RESUMO
OBJECTIVE: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA. DESIGN: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1. RESULTS: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo. CONCLUSION: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.
Assuntos
Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Predisposição Genética para Doença/genética , Osteoartrite/genética , Osteoartrite/fisiopatologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Condrócitos/patologia , Cromossomos Artificiais Bacterianos/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/fisiologia , Colágeno Tipo IX/genética , Colágeno Tipo IX/metabolismo , Colágeno Tipo IX/fisiologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Colágeno Tipo X/fisiologia , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Hipertrofia/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Type IX collagen is a structural protein which consists of the cartilage collagen II/IX/XI heteropolymer. Transgenic mice overexpressing a mutant alpha 1 (IX) collagen chain in cartilage develop a chondrodysplasia-like phenotype associated with early-onset osteoarthritis and spondylosis. Subsequent identifications of the human type IX collagen diseases proved the correctness of the work on mice. Now we know that type IX collagen gene mutations cause multiple epiphyseal dysplasia, an osteochondrodysplasia which frequently features early onset osteoarthritis. Polymorphic variants of the type IX collagen genes are associated with lumbar disc diseases. It is getting more important to understand the biological roles of collagen IX in the musculoskeletal system.
Assuntos
Doenças do Colágeno/genética , Colágeno Tipo IX/genética , Doenças Musculoesqueléticas/genética , Mutação , Animais , Colágeno Tipo IX/fisiologia , Humanos , Vértebras Lombares , Camundongos , Camundongos Transgênicos , Osteocondrodisplasias/genética , Polimorfismo Genético , Doenças da Coluna Vertebral/genética , Triptofano/genéticaRESUMO
cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.
Assuntos
Cóclea/fisiologia , Colágeno Tipo IX/fisiologia , Audição/fisiologia , Pró-Colágeno/fisiologia , Animais , Limiar Auditivo/fisiologia , Northern Blotting/métodos , Cóclea/citologia , Colágeno Tipo II/metabolismo , Colágeno Tipo IX/deficiência , Testes Auditivos/métodos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Colágeno/deficiência , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.
Assuntos
Cartilagem/citologia , Cartilagem/ultraestrutura , Condrócitos/metabolismo , Colágeno Tipo IX/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/genética , Glicoproteínas/fisiologia , Cálcio/metabolismo , Cartilagem/metabolismo , Cartilagem/patologia , Proteína de Matriz Oligomérica de Cartilagem , Condrócitos/citologia , Colágeno Tipo II/química , Colágeno Tipo IX/química , Nanismo/genética , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/química , Humanos , Proteínas Matrilinas , Microscopia de Fluorescência , Mutação , Fenótipo , Ligação Proteica , Dobramento de Proteína , Fatores de TempoRESUMO
Type IX collagen is one of the important components, together with type II, V, and XI collagens, in the tectorial membrane of the organ of Corti. To confirm the significance of type IX collagen for normal hearing, we assessed the detailed morphological and electrophysiological features of type IX collagen knock-out mice, which have recently been reported as a deafness model. Through assessment by auditory brainstem response (ABR), knock-out mice were shown to have progressive hearing loss. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape. These morphological changes started in the basal turn and were progressive toward the apical turn. Electron microscopy confirmed disturbance of organization of the collagen fibrils. These results suggest that mutations in type IX collagen genes may lead to abnormal integrity of collagen fibers in the tectorial membrane.
Assuntos
Colágeno Tipo IX/fisiologia , Perda Auditiva/genética , Camundongos Knockout/fisiologia , Estimulação Acústica/métodos , Animais , Limiar Auditivo/fisiologia , Tronco Encefálico/fisiopatologia , Colágeno Tipo IX/deficiência , Colágeno Tipo IX/genética , Progressão da Doença , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão/métodos , Membrana Tectorial/patologia , Membrana Tectorial/ultraestrutura , Fatores de TempoRESUMO
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.
Assuntos
Cartilagem/metabolismo , Colágeno Tipo IX/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cartilagem/química , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Condrócitos/metabolismo , Condrossarcoma/metabolismo , Colágeno/química , Colágeno/metabolismo , Colágeno Tipo IX/química , Colágeno Tipo IX/metabolismo , Cricetinae , Escherichia coli/metabolismo , Humanos , Imunoprecipitação , Cadeias alfa de Integrinas/biossíntese , Integrina alfa1/biossíntese , Integrina alfa2/biossíntese , Ligantes , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosAssuntos
Osteoartrite do Quadril/genética , Osteoartrite/genética , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Artroplastia de Substituição/métodos , Artroplastia de Substituição/tendências , Cartilagem Articular/química , Cartilagem Articular/metabolismo , Colágeno Tipo IX/genética , Colágeno Tipo IX/fisiologia , Proteínas da Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica/tendências , Genes/genética , Genes/fisiologia , Predisposição Genética para Doença/genética , Humanos , Masculino , Proteínas Matrilinas , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Osteoartrite/reabilitação , Osteoartrite/cirurgia , Osteoartrite/terapia , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/fisiologia , Proteínas/genética , Proteínas/fisiologia , Proteômica/tendências , Distribuição por SexoRESUMO
OBJECTIVE: The extracellular matrix (ECM) of hyaline cartilage contains an elaborated collagen fibrillar network, which is essential for the mechanical stability and the proper function of the tissue. Cartilage collagen fibrils consist of collagen II, the quantitatively minor collagens IX and XI, and several non-collagenous fibril-associated proteins. To understand the role some of these molecules in skeletal development, we have generated transgenic mouse strains harboring ablated genes for collagens II and IX, and matrilin-1. DESIGN: Mice lacking collagen II, collagen IX and matrilin-1 have been established earlier in our laboratory using standard techniques. To determine the consequences of the null mutations we used skeletal staining, histochemical and immunohistochemical assays, in situ hybridization and ultrastructural analysis. RESULTS: Transgenic mice deficient in collagen II (Col2a1-/-) die at birth and display a severely malformed skeleton characterized by abnormal endochondral ossification and impaired intervertebral disc development. Mice lacking collagen IX (Col9a1-/-) are viable and develop an osteoarthritis-like phenotype in knee joints between 9-12 months of age. To test the possibility that the reduction in collagen II content has an influence on the onset of degenerative changes of articular cartilage, we have generated mice, which are heterozygous for the collagen II null mutation and homozygous for the collagen IX null mutation. Col2a1+/- Col9a1-/- mice show no accelerated development of osteoarthritis compared with the collagen IX knockout animals. Finally, mice lacking matrilin-1, a non-collagenous glycoprotein that binds to both collagen fibrils and aggrecan, develop normally without detectable abnormalities in their skeleton. CONCLUSIONS: Our transgenic mouse strains carrying null mutations in genes encoding cartilage ECM proteins demonstrate that these proteins have different roles during skeletal development. Collagen II is important for cartilage formation, collagen IX for cartilage maintenance and matrilin-1 is redundant.
Assuntos
Cartilagem Articular/crescimento & desenvolvimento , Colágeno Tipo II/fisiologia , Colágenos Associados a Fibrilas/fisiologia , Animais , Colágeno Tipo IX/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Membro Anterior/anormalidades , Glicoproteínas/fisiologia , Membro Posterior/anormalidades , Proteínas Matrilinas , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Inclusão em Parafina , Fenótipo , Inclusão em Plástico , Coluna Vertebral/anormalidadesRESUMO
OBJECTIVE: Chondrocytes in the growth plate at different stages of differentiation synthesize characteristic extracellular matrix (ECM) components. Mutations in some ECM genes result in chondrodysplasia in humans and mice. We aimed to evaluate the impact of loss- and gain-of-function mutations of ECM genes on matrix structure, gene expression and formation of the growth plate. DESIGN: We review information on the impact of deficiencies in proteoglycans, and types X and II collagens on skeletal development. Additionally, we compare the impact of a glycine904 to cysteine (G904C) mutation in the triple helical coding domain of mouse Col2a1 with two previously reported Col2a1 mutations (exon7 deletion (Del1) and G85C). The G904C Col2a1 gene was introduced as a transgene into mice. Transgenic newborn mice were examined for skeletal development. The histology of the epiphyseal cartilage and the growth plate, and the ultrastructure of chondrocytes and collagen fibrillar morphology in the ECM were studied in 18.5-day transgenic and wild-type fetuses. The distribution of the mRNAs for Col2a1, Col11a1, Col9a1, Matn1, Agc and Ihh in the growth plate of 18.5-day G904C/G904C and wild type fetuses were compared by in situ hybridization. RESULTS: Heterozygous transgenic mice harbouring five copies of the G904C Col2a1 transgene developed skeletal abnormalities and dwarfism. Homozygous G904C/G904C mice died at birth, showing cleft palate, disrupted zonation of chondrocytes and reduction of the zone of hypertrophic chondrocytes. Fewer collagen fibrils were found in ECM of the cartilage. Rough endoplasmic reticulum of the chondrocytes of G904C/+ and G904C/G904C mice was distended. In G904C/G904C mutant mice, Agc gene activity was extended to the hypertrophic zone. Expression of the other genes studied was unchanged. Calcified materials that were not found normally in the maturing and only at low abundance in the hypertrophic zones of the wild type growth plate, were present in these zones in G904C/G904C mice. Despite phenotypic similarities for the G904C and Del1 mice, reduced expression of types I, II, IX, X collagens and aggrecan were reported for the latter mutation. Changes in gene activity and matrix organization in the growth plate also accompanied deficiencies in aggrecan, perlecan and collagen II. CONCLUSIONS: The data suggest that a single amino acid alteration in collagen II could lead to skeletal abnormalities through multiple secondary effects on the synthesis and assembly of ECM components. The functional impact of mutations of ECM genes reveals that chondrodysplasia is caused not just by the formation of abnormal matrix molecules, but that the alteration of one ECM component may lead to a cascade of disruption of other gene activities in chondrocytes which collectively contribute to the pathological changes in the architecture of the growth plate.
Assuntos
Cartilagem Articular/fisiologia , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/fisiologia , Mutação/genética , Agrecanas , Animais , Condrócitos/ultraestrutura , Colágeno Tipo I/fisiologia , Colágeno Tipo I/ultraestrutura , Colágeno Tipo II/fisiologia , Colágeno Tipo II/ultraestrutura , Colágeno Tipo IX/fisiologia , Colágeno Tipo IX/ultraestrutura , Colágeno Tipo X/fisiologia , Colágeno Tipo X/ultraestrutura , Expressão Gênica , Lâmina de Crescimento/anatomia & histologia , Hibridização In Situ , Lectinas Tipo C , Camundongos , Camundongos Transgênicos , Proteoglicanas/fisiologiaRESUMO
OBJECTIVE: Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation. This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions. We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes. DESIGN: Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources. The culture media either contained or lacked FBS. Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype). RESULTS: The cells often retained their differentiated phenotype only if cultured with serum. Under serum-free conditions, cartilage characteristics were lost. The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens. Shape changes were influenced by beta1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns. Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability. CONCLUSIONS: Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment. Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype. However, other suprastructures, e.g. in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype. The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage. If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further.