RESUMO
BACKGROUND: In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated. RESULTS: Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice. CONCLUSIONS: The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.
Assuntos
Decorina/metabolismo , Dermatan Sulfato/metabolismo , Desenvolvimento Embrionário , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Cartilagem/patologia , Cartilagem/ultraestrutura , Decorina/química , Decorina/genética , Técnicas de Introdução de Genes , Homozigoto , Camundongos Endogâmicos C57BL , CicatrizaçãoRESUMO
BACKGROUND: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals. METHODS: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts. RESULTS: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent. CONCLUSIONS: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.
Assuntos
Agrecanas/análise , Cartilagem Articular/química , Osteoartrite do Joelho/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/metabolismoRESUMO
BACKGROUND: Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of aggrecan result in impairment of disc function and the onset of degeneration. SCOPE OF REVIEW: This review summarizes current knowledge concerning the structure and function of aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc. MAJOR CONCLUSIONS: Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties. GENERAL SIGNIFICANCE: This review provides a basis for scientists and clinicians to understand and appreciate the central role of aggrecan in the function, degeneration and repair of the intervertebral disc.
Assuntos
Agrecanas/metabolismo , Envelhecimento/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Envelhecimento/patologia , Animais , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Relação Estrutura-AtividadeRESUMO
Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca(2+) signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Osteoclastos/metabolismo , Adolescente , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibrilina-1 , Fibrilinas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Osteoclastos/citologia , Fosforilação , Transdução de SinaisRESUMO
OBJECTIVES: Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo. METHODS: Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling. RESULTS: mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage. CONCLUSIONS: This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.
Assuntos
Autofagia/fisiologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Osteoartrite/metabolismo , Osteoartrite/prevenção & controle , Serina-Treonina Quinases TOR/deficiência , Regulação para Cima/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Cartilagem Articular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Cães , Inativação Gênica , Humanos , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVES: We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. METHODS: Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. RESULTS: Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. SIGNIFICANCE: PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.
Assuntos
Cartilagem Articular/metabolismo , Osteoartrite do Joelho/metabolismo , PPAR gama/genética , Serina-Treonina Quinases TOR/genética , Animais , Modelos Animais de Doenças , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , PPAR gama/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.
Assuntos
Envelhecimento , Proteínas da Matriz Extracelular/metabolismo , Degeneração do Disco Intervertebral/enzimologia , Serina Endopeptidases/metabolismo , Adolescente , Fatores Etários , Sítios de Ligação , Diagnóstico Diferencial , Matriz Extracelular/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Inflamação , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Pessoa de Meia-Idade , Peptídeo Hidrolases/metabolismoRESUMO
Metabolic bone disorders in children frequently are heritable, but the expanding number of genes associated with these conditions makes it difficult to perform molecular diagnosis. In the present study, we therefore evaluated a semiconductor (SC)-based sequencing system for this purpose. A total of 65 DNA samples were analyzed comprising 24 samples from patients with 27 known pathogenic mutations, 6 samples from patients with prior negative Sanger sequencing, and 35 consecutive samples from patients with suspected heritable metabolic bone disorders who had not had prior molecular diagnosis. In the samples with known pathogenic mutations, 26 of 27 mutations were identified by SC sequencing. All single nucleotide variants were correctly identified, but a 7-nucleotide duplication in CYP27B1 was not detected. SC sequencing revealed two pathogenic mutations in the six samples where prior Sanger sequencing had failed to identify a mutation. Finally, pathogenic mutations were found in 27 samples of patients with unknown mutation status (15 in COL1A1, 9 in COL1A2, 1 in LEPRE1, 1 in LRP5, 1 in PHEX). Subsequent Sanger sequencing confirmed the mutations in all 27 samples. In conclusion, we found that SC sequencing is suitable for the diagnosis of heritable metabolic bone disorders in children.
Assuntos
Osso e Ossos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Criança , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , DNA/genética , Predisposição Genética para Doença , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Glicoproteínas de Membrana/genética , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Pediatria , Prolil Hidroxilases , Proteoglicanas/genética , Semicondutores , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: Osteogenesis imperfecta (OI) type V is an autosomal dominant bone fragility disorder that we had described a decade ago. Recent research has shown that OI type V is caused by a recurrent c.-14C>T mutation in IFITM5. In the present study, we assessed all patients diagnosed with OI type V at our institutions for the presence of the IFITM5 mutation. METHODS: IFITM5 exon 1 was analysed by Sanger sequencing in genomic DNA from 42 patients with OI type V (age: 2-67 years; 18 female). RESULTS: The c.-14C>T mutation of IFITM5 was detected in all individuals. Indicators of disease severity varied widely: Height z-scores (n=38) ranged from -8.7 to -0.1, median -3.5. Median final height was 147 cm in men (N=15) and 145 cm in women (N=10). Lumbar spine areal bone mineral density z-scores in the absence of bisphosphonate treatment (n=29) were between -7.7 and -0.7, median -5.3. Scoliosis was present in 57%, vertebral compression fractures in 90% of patients. CONCLUSIONS: Even though the disease-causing mutation is identical among patients with OI type V, the interindividual phenotypic variability is considerable.
Assuntos
Proteínas de Membrana/genética , Mutação , Osteogênese Imperfeita/genética , Fenótipo , Adolescente , Adulto , Idoso , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese Imperfeita/diagnóstico , Radiografia , Adulto JovemRESUMO
The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.
Assuntos
Agrecanas/metabolismo , Envelhecimento/fisiologia , Cartilagem/metabolismo , Glicosaminoglicanos/metabolismo , Adulto , Agrecanas/ultraestrutura , Condroitina Liases/metabolismo , Glicosaminoglicanos/ultraestrutura , Glicosídeo Hidrolases/metabolismo , Humanos , Técnicas In Vitro , Recém-Nascido , Microscopia de Força AtômicaRESUMO
Intervertebral disc (IVD) degeneration is the most common cause of back pain. Presently there is no medical treatment, leaving surgery as the only offered option. Here we evaluate the potential of Link-N to promote extracellular matrix regeneration in human IVDs. Human disc cells cultured in alginate and intact human discs were exposed to a combination of Link-N and ³5SO4 in the presence or absence of interleukin (IL)-1, and the effect on proteoglycan synthesis was evaluated. In addition, message levels of aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, a Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS)-4 and ADAMTS-5 were evaluated in alginate cultures. Human disc cells responded in a dose dependent manner with maximal proteoglycan synthesis at 1 µg/mL Link-N. Link-N treatment also induced proteoglycan synthesis in intact human discs, and a prolonged effect was found up to one week after Link-N treatment. Message levels of proteinases were decreased by Link-N in the presence of IL-1. Thus, Link-N can promote proteoglycan synthesis and deplete proteinase expression in adult human discs. Link-N could therefore be a promising candidate for biologically-induced disc repair, and could provide an alternative to surgical intervention for early stage disc degeneration.
Assuntos
Proteínas da Matriz Extracelular/farmacologia , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Proteoglicanas/farmacologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Adolescente , Adulto , Idoso , Agrecanas/genética , Agrecanas/metabolismo , Animais , Bovinos , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Disco Intervertebral/efeitos dos fármacos , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/biossíntese , Proteoglicanas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al. (2000) Dev Biol 224(2):299-311; Stickens et al. (2005) Development 132(22):5055-5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.
Assuntos
Neoplasias Ósseas/etiologia , Condrócitos/metabolismo , Exostose Múltipla Hereditária/etiologia , N-Acetilglucosaminiltransferases/genética , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proliferação de Células , Condrócitos/patologia , Modelos Animais de Doenças , Éxons , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/patologia , Marcação de Genes , Homozigoto , Humanos , Perda de Heterozigosidade , Camundongos , Camundongos Transgênicos , Mutação , N-Acetilglucosaminiltransferases/antagonistas & inibidores , FenótipoRESUMO
Extracellular matrix (ECM) composition has an important role in determining airway structure. We postulated that ECM lung composition of chronic obstructive pulmonary disease (COPD) patients differs from that observed in smoking and nonsmoking subjects without airflow obstruction. We determined the fractional areas of elastic fibres, type-I, -III and -IV collagen, versican, decorin, biglycan, lumican, fibronectin and tenascin in different compartments of the large and small airways and lung parenchyma in 26 COPD patients, 26 smokers without COPD and 16 nonsmoking control subjects. The fractional area of elastic fibres was higher in non-obstructed smokers than in COPD and nonsmoking controls, in all lung compartments. Type-I collagen fractional area was lower in the large and small airways of COPD patients and in the small airways of non-obstructed smokers than in nonsmokers. Compared with nonsmokers, COPD patients had lower versican fractional area in the parenchyma, higher fibronectin fractional area in small airways and higher tenascin fractional area in large and small airways compartments. In COPD patients, significant correlations were found between elastic fibres and fibronectin and lung function parameters. Alterations of the major ECM components are widespread in all lung compartments of patients with COPD and may contribute to persistent airflow obstruction.
Assuntos
Matriz Extracelular/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adulto , Idoso , Biglicano/metabolismo , Estudos de Casos e Controles , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Decorina/metabolismo , Feminino , Fibronectinas/metabolismo , Humanos , Imuno-Histoquímica/métodos , Sulfato de Queratano/metabolismo , Lumicana , Pulmão/metabolismo , Pulmão/cirurgia , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Fumar/efeitos adversos , Tenascina/metabolismoRESUMO
Osteogenesis imperfecta type VI (OI type VI) is a rare autosomal recessive disorder caused by mutations in the SERPINF1 gene that encodes pigment epithelium-derived factor (PEDF). Cystinosis is an autosomal recessive lysosomal transport disorder caused by mutations in the CTNS gene. Both SERPINF1 and CTNS are located on chromosome 17p13.3. We describe an individual presenting with both OI type VI and cystinosis. The patient was diagnosed with cystinosis at the age of 11 months and OI type VI on bone biopsy at the age of 8 years. He has sustained over 30 fractures during his lifetime, and at the age of 19 years entered end-stage renal disease and subsequent renal transplant. An Affymetrix 6.0 array was used to look for areas of loss of heterozygosity on chromosome 17. Sequencing of the SERPINF1 and CTNS genes was performed, followed by quantitative PCR and Western blot of PEDF to characterize the identified mutation. A 6.58 Mb region of homozygosity was identified on the Affymetrix 6.0 array, encompassing both the SERPINF1 and CTNS genes. Sequencing of the genes identified homozygosity for a known pathogenic CTNS mutation and for a novel in-frame duplication in SERPINF1. Skin fibroblasts produced a markedly reduced amount of SERPINF1 transcript and PEDF protein. This patient has the concurrent phenotype of two rare recessive diseases, cystinosis and OI type VI. We identified for the first time an in-frame duplication in SERPINF1 that is responsible for the OI type VI phenotype in this patient.
Assuntos
Cistinose/complicações , Osteogênese Imperfeita/complicações , Adolescente , Adulto , Sistemas de Transporte de Aminoácidos Neutros/genética , Sequência de Bases , Criança , Cistinose/diagnóstico , Cistinose/genética , Proteínas do Olho/genética , Duplicação Gênica , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Fatores de Crescimento Neural/genética , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/genética , Serpinas/genética , Adulto JovemRESUMO
PURPOSE: Proteoglycans are important to the functioning of the intervertebral disc. In addition to aggrecan there are the small leucine-rich proteoglycans (SLRPs). These are less common but in other locations their functions include collagen organisation, sequestering growth factors and stimulating inflammation. We have performed a comparative analysis of the SLRP core protein species present in intervertebral discs with various pathologies. METHODS: Eighteen intervertebral discs from patients with scoliosis (n = 7, 19-53 years), degenerative disc disease (n = 6, 35-51 years) and herniations (n = 5, 33-58 years) were used in this study. Proteoglycans were dissociatively extracted from disc tissues and the SLRPs (biglycan, decorin, fibromodulin, keratocan and lumican) assessed by Western blotting following deglycosylation with chondroitinase ABC and keratanase. RESULTS: Intact SLRP core proteins and a number of core protein fragments were identified in most of the discs examined. Biglycan and fibromodulin were the most extensively fragmented. Keratocan generally occurred as two bands, one representing the intact core protein, the other a smaller fragment. The intact core protein of lumican was detected in all samples with fragmentation evident in only one of the older scoliotic discs. Decorin was less obvious in the disc samples and showed little fragmentation. CONCLUSION: In this cohort of pathological intervertebral discs, fragmentation of certain SLRP core proteins was common, indicating that some SLRPs are extensively processed during the pathological process. Identification of specific SLRP fragments which correlate with disc pathology may not only help understand their aetiopathogeneses, but also provide biomarkers which can be used to monitor disease progression or to identify particular disc disorders.
Assuntos
Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Leucina/metabolismo , Proteoglicanas/metabolismo , Escoliose/metabolismo , Adulto , Biglicano/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Estudos de Coortes , Decorina/metabolismo , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Humanos , Degeneração do Disco Intervertebral/patologia , Deslocamento do Disco Intervertebral/patologia , Sulfato de Queratano/metabolismo , Lumicana , Masculino , Pessoa de Meia-Idade , Escoliose/patologiaRESUMO
Mice possessing no Has2 expression in chondrocytes died near birth and displayed abnormalities throughout their skeleton. By embryonic day 18.5, the long bones were short and wide, and possessed excessive mineralization within their diaphysis, with little evidence of diaphyseal bone modeling. However, this does not appear to be associated with an absence of blood vessel invasion or the reduced presence of osteoclasts. There was no evidence for the formation of an organized growth plate between the epiphysis and diaphysis, and while hypertrophic chondrocytes were present in this region they were abnormal in both appearance and organization. There was also increased cellularity in the epiphyseal cartilage and a corresponding decrease in the abundance of extracellular matrix, but aggrecan was still present. Thus, hyaluronan production by chondrocytes is not only essential for formation of an organized growth plate and subsequent long bone growth but also for normal modeling of the diaphyseal bone.
Assuntos
Desenvolvimento Ósseo/fisiologia , Condrócitos/fisiologia , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Ácido Hialurônico/metabolismo , Animais , Osso e Ossos/anatomia & histologia , Osso e Ossos/patologia , Osso e Ossos/fisiologia , Cartilagem/citologia , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/citologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Hialuronan Sintases , Camundongos , Camundongos Knockout , FenótipoRESUMO
The present work describes a novel bovine disc organ culture system with long-term maintenance of cell viability, in which degenerative changes can be induced as a prelude to studying repair. Discs were isolated with three different techniques: without endplates (NEP), with bony endplates (BEP) and with intact cartilage endplates (CEP). Swelling, deformation, and cell viability were evaluated in unloaded cultures. Degeneration was induced by a single trypsin injection into the center of the disc and the effect on cell viability and matrix degradation was followed. Trypsin-treated discs were exposed to TGFß to evaluate the potential to study repair in this system. NEP isolated discs showed >75% maintained cell viability for up to 10 days but were severely deformed, BEP discs on the other hand maintained morphology but failed to retain cell viability having only 27% viable cells after 10 days. In CEP discs, both cell viability and morphology were maintained for at least 4 weeks where >75% of the cells were still viable. To mimic proteoglycan loss during disc degeneration, a single trypsin injection was administered to the center of the disc. This resulted in 60% loss of aggrecan, after 7 days, without affecting cell viability. When TGFß was injected to validate that the system can be used to study a repair response following injection of a bio-active substance, proteoglycan synthesis nearly doubled compared to baseline synthesis. Trypsin-treated bovine CEP discs therefore provide a model system for studying repair of the degenerate disc, as morphology, cell viability and responsiveness to bio-active substances were maintained.
Assuntos
Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/cirurgia , Disco Intervertebral/fisiologia , Disco Intervertebral/cirurgia , Técnicas de Cultura de Órgãos/métodos , Tripsina/farmacologia , Animais , Cartilagem Articular/patologia , Cartilagem Articular/fisiologia , Cartilagem Articular/cirurgia , Bovinos , Sobrevivência Celular/fisiologia , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/induzido quimicamente , MasculinoRESUMO
PURPOSE: The intervertebral discs become wedged and narrowed in scoliosis, and this may result from altered biomechanical environment. The effects of four permutations of disc compression, angulation and reduced mobility were studied to identify possible causes of progressive disc deformity in scoliosis. The purpose of this study was to document morphological and biomechanical changes in four different models of altered mechanical environment in intervertebral discs of growing rats and in a sham and control groups. METHODS: External rings were attached by percutaneous pins transfixing adjacent caudal vertebrae of 5-week-old Sprague-Dawley rats. Four experimental Groups of animals underwent permutations of the imposed mechanical conditions (A) 15° disc angulation, (B) angulation with 0.1 MPa compression, (C) 0.1 MPa compression and (R) reduced mobility (N = 20 per group), and they were compared with a sham group (N = 12) and control group (N = 8) (total of 6 groups of animals). The altered mechanical conditions were applied for 5 weeks. Intervertebral disc space was measured from micro-CT images at weeks 1 and 5. Post euthanasia, lateral bending stiffness of experimental and within-animal control discs was measured in a mechanical testing jig and collagen crimp was measured from histological sections. RESULTS: After 5 weeks, micro-CT images showed disc space loss averaging 35, 53, 56 and 35% of the adjacent disc values in the four intervention groups. Lateral bending stiffness was 4.2 times that of within-animal controls in Group B and 2.3 times in Group R. The minimum stiffness occurred at an angle close to the in vivo value, indicating that angulated discs had adapted to the imposed deformity, this is also supported by measurements of collagen crimping at concave and convex sides of the disc annuli. CONCLUSION: Loss of disc space was present in all of the instrumented discs. Thus, reduced mobility, that was common to all interventions, may be a major source of the observed disc changes and may be a factor in disc deformity in scoliosis. Clinically, it is possible that rigid bracing for control of scoliosis progression may have secondary harmful effects by reducing spinal mobility.
Assuntos
Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/fisiologia , Amplitude de Movimento Articular/fisiologia , Escoliose/diagnóstico por imagem , Escoliose/fisiopatologia , Fatores Etários , Animais , Fenômenos Biomecânicos/fisiologia , Microambiente Celular/fisiologia , Colágeno/fisiologia , Força Compressiva/fisiologia , Modelos Animais de Doenças , Disco Intervertebral/patologia , Ratos , Ratos Sprague-Dawley , Escoliose/patologia , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/patologia , Coluna Vertebral/fisiopatologia , Estresse Mecânico , Microtomografia por Raio-X/métodosRESUMO
We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function.
Assuntos
Densidade Óssea/genética , Osso e Ossos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Fator 4 Ativador da Transcrição , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Osso e Ossos/citologia , Células COS , Calcificação Fisiológica/genética , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proliferação de Células , Chlorocebus aethiops , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Reguladores/genética , Zíper de Leucina/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Osteocalcina/biossíntese , Osteocalcina/genética , Osteoclastos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificaçãoRESUMO
Osteogenesis imperfecta (OI) is a heritable bone disorder characterized by fractures with minimal trauma. Intracranial hemorrhage has been reported in a small number of OI patients. Here we describe three patients, a boy (aged 15 years) and two girls (aged 17 and 7 years) with OI type III who suffered intracranial hemorrhage and in addition had brachydactyly and nail hypoplasia. In all of these patients, OI was caused by glycine mutations affecting exon 49 of the COL1A2 gene, which codes for the most carboxy-terminal part of the triple-helical domain of the collagen type I alpha 2 chain. These observations suggest that mutations in this region of the collagen type I alpha 2 chain carry a high risk of abnormal limb development and intracranial bleeding.