RESUMEN
Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.
Asunto(s)
Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteoglicanos/metabolismo , Animales , Sitios de Unión , Bovinos , Colágeno/química , Colágeno/ultraestructura , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/deficiencia , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Unión Proteica , Proteoglicanos/química , Proteoglicanos/deficiencia , Cuerpo Vítreo/química , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/ultraestructuraRESUMEN
Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of ß1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1ß1-, α2ß1- and α10ß1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.
Asunto(s)
Condrocitos/fisiología , Colágenos Fibrilares/química , Cadenas alfa de Integrinas/química , Integrina alfa1beta1/química , Integrina alfa2beta1/química , Animales , Cartílago Articular/citología , Bovinos , Adhesión Celular , Células Cultivadas , Embrión de Pollo , Receptores con Dominio Discoidina/fisiología , Colágenos Fibrilares/fisiología , Humanos , Proteínas Inmovilizadas/química , Cadenas alfa de Integrinas/fisiología , Integrina alfa1beta1/fisiología , Integrina alfa2beta1/fisiología , Unión ProteicaRESUMEN
OBJECTIVE: Interterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor ß (TGFß), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFß in decorin-deficient (Dcn-/-) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA). METHODS: Unchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor ß-1 (TGFß1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFß1 treatment. In addition, OA was induced in Dcn-/- and wild-type (WT) mice via forced exercise on a treadmill. RESULTS: AFM analysis revealed a strikingly higher compressive stiffness in Dcn-/- than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFß1. Increased TGFß signalling led to enhanced Chst11 sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice. CONCLUSIONS: Our study demonstrates that the disruption of decorin-restricted TGFß signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.
Asunto(s)
Artritis Experimental/genética , Cartílago Articular/metabolismo , Decorina/genética , Osteoartritis/genética , Condicionamiento Físico Animal/métodos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Artritis Experimental/etiología , Artritis Experimental/metabolismo , Fenómenos Biomecánicos , Decorina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glicosaminoglicanos/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Osteoartritis/etiología , Osteoartritis/metabolismo , Condicionamiento Físico Animal/efectos adversos , ARN Mensajero/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage.
Asunto(s)
Cartílago Articular/ultraestructura , Colágenos Fibrilares/química , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patología , Cartílago Articular/patología , Articulación de la Cadera/metabolismo , Articulación de la Cadera/patología , Humanos , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Microscopía Electrónica de Rastreo , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Rodilla/metabolismoRESUMEN
Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.
Asunto(s)
Cartílago/enzimología , Condrocitos/enzimología , Estrés del Retículo Endoplásmico , Placa de Crecimiento/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Maduración Sexual , Animales , Cartílago/patología , Condrocitos/patología , Placa de Crecimiento/patología , Ratones , Ratones Noqueados , Proteína Disulfuro Isomerasas/genética , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND AND PURPOSE: The chronological development and natural history of cerebral aneurysms (CAs) remain incompletely understood. We used (14)C birth dating of a main constituent of CAs, that is, collagen type I, as an indicator for biosynthesis and turnover of collagen in CAs in relation to human cerebral arteries to investigate this further. METHODS: Forty-six ruptured and unruptured CA samples from 43 patients and 10 cadaveric human cerebral arteries were obtained. The age of collagen, extracted and purified from excised CAs, was estimated using (14)C birth dating and correlated with CA and patient characteristics, including the history of risk factors associated with atherosclerosis and potentially aneurysm growth and rupture. RESULTS: Nearly all CA samples contained collagen type I, which was <5 years old, irrespective of patient age, aneurysm size, morphology, or rupture status. However, CAs from patients with a history of risk factors (smoking or hypertension) contained significantly younger collagen than CAs from patients with no risk factors (mean, 1.6±1.2 versus 3.9±3.3 years, respectively; P=0.012). CAs and cerebral arteries did not share a dominant structural protein, such as collagen type I, which would allow comparison of their collagen turnover. CONCLUSIONS: The abundant amount of relatively young collagen type I in CAs suggests that there is an ongoing collagen remodeling in aneurysms, which is significantly more rapid in patients with risk factors. These findings challenge the concept that CAs are present for decades and that they undergo only sporadic episodes of structural change.
Asunto(s)
Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Colágeno Tipo I/metabolismo , Aneurisma Intracraneal/metabolismo , Aneurisma Intracraneal/patología , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Hipertensión/metabolismo , Hipertensión/patología , Masculino , Persona de Mediana Edad , Factores de Riesgo , Fumar/efectos adversos , Fumar/metabolismoRESUMEN
The prevalence of unruptured intracranial aneurysms (UIAs) in the general population is up to 3%. Existing epidemiological data suggests that only a small fraction of UIAs progress towards rupture over the lifetime of an individual, but the surrogates for subsequent rupture and the natural history of UIAs are discussed very controversially at present. In case of rupture of an UIA, the case fatality is up to 50%, which therefore continues to stimulate interest in the pathogenesis of cerebral aneurysm formation and progression. Actual data on the chronological development of cerebral aneurysm has been especially difficult to obtain and, until recently, the existing knowledge in this respect is mainly derived from animal or mathematical models or short-term observational studies. Here, we review the current data on cerebral aneurysm formation and progression as well as a novel approach to investigate the developmental chronology of cerebral aneurysms.
Asunto(s)
Aneurisma Intracraneal/etiología , Aneurisma Intracraneal/patología , Progresión de la Enfermedad , Humanos , Modelos Teóricos , Factores de RiesgoRESUMEN
Corneal stroma contains an extracellular matrix of orthogonal lamellae formed by parallel and equidistant fibrils with a homogeneous diameter of ~35 nm. This is indispensable for corneal transparency and mechanical functions. However, the mechanisms controlling corneal fibrillogenesis are incompletely understood and the conditions required for lamellar stacking are essentially unknown. Under appropriate conditions, chick embryo corneal fibroblasts can produce an extracellular matrix in vitro resembling primary corneal stroma during embryonic development. Among other requirements, cross-links between fibrillar collagens, introduced by tissue transglutaminase-2, are necessary for the self-assembly of uniform, small diameter fibrils but not their lamellar stacking. By contrast, the subsequent lamellar organization into plywood-like stacks depends on lysyl aldehyde-derived cross-links introduced by lysyl oxidase activity, which, in turn, only weakly influences fibril diameters. These cross-links are introduced at early stages of fibrillogenesis. The enzymes are likely to be important for a correct matrix deposition also during repair of the cornea.
Asunto(s)
Proteínas Aviares/metabolismo , Colágeno/metabolismo , Sustancia Propia/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Transglutaminasas/metabolismo , Aminopropionitrilo/farmacología , Animales , Proteínas Aviares/química , Técnicas de Cultivo de Célula , Células Cultivadas , Embrión de Pollo , Colágeno/química , Sustancia Propia/citología , Sustancia Propia/embriología , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/química , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/químicaRESUMEN
BACKGROUND AND PURPOSE: There is a controversy about the time span over which cerebral aneurysms develop. In particular, it is unknown whether collagen in ruptured aneurysms undergoes more rapid turnover than in unruptured aneurysms.(14)C birth dating of collagen could be used to address this question. METHODS: Aneurysmal domes from patients undergoing surgical treatment for ruptured or unruptured aneurysms were excised. Aneurysmal collagen was isolated and purified after pepsin digestion. Collagen from mouse tendons served as controls. F(14)C levels in collagen were analyzed by accelerator mass spectrometry and correlated with patient age and aneurysm size. RESULTS: Analysis of 10 aneurysms from 9 patients (6 ruptured, 3 unruptured) revealed an average aneurysm collagen age of <5 years, generally irrespective of patient age and aneurysm size or rupture status. Interestingly, F(14)C levels correlated with patient age as well as aneurysm size in ruptured aneurysm collagen samples. CONCLUSIONS: Our preliminary data suggest that collagen extracted from intracranial aneurysms generally has a high turnover, associated with aneurysm size and patient age. The correlation of patient age and aneurysm F(14)C levels could explain models of aneurysm development. Although preliminary, our findings may have implications for the biological and structural stability of ruptured and unruptured intracranial aneurysms.
Asunto(s)
Aneurisma Roto/diagnóstico , Carbono/análisis , Colágeno/química , Aneurisma Intracraneal/diagnóstico , Datación Radiométrica/métodos , Factores de Edad , Anciano , Aneurisma Roto/metabolismo , Radioisótopos de Carbono , Colágeno/metabolismo , Estudios de Factibilidad , Humanos , Aneurisma Intracraneal/metabolismo , Espectrometría de Masas , Persona de Mediana Edad , Proyectos Piloto , Factores de TiempoRESUMEN
OBJECTIVE: Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component. METHODS: Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using ß-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed. RESULTS: In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression. CONCLUSION: Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.
Asunto(s)
Desarrollo Óseo/fisiología , Fracturas del Fémur/fisiopatología , Curación de Fractura/fisiología , Sindecano-4/fisiología , Animales , Diferenciación Celular/fisiología , Condrocitos/citología , Condrocitos/fisiología , Femenino , Fémur/citología , Fémur/embriología , Fémur/fisiología , Placa de Crecimiento/citología , Placa de Crecimiento/embriología , Placa de Crecimiento/fisiología , Inflamación/fisiopatología , Operón Lac/genética , Masculino , Ratones , Ratones Noqueados , Osteogénesis/fisiología , Embarazo , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Sindecano-2/genética , Sindecano-2/fisiología , Sindecano-4/genética , Tibia/citología , Tibia/embriología , Tibia/fisiologíaRESUMEN
The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.
Asunto(s)
Colágeno Tipo IV/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Adulto , Membrana Basal/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Collagen VI and WARP are extracellular structural macromolecules present in cartilage and associated with BM suprastructures in non-skeletal tissues. We have previously shown that in WARP-deficient mice, collagen VI is specifically reduced in regions of the peripheral nerve ECM where WARP is expressed, suggesting that both macromolecules are part of the same suprastructure. The object of this study was to conduct a detailed analysis of WARP-collagen VI interactions in vitro in cartilage, a tissue rich in WARP and collagen VI. Immunohistochemical analysis of mouse and human articular cartilage showed that WARP and collagen VI co-localize in the pericellular matrix of superficial zone articular chondrocytes. EM analysis on extracts of human articular cartilage showed that WARP associates closely with collagen VI-containing suprastructures. Additional evidence of an interaction is provided by immunogold EM and immunoblot analysis showing that WARP was present in collagen VI-containing networks isolated from cartilage. Further characterization were done by solid phase binding studies and reconstitution experiments using purified recombinant WARP and isolated collagen VI. Collagen VI binds to WARP with an apparent K(d) of approximately 22 nM and the binding site(s) for WARP resides within the triple helical domain since WARP binds to both intact collagen VI tetramers and pepsinized collagen VI. Together, these data confirm and extend our previous findings by demonstrating that WARP and collagen VI form high affinity associations in vivo in cartilage. We conclude that WARP is ideally placed to function as an adapter protein in the cartilage pericellular matrix.
Asunto(s)
Condrocitos/metabolismo , Colágeno Tipo VI/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Microfibrillas/metabolismo , Animales , Cartílago Articular/citología , Cartílago Articular/metabolismo , Cartílago Articular/ultraestructura , Bovinos , Colágeno Tipo VI/química , Colágeno Tipo VI/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/química , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Inmunoprecipitación , Ratones , Microfibrillas/química , Microfibrillas/ultraestructura , Unión ProteicaRESUMEN
OBJECTIVE: Mutations in human cartilage oligomeric matrix protein (COMP) cause multiple epiphyseal dysplasia or pseudoachondroplasia. Electron microscopic analyses of patient biopsy tissue have shown that, in most cases, mutated COMP is retained in granular or lamellar inclusions in the endoplasmic reticulum of chondrocytes. However, some mutations that do not interfere with protein trafficking, resulting in normal secretion of the mutated protein, have been identified. These mutations are likely to cause the chondrodysplasia phenotype, via events that occur after secretion. The aim of the present study was to identify such extracellular mechanisms associated with the pathogenesis of chondrodysplasias. METHODS: A mutated but secreted COMP variant, p.H587R, as well as wild-type COMP were recombinantly expressed and purified from cell culture supernatants. Since recent studies have shown that COMP can facilitate collagen fibrillogenesis in vitro, the effect of the p.H587R mutation on this process was determined by analyzing the kinetics of fibrillogenesis in vitro and determining the structure of the collagen fibrils formed by immunogold electron microscopy. RESULTS: Mutated p.H587R COMP accelerated fibril formation by type I collagen in vitro to a slightly greater extent than that with wild-type COMP. However, p.H587R COMP induced aggregation and disorganization of fibril intermediates and end products. Mixtures of cartilage collagens or of type XI collagen alone produced similar results. The addition of p.H587R COMP to preformed fibrils induced aggregation and fusion of the fibrils, whereas wild-type COMP had little effect. CONCLUSION: The mutant COMP variant p.H587R generally interferes with normal collagen organization during fibrillogenesis. This constitutes a novel pathogenetic mechanism of COMP-associated chondrodysplasias.
Asunto(s)
Colágeno/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Acondroplasia/genética , Acondroplasia/metabolismo , Proteína de la Matriz Oligomérica del Cartílago , Células Cultivadas , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Proteínas Matrilinas , Microscopía Electrónica , MutaciónRESUMEN
Extracellular matrices (ECM) not only serve as structural scaffolds in organs and tissues, but also determine critical cellular functions through cell-matrix interactions. These are mediated by cell surface receptors that recognise specific structural features of ECMs and, hence, overall physical properties of the acellular environment. ECM structures are subject to hierarchic organisations, which are tightly adapted to the functions of tissues and organs. Only a few specialised tasks are reserved for isolated ECM macromolecules. Instead, molecular ECM components attain their prominent functions only after polymerising into insoluble suprastructural elements, i.e. fibrils, microfibrils, or networks that, in turn, are assembled into regional tissue structures, such as fibres or basement membranes. As an outstanding feature, most, if not all, ECM suprastructures are co-polymers of more than one molecular species that differ in their identity and relative abundance. Thus, ECM suprastructures are composite biological amalgamates. The analogy to metal alloys refers to structural and functional characteristics of ECM composites, which differ from those of each homo-polymeric aggregate. At the tissue level, biological alloys can themselves be assembled into conglomerates that again assume properties distinct from those of each individual alloy. Nevertheless, most studies in matrix biology solely focus on molecular features and mechanisms. Progress has however been made in identifying principles of interactions within suprastructural elements and their functional consequences. We are now only beginning to understand the impact of suprastructural organisation on the assembly and the functions of whole tissues and many fundamental issues in this almost pristine field await discovery.
Asunto(s)
Membrana Basal/metabolismo , Matriz Extracelular/metabolismo , Animales , Membrana Basal/ultraestructura , Matriz Extracelular/ultraestructura , HumanosRESUMEN
The role of endogenous inducers of inflammation is poorly understood. To produce the proinflammatory master cytokine interleukin (IL)-1beta, macrophages need double stimulation with ligands to both Toll-like receptors (TLRs) for IL-1beta gene transcription and nucleotide-binding oligomerization domain-like receptors for activation of the inflammasome. It is particularly intriguing to define how this complex regulation is mediated in the absence of an infectious trigger. Biglycan, a ubiquitous leucine-rich repeat proteoglycan of the extracellular matrix, interacts with TLR2/4 on macrophages. The objective of this study was to define the role of biglycan in the synthesis and activation of IL-1beta. Here we show that in macrophages, soluble biglycan induces the NLRP3/ASC inflammasome, activating caspase-1 and releasing mature IL-1beta without the need for additional costimulatory factors. This is brought about by the interaction of biglycan with TLR2/4 and purinergic P2X(4)/P2X(7) receptors, which induces receptor cooperativity. Furthermore, reactive oxygen species formation is involved in biglycan-mediated activation of the inflammasome. By signaling through TLR2/4, biglycan stimulates the expression of NLRP3 and pro-IL-1beta mRNA. Both in a model of non-infectious inflammatory renal injury (unilateral ureteral obstruction) and in lipopolysaccharide-induced sepsis, biglycan-deficient mice displayed lower levels of active caspase-1 and mature IL-1beta in the kidney, lung, and circulation. Our results provide evidence for direct activation of the NLRP3 inflammasome by biglycan and describe a fundamental paradigm of how tissue stress or injury is monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas de la Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Receptores Purinérgicos P2/metabolismo , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Biglicano , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/inmunología , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Riñón/inmunología , Riñón/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Estructura Terciaria de Proteína/genética , Proteoglicanos/genética , Proteoglicanos/inmunología , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Obstrucción Ureteral/genética , Obstrucción Ureteral/inmunología , Obstrucción Ureteral/metabolismoRESUMEN
We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MS(n)). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di- and hexasaccharides were analyzed by ESI MS(n). MS(2) on bisulfated 4,5-Delta-HexAGalNAc revealed an additional sulfate ester group at 4,5-Delta-HexA. MS(2) data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5-Delta-HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS(1) mode indicated direct correlation between the sulfate distribution and HexA epimerization. MS(n) performed on ions that, according to mass calculation, correspond to pentasulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)], trisulfated [4,5-Delta-HexAGalNAc(GlcAGalNAc)(2)] with IdoA-derived 4,5-Delta-HexA at the nonreducing end, tetrasulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] and monosulfated [4,5-Delta-HexAGalNAc(IdoAGalNAc)(2)] with GlcA-derived 4,5-Delta-HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over-, regular, and under-sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.
Asunto(s)
Condroitín Liasas/metabolismo , Glicosaminoglicanos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Azufre/química , Espectrometría de Masas en Tándem/métodos , Secuencia de Carbohidratos , Decorina , Proteínas de la Matriz Extracelular/aislamiento & purificación , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Humanos , Oligosacáridos/análisis , Oligosacáridos/química , Oligosacáridos/metabolismo , Proteoglicanos/aislamiento & purificaciónRESUMEN
The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.
Asunto(s)
Colágeno Tipo I/química , Colágeno Tipo VII/química , Dermis/química , Epidermis/química , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Secuencias de Aminoácidos/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/patología , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica/genéticaRESUMEN
Spontaneous cervical artery dissection (sCAD) is a common cause of stroke in patients below 55 years of age. Hereditary connective tissue disorders, including Ehlers-Danlos syndrome type IV, have been associated with sCAD and suprastructural abnormalities of both collagen fibrils and elastic fibers have been found by transmission electron microscopy in the dermis of about 50% of sCAD patients. Here, we investigated dermal connective tissue abnormalities using a novel method. Transmission and immunogold electron microscopy were used to study mechanically generated fragments of dermal matrix suprastructures, in particular collagen fibrils. Analysis of dermal tissue of sCAD patients revealed structurally abnormal collagen fibrils with irregularly contoured surfaces and increased diameters, often associated with a faint or absent banding pattern. Interestingly, only a small number of fibrils displayed short abnormal sections along the length of the fibril. Collagens I and III were present in normal as well as abnormal sections of the fibrils.However, immunogold labeling for the two proteins was strongly increased in abnormal sections.A systematic blinded investigation of skin biopsies of 31 sCAD patients and 17 controls revealed abnormal collagen fibrils in 7 sCAD patients but none of the controls. We conclude that approximately 20% of sCAD patients show collagen fibril alterations, establishing a promising basis for further investigation of connective tissue aberrations in skin biopsies of sCAD patients.
Asunto(s)
Arterias Cerebrales/patología , Trastornos Cerebrovasculares/patología , Tejido Conectivo/patología , Piel/patología , Adulto , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Miofibrillas/patología , Extractos de Tejidos/químicaRESUMEN
For a large part, skeletal development, growth, and repair occur by endochondral ossification which comprises an orderly sequence of consecutive steps of proliferation and late differentiation of chondrocytes. After vascular invasion into hypertrophic cartilage, the tissue is remodelled into bone. At all stages, the process is under tight environmental control exerted by a combination of regulators, including nutritional supply and signalling through growth factors, hormones, and cell-matrix-interactions. Therefore, genetic elimination of collagen IX, a stabilizing component of the periphery of thin cartilage fibrils, is expected to compromise extracellular matrix properties and, hence, the chondrocyte environment required for normal cartilage development and homeostasis. Here, we have shown that growth plate cartilage morphology is markedly disturbed in mice lacking collagen IX. Abnormalities were most prominent in late proliferative, pre-hypertrophic, and hypertrophic zones whereas resting and early proliferative zones were less affected. In central epiphyseal regions of long bones, newborn animals show grossly abnormal areas with strongly reduced cell numbers, irregular distribution of glycosaminoglycans in the extracellular matrix, and a profoundly disturbed columnar arrangement of chondrocytes with an irregular beta1 integrin immunostaining. As a result, all long bones are shorter and broader in newborn Col9a1-/- mice. Remarkably, these abnormalities are attenuated in adult mice, but the number of cells per area still is too low due to reduced cell proliferation.
Asunto(s)
Cartílago/anomalías , Cartílago/crecimiento & desarrollo , Colágeno Tipo IX/deficiencia , Colágeno Tipo IX/metabolismo , Envejecimiento/fisiología , Animales , Cartílago/citología , Cartílago/metabolismo , Proliferación Celular , Forma de la Célula , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo IX/genética , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Integrina beta1/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Endochondral ossification comprises a cascade of cell differentiation culminating in chondrocyte hypertrophy and is negatively controlled by soluble environmental mediators at several checkpoints. Proteinases modulate this control by processing protein signals and/or their receptors. Here, we show that insulin-like growth factor I can trigger hypertrophic development by stimulating production and/or activation of proteinases in some populations of chick embryo chondrocytes. Cell surface targets of the enzymes include 1,25-dihydroxyvitamin D3 membrane-associated rapid response steroid receptor (1,25 D3 MARRS receptor), also known as ERp57/GRp58/ERp60. This protein is anchored to the outer surface of plasma membranes and inhibits late chondrocyte differentiation after binding of 1,25-dihydroxyvitamin D3. Upon treatment with insulin-like growth factor I, 1,25 D3 MARRS receptor is cleaved into two fragments of approximately 30 and 22 kDa. This process is abrogated along with hypertrophic development by E-64 or cystatin C, inhibitors of cysteine proteinases. Cell differentiation is enhanced by treatment with antibodies to 1,25 D3 MARRS receptor that either block binding of the inhibitory ligand 1,25-dihydroxyvitamin D3 or inactivate 1,25 D3 MARRS receptor left intact after treatment with proteinase inhibitors. Therefore, proteolytic shedding of 1,25 D3 MARRS receptor constitutes a molecular mechanism eliminating the 1,25-dihydroxyvitamin D3-induced barrier against late cartilage differentiation and is a potentially important step during endochondral ossification or cartilage degeneration in osteoarthritis.