Biomarcadores , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/etiologia , Neoplasias Ósseas/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Humanos , Anotação de Sequência Molecular , Osteocondroma/tratamento farmacológico , Osteocondroma/etiologia , Osteocondroma/patologia , Transdução de Sinais , Técnicas de Cultura de Tecidos , Transcriptoma , Receptor gama de Ácido RetinoicoRESUMO
Osteogenesis imperfecta (OI) is a hereditary bone disorder most commonly caused by autosomal dominant mutations in genes encoding type I collagen. In addition to bone fragility, patients suffer from impaired longitudinal bone growth. It has been demonstrated that in OI, an accumulation of mutated type I collagen in the endoplasmic reticulum (ER) induces ER stress in osteoblasts, causing osteoblast dysfunction leading to bone fragility. We hypothesize that ER stress is also induced in the growth plate where bone growth is initiated, and examined a mouse model of dominant OI that carries a G610C mutation in the procollagen α2 chain. The results demonstrated that G610C OI mice had significantly shorter long bones with growth plate abnormalities including elongated total height and hypertrophic zone. Moreover, we found that mature hypertrophic chondrocytes expressed type I collagen and ER dilation was more pronounced compared to wild type littermates. The results from in vitro chondrocyte cultures demonstrated that the maturation of G610C OI hypertrophic chondrocytes was significantly suppressed and ER stress related genes were upregulated. Given that the alteration of hypertrophic chondrocyte activity often causes dwarfism, our findings suggest that hypertrophic chondrocyte dysfunction induced by ER stress may be an underlying cause of growth deficiency in G610C OI mice.
Assuntos
Condrócitos/patologia , Colágeno Tipo I/genética , Estresse do Retículo Endoplasmático , Lâmina de Crescimento/anormalidades , Osteogênese Imperfeita/genética , Mutação Puntual , Animais , Condrócitos/metabolismo , Modelos Animais de Doenças , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Masculino , Camundongos Endogâmicos C57BL , Osteogênese Imperfeita/patologiaRESUMO
Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.
Assuntos
Artrite Experimental/metabolismo , Cartilagem Articular/metabolismo , Receptores ErbB/metabolismo , Osteoartrite/metabolismo , Animais , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese , Receptores ErbB/deficiência , Receptores ErbB/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Osteoartrite/patologia , Osteoartrite/prevenção & controle , Proteoglicanas/metabolismo , Transdução de SinaisRESUMO
Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.
Assuntos
Cartilagem Articular , Tamanho Celular , Condrogênese/fisiologia , Traumatismos do Joelho/metabolismo , Articulação do Joelho/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Animais , Cartilagem Articular/citologia , Cartilagem Articular/embriologia , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/lesões , Diferenciação Celular/fisiologia , Linhagem da Célula , Proliferação de Células , Condrócitos/citologia , Colágeno/metabolismo , Fator 5 de Diferenciação de Crescimento/metabolismo , Articulação do Joelho/citologia , Camundongos , Camundongos Transgênicos , Membrana Sinovial/citologiaRESUMO
Cartilage not only plays essential roles in skeletal development and growth during pre- and postnatal stages but also serves to provide smooth movement of skeletons throughout life. Thus, dysfunction of cartilage causes a variety of skeletal disorders. Results from animal studies reveal that ß-catenin-dependent canonical and independent non-canonical Wnt signaling pathways have multiple roles in regulation of cartilage development, growth, and maintenance. ß-Catenin-dependent signaling is required for progression of endochondral ossification and growth of axial and appendicular skeletons, while excessive activation of this signaling can cause severe inhibition of initial cartilage formation and growth plate organization and function in mice. In contrast, non-canonical Wnt signaling is important in columnar organization of growth plate chondrocytes. Manipulation of Wnt signaling causes or ameliorates articular cartilage degeneration in rodent osteoarthritis models. Human genetic studies indicate that Wnt/ß-catenin signaling is a risk factor for osteoarthritis. Accumulative findings from analysis of expression of Wnt signaling molecules and in vivo and in vitro functional experiments suggest that Wnt signaling is a therapeutic target for osteoarthritis. The target tissues of Wnt signaling may be not only articular cartilage but also synovium and subchondral bone.
Assuntos
Condrogênese , Via de Sinalização Wnt , Animais , Doenças das Cartilagens , Humanos , CamundongosRESUMO
Hereditary multiple exostoses is a pediatric skeletal disorder characterized by benign cartilaginous tumors called exostoses that form next to growing skeletal elements. Hereditary multiple exostoses patients carry heterozygous mutations in the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2, but studies suggest that EXT haploinsufficiency and ensuing partial HS deficiency are insufficient for exostosis formation. Searching for additional pathways, we analyzed presence and distribution of heparanase in human exostoses. Heparanase was readily detectable in most chondrocytes, particularly in cell clusters. In control growth plates from unaffected persons, however, heparanase was detectable only in hypertrophic zone. Treatment of mouse embryo limb mesenchymal micromass cultures with exogenous heparanase greatly stimulated chondrogenesis and bone morphogenetic protein signaling as revealed by Smad1/5/8 phosphorylation. It also stimulated cell migration and proliferation. Interfering with HS function both with the chemical antagonist Surfen or treatment with bacterial heparitinase up-regulated endogenous heparanase gene expression, suggesting a counterintuitive feedback mechanism that would result in further HS reduction and increased signaling. Thus, we tested a potent heparanase inhibitor (SST0001), which strongly inhibited chondrogenesis. Our data clearly indicate that heparanase is able to stimulate chondrogenesis, bone morphogenetic protein signaling, cell migration, and cell proliferation in chondrogenic cells. These properties may allow heparanase to play a role in exostosis genesis and pathogenesis, thus making it a conceivable therapeutic target in hereditary multiple exostoses.
Assuntos
Cartilagem/metabolismo , Condrogênese/efeitos dos fármacos , Exostose Múltipla Hereditária/metabolismo , Glucuronidase/metabolismo , Animais , Cartilagem/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Criança , Condrócitos/metabolismo , Condrogênese/fisiologia , Exostose Múltipla Hereditária/genética , Glucuronidase/farmacologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Humanos , Camundongos , Regulação para CimaRESUMO
Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule - we named Kartogenin (KGN) - that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFß superfamily members, particularly TFGß1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFß1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFß1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFß and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFß signaling, working in addition to and/or in concert with the filamin A/CBFß/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.
Assuntos
Anilidas/farmacologia , Condrogênese/efeitos dos fármacos , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cápsula Articular/embriologia , Mesoderma/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Animais , Primers do DNA/genética , Processamento de Imagem Assistida por Computador , Immunoblotting , Hibridização In Situ , Cápsula Articular/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Proteoglicanas/metabolismo , Regeneração/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Vermelha FluorescenteRESUMO
Tendon injuries are common, and the damaged tendon often turns into scar tissue and never completely regains the original biomechanical properties. Previous studies have reported that the mRNA levels of inflammatory cytokines such as IL-1ß are remarkably up-regulated in injured tendons. To examine how IL-1ß impacts tendon repair process, we isolated the injured tendon-derived progenitor cells (inTPCs) from mouse injured Achilles tendons and studied the effects of IL-1ß on the inTPCs in vitro. IL-1ß treatment strongly reduced expression of tendon cell markers such as scleraxis and tenomodulin, and also down-regulated gene expression of collagen 1, collagen 3, biglycan and fibromodulin in inTPCs. Interestingly, IL-1ß stimulated lactate production with increases in hexokinase II and lactate dehydrogenase expression and a decrease in pyruvate dehydrogenase. Inhibition of lactate production restored IL-1ß-induced down-regulation of collagen1 and scleraxis expression. Furthermore, IL-1ß significantly inhibited adipogenic, chondrogenic and osteogenic differentiation of inTPCs. Interestingly, inhibition of tenogenic and adipogenic differentiation was not recovered after removal of IL-1ß while chondrogenic and osteogenic differentiation abilities were not affected. These findings indicate that IL-1ß strongly and irreversibly impairs tenogenic potential and alters glucose metabolism in tendon progenitors appearing in injured tendons. Inhibition of IL-1ß may be beneficial for maintaining function of tendon progenitor cells during the tendon repair process.
Assuntos
Diferenciação Celular/fisiologia , Interleucina-1beta/fisiologia , Células-Tronco/metabolismo , Tendões/metabolismo , Animais , Sequência de Bases , Primers do DNA , Feminino , Glucose/metabolismo , Técnicas In Vitro , Camundongos , Reação em Cadeia da Polimerase , Tendões/citologiaRESUMO
To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification. Surprisingly, the micromass culture of the inTPCs rapidly underwent chondrogenic differentiation even in the absence of exogenous bone morphogenetic proteins or TGFßs. The cells isolated from human ruptured tendon tissues also showed connective tissue progenitor properties and exhibited stronger chondrogenic ability than bone marrow stromal cells. The mouse inTPCs contained two subpopulations one positive and one negative for CD105, a coreceptor of the TGFß superfamily. The CD105-negative cells showed superior chondrogenic potential in vitro and induced larger chondroid degenerative lesions in mice as compared to the CD105-positive cells. These findings indicate that tendon progenitor cells are recruited to the injured site of tendons and have a strong chondrogenic potential and that the CD105-negative population of these cells would be the cause for chondroid degeneration in injured tendons. The newly identified cells recruited to the injured tendon may provide novel targets to develop therapeutic strategies to facilitate tendon repair.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Tendões/citologia , Animais , Células Cultivadas , Condrogênese/fisiologia , Endoglina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Osteogênese/fisiologia , Tendões/metabolismoRESUMO
The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/ß-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active ß-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced ß-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the ß-catenin pathway.
Assuntos
Condrócitos/metabolismo , Receptores ErbB/metabolismo , Lâmina de Crescimento/embriologia , Via de Sinalização Wnt/fisiologia , Animais , Colagenases/biossíntese , Colagenases/genética , Receptores ErbB/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Lâmina de Crescimento/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/fisiologia , Ligante RANK/genética , Ligante RANK/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of ß-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of ß-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after ß-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of ß-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a ß-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable ß-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of ß-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.
Assuntos
Neoplasias Ósseas/patologia , Condroma/patologia , Periósteo/patologia , beta Catenina/deficiência , Fosfatase Ácida/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/metabolismo , Cartilagem/diagnóstico por imagem , Cartilagem/patologia , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/transplante , Condroma/diagnóstico por imagem , Condroma/metabolismo , Coristoma/diagnóstico por imagem , Coristoma/patologia , Colágeno Tipo II/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Humanos , Marcação In Situ das Extremidades Cortadas , Indóis/farmacologia , Integrases/metabolismo , Isoenzimas/metabolismo , Camundongos , Osteocondroma/metabolismo , Osteocondroma/patologia , Oximas/farmacologia , Periósteo/diagnóstico por imagem , Periósteo/efeitos dos fármacos , Periósteo/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Radiografia , Costelas/patologia , Tamoxifeno/farmacologia , Fosfatase Ácida Resistente a Tartarato , beta Catenina/metabolismoRESUMO
Heparan sulfate (HS) is an essential component of cell surface and matrix-associated proteoglycans. Due to their sulfation patterns, the HS chains interact with numerous signaling proteins and regulate their distribution and activity on target cells. Many of these proteins, including bone morphogenetic protein family members, are expressed in the growth plate of developing skeletal elements, and several skeletal phenotypes are caused by mutations in those proteins as well as in HS-synthesizing and modifying enzymes. The disease we discuss here is hereditary multiple exostoses (HME), a disorder caused by mutations in HS synthesizing enzymes EXT1 and EXT2, leading to HS deficiency. The exostoses are benign cartilaginous-bony outgrowths, form next to growth plates, can cause growth retardation and deformities, chronic pain and impaired motion, and progress to malignancy in 2-5% of patients. We describe recent advancements on HME pathogenesis and exostosis formation deriving from studies that have determined distribution, activities and roles of signaling proteins in wild-type and HS-deficient cells and tissues. Aberrant distribution of signaling factors combined with aberrant responsiveness of target cells to those same factors appear to be a major culprit in exostosis formation. Insights from these studies suggest plausible and cogent ideas about how HME could be treated in the future.
Assuntos
Osso e Ossos , Exostose Múltipla Hereditária , Heparitina Sulfato , Desenvolvimento Musculoesquelético/genética , N-Acetilglucosaminiltransferases , Animais , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Exostose Múltipla Hereditária/enzimologia , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/patologia , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismoRESUMO
OBJECTIVES: Back pain subsequent to intervertebral disc (IVD) injury is a common clinical problem. Previous work examining early molecular changes post injury mainly used a candidate marker approach. In this study, gene expression in the injured and intact mouse tail IVDs was determined with a nonbiased whole transcriptome approach. DESIGN: Mouse tail IVD injury was induced by a needle puncture. Whole murine transcriptome was determined by RNASeq. Transcriptomes of injured IVDs were compared with those of intact controls by bioinformatic methods. RESULTS: Among the 18,078 murine genes examined, 592 genes were differentially expressed (P.adj < 0.01). Novel genes upregulated in injured compared with intact IVDs included Chl1, Lum, etc. Ontology study of upregulated genes revealed that leukocyte migration was the most enriched biological process, and network analysis showed that Tnfa had the most protein-protein interactions. Novel downregulated genes in the injured IVDs included 4833412C05Rik, Myoc, etc. The most enriched downregulated pathways were related to cytoskeletal organization. CONCLUSION: Novel genes highly regulated post disc injury were identified with an unbiased approach; they may serve as biomarkers of injury and response to treatments in future experiments. Enriched biological pathways and molecules with high numbers of connections may be targets for treatments post injury.
RESUMO
Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra-articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site.
Assuntos
Cartilagem Articular/fisiologia , Articulações/fisiologia , Regeneração , Animais , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese/fisiologia , Humanos , Articulações/transplante , Modelos Animais , Fenótipo , Transplante de Células-Tronco/métodos , Células-TroncoRESUMO
Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2CreERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.
RESUMO
Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1(f/f) and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1(f/f) mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1(f/f) mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;ß-catenin(f/f) mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/ß-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth.
Assuntos
Extremidades/embriologia , Heparitina Sulfato/biossíntese , Articulações/embriologia , N-Acetilglucosaminiltransferases/fisiologia , Coluna Vertebral/embriologia , Membrana Sinovial/embriologia , Animais , Apoptose , Diferenciação Celular , Colágeno/genética , Camundongos , N-Acetilglucosaminiltransferases/análiseRESUMO
OBJECTIVE: Although mouse osteoarthritis (OA) models are widely used, their histological analysis may be susceptible to arbitrariness and inter-examiner variability in conventional methods. Therefore, a method for the unbiased scoring of OA histology is needed. In this study, as the first step for establishing this system, we developed a computer-vision algorithm that automatically detects the medial and lateral compartments of mouse knee sections in a rigorous and unbiased manner. DESIGN: A total of 706 images of coronal sections of mouse knee joints stained by hematoxylin and eosin, safranin O, or toluidine blue were randomly divided into training and validation images at a ratio of 80:20. A model to detect both compartments automatically was built by machine learning using a single-shot multibox detector (SSD) algorithm with training images. The model was tested to determine whether it could accurately detect both compartments by analyzing the validation images and 52 images of sections stained with Picrosirius red, a method not used for the training images. RESULTS: The trained model accurately detected both medial and lateral compartments of all 140 validation images regardless of the staining method employed, severity of articular cartilage defects, and the anatomical positions and conditions of the sections. Our model also correctly detected both compartments of 50 of 52 Picrosirius red-stained images. CONCLUSIONS: By applying deep learning based on the SSD algorithm, we successfully developed a model that detects the locations of the medial and lateral compartments of tissue sections of mouse knee joints with high accuracy.